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[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "developmental biology" ]

[ [ "Longitudinal growth of limbs and vertebrae depends on division and differentiation of chondrocytes within the cartilage growth plates located at each end of the growing bone ( see Figure 2B ) , resulting in formation of a scaffold that is subsequently mineralized ( Kozhemyakina et al . , 2015 ) .", "These processes are tightly controlled by multiple regulatory pathways .", "One such regulator is the natriuretic peptide-stimulated guanylyl cyclase , natriuretic peptide receptor 2 ( NPR2 ) , also known as guanylyl cyclase B , which is found in growth plate chondrocytes and promotes bone elongation ( Yasoda et al . , 1998; Yamashita et al . , 2000; Chusho et al . , 2001; Tamura et al . , 2004; Bartels et al . , 2004 ) .", "Inactivating mutations in NPR2 , which reduce cGMP , result in severe shortening of bones in mice and people , causing the condition acromesomelic dysplasia type Maroteaux ( AMDM ) , in which height is reduced by ~30% ( Maroteaux et al . , 1971; Tamura et al . , 2004; Bartels et al . , 2004; Khan et al . , 2012; Geister et al . , 2013; Nakao et al . , 2015 ) .", "Conversely , activating mutations of NPR2 result in longer bones ( Miura et al . , 2012; Hannema et al . , 2013; Miura et al . , 2014 ) , opposite to what is seen with the AMDM patients .", "Activation of the NPR2 guanylyl cyclase requires the extracellular binding of C-type natriuretic peptide ( CNP ) and also the phosphorylation of multiple intracellular juxtamembrane serines and threonines ( Potter , 1998; Potter , 2011 ) ( Figure 1A ) .", "Previous studies have established a close correlation between NPR2 phosphorylation and NPR2 activity ( Abbey-Hosch et al . , 2005; Egbert et al . , 2014; Robinson et al . , 2017 ) , and if these phosphorylation sites are mutated to alanine , CNP-dependent guanylyl cyclase activity is reduced to only 6% of that of the wild type protein ( Potter and Hunter , 1998 ) .", "Correspondingly , if the seven serines and threonines are mutated to the phosphomimetic amino acid glutamate ( NPR2-7E ) , the maximal velocity of the CNP-dependent guanylyl cyclase activity is the same as that of the wild type enzyme , but does not decrease in response to stimuli that dephosphorylate and inactivate the wild type protein ( Yoder et al . , 2012; Shuhaibar et al . , 2016; Robinson et al . , 2017 ) ( Figure 1A ) .", "Our previous analysis of mice in which both copies of Npr2 were globally replaced with a sequence encoding NPR2-7E ( Npr27E/7E ) demonstrated that dephosphorylation of NPR2 is a physiological mediator of hormonal signaling in ovarian follicles ( Shuhaibar et al . , 2016 ) .", "These findings led us to investigate whether the phosphorylation of NPR2 could also be a mediator of growth factor signaling in bones .", "Another essential regulator of bone elongation is FGF receptor 3 ( FGFR3 ) ; activating mutations of FGFR3 inhibit bone growth , causing achondroplasia , in which human height is reduced by ~25% ( Horton et al . , 1978; Rousseau et al . , 1994; Shiang et al . , 1994; Naski et al . , 1998; Wang et al . , 1999; Lorget et al . , 2012; Lee et al . , 2017; Ornitz and Legeai-Mallet , 2017 ) .", "Conversely , mice and people lacking functional FGFR3 have longer bones ( Colvin et al . , 1996; Deng et al . , 1996; Makrythanasis et al . , 2014 ) .", "Intriguingly , the greatly reduced bone length seen with activating mutations of FGFR3 , resembles that seen with inactivating mutations of NPR2 , and activation of NPR2 by increasing CNP opposes the decrease in bone growth caused by activating mutations of FGFR3 ( Yasoda et al . , 2004; Yasoda et al . , 2009; Lorget et al . , 2012; Wendt et al . , 2015 ) .", "Likewise , the increased bone length seen with mice lacking FGFR3 resembles that seen with activating mutations of NPR2 ( Miura et al . , 2012; Hannema et al . , 2013; Miura et al . , 2014 ) .", "Furthermore , studies of fibroblasts and chondrogenic cells derived from embryonic carcinomas and chondrosarcomas have indicated that FGF signaling decreases NPR2 activity ( Chrisman and Garbers , 1999; Ozasa et al . , 2005; Robinson et al . , 2017 ) , and that the inactivation is due to dephosphorylation of NPR2 ( Robinson et al . , 2017 ) .", "These studies suggested to us that dephosphorylation and inactivation of NPR2 could be a mechanism by which FGF decreases elongation of bones in vivo .", "Here we tested this hypothesis by investigating the growth of bones in Npr27E/7E mice in which NPR2 cannot be inactivated by dephosphorylation , and found that their bones are longer than those of wild type mice .", "We then developed a live tissue imaging system for monitoring the guanylyl cyclase activity of NPR2 in intact growth plates from wild type and Npr27E/7E mice that express a FRET sensor for cGMP .", "Using this system , we found that FGF-induced dephosphorylation and inactivation of NPR2 decreases cGMP production in growth plate chondrocytes , thus contributing to FGF-dependent decreases in bone growth ." ], [ "Npr27E/7E mice , in which NPR2 is modified to mimic a constitutively phosphorylated protein ( Shuhaibar et al . , 2016 ) ( Figure 1A ) , have longer bones .", "Measurements at 8–16 weeks of age showed that the tails , femurs , tibias , and bodies of Npr27E/7E mice were 8% to 14% longer than wild type ( Figure 1B , E , F , G; Figure 1—source data 1 ) .", "Differences in limb and body length were seen as early as 4 weeks ( Figure 1E–G ) .", "The fifth caudal vertebra was 18% longer , as measured at 18 weeks ( Figure 1C , D; Figure 1—source data 1 ) .", "These results indicate that when NPR2 cannot be inactivated by dephosphorylation , endochondral bone growth is increased .", "The width of the cranium , which is dependent on membranous , not endochondral , bone growth was unaffected ( Figure 1H; Figure 1—source data 1 ) .", "The increases in body and bone length , which resulted from an inability of NPR2 to be inactivated by dephosphorylation , phenocopied those previously reported for mice in which NPR2 activity was increased by overexpression of the NPR2 agonist CNP or by an activating mutation in the NPR2 guanylyl cyclase domain ( Yasoda et al . , 2004; Miura et al . , 2012 ) and in a human patient with the same activating mutation in NPR2 , who was 14% taller than average at 15 years of age ( Miura et al . , 2012 ) .", "Because NPR2 is expressed in tissues outside of the bone growth plate , including the pituitary and gastro-intestinal tract ( Tamura and Garbers , 2003; Sogawa et al . , 2010 ) , we tested whether the longer bones of the Npr27E/7E mice resulted from a direct action on bone rather than an indirect action on other tissues .", "To do this , we cultured isolated tibia from 3 to 4 day old mice , such that we could measure growth rates in the absence of extrinsic hormonal or nutritional effects .", "When first dissected , Npr27E/7E and wild type tibia did not differ in length ( Figure 2A–C; Figure 2—source data 1 ) .", "Measurements were made by adding the lengths of the proximal and distal epiphyses and the calcified ossification center ( Figure 2B ) .", "The absence of a detectable difference in length is consistent with previous studies showing no difference in body or tibia length at birth comparing mice with inactivated NPR2 and wild type mice ( Tamura et al . , 2004 ) .", "Likewise , little or no difference in body or bone length is seen at birth of humans with inactivating NPR2 mutations ( Bartels et al . , 2004 ) .", "These previous studies indicate that NPR2 activity is not required for prenatal bone elongation .", "However , when bones of newborn mice were placed in culture , the rate of bone elongation over a 6 day period was reduced if the Npr2 gene was disrupted ( Tamura et al . , 2004 ) .", "Correspondingly , when cultured in the presence of 1 μM CNP and measured over a period of 6 days , the rate of bone elongation was greater for Npr27E/7E compared with wild type tibia ( Figure 2A , D; Figure 2—source data 1 ) .", "Because the growth plate is located within the epiphysis region ( Figure 2B ) , we also measured the rate of elongation within this specific region .", "For the proximal epiphysis , there was a 14% greater elongation rate for Npr27E/7E compared to wild type ( Figure 2E; Figure 2—source data 1 ) .", "These results show that Npr27E/7E mice have longer bones due to a direct effect of the mutations on NPR2 function within the bone itself .", "Interestingly , there was no effect of the Npr27E/7E mutations on bone elongation in medium without added CNP ( Figure 2F , G; Figure 2—source data 1 ) .", "This result suggests CNP produced by the bones of 3–10 day old mice is not sufficient to activate NPR2 , at least under our culture conditions .", "To establish a method to investigate the signaling mechanisms underlying the increased bone growth in Npr27E/7E mice , we developed a live imaging technique for monitoring NPR2 guanylyl cyclase activity in intact growth plates , using mice that globally express a sensor for cGMP , cGi500 ( Russwurm et al . , 2007; Thunemann et al . , 2013; Shuhaibar et al . , 2015 ) .", "NPR2 activity has been previously measured using homogenates of fetal tibia ( Yasoda et al . , 1998 ) and membrane preparations from chondrocyte cell lines ( Ozasa et al . , 2005; Robinson et al . , 2017 ) .", "The availability of mice expressing a sensor for cGMP , combined with the development of a new method to visualize chondrocytes in live and intact growth plates by confocal microscopy , allowed us to measure NPR2 activity under physiological conditions and with high spatial and temporal resolution .", "cGi500 is comprised of cyan fluorescent protein ( CFP ) and yellow fluorescent protein ( YFP ) , linked by a cGMP-binding domain; the sensitivity of cGi500 to cGMP is based on Förster resonance energy transfer ( FRET ) .", "Binding of cGMP to the linker domain causes the CFP and YFP domains to move farther apart .", "The proximity of CFP and YFP can be detected by imaging cells using a 440 nm laser to excite CFP; the energy emitted by CFP then excites YFP in proportion to the distance between the two fluorophores .", "Thus measurement of the relative intensities of the light emitted by CFP and YFP provides an indicator of the cGMP concentration .", "The CFP/YFP emission ratio is higher when the cGMP concentration is higher , with a half-maximal response at 500 nM cGMP , and a dynamic range of ~100 nM to ~3 μM cGMP ( Russwurm et al . , 2007 ) .", "Tibias were removed from newborn mice expressing cGi500 , and placed in culture on a Millicell membrane .", "After 1–2 days in culture , the growth plate was exposed by cutting away the overlying tissue ( Figure 3A ) , thus allowing imaging and rapid exchange of solutions .", "A tibia was then placed in a perfusion slide ( Figure 3B , C ) on the stage of a confocal microscope .", "Excitation of CFP resulted in fluorescence in all regions of the growth plate ( Figure 3D ) , allowing identification of chondrocytes at different stages of development , based on the shape of the cells: round ( also called resting ) , columnar ( also called proliferating ) , and hypertrophic ( Kozhemyakina et al . , 2015; Ornitz and Legeai-Mallet , 2017 ) .", "Columnar cells that are beginning to increase their volume , referred to as ‘prehypertrophic’ , express both NPR2 and FGFR3 ( Yamashita et al . , 2000; Chusho et al . , 2001; de Frutos et al . , 2007; Kozhemyakina et al . , 2015 ) .", "We used this columnar/prehypertrophic region for the FRET measurements ( Figure 3E ) , such that FGF effects on NPR2 activity could be investigated .", "To measure the CNP-dependent guanylyl cyclase activity of NPR2 , we first recorded baseline images of CFP and YFP emission in the absence of CNP .", "After establishing the baseline ratio , we perfused 100 nM CNP through the imaging chamber .", "100 nM is close to the EC50 for activation of NPR2 expressed in a HEK cell line ( Shuhaibar et al . , 2016 ) .", "In response to CNP , the CFP emission intensity increased and the YFP emission intensity decreased ( Figure 3F ) , resulting in a higher CFP/YFP ratio ( Figure 3G ) and indicating an increase in cGMP due to activation of NPR2 .", "Thus , confocal microscopy of isolated tibia expressing cGi500 allowed us to monitor NPR2 activity in the live and intact growth plate for the first time .", "The methods described here are useful for comparing rates of cGMP production by NPR2 under different experimental conditions as will be described below .", "However , they cannot be interpreted as a measure of cGMP levels in chondrocytes in an intact mouse , because CNP concentrations in the extracellular space around the cells of the growth plate in vivo are unknown .", "In our isolated tissue preparation , in which the tibia is sliced to expose the growth plate , CNP can diffuse between the extracellular space into the large volume of culture medium .", "Therefore the cGMP level in the absence of added CNP in the medium is not an indicator of the cGMP concentration in vivo .", "To investigate whether FGF signaling reduces NPR2 activity within growth plate chondrocytes , we incubated cGi500-expressing tibias with or without FGF18 , and imaged the fluorescence of chondrocytes in the columnar/prehypertrophic region .", "Among the many FGF isoforms , we used FGF18 because it is one of the two redundant FGF isoforms that function to activate FGFR3 in the growth plate ( Ornitz and Marie , 2015; Hung et al . , 2016 ) .", "In tibias without FGF18 pretreatment , perfusion of CNP through the imaging chamber caused a large increase in the concentration of cGMP ( Figure 4A; Figure 4—source data 1 ) .", "However , if the tibias were pretreated with FGF18 , the CNP-induced increase in cGMP was smaller ( Figure 4A , G; Figure 4—source data 1 ) .", "These results indicate that in chondrocytes within a living growth plate , FGF signaling reduces the activity of the NPR2 guanylyl cyclase .", "Similar results were obtained with FGF18 or control pretreatment times of 80–140 min ( Figure 4A , G ) , or 30–50 min ( Figure 4—figure supplement 1; Figure 4—source data 1 ) .", "The baseline CFP/YFP ratio before CNP perfusion was the same with and without FGF pretreatment , suggesting that FGF had no effect on the baseline cGMP concentration ( Figure 4A ) .", "However , since it was possible that the baseline cGMP level was lower than could be detected by the cGi500 sensor , we isolated epiphyseal regions from tibias prepared and cultured under the same conditions that were used for cGi500 imaging , and measured cGMP content using an ELISA .", "At this stage of limb development , almost all of the epiphysis is comprised of growth plate chondrocytes .", "Before CNP treatment , the cGMP content of the epiphysis was only 1–2% of that after CNP addition ( Figure 5A; Figure 5—source data 1 ) , and no significant effect of an 80 min preincubation with FGF was detected ( Figure 5B; Figure 5—source data 1 ) .", "These cGMP ELISA measurements also showed that in growth plates assayed after CNP addition , FGF pretreatment for 80 min decreased cGMP content to 40% of the initial level ( Figure 5A ) , consistent with the cGi500 measurements .", "Studies of the RCS chondrocyte cell line have shown that FGF signaling decreases NPR2 phosphorylation and activity , and that the decrease in activity depends on the decrease in phosphorylation ( Robinson et al . , 2017 ) .", "So far , we have not succeeded in establishing methods to measure the phosphorylation state of NPR2 from growth plate tissue , and thus have not been able to determine whether the dephosphorylation seen in the chondrocyte cell line also occurs in vivo .", "However , we were able to use the Npr27E/7E mice to test whether FGF reduces the guanylyl cyclase activity of NPR2 in growth plate chondrocytes by dephosphorylating NPR2 , as diagrammed in Figure 4B , C .", "As with wild type , growth plates from Npr27E/7E mice showed an increase in cGMP when exposed to CNP ( Figure 4D; Figure 4—source data 1 ) .", "In fact , the CNP-stimulated cGMP increase was greater for Npr27E/7E vs wild type ( Figure 4G; Figure 4—source data 1 ) , as would be expected if NPR2 in the wild type growth plate was partially dephosphorylated as a result of signaling by endogenous hormones or growth factors ( compare diagrams in Figure 4B and E ) .", "The higher NPR2 activity measured in the Npr27E/7E vs wild type growth plate was not explained by a difference in the amount of NPR2 protein , which was similar for the growth plate region of the two genotypes ( Figure 4—figure supplement 2 ) .", "Another possible explanation of the higher NPR2 activity observed for the Npr27E/7E genotype compared to wild type is that the 7E enzyme has a somewhat lower Km for GTP than the wildtype enzyme , although at the GTP concentrations found in most intact tissues ( Traut , 1994 ) , the difference in enzyme activity would be small .", "The 7E mutations also have no effect on the CNP concentration required to activate NPR2 to half its maximum value ( Shuhaibar et al . , 2016 ) .", "Importantly , in growth plates from Npr27E/7E mice , FGF pretreatment did not cause a decrease in CNP-stimulated cGMP production ( Figure 4D , G ) .", "This lack of response to FGF contrasts with the FGF-induced decrease in NPR2 activity that is seen with wild type ( Figure 4A , G ) , and indicates that because the NPR2-7E protein cannot be dephosphorylated , FGF signaling does not reduce its enzyme activity ( Figure 4F ) .", "These results support the conclusion that FGF signaling reduces cGMP production by dephosphorylating NPR2 in the growth plate .", "Our previous studies of NPR2 activity in ovarian follicles indicated that a PPP-family phosphatase mediates the dephosphorylation of NPR2 in response to luteinizing hormone ( Egbert et al . , 2014 ) .", "To test if a PPP-family phosphatase also mediates the FGF-induced dephosphorylation of NPR2 in the growth plate , we preincubated tibias from newborn mice with the phosphatase inhibitor cantharidin .", "At 100 μM , cantharidin selectively inhibits the PPP family phosphatases , PPP1 , PPP2 ( also known as PP2A ) , PPP4 , and PPP5 ( Swingle et al . , 2007; Pereira et al . , 2011 ) but does not inhibit PPP3 ( also known as PP2B or calcineurin ) ( Honkanen , 1993; Pereira et al . , 2011 ) or PPM ( also known as PP2C ) ( Li et al . , 1993 ) .", "Tibias from newborn mice expressing cGi500 were pre-incubated with or without 100 μM cantharidin , then with or without FGF-18 , then imaged before and after perfusion of CNP ( Figure 6 ) .", "Control tibias without cantharidin treatment showed the expected decrease in CNP-dependent cGMP production in response to FGF-18 ( Figure 6A , C; Figure 6—source data 1 ) .", "However , tibias that had been pre-incubated with cantharidin showed no difference in NPR2 activity comparing those that had or had not been treated with FGF-18 ( Figure 6B , C; Figure 6—source data 1 ) .", "These results show that like the Npr27E/7E mutation , cantharidin inhibits the decrease in cGMP production in response to FGF .", "These experiments provide independent evidence that preventing the dephosphorylation of NPR2 prevents the decrease in NPR2 activity , and also indicate that a PPP family phosphatase is responsible for the dephosphorylation ." ], [ "Our findings identify two new components of the signaling network by which FGFR3 and NPR2 signaling regulate bone growth .", "Firstly , our results indicate that phosphorylation of the NPR2 guanylyl cyclase promotes bone elongation; this conclusion is based on our finding that a mutation that prevents NPR2 dephosphorylation results in longer bones .", "Secondly , our results indicate that part of the mechanism by which FGF signaling inhibits bone elongation is by decreasing NPR2 phosphorylation and activity .", "The dephosphorylation and inactivation of NPR2 is mediated by a PPP-family phosphatase .", "However , mice lacking the FGFR3 receptor ( Colvin et al . , 1996; Deng et al . , 1996 ) show a greater increase in bone length ( ~7–40% ) than we report here for mice with the mutation that prevents dephosphorylation of NPR2 ( 8–14% ) , consistent with previous studies identifying multiple mechanisms by which FGF signaling opposes bone elongation ( Ornitz and Legeai-Mallet , 2017 ) .", "The working model in Figure 7 shows our findings in the context of other knowledge about the cross-talk between FGFR3 and NPR2 signaling .", "An important pathway by which FGFR3 signaling decreases bone elongation is by activation of MAP kinase ( see Ornitz and Legeai-Mallet , 2017 ) .", "An important pathway by which NPR2 guanylyl cyclase activity increases bone elongation is by elevating cGMP and activating the cGMP-dependent protein kinase PRKG2 ( Pfeifer et al . , 1996; Chikuda et al . , 2004 ) .", "Previous studies have established that application of CNP to increase NPR2 guanylyl cyclase activity in chondrocytes , thus elevating cGMP and stimulating cGMP-dependent protein kinase , inhibits FGF-induced MAP kinase activation ( Yasoda et al . , 2004; Krejci et al . , 2005; Ozasa et al . , 2005; Kamemura et al . , 2017 ) , which would counteract FGF-inhibition of bone growth ( solid blue line in Figure 7 ) .", "Other mechanisms ( for example , see Kawasaki et al . , 2008 ) may also contribute to how PRKG2 activity increases bone growth ( dotted blue line in Figure 7 ) .", "Our present findings identify another level of crosstalk between FGFR3 and NPR2 signaling , indicated by the orange box in Figure 7 .", "By causing dephosphorylation of NPR2 , FGF signaling reduces cGMP production , thus opposing bone elongation .", "How FGF signaling causes dephosphorylation of NPR2 remains to be determined; this could occur by inactivation of the kinase ( s ) that phosphorylate NPR2 , and/or by activation of the phosphatase ( s ) that dephosphorylate it .", "Our results indicate that the phosphatase ( s ) belong to the PPP family , but do not identify the particular PPP family member ( s ) .", "Activation of PPP2 is one possibility , since PPP2 is activated by FGF signaling in the RCS chondrocyte cell line ( Kolupaeva et al . , 2013 ) , and purified PPP2 can dephosphorylate NPR2 in vitro ( Potter , 1998 ) .", "Other groups have used fluorescence microscopy to quantify chondrocyte movement , division , and volume in live avian growth plate cartilage ( Li et al . , 2015 ) , and to measure chondrocyte density in fixed mouse mandibular cartilage ( He et al . , 2017 ) .", "However , the methods that we have developed for imaging the growth plate of mammalian bones are unique in that they allow rapid manipulation of the chemical environment surrounding the growth plate and real-time measurements of changes in signaling pathways within the intact tissue .", "These methods are broadly applicable to studies of signaling by other hormones and growth factors that might affect cGMP levels in growth plate chondrocytes .", "With mice expressing related FRET sensors for cAMP ( Calebiro et al . , 2009 ) , these methods could also be readily applied to studies of signaling by hormones such as parathyroid hormone related protein that regulate chondrocyte and osteoblast growth and differentiation by way of cAMP ( Chagin et al . , 2014; Kozhemyakina et al . , 2015; Esbrit et al . , 2016 ) .", "Ongoing clinical trials have shown that in patients with achondroplasia , in which skeletal dysplasia is caused by a constitutively active form of FGFR3 , stimulation of NPR2 by subcutaneous injection of a hydrolysis-resistant analog of CNP increases bone length ( Klag and Horton , 2016 ) .", "These results are consistent with the increased bone growth that results from increasing CNP in mouse models of achondroplasia ( Yasoda et al . , 2004; Yasoda et al . , 2009; Lorget et al . , 2012; Wendt et al . , 2015 ) .", "Studies using a mouse model overexpressing a constitutively active form of FGFR3 showed that applying CNP or a CNP analog in vivo or in vitro completely rescued the growth defect ( Yasoda et al . , 2004; Yasoda et al . , 2009; Wendt et al . , 2015 ) .", "However , in a study involving mice in which one allele of the Fgfr3 gene was replaced with a constitutively active form , as occurs in achondroplasia , it was found that treatment with the CNP analog increased the growth rate of cultured tibia only partially , from ~40% to~70% of wild type ( Lorget et al . , 2012 ) .", "Such incomplete rescue might be expected , considering that NPR2 guanylyl cyclase activity depends not only on the presence of CNP but also on phosphorylation of multiple regulatory serines and threonines of NPR2 ( Figure 1A ) .", "CNP levels are elevated in patients with achondroplasia , suggesting that NPR2 in their chondrocytes is resistant to CNP ( Olney et al . , 2015 ) , which could be due to NPR2 dephosphorylation .", "Thus , if NPR2 phosphorylation could be increased in chondrocytes of these patients , by inhibiting the phosphatase that dephosphorylates NPR2 , this could potentially enhance the therapeutic stimulation of NPR2 activity by CNP as a treatment for achondroplasia .", "Our findings with the phosphatase inhibitor cantharidin support the concept of using related phosphatase inhibitors ( Lu et al . , 2009; Chung et al . , 2017 ) for treatment of achondroplasia ." ], [ "Two mouse lines were used for this study: Npr2-7E ( Shuhaibar et al . , 2016 ) , and cGi500 ( Thunemann et al . , 2013 ) .", "The genetic background of the Npr2-7E line was 75% C57BL/6 and 25% 129Sv .", "All of the mice that were used for Figure 1 were from the Npr2-7E line; about half of these mice were littermates from heterozygote x heterozygote breeding pairs .", "The genetic background of the cGi500 line was >90% C57BL/6 .", "All experiments were conducted as approved by the University of Connecticut Health Center and the University of Minnesota animal care committees .", "Tail lengths of live mice were measured at 3 week intervals .", "The lengths of the fifth caudal vertebrae were measured from euthanized mice , using a Faxitron cabinet x-ray system .", "Body lengths were measured from the tip of the nose to the base of the tail , using euthanized mice and a digital caliper .", "Femur and tibia lengths and cranial width were measured using excised bones and a digital caliper .", "For all measurements , there were approximately equal numbers of males and females of each genotype .", "Tibias were dissected from 3 to 4 day old mice , and cultured as previously described ( Tamura et al . , 2004 ) on Millicell organotypic membranes ( PICMORG50; Merck Millipore Ltd , Cork , IRL ) , in BGJb medium ( Fitton-Jackson modification ) ( Life Technologies , Grand Island , NY ) with 0 . 1% BSA , 100 units/ml each of penicillin and streptomycin , with or without 1 μM CNP ( Phoenix Pharmaceuticals , Burlingame , CA ) .", "The medium was changed every 2 days .", "Tibias were photographed using a Leica stereoscope , for measurement of length before and after a 6 day culture period , as shown in Figure 2B .", "Measurements were done using ImageJ software ( National Institutes of Health , Bethesda , MD ) .", "Cyclic GMP production was measured using tibias dissected from newborn mice ( day 0–1 ) that globally expressed one or two copies of the cGi500 FRET sensor inserted into the Rosa26 locus ( Thunemann et al . , 2013 ) .", "Isolated tibias were placed on Millicell membranes , in the medium described above , without CNP , and were used for imaging over the next 2 days .", "Prior to imaging , the epiphysis region of the proximal end of the tibia was slit with a razor blade to expose the growth plate ( Figure 3A ) .", "This was accomplished by placing the tibia , dorsal side up , in a 500 µm deep channel in a plastic slide ( ibidi USA , Fitchburg , WI; cat . no . 80176 , special order with no adhesive ) .", "The depth of the channel was modified from 400 to 500 µm by adding a piece of tape on each side of the channel .", "The epiphysis region was then slid through the edge of a razor blade on the slide surface .", "This procedure resulted in a 500 µm thick piece of tissue underlying the growth plate surface .", "The overlying flap of tissue was trimmed away , and the tibia was placed in a 4-well plate ( Nunc 176740; Thermo Scientific , Rochester , NY ) containing 0 . 5–1 ml of medium/well at 37°C with 5% CO2 , on a rotating platform .", "Where indicated , FGF18 ( PeproTech , Rocky Hill , NJ ) was added at a saturating concentration ( 0 . 5 µg/ml ) , along with 1 µg/ml heparin ( Sigma-Aldrich , H4784 , St . Louis , MO ) .", "Heparin was included because it enhances FGF receptor activation ( Ornitz and Marie , 2015 ) .", "Control samples were incubated with heparin only .", "For phosphatase inhibitor experiments , cantharidin ( Tocris BioScience , Bristol UK ) was dissolved at 50 mM in DMSO and diluted in media to 100 μM .", "Control tibias were incubated with 0 . 2% DMSO .", "After the indicated pre-incubations , the distal half of the tibia was cut off , and the proximal piece placed , cut edge up , in a 600 µm deep channel in a plastic slide with access wells for perfusion ( ibidi USA; cat . no . 80186; special order with no adhesive ) ( Figure 3B ) .", "Silicon grease was applied around the rim of the channel .", "A coverslip was prepared by adhering to its surface two 200 μm thick pads that were separated by a 1–1 . 5 mm space .", "The pads were made from 2 layers of Scotch double sided tape , each 100 µm thick .", "The coverslip was then placed over the tibia , such that the bone spanned the two pads and the surface of the growth plate was separated by ~200 µm from the surface of the coverslip ( Figure 3C ) .", "The coverslip was pressed down gently against the silicon grease on the perfusion slide , such that the uncut surface of the tibia was held against the perfusion slide .", "The slide was then inverted and filled with media , by way of ports on the slide .", "This resulted in an assembly in which media could be flowed under the bridge formed by the tibia resting on the tape pads .", "For each experiment , the separation of the growth plate surface from the coverslip was confirmed by measuring the distance between these surfaces using the confocal microscope software .", "The growth plate was imaged using a Pascal confocal system ( Carl Zeiss Microscopy , Thornwood , NY ) .", "Imaging was performed at ~34°C .", "The whole growth plate was imaged with a 10x/0 . 45 N . A . water immersion objective ( Figure 3D ) .", "For FRET measurements , columnar/prehypertrophic chondrocytes were imaged using a 25x/0 . 8 NA water immersion objective ( Figure 3E ) .", "To avoid evaporation during time-lapse imaging , Immersol ( Carl Zeiss Microscopy ) was used to form the contact with the coverslip .", "The pinhole was set at maximum , resulting in an optical section thickness of ~24 µm .", "FRET measurements were performed and analyzed as previously described ( Norris et al . , 2009; Shuhaibar et al . , 2015 ) .", "Images were collected at zoom 0 . 7 , using 3 . 2 s scans at 30 s intervals , for 5 min before CNP perfusion and 15 min afterwards .", "Files were saved as 12-bit images .", "The fluorescence signal was 2–6 times the background level .", "Measurements were corrected for background and for spectral bleedthrough of light emitted by CFP into the YFP channel .", "Graphs show measurements from 490 × 490 μm regions .", "To prepare samples for cGMP ELISA measurements , tibias from newborn mice were treated as described in the legend for Figure 5 , and after the indicated incubations , 2–3 proximal epiphyses were cut off with a scissors and placed in a tube containing 200 μl of 0 . 1 M HCl .", "The samples were sonicated with a probe sonicator ( model 60 Sonic Dismembrator , ThermoFisher Scientific; 3 × 10 pulses ) , heated for 3 min at 95°C , sonicated again ( 3 × 10 pulses ) , stored at −80°C , and analyzed for protein content ( ~10 μg per epiphysis; Pierce BCA assay , ThermoScientific , Rockford IL ) and cGMP content ( ADI-900–014 cGMP ELISA kit , Enzo Life Sciences , Farmingdale NY ) .", "To prepare samples for western blotting ( Figure 4—figure supplement 2 ) , epiphyses were dissected from femurs and tibias of newborn mice ( day 0 ) , and slit to expose the growth plate .", "Protein was solubilized by heating the tissue in 1% SDS at 95°C for 10 min .", "Protein content was determined by a BCA assay ( ~10 μg per epiphysis ) .", "25 μg of protein was separated by SDS-PAGE electrophoresis , transferred to a nitrocellulose membrane , and probed with an antibody made in guinea pig against the extracellular domain of mouse NPR2 ( Ter-Avetisyan et al . , 2014 ) .", "The antibody was a gift from Hannes Schmidt ( University of Tübingen ) ." ] ]

[ "Activating mutations in fibroblast growth factor ( FGF ) receptor 3 and inactivating mutations in the NPR2 guanylyl cyclase both cause severe short stature , but how these two signaling systems interact to regulate bone growth is poorly understood .", "Here , we show that bone elongation is increased when NPR2 cannot be dephosphorylated and thus produces more cyclic GMP .", "By developing an in vivo imaging system to measure cyclic GMP production in intact tibia , we show that FGF-induced dephosphorylation of NPR2 decreases its guanylyl cyclase activity in growth plate chondrocytes in living bone .", "The dephosphorylation requires a PPP-family phosphatase .", "Thus FGF signaling lowers cyclic GMP production in the growth plate , which counteracts bone elongation .", "These results define a new component of the signaling network by which activating mutations in the FGF receptor inhibit bone growth ." ]

[ "Between birth and puberty , the bones of mammals grow drastically in length .", "This process is controlled by many proteins , and mutations affecting these proteins can cause bones to either be too long or too short .", "For example , mutations of a protein called the fibroblast growth factor receptor , or FGF for short , and a protein called NPR2 , can cause similar forms of dwarfism – a condition characterized by short stature .", "The FGF protein controls bone growth , and people with overactive receptors for FGF suffer from a form of dwarfism known as achondroplasia , while people that lack FGF receptors have longer bones .", "The NPR2 protein , on the other hand , produces a molecule called cGMP , which is necessary for the bones to grow .", "When NPR2 is blocked , less cGMP is produced , which results in shorter limbs .", "Previous studies of bone cells grown in the laboratory have shown that these two proteins are linked by a chain of chemical messages .", "When the FGF receptor is active , phosphate molecules are removed from the NPR2 protein , which reduces the amount of GMP produced .", "However , until now it was not known whether this mechanism also controls growth in actual bones .", "Here , Shuhaibar et al . used genetically modified mice in which the phosphate group could not be removed from their NPR2 enzyme .", "As a result , the bones of these mice were longer than usual .", "Shuhaibar et al . then developed an imaging technique to examine the region in the bone were growth happens .", "To see whether FGF reduces the amount of cGMP produced by NPR2 in these areas , cGMP was detected with a fluorescent sensor in order to be tracked .", "In normal mice , the FGF receptor reduced the rate at which cGMP was produced , but in mice with mutated NPR2 , this did not happen .", "When the cells could not remove the phosphates from NPR2 , cGMP levels stayed high and the bones grew longer .", "These findings reveal new insights into the molecular causes of dwarfism .", "The next step will be to identify the enzyme responsible for removing phosphate from NPR2 .", "Blocking its activity could help to enhance bone growth .", "In the future , this could lead to new drug treatments for achondroplasia ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "chromosomes and gene expression", "neuroscience" ]

[ [ "Compared to other cells , neurons undergo an unusually long period of maturation as they differentiate .", "In rodents , the time required for neuronal progenitors to become fully functional neurons can be many days , during which cells will exit mitosis , migrate to a proper location , grow initial neurites to be defined in their polarity , fully extend these axons and dendrites to their proper targets , and finally assemble active synaptic connections and circuits ( Waites et al . , 2005; Craig et al . , 2006; Hamby et al . , 2008; Barnes and Polleux , 2009 ) .", "A wide variety of genetic mechanisms ensure that these events occur in proper sequence and induce cell death when a process is incorrect .", "This developmental pathway is best understood at the level of transcriptional regulation where important signaling molecules , transcription factors , and epigenetic modifications are implicated in lineage commitment , cell survival , and establishment of synaptic connections .", "However , many aspects of the genetic control of neuronal development are not understood .", "Alternative pre-messenger RNA splicing is a common mechanism for genetic control in the nervous system , where many proteins important for neuronal differentiation and function are made in diverse isoforms through changes in splice site choice ( Licatalosi and Darnell , 2006; Li et al . , 2007; Norris and Calarco , 2012; Yap and Makeyev , 2013; Zheng and Black , 2013 ) .", "Changes of splicing pattern are regulated by trans-acting RNA-binding proteins that can be expressed in a temporal- or cell-type-specific manner to enhance or silence particular splicing events ( Black , 2003; Matlin et al . , 2005; Chen and Manley , 2009; Kalsotra and Cooper , 2011; Braunschweig et al . , 2013; Kornblihtt et al . , 2013 ) .", "Splicing regulators generally control large numbers of target transcripts and several have been shown to be essential for proper brain development or function ( Jensen et al . , 2000; Gehman et al . , 2011 , 2012; Yano et al . , 2010; Iijima et al . , 2011; Charizanis et al . , 2012; Ince-Dunn et al . , 2012 ) .", "At the onset of neuronal differentiation , there is a change in the expression of two polypyrimidine tract binding proteins , PTBP1 ( also known as PTB or HnRNP I ) and PTBP2 ( also known as nPTB or brPTB ) ( Keppetipola et al . , 2012 ) .", "Early in development , PTBP2 is present at low levels in neuronal progenitor cells ( NPC ) , but is sharply upregulated when NPCs are induced to differentiate into post-mitotic neurons ( Boutz et al . , 2007; Tang et al . , 2011; Zheng et al . , 2012 ) .", "In contrast , PTBP1 is expressed at high levels in neural progenitor cells , glia , and other non-neuronal cells , but is repressed in differentiating neurons coincident with the increase in PTBP2 concentration ( Polydorides et al . , 2000; Boutz et al . , 2007; Makeyev et al . , 2007; Zheng et al . , 2012 ) .", "The repression of PTBP1 is mediated by the miRNA miR124 ( Makeyev et al . , 2007 ) , which leads to the induction of PTBP2 expression through additional post-transcriptional mechanisms ( Boutz et al . , 2007; Makeyev et al . , 2007; Spellman et al . , 2007 ) .", "SiRNA mediated depletion of PTBP1 stimulates PTBP2 expression and can be sufficient to induce neuronal differentiation of fibroblasts ( Xue et al . , 2013 ) .", "However , the role of PTBP2 in differentiating neurons is not understood .", "PTBP1 and PTBP2 are encoded by paralogous genes and are very similar in peptide sequence , especially in their four RNA recognition motifs ( RRMs ) , giving them similar RNA-binding properties ( Oberstrass et al . , 2005 ) .", "The best-characterized function of the two PTB proteins is in the regulation of alternative splicing patterns .", "PTBP1 and PTBP2 bind to CU-rich regulatory sequences within or adjacent to many alternative exons to repress or activate their splicing , or sometimes to cause intron retention ( Xue et al . , 2009; Llorian et al . , 2010; Kafasla et al . , 2012; Keppetipola et al . , 2012 ) .", "However the two proteins differ in their splicing regulatory activities .", "Some exons , such as exon 8A of the CaV1 . 2 calcium channel transcript ( Cacna1c ) , are more strongly repressed by PTBP1 than PTBP2 , whereas other exons , such as exon 18 of the PSD95 transcript ( Dlg4 ) , are affected by both proteins ( Markovtsov et al . , 2000; Tang et al . , 2011; Zheng et al . , 2012 ) .", "Neither the mechanisms underlying the different responses of exons to the two PTB proteins , nor the roles of their partially overlapping splicing programs in neuronal development are known .", "To identify roles of PTBP2 in the developing mouse brain , we used conditional gene targeting .", "We show that the loss of PTBP2 leads to a catastrophic failure in neuronal maturation and to the misexpression of many protein isoforms affecting neurite growth , synapse formation and synaptic transmission .", "We find that PTBP2 and the program of splicing it controls are critical to both embryonic and postnatal brain development ." ], [ "PTBP2 is induced as nestin-positive neuronal progenitors exit mitosis and begin to differentiate ( Boutz et al . , 2007 ) .", "This can be seen in the embryonic cortex and olfactory bulb where proliferating cells in the subventricular zone are stained for nestin , but more mature cells populating the outer cortical layers have lost nestin expression but gained PTBP2 immunoreactivity ( Figure 1A ) .", "To investigate the function of PTBP2 in the developing brain , we generated a conditional null allele carrying loxP sites flanking exon 4 of the Ptbp2 gene ( Ptbp2loxP ) ( Figure 1B ) .", "In targeted tissues , Cre-mediated deletion of exon 4 introduces a reading frame shift to render the allele null .", "Correct targeting in ES cells and germ line transmission were confirmed by Southern blot and PCR of genomic DNA ( Figure 1C , D ) .", "A Ptbp2 null allele ( Ptbp2− ) was generated in crosses of Ptbp2loxP/+ mice to a germline-active Cre transgenic line EIIaCre ( Lakso et al . , 1996 ) .", "Homozygous Ptbp2 null pups generated in subsequent crosses failed to initiate breathing and died due to apparent respiratory failure at birth ( Figure 1E ) .", "Unlike their wild-type or heterozygous littermates , they do not respond to touch and appear completely paralyzed .", "Immunoblots of E18 . 5 whole brain lysates confirm the complete absence of PTBP2 in homozygous mutant mice and reduced protein in heterozygous mice compared to wild type ( Figure 1F ) .", "We also do not observe a truncated PTBP2 protein arising from the mutant allele ( Figure 1—figure supplement 1 ) .", "This phenotype is consistent with that of another full Ptbp2 null allele ( Licatalosi et al . , 2012 ) .", "Since Ptbp2 is also expressed in cardiac and skeletal muscle , defects in these tissues may contribute to the paralysis and death of the germline mutant mice . 10 . 7554/eLife . 01201 . 003Figure 1 . Generation and validation of the Ptbp2 conditional mutation .", "( A ) Expression of PTBP2 in maturing neurons of E15 . 5 brain .", "Nestin-positive cells ( green , middle panel ) of the subventricular zone express only limited PTBP2 ( red , right panel ) , but as these cells mature and migrate to outer layers , PTBP2 is induced ( Overlay panel to the left ) .", "V indicates the ventricle .", "( B ) The targeting construct carrying loxP sites flanking exon 4 of Ptbp2 was integrated into the endogenous Ptbp2 locus by homologous recombination .", "The neomycin ( Neo ) selection cassette flanked by Frt sites was removed by crossing founder mice ( Ptbp2Neo-loxP/+ ) with 129S4/SvJaeSor-Gt ( ROSA ) 26Sortm1 ( FLP1 ) Dym/J mice ( Jackson ) carrying Flp recombinase .", "The resulting Ptbp2loxP/+ mice were bred to C57Bl/6 Cre transgenic strains to generate Ptbp2+/− animals in targeted tissues ( C and D ) .", "Genotypes were verified by Southern blot of genomic DNA digested with XbaI ( C ) , and by PCR ( D ) .", "( E ) Ptbp2 null mice display cyanosis and die immediately after birth due to respiratory failure .", "( F ) Immunoblot for PTBP2 confirms that the disrupted allele eliminates expression of PTBP2 . DOI: http://dx . doi . org/10 . 7554/eLife . 01201 . 00310 . 7554/eLife . 01201 . 004Figure 1—figure supplement 1 . Immunoblot of PTBP2 in Emx-knockout and wild-type brain .", "Truncated proteins are not observed . DOI: http://dx . doi . org/10 . 7554/eLife . 01201 . 004 To examine PTBP2 function specifically in the nervous system , Ptbp2LoxP/LoxP mice were crossed to a Nestin-Cre transgenic line , expressing Cre in all neuronal progenitor cells , and bred to obtain homozygous Ptbp2LoxP/LoxP/Nestin-Cre+/− ( Ptbp2-NesKO ) mice ( Tronche et al . , 1999 ) .", "Unlike the full null , these pups initiated breathing , although at a lower rate than wild type and appeared cyanotic , dying within 1 hr after birth .", "Immunoblots of E18 . 5 whole brain from Ptbp2-NesKO pups confirmed the absence of PTBP2 protein ( Figure 1F , left two lanes ) .", "Both Ptbp2 null and Ptbp2-NesKO embryos were recovered at Mendelian ratios ( 27 out of 91 , and 12 out of 50 ) at the late embryonic stage E18 . 5 , suggesting that the lethality was specific to neonates .", "Immunohistochemistry with PTBP2 antibody on sections of E18 . 5 Ptbp2LoxP/LoxP/Nestin-Cre brain showed that the vast majority of the neurons in the CNS had lost PTBP2 expression ( Figure 2A ) , while PTBP2 expression in heart and skeletal muscle was unaffected ( data not shown ) .", "Expression of Ptbp1 , a close paralog of Ptbp2 expressed in neural progenitor cells as well as astrocytes , ependymal cells , and other non-neuronal cell types in the brain , was not changed in the absence of PTBP2 ( data not shown ) .", "This suggests that , although PTBP1 can repress PTBP2 expression in cells where PTBP1 is predominant , PTBP2 is not required to maintain PTBP1 repression in post-mitotic neurons where PTBP2 predominates .", "These results demonstrate that neonatal survival requires PTBP2 expression specifically in the CNS . 10 . 7554/eLife . 01201 . 005Figure 2 . Embryonic brain development appears largely normal in the absence of PTBP2 . ( A and B ) Sagittal sections of E18 . 5 cortex from control and NesKO mice .", "( A ) Staining with anti-PTBP2 antibody , control cortex shows PTBP2 expression in all neuronal nuclei , whereas NesKO cortex has lost PTBP2 expression in the vast majority of neurons .", "( B ) H&E staining showing that NesKO cortex has similar thickness and largely normal morphology .", "Scale bar = 100 μM .", "( C ) Nissl stained coronal sections of E18 . 5 cortex of wild-type and NesKO brain at two rostral–caudal planes .", "In the knockout brain , several major axonal tracks including the lateral olfactory tract ( lot ) , internal capsule ( ic ) and external capsule ( ec ) were reduced , and the anterior commisure ( ac ) was missing .", "Corpus collosum , the major axon bundle connecting the two hemispheres , was present in the knockout brain .", "Scale bar = 250 μM .", "Staining was done on at least four knockout mice and four wild-type littermate controls from two litters . DOI: http://dx . doi . org/10 . 7554/eLife . 01201 . 005 To assess if loss of PTBP2 leads to developmental defects in the brain , we examined Ptbp2-null brains at late embryonic stage E18 . 5 .", "When stained by Nissl or hematoxylin and eosin ( H&E ) , mutant embryos showed largely normal morphology for major brain structures such as the neocortex , striatum , hippocampus , and thalamus ( Figure 2BC ) .", "The neocortex showed normal thickness and laminar organization ( Figure 2B ) .", "Immunostaining for a variety of neuronal markers , including pan-neuronal markers ( Map2 , TuJ1 ) , interneuron subtype markers ( calbindin and calrectinin ) , a marker for proliferating nuclei ( phosphoHistone3 ) , and for glial and radial glial markers ( GFAP and RC2 ) all showed no significant differences between mutant and control littermate brains ( data not shown ) .", "To assess possible defects in neuronal migration , we examined cortical layer markers ( Tbr2 , Brn2 and Cux1 ) and again did not observe any significant differences between mutant and control brains ( data not shown ) .", "Notably , we did not observe ectopic subventricular mitotic cells in either the full null or the Ptbp2-NesKO brains .", "Such cells were previously reported as sporadically occurring in another full germline Ptbp2 null mouse ( Licatalosi et al . , 2012 ) .", "These different observations may result from different sensitivity in staining methods or differences in genetic background .", "Although the grey matter of the Ptbp2 mutant brain appears largely normal in its overall patterning and the specification of major neuronal subtypes , examination of Nissl stained coronal sections revealed some white matter defects ( Figure 2C ) .", "Several major axonal tracts were either absent or significantly diminished in the Ptbp2 mutant brain; the internal capsule , external capsule , and the lateral olfactory tract were reduced , and the anterior commissure was absent in the Ptbp2 mutant .", "These major axonal tracts connect distant regions of the developing brain , such as cortex , thalamus , and the brainstem , and are essential to basic physiological functions such as respiratory control .", "These axonal defects may thus contribute to the neonatal lethal phenotype .", "However , quantification of this phenotype and linking it to neonatal death will require more extensive analysis of mice at a variety of developmental timepoints .", "These observations are consistent with a role for PTBP2 in axonogenesis or myelination ( See Figures 3 and 4 below ) .", "PTBP2 expression goes down during the first postnatal week of brain development with moderate levels maintained into adulthood .", "Identified PTBP2 targets such as PSD95 ( Dlg4 ) show changes in splicing coincident with this postnatal depletion of PTBP2 ( Zheng et al . , 2012 ) .", "Because of their neonatal lethal phenotype , the postnatal effects of PTBP2 loss cannot be examined in either the germline or pan-CNS null mice .", "To look at PTBP2 function later in development , we crossed Ptbp2loxP/loxP mice with Emx-Cre transgenic mice , where Cre recombinase is expressed in projecting neurons of the higher forebrain , allowing selective gene disruption in the cerebral cortex , hippocampus , and olfactory bulb ( Iwasato et al . , 2000 ) .", "In Ptbp2loxP/loxP:Emx1-Cre+/− mice ( Ptbp2-EmxKO ) , Ptbp2 is highly depleted from the cerebral cortex , hippocampus , and olfactory bulb , but still expressed at high levels in other brain regions , such as cerebellum , striatum , and brain stem ( Figure 3AB and data not shown ) . 10 . 7554/eLife . 01201 . 006Figure 3 . PTBP2 is required for postnatal cortical development .", "( A ) Ptbp2 EmxKO mutants display slow growth ( shown at P11 ) and die around weaning .", "( B ) Immunoblot for PTBP2 at P21 confirms that its expression is largely eliminated in cortex , but remains unchanged in other structures such as brain stem .", "( C ) Postnatal development of the cortex was disrupted in EmxKO mice .", "Mutant cortical tissue failed to thicken as in wild type and degenerated .", "Scale bar = 1 cm .", "( D ) Nissl stained coronal sections of control and EmxKO brain at P5 showing loss of cell density and degeneration .", "Enlarged panels show a loss of nuclei in the EmxKO tissue .", "( E ) Coronal sections at P11 , similar to ( D ) .", "Scale bars in D and E indicate 0 . 5 mm .", "Staining was done on at least four knockout mice and four wild-type littermate controls from two litters . DOI: http://dx . doi . org/10 . 7554/eLife . 01201 . 006 Unlike the Nestin and Germline-Cre knockouts , Ptbp2-EmxKO pups were viable , with body weights similar to control newborns at P0 .", "However , these mutant mice displayed slower growth than their littermates and could be identified by their small size and low body weight as early as postnatal day 3 ( Figure 3A ) .", "By P14 , their average weight was less than half of control pups , and they all died around weaning age ( P18-21 ) .", "The cause of the Ptbp2-EmxKO pups’ failure to thrive was apparent upon dissection of their brains .", "The Ptbp2-EmxKO cortex appeared relatively normal at birth .", "However , while the control cortices thicken and grow in size during the first three postnatal weeks , largely due to development of the neuropil and white matter , the EmxKO cortex displayed widespread neuronal death and degeneration ( Figure 3C–E ) .", "Brain structures not subject to PTBP2 loss , such as striatum , developed normally .", "The knockout tissue showed a progressive loss of structural integrity making it difficult to section and stain .", "Comparing coronal sections of mutant and control brains at P5 , the EmxKO cortex was substantially thinner than control cortices .", "Nissl staining revealed widespread pyknotic nuclei within the EmxKO cortical plate as a result of neuronal cell death ( Figure 3D ) .", "Further staining with more specific cell death markers showed evidence of both necrotic and apoptotic cell death but was difficult to quantify in the degenerating tissue ( data not shown ) .", "Understanding the mechanisms initiating cell death in these tissues will require additional analyses .", "By P11 , when control cortex had substantially thickened with well-defined cellular layers , the EmxKO cortex had further degenerated with additional loss of tissue integrity .", "The lateral ventricles were enlarged in EmxKO brains at both P5 and P11 , and the corpus callosum was missing ( Figure 3E and data not shown ) .", "Other brain structures where Ptbp2 was inactivated were also defective in the EmxKO; the mutant hippocampus was significantly reduced in size and the lamination in the olfactory bulb was highly disrupted , with the Mitral cell layer entirely absent ( data not shown ) .", "The postnatal lethality of Ptbp2-EmxKO mice demonstrates a continued requirement for PTBP2 to allow neuronal maturation and survival in the developing postnatal cortex .", "The survival of these mice past birth suggests that the neonatal lethality of Ptbp2 null and NesKO mice is due to loss of PTBP2 in early maturing regions of the CNS critical for basic physiological function in the neonate .", "While initial neurogenesis , as well as patterning and specification of the brain , appeared largely normal in the absence of PTBP2 , the phenotype of the EmxKO mice indicated a failure of later neuronal development .", "The paralysis phenotype of Ptbp2 pan-neural knockout pups also implied defects in synaptic transmission .", "To assess whether neural development proceeded to later stages , such as synapse formation , in the absence of PTBP2 , we examined the expression of several important synaptic proteins in the brains of E18 Ptbp2 null mice .", "The mutant brains exhibited strikingly lower levels of multiple synaptic markers ( Figure 4A ) , including PSD95 , the neurotransmitter receptor NR2B , the major synaptic vesicle protein Synaptophysin ( SYP ) , and the trans-synaptic scaffolding protein Neuroligin1 ( NLG1 ) .", "Other synaptic proteins , such as the calcium/calmodulin-dependent protein kinase Cask and the Glutamate Receptor GluR2 also declined .", "These results indicate that cells may be arrested or delayed in their maturation or that synapse formation may be otherwise defective in the absence of PTBP2 . 10 . 7554/eLife . 01201 . 007Figure 4 . Loss of PTBP2 leads to reduced synaptic protein expression in vivo and to early cell death in primary cell culture .", "( A ) Immunoblots of whole brain lysates from wild type , heterozygous ( +/− ) , and knockout ( −/− ) mouse at E18 . 5 show substantial reduction of synaptic proteins in knockout brain .", "( B ) Dissociated cortical cultures ( Days in vitro , DIV6 ) of wild-type and Ptbp2-knockout neurons stained for PTBP2 and the neurite marker MAP2 .", "Neurons lacking PTBP2 survive plating and short-term culture with similar efficiency to wild type , and extend multiple MAP2-positive neurites .", "( C ) PTBP2-deficient hippocampal cultures show increasing neuronal death starting from second week of culture and do not survive past 3 weeks .", "A phase contrast image of cells at DIV15 is shown , with the quantification of cell numbers plotted to the right .", "Starting viable cell numbers averaged 417 cells from wt and 442 cells from knockout based on cultures from four embryos of each genotype at DIV1 .", "Percent Survival is the fraction of live cells at each time point relative to the wild type average at DIV1 .", "Error bars are standard deviations derived from four cultures of each genotype at each time point . DOI: http://dx . doi . org/10 . 7554/eLife . 01201 . 007 Interestingly , heterozygous Ptbp2+/− mouse brains express the PTBP2 protein at about half normal levels ( Figure 1F ) .", "The effect of this partial depletion on synaptic protein expression and on splicing of target transcripts was variable ( Figure 4A , and see below ) .", "For some targets , protein levels in the heterozygous brains were intermediate between the wild type and homozygous knockout ( GluR2 and NR2B , Figure 4A , also see CamKinase 2b below ) .", "In other cases , the heterozygote appeared similar to the wild-type mice , expressing close to normal levels of PSD95 , Synaptophysin , Neuroligin1 , and Cask .", "Given that Ptbp2 heterozygous mice are viable , a single copy of Ptbp2 is apparently sufficient to support development .", "It will be interesting to examine the behavior and physiology of these heterozygous mice .", "To investigate how developing neurons are affected by the loss of PTBP2 , we cultured neurons from knockout and wild-type embryos .", "Dissociated mouse neurons cultured in vitro progress through several stages of maturation that parallel their in vivo development ( Dotti et al . , 1988; Palmer et al . , 1997; Arimura and Kaibuchi , 2007 ) .", "These include the initial extension of multiple immature neurites ( days in vitro , DIV1-2 ) , establishment of axon–dendrite polarity ( DIV2-4 ) , axon and dendrite outgrowth ( DIV4-15 ) , and finally synapse formation and maturation ( after DIV10 ) .", "Immediately after dissociation and plating , cortical neurons from E15-16 Ptbp2-NesKO mice exhibited similar viability to control neurons .", "The Ptbp2-KO neurons grew extensive neurites during the first week in culture and were indistinguishable from control cultures when stained with the dendritic marker MAP2 ( Figure 4B ) .", "However during the second week , increased signs of neuronal death appear in cultures of Ptbp2-KO neurons , including neurite retraction , membrane blebbing and shrinkage , and nuclear condensation .", "All neurons lacking PTBP2 die by the end of third week of culture .", "A similar catastrophic cell death occurred during the third week of culture in hippocampal neurons cultured from E18 Ptbp2 null mice ( Figure 4C ) .", "The timing and scale of the neuronal death in culture is similar to the wide-spread degeneration seen in the postnatal ( P5-P11 ) Emx-KO brains .", "Both in vivo and in vitro , PTBP2 is clearly required for the survival of forebrain neurons .", "Splicing regulators generally have many target transcripts and it is expected that the dramatic phenotype of PTBP2 loss will be derived from multiple deficits .", "To assess transcriptome changes caused by the loss of PTBP2 , we carried out genomewide expression and splicing analyses of the nesKO mice , initially using splicing-sensitive exon junction microarrays ( MJAY ) analyzed by Omniviewer , and later using RNAseq and SpliceTrap analysis ( Srinivasan et al . , 2005; Wu et al . , 2011; Shen et al . , 2012 ) .", "These analyses identified a large set of cassette exons and other alternative splicing events that are mis-regulated in developing ( E18 ) Ptbp2−/− brains ( Supplementary files 1–3 in Dryad [Li et al . , 2014] ) .", "Alternative splicing events that changed between wild type and mutant brains were ranked by Sepscore and q-value for microarray analysis , and by delta PSI ( percent spliced in ) and false discovery rate ( FDR ) for RNAseq ( ‘Materials and methods’ ) .", "Using cutoffs of a Sepscore of 0 . 345 for the arrays and a 10% change in PSI for the RNAseq , we identified 359 PTBP2 target cassette exons by array ( Supplementary file 2 in Dryad [Li et al . , 2014] ) ) and 597 exons by RNAseq ( Supplementary file 3 in Dryad [Li et al . , 2014] ) .", "These included 92 exons found by both methods .", "Exons identified by RNAseq but not microarray included exons that were not represented on the array , exons exhibiting complex patterns of splicing , and a few exons where particular exon probes seemed to fail on the array .", "Alternative splicing events identified by array but not RNAseq included exons that did not pass the deltaPSI and/or overall expression filters , and a few exons that were absent from the SpliceTrap database .", "Combining the two methods provided a significant increase in the total set of splicing changes identified in the mutant mice .", "Assaying additional control and mutant mice as biological replicates , a large set of splicing events was further validated by RT-PCR ( 77 out of 88; Figure 5A; Supplementary file 1 in Dryad [Li et al . , 2014] ) .", "These included exons identified by either of the two profiling methods , as well as several exons previously identified as PTBP1 targets .", "Both the array and RNAseq methods yielded validation rates above 70% .", "The majority of identified cassette exons ( 379 of 597 from the RNAseq analysis ) showed increased splicing in the absence of PTBP2 ( Supplementary file 3 in Dryad [Li et al . , 2014] ) , suggesting that PTBP2 functions more often as a splicing repressor than as a splicing activator , similar to the observations of PTBP1 ( Keppetipola et al . , 2012 ) . 10 . 7554/eLife . 01201 . 008Figure 5 . PTBP2 regulates a large set of splicing targets important to neuronal development and function .", "( A ) Sample RT-PCR gels showing aberrant splicing ratios for selected targets important for neuronal development and function .", "( B ) Two alternative exons in Camk2b are developmentally regulated .", "In plots of RT/PCR measurements , normal mouse brain shows low inclusion of these exons in the embryo ( E18 ) and a postnatal increase ( P5 , P11 and P22 ) in splicing to near full inclusion in adult brain .", "In the PTBP2 null brain these exons exhibit adult levels of splicing at E18 .", "( C ) The premature switch in CAMK2B protein isoforms is observed by immunoblot in both Ptbp2 null ( KO ) and NesKO mice ( E18 . 5-P0 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 01201 . 00810 . 7554/eLife . 01201 . 009Figure 5—figure supplement 1 . Quantification of RT/PCR analysis for multiple exons mis-regulated in Ptbp2 Nes-KO brain . In many cases , exon inclusion is abnormally high in the embryo for an exon that normally increases in postnatal development . DOI: http://dx . doi . org/10 . 7554/eLife . 01201 . 009 Altered splicing was observed in many known direct PTBP1/PTBP2 targets including PSD95 ( Dlg4 ) , Atp2b1 , Bin1 , Dst and Fn1 ( Boutz et al . , 2007; Makeyev et al . , 2007; Zheng et al . , 2012 ) .", "Direct targeting by PTBP2 can also be inferred from crosslinking-immunoprecipitation ( CLIPseq ) data ( Licatalosi et al . , 2012; Ule et al . , 2003 ) .", "For exons whose percent-spliced-in value ( PSI ) changed by more than 20% in the knockout , approximately 70% are reported to have significant PTBP2 CLIP clusters in their adjacent introns in E18 mouse brain ( Licatalosi et al . , 2012 ) ( SZ unpublished data ) .", "Exons in Agrn , Arhgef7 , Dbn1 , Erc1 and Sorbs2 , whose splicing is altered in the PTBP2−/− brain and which are also expressed in HeLa cells , were also identified as PTBP1 targets in a HeLa CLIP analysis ( Xue et al . , 2009 ) .", "These data indicate that the majority of isoform changes identified in the knockout are likely to be directly regulated by PTBP2 .", "Nevertheless , it should be noted that the analyzed brains developed to E18 in the absence of PTBP2 and it is expected that some splicing changes will be indirect effects of other changes in the developing mice .", "Validated PTBP2-dependent splicing events include many transcripts with important neuronal functions , including the postsynaptic scaffolding proteins PSD95 ( Dlg4 ) and gephryn ( Gphn ) , the neurotransmitter receptor GABAA Rγ2 ( Gabg2 ) , and the key calcium signaling molecules CamK2a and CamK2b ( Figure 5AB , Figure 5—figure supplement 1; Li et al . , 2014 ) .", "As a general assessment of the PTBP2 regulatory network , gene ontology analyses were performed on the set of PTBP2-dependent splicing changes relative to the set of transcripts identified by RNAseq as expressed in mouse brain .", "Biological Process annotations that were significantly enriched in the PTBP2 target set ( p<0 . 001 ) included ‘regulation of transcription’ , ‘synaptic transmission’ , ‘transmission of nerve impulse’ , ‘synapse organization’ , ‘cell morphogenesis’ , ‘endocytosis’ , and ‘neuron projection development’ ( Supplementary file 4 in Dryad [Li et al . , 2014] ) .", "All of these functions are dynamically changing during the period of neuronal maturation when PTBP2-dependent defects are observed .", "We also analyzed the RNA-seq data using CuffDiff to identify changes in overall transcript expression ( rather than splicing ) in the Ptbp2 mutant brain ( Supplementary file 5 in Dryad [Li et al . , 2014] ) .", "Some of these transcripts are likely to be reduced through direct PTBP2 effects on post-transcriptional processes .", "These include five transcripts containing PTBP2-dependent exons with mapped CLIP clusters whose change in splicing is predicted to lead to nonsense mediated decay .", "Other changes in transcript expression are likely to be an indirect result of developmental defects associated with the loss of PTBP2 .", "Gene ontology analyses were performed on genes exhibiting greater than a twofold change in the knockout brain ( Supplementary files 6 and 7 in Dryad [Li et al . , 2014] ) .", "Relative to all transcripts expressed in wild-type brains , upregulated transcripts were highly enriched for GO terms associated with innate immunity and the inflammatory response ( p<0 . 001 ) .", "Given the phenotype of the mice , these functional enrichments likely result from the degeneration seen in the Ptbp2 knockout brains , rather than direct PTBP2 targeting of the transcripts .", "Down-regulated transcripts were only weakly enriched for particular bioprocess terms .", "Only terms related to cell adhesion had enrichment p values below 0 . 01 .", "Terms related to axonogenesis and axon choice point recognition were less significantly enriched among the down-regulated transcripts .", "We find that many of the identified PTBP2 targets display a common pattern of temporal regulation in the developing wild-type brain .", "These transcripts contain exons that are mostly excluded in the embryonic brain and whose splicing increases during the first 3 weeks after birth ( Figure 5B , Figure 5—figure supplement 1 in Dryad [Li et al . , 2014] ) .", "For much of the brain , this time period represents a key phase of neuronal maturation when neurons project processes and establish synapses .", "The normal shift in splicing of these transcripts coincides with the known postnatal decrease in PTBP2 levels in the brain ( Zheng et al . , 2012 ) , and the mutation of Ptbp2 leads to a premature switch from embryonic to adult isoforms .", "In one example , the Camk2b gene contains two alternative exons that are mostly excluded from embryonic Camk2b transcripts , but are included in the adult isoform ( Brocke et al . , 1995 ) .", "These two cassette exons insert short peptide sequences into the variable domain of the protein to alter its autophosphorylation , its response to Ca2+ oscillations , and its association with F-actin ( Figure 5B ) ( Brocke et al . , 1999; Bayer et al . , 2002; O’Leary et al . , 2006 ) .", "In the Ptbp2 mutant brains , the two adult exons are nearly completely included by embryonic day 18 ( Figure 5B ) .", "These Camk2b protein isoforms can also be distinguished in immunoblots that demonstrate a dramatic switch from the embryonic ( β’ , βe and β’e ) proteins to the adult ( β ) isoform ( Figure 5C ) .", "Multiple proteins important to neuronal differentiation and synaptic function show a precocious switch to adult isoforms in the Ptbp2 knockout mice ( Figure 5—figure supplement 1 ) .", "Like CamK2b , the adult and embryonic isoforms of the postsynaptic protein Drebrin ( Dbn1 ) and the neural cell adhesion molecule NCAM have been shown to be functionally different ( Polo-Parada et al . , 2005; Hata et al . , 2007; Mizui et al . , 2009; Kojima et al . , 2010 ) .", "For these genes , adult RNA and protein products are both expressed much earlier in the Ptbp2 mutant than in wild-type mice ( Figure 5A and data not shown ) .", "Other key proteins targeted by PTBP2 include dynamin1 ( Dnm1 ) , an essential protein in synaptic vesicle endocytosis and trafficking , Magi1 , a scaffolding protein at the postsynaptic density , the potassium channel Kcnq2 , a critical regulator of neuronal excitability , and calcineurin A ( Ppp3ca ) , an important phosphatase affecting calcium signaling ( Figure 6A , Figure 5—figure supplement 1 ) .", "The adult isoforms of these proteins are presumably needed in mature synaptically active neurons . 10 . 7554/eLife . 01201 . 010Figure 6 . Premature and aberrantly high splicing of PTBP2 target exons in E18 Nes-KO whole brain and P5 Emx-KO cortex .", "( A ) RT/PCR of Dynamin1 .", "Dynamin1 contains a pair of mutually exclusive exons 9a and 9b that normally switch from 9b to 9a during development .", "In both the Nes-KO and Emx-KO there is a premature switch to the 9a isoform .", "Note that the heterozygous Nes-KO exhibits an intermediate level of exon 9a splicing .", "The high level of 9a splicing can be seen in the wild-type adult cortex ( Ad WT ) .", "( B ) RT/PCR of Braf ( left ) and Pitpnb ( right ) .", "Braf and Pitpnb each contain a PTBP2 repressed exon that is spliced at aberrantly high levels in the Ptbp2 KO mice .", "Braf exon 9 and Pitpnb exon 11 are spliced into the mRNAs two to three times more frequently in the embryonic ( E18 ) or postnatal ( P5 ) mutant mice ( NKO and EKO ) , and at higher levels than normally seen in the wild-type adult ( Ad WT ) .", "The percent exon inclusion is shown at the bottom . DOI: http://dx . doi . org/10 . 7554/eLife . 01201 . 010 Adult brain exhibits many splicing changes from the embryo that result from changes in a wide variety of regulators .", "We find that within a set of 1143 exons whose splicing increases between the embryo and adult , 17% are derepressed by the loss of PTBP2 ( AH unpublished observations ) .", "Thus , the PTBP2 regulatory program accounts for a substantial fraction of the regulated splicing events controlled during brain development .", "The defective development of the Ptbp2−/− brain indicates that early switching to these adult isoforms is deleterious to neurons as they mature .", "RNA isolated from embryonic brain at E18 will be derived from a variety of structures at different stages of development .", "To examine splicing within a more defined population of cells coincident with the normal downregulation of PTBP2 , we isolated RNA from the EmxKO cortex for RNAseq at P1 .", "These analyses identified splicing changes in many of the targets previously identified in the NesKO mice ( Supplementary file 8 in Dryad [Li et al . , 2014] ) .", "As seen in the NesKO at E18 , the mutually exclusive exons 9a and 9b of the dynamin1 ( Dnm1 ) transcript also display aberrant splicing in the EmxKO cortex ( Figures 6A and 7A ) .", "The adult exon 9a was spliced at significantly higher levels ( 79% ) in the EmxKO than in control mice ( 56% ) .", "The embryonic and adult isoforms of dynamin1 are functionally distinct and their activity has been investigated in mutant mice , where a missense mutation in exon 9a results in impaired synaptic function , seizures and other behavioral defects ( Boumil et al . , 2010 ) .", "Thus , PTBP2 is playing a key role in the regulation in this postnatal switch in Dnm1 isoforms . 10 . 7554/eLife . 01201 . 011Figure 7 . Exons can respond to the loss of PTBP2 differently in E18 whole brain or P1 cortex . Genome browser tracks of aligned RNAseq reads from E18 whole brain , NesKO ( green ) and wild type ( pink ) , and from P1 cortex , EmxKO ( blue ) and wild type ( yellow ) .", "( A ) For Dynamin 1 , exons 9a and 9b are boxed .", "The two knockout samples show a different degree of shift towards the use of downstream exon 9a .", "( B ) An exon in the Zinc finger protein Zfp277 ( boxed ) is entirely excluded in E18 brain .", "In postnatal cortex this exon has been strongly induced , but is still regulated by PTBP2 as indicated by the stronger induction of the exon in the Emx-KO .", "( C ) An exon in the microtubule associated monooxygenase Mical3 is fully spliced at E18 , regardless of the presence of PTBP2 .", "In postnatal cortex this exon becomes repressed and this repression is in part dependent on PTBP2 . DOI: http://dx . doi . org/10 . 7554/eLife . 01201 . 011 Several exons were found to be included in the Ptbp2 knockout brain at levels much higher than those observed at any point of normal development .", "These include an exon in B-raf ( Braf ) , a protein kinase in the Ras-Raf-MEK-ERK signaling pathway ( Maurer et al . , 2011 ) .", "This exon is normally included in no more than 25% of the total Braf transcript .", "In contrast , 60% of Braf mRNA in E18 null brain , and 41% of the mRNA in P5 EmxKO cortex contains the exon ( Figure 6B ) .", "The PITPbeta ( phosphatidylinositol transfer protein beta , Pitpnb ) transcript , involved in inter-organelle lipid transfer and inositol signaling , contains an exon that can be skipped or included to produce alternative C-termini of the final protein ( Cockcroft , 2001 ) .", "In wild-type mouse brain , this exon is included in less than 10% of the Pitpnb mRNA at all points of normal development .", "However , in the absence of PTBP2 splicing of this exon increases to more than 25% ( Figure 6B ) .", "These and other exons appear to be especially sensitive to loss of PTBP2 ( Figure 5—figure supplement 1 ) .", "Although reduced in expression after differentiation , PTBP2 continues to be present at moderate levels into adulthood .", "This remaining PTBP2 may serve to maintain the partial repression of some exons .", "In addition to the precocious expression of adult isoforms , the deleterious effect of PTBP2 loss may also result from the aberrantly high expression of isoforms normally present at lower levels .", "In addition to splicing changes seen in the NesKO whole brain , new PTBP2-dependent splicing events were identified in EmxKO cortex ( Figure 7BC , Supplementary file 8 in Dryad [Li et al . , 2014] ) .", "The zinc finger protein transcript Zfp277 contains two exons whose splicing increases substantially in postnatal cortex compared to E18 whole brain .", "Interestingly , one of these exons shows greater induction in the Emx-cre KO than in wildtype , indicating that it is partially repressed by PTBP2 in this tissue .", "In contrast , this exon remains fully repressed in E18 brain even in the absence of PTBP2 ( Figure 7B ) .", "Conversely , several exons in the Mical3 transcript are repressed in P1 cortex but not E18 whole brain .", "One of these exons loses this cortical repression in the absence of PTBP2 , even though it is unaffected by PTBP2 depletion at E18 ( Figure 7C ) .", "These exons showing PTBP2 dependence in P1 cortex but not E18 whole brain indicate that the PTBP2 target transcripts are subject to other splicing regulatory programs that are also changing during development , with exons exhibiting different regulatory factor dependencies at different developmental stages .", "The many adult isoforms misregulated in the Ptbp2−/− embryos greatly expands earlier observations of exon 18 of the Dlg4 ( PSD95 ) transcript , whose skipping generates a non-productive transcript subject to nonsense-mediated decay .", "The splicing of this exon is required for PSD95 protein expression , and its repression by PTBP2 keeps PSD95 levels low until late in neuronal maturation ( Zheng et al . , 2012 ) .", "We now find that this exon is part of a large program of coordinated splicing changes .", "This PTBP2-driven splicing transition takes place subsequent to the earlier splicing switch driven by PTBP1 depletion as neurons are born ( Figure 8 ) . 10 . 7554/eLife . 01201 . 012Figure 8 . Changes in the expression of the two PTB proteins define three splicing regulatory states during neuronal differentiation . Neuronal progenitor cells primarily express PTBP1 .", "When these cells are induced to differentiate , PTBP1 is repressed and PTBP2 is induced .", "This switch in RNA binding proteins causes changes in the splicing of exons that are more sensitive to PTBP1 such as exon 8 of the Cacna1c calcium channel transcript .", "Other exons that are sensitive to both proteins maintain their repression during the early stages of neuron differentiation , when PTBP2 is high .", "When these cells finally mature and form synapses , PTBP2 is downregulated .", "This leads to changes in a splicing regulatory program that includes exons in Cam Kinase 2 and Dynamin 1 .", "These exons are found in mRNA isoforms associated with adult brain and are precociously expressed in PTBP2 knockout brains , leading to cell death prior to final maturation . DOI: http://dx . doi . org/10 . 7554/eLife . 01201 . 012" ], [ "We find that the splicing regulator PTBP2 is required for pre and postnatal brain development .", "The loss of PTBP2 in the complete CNS or in particular neuronal lineages does not greatly affect lineage commitment or developmental patterning .", "However , postmitotic neuronal maturation and survival are severely impaired .", "Mice with Emx-cre driven depletion of PTBP2 from developing cortex survive through about 3 weeks of postnatal development during which time the cortex degenerates .", "Neuronal cell death is also seen in Ptbp2 knockout neurons in culture .", "These cells initiate differentiation from progenitor cells and progress through the first stages of post-mitotic differentiation , with the extension of neuritic processes and expression of early neuronal markers .", "However , they subsequently undergo a catastrophic failure in the second to third week of culture and die prior to synaptogenesis .", "Thus , the PTBP2-driven splicing program plays a key role in the maturation and survival of neurons .", "In keeping with the neuronal maturation defect , we find PTBP2 target exons in many transcripts affecting neurite outgrowth , axon guidance , synaptic assembly , and synaptic function .", "In the mutant embryos , a large number of genes exhibit early expression of spliced isoforms that are normally expressed only in the adult brain .", "For these genes , the presence of PTBP2 in developing cells serves to prevent expression of a functionally distinct adult isoform until late in neuronal maturation .", "This pattern of regulation is seen for NCAM , Cam Kinase II , drebrin , dynamin1 , Magi1 , Kcnq2 , Calcineurin and the previously identified PTBP2 target PSD95 ( Dlg4 ) .", "These proteins all play key roles in the development and function of the neuron including both its pre and postsynaptic specializations .", "PTBP2 is present at low levels in nestin-positive neuronal progenitor cells .", "When these cells exit mitosis and begin differentiation , PTBP1 expression is repressed and PTBP2 expression is induced ( Figure 1A; Boutz et al . , 2007 ) .", "PTBP2 levels remain high in the differentiating cells and then drop late in maturation , with adult neurons expressing moderate levels .", "It was previously shown that some exons are more strongly affected by PTBP1 than by PTBP2 , whereas for other exons the regulatory effect of the two proteins is nearly equal ( Boutz et al . , 2007; Makeyev et al . , 2007; Llorian et al . , 2010; Tang et al . , 2011; Keppetipola et al . , 2012 ) .", "The expression profile of the PTB proteins thus creates three regulatory states during neuronal differentiation ( Figure 8 ) .", "PTBP1 maintains repression of many exons in neuronal progenitor cells .", "As cells begin to differentiate and shift to PTBP2 expression , exons that are more sensitive to PTBP1 begin to be spliced .", "These include the c-src N1 exon and exon 8A from the Cacna1c calcium channel transcript ( Markovtsov et al . , 2000; Tang et al . , 2011 ) .", "Later in neuronal maturation when PTBP2 levels drop , a second transition occurs with changes in the splicing of a new group of transcripts .", "We previously found that PSD95 exon 18 is controlled during this second transition ( Zheng et al . , 2012 ) .", "We now identify a large network of exon targets that have this common regulatory profile and are precociously spliced in the Ptbp2−/− mice .", "Thus , one function of the PTBP2 protein is to maintain repression of a subset of PTBP1 target exons after PTBP1 is depleted in early differentiation .", "The differing sensitivity of target exons either to just PTBP1 or to both PTBP1 and PTBP2 presents an interesting mechanistic question of how an exon can be structured to respond to one protein or to both .", "A recent paper described the phenotype of a full germline Ptbp2 null mouse ( Licatalosi et al . , 2012 ) .", "As we observe with both germline and pan-CNS conditional knockouts , this mouse exhibited neonatal lethality , precluding examination of later neuronal maturation .", "Perhaps because of this neonatal lethality , the authors focused on PTBP2 expression in neuronal progenitor cells .", "Although we also see low expression in these cells ( Figure 1A ) , we find the primary period of PTBP2 expression to be during neuronal maturation ( Boutz et al . , 2007; Tang et al . , 2011; Zheng et al . , 2012 ) .", "This study reported sporadic observation of cortical progenitors undergoing ectopic mitoses away from the apical edge of the ventricular zone in Ptbp2 null mice ( Licatalosi et al . , 2012 ) .", "This defect was in keeping with an observed change in Numb pre-mRNA splicing .", "We have not observed ectopic mitoses in either the germline null or the pan-CNS conditional null mice , and found only minor changes in Numb splicing .", "However , this phenotype was reported to be sporadic and a careful comparison of the phenotypes from the different mutations will require matching their genetic backgrounds .", "Licatalosi et al . also report microarray identification of PTBP2 target exons in germline null brain that largely overlap with exons we identify in the pan-CNS knockout brain by microarray and by RNAseq .", "They suggest a role for the PTBP2 splicing program in neurogenesis .", "We do not see defects in neurogenesis and commitment to neuronal lineages , but find a dramatic defect in the later maturation of neurons after their birth .", "This is in keeping with both the developmental timing of PTBP2 expression and the precocious expression of adult isoforms observed by both groups in the knockout mice .", "Interestingly , the later Emx-knockout mice show some differences from the NesKO in their ensemble of PTB2 target exons .", "These transcripts may be subject to regulatory events that occur after birth and are thus not observable in mice subject to neonatal lethality .", "Exons observed to change in the EmxKO but not in the NesKO mice point to the dynamic nature of splicing regulation during neuronal development .", "Most exons are controlled by combinations of factors , and there is likely to be extensive intersection between the PTBP2 regulatory program and splicing programs driven by other splicing factors ( Calarco et al . , 2011 ) .", "In the future , it will be interesting to focus on the development of particular circuits and assess the temporal expression profiles of multiple other splicing regulators .", "Such studies will allow examination of how the different splicing regulatory networks are integrated .", "Our analysis has focused on the dramatic changes in alternative splicing caused by loss of PTBP2 .", "PTBP1 is known to also affect miRNA function and cytoplasmic translation ( Sawicka et al . , 2008; Fred and Welsh , 2009; Gorospe et al . , 2011; Kafasla et al . , 2012; Xue et al . , 2013 ) .", "PTBP2 will likely also serve similar roles , and a portion of the Ptbp2 null phenotype may also derive from the loss of these other functions .", "In the CAD neuronal cell line , PTBP1 has been reported to cause retention of selected introns in transcripts for several important synaptic proteins , leading to their downregulation through nuclear RNA decay ( Yap et al . , 2012; Yap and Makeyev , 2013 ) .", "These introns appear to be efficiently spliced in embryonic brain , as might be expected after the downregulation of PTBP1 .", "PTBP2 may have similar activity on a different set of target transcripts .", "So far we have not seen a clear example of this , but one possibility is Ngrn , which contains a 3′-terminal intron that is spliced more efficiently in the Emx-KO than in wild type ( data not shown ) .", "As seen with other splicing regulators , the Ptbp2 mutation is highly pleiotropic .", "Future studies will examine the functional roles of individual PTBP2 target transcripts .", "We are particularly interested in two processes .", "The establishment of axonal/dendritic polarity that occurs early in differentiation may coincide with the loss of PTBP1 protein and gain of PTBP2 .", "In contrast , synaptic assembly occurs late in maturation and requires the downregulation of PTBP2 .", "Many identified synaptic proteins change their splicing at this stage and it will be very interesting to examine how their isoform switching affects synaptic architecture and function ." ], [ "The Ptbp2 conditional null allele was generated using homologous recombination in embryonic stem ( ES ) cells .", "The pFlox-PGK-Neo vector , containing a neomycin resistance gene ( Neo ) flanked by FRT and loxP sites and a diphtheria toxin gene cassette , was used for Ptbp2 targeting .", "The 5′ and 3′ knockout arms of the targeting construct were generated by high-fidelity PCR amplification with Pfu polymerase of 129S2 genomic DNA .", "The targeting vector was linearized with Xba I and electroporated into 129S2-derived ES cells .", "Isolated ES cell clones were analyzed for homologous recombination .", "Incorporation of the 5′ and 3′ loxP sites was confirmed by Southern blotting using 5′ and 3′ probes following digestion with XbaI .", "Clones with the targeted Ptbp2 allele were injected into 3 . 5-day C57BL/6 blastocysts , and the resulting chimeras were crossed to C57BL/6 females to achieve germline transmission of the targeted ( PTBP2Neo-loxP ) allele .", "The Ptbp2Neo-loxP mice were crossed to FLPe transgenic mice ( 129S4/SvJaeSor-Gt ( ROSA ) 26Sortm1 ( FLP1 ) Dym/J; Jackson , Bar Harbor , ME ) to remove the Neo cassette .", "Ptbp2loxP was crossed to the germline-active EIIaCre transgenic mice ( gift of Stephen Smale ) to obtain the Ptbp2KO alleles .", "Conditional targeting in the CNS or forebrain projection neurons was achieved in crosses with Nestin-Cre ( Jackson ) and Emx-Cre ( gift of William Yang ) mice respectively .", "The Cre-transgenes were all in the C57Bl/6 background .", "Analyzed knockouts had been backcrossed to this background for three to eight generations .", "At least 12 litters were generated for each Cre genotype to assess the phenotypes of each homozygous mutation .", "Genotyping PCR primers were TCTACTTCATTGTGTTGTTTTG ( F1 ) , AGGCATCCATAATTACACAGTGT ( F2 ) , GATACAGCAGGCTCCCCTCA ( R1 ) , AAGTGATACAGCAGGCTCCC ( R1 . 1 ) , ATAAGCATTTTCTAGCACCAA ( R2 ) and GCGCGTATCCTCAAACAAGAC ( R3 ) .", "50-100 ng of purified genomic DNA was amplified by PCR using 5 μL of 2x GoTaq Green Master Mix ( PromegaTM M7123 ) with 0 . 5 uM of each PCR primer in a total 10 μL reaction .", "The F2 and R1 . 1 pair yields a 302 bp product for the WT and a 413 bp product for the loxP allele .", "Initial denaturation was at 95°C for 2 min , followed by 30 cycles of denaturation at 95°C for 30 sec , annealing at 53°C for 30 sec and extension at 72°C for 40 sec , and final extension at 72°C for 6 min .", "The F2 and R3 pair yields a 709 bp product for the loxP deleted allele ( KO ) .", "For these primers , initial denaturation was at 95°C for 2 min , followed by 31 cycles of denaturation at 95°C for 30 sec , annealing at 60°C for 30 sec and extension at 72°C for 40 sec , and final extension at 72°C for 6 min .", "The F1 and R1 primer pair yields a 184 bp product for the WT allele and 295 bp product for the loxP allele .", "The F1 and R2 pair yields a 455 bp product for the loxP deleted allele ( KO ) .", "For these primer pairs , initial denaturation was at 94°C for 4 min , followed by 35 cycles of denaturation at 94°C for 30 sec , annealing at 55°C for 30 sec and extension at 72°C for 30 sec , and final extension at 72°C for 5 min .", "Animals were housed in a 12-hr light/dark cycle with food and water available ad libitum and were maintained by the University of California , Los Angeles ( UCLA ) Division of Laboratory Medicine , as accredited by the Association for Assessment and Accreditation of Laboratory Animal Care .", "All experiments were approved by the UCLA Animal Research Committee .", "Fluorescent Immunoblots were performed on total protein from embryonic or postnatal mouse brain lysed in RIPA buffer and sonicated to homogenize samples using a W-385 Ultrasonic Processor ( Heat Systems/Qsonica , Newtown , CT ) at 10 Hz .", "After a 30 min incubation at 4°C , samples were frozen at −20°C until further processing .", "Lysates were diluted in 2× SDS loading buffer , heated at 95°C for 10 min , and loaded onto 10% polyacrylamide Laemmli SDS-PAGE gels .", "For blotting with fluorophore-conjugated secondary antibodies , transfers were performed on a Novex X-Cell mini-cell transfer apparatus ( Invitrogen/Life Technologies , Grand Island , NY ) onto Immobilon-FL PVDF membranes ( Millipore , Billerica , MA ) .", "The membranes were blotted under standard conditions with primary antibodies overnight at 4°C , followed by washes and incubation with ECL Plex Cy5-conjugated goat α-mouse and goat α-rabbit secondary antibodies ( 1:2500; GE Healthcare , Pittsburgh , PA ) , and then scanned on a Typhoon Phosphorimager ( GE Healthcare ) .", "Quantification of fluorescent signal was performed using ImageQuant 5 . 1 software ( Molecular Dynamics/GE Healthcare , Pittsburgh , PA ) .", "The following primary antibodies were used: α-GAPDH 6C5 ( Research Diagnostics , Inc . , Flanders , NJ ) , α-PTBP2 IS2 ( Sharma et al . , 2005 ) , α-U1 70 k , α-PSD95 and α-Synaptophysin ( Millipore ) , CamKIIbeta ( AbCam , Cambridge , UK ) , Drebrin ( Assay Designs/ENZO , Farmingdale , NY ) , SV2 ( Developmental Studies Hybridoma Bank , Iowa City , IA ) , Cask , GluR2 , Synapsin , NLG1 , NR2B ( NeuroMab , Davis , CA ) .", "To generate histological sections , adult mice were perfused transcardially with ice-cold PBS , followed by ice-cold 4% PFA/PBS .", "The brains were removed , post-fixed in 4% PFA/PBS overnight , cryoprotected in 20% sucrose-PBS , frozen in 4-methyl-butane , and stored at −80°C until use .", "10-µm sections were cut on a cryostat and collected onto Superfrost plus slides ( Thermo Fisher , San Jose , CA ) at −80°C until use .", "The sections were thawed , post-fixed in 10% formalin for 10 min , rinsed twice in PBS , permeablized with 0 . 5% Triton in PBS for 10 min and incubated in 1% normal goat serum and 2 mg/ml BSA in PBS for 1 hr .", "The sections were incubated at room temperature overnight with primary antibodies , rinsed in PBS with 0 . 1%Triton ( PBST ) before incubation with Alexa-conjugated secondary antibodies for 2 hr at room temperature .", "Slides were rinsed in PBST and mounted with a Prolong Gold AntiFade mounting medium containing nuclear stain DAPI ( Molecular Probes/Life Technologies , Grand Island , NY ) .", "In all cases , controls with no primary antibody yielded no labeling .", "Similar procedures were used for immunofluorescence staining of cortical precursors and cerebellar cells , except that they are fixed with 4% PFA/PBS for 20 min and incubated with primary antibody for 2 hr .", "Primary antibodies were used at the following concentrations: PTBP2 ( Boutz et al . , 2007 ) , 1:200; MAP2 ( Millipore ) 1:500: tau-1 ( clone PC1C6; Millipore ) 1:200; AnkG ( NeuroMab ) 1:100 .", "Secondary antibodies , Alexa488-anti-mouse IgG and Alexa568-anti-rabbit IgG ( Molecular Probes ) were used at 1:1000 dilutions .", "Mouse brain RNA was extracted in Trizol after tissue homogenization with a Tissue Tearor ( Biospec , Bartlesville , OK ) , and quantified by absorbance ( A260 ) using a Nanodrop-1000 spectrophotometer ( Nanodrop Technologies/Thermo Fisher , San Jose , CA ) .", "Each RT reaction contained 0 . 5–1 μg total RNA in a 10 μl reaction with 0 . 25 μl Superscript II reverse transcriptase ( Invitrogen ) .", "1/10th volume of the RT reaction was subjected to PCR amplification in a 25 μl PCR reaction containing 200 , 000–500 , 000 cpm of 32P end-labeled reverse-strand primer; PCR reactions were run on an MJ Research PTC-200 thermocycler for 18–22 cycles with an annealing temperature of 55°C .", "A half of each PCR reaction was mixed 1:1 with 95% formamide containing 5% 10 mM Tris pH 8 . 0 with bromophenol blue and xylene cyanol .", "RT-PCR reactions were loaded onto 8% polyacrylamide , 7 . 5 M urea gels and electrophoresed .", "Gels were dried , imaged on a Typhoon Phosphorimager ( GE Lifesciences ) , and bands were quantified using ImageQuant 5 . 1 software ( Molecular Dynamics ) .", "For microarray hybridization , Trizol extracted total RNA from mouse brain was further treated with DnaseI and purified by phenol/chloroform extraction and ethanol precipitation .", "For transcriptome-wide splicing analysis using Affymetrix MJAY splice junction arrays , ribosomal RNAs were removed from samples using the RiboMinus Transcriptome Isolation Kit ( Invitrogen ) according to the manufacturer’s instructions .", "Amplified , biotinylated cDNA samples were produced using the GeneChip Whole Transcript Sense Target Labeling and Control Reagents kit ( Affymetrix , Santa Clara , CA ) according to the manufacturer’s instructions .", "RNA from three animals for each genotype ( wild type and knockout ) was used as biological triplicates .", "Labeled samples were hybridized overnight to a Mouse GeneSplice Array ( Affymetrix PN 540092 ) .", "Hybridized arrays were processed using the Affymetrix Fluidics Station 450 and scanned with an Affymetrix GeneChip scanner .", "Data were analyzed as previously described ( Srinivasan et al . , 2005 ) .", "RNAseq analysis was carried out on polyA + RNA from two brains each of Nes-KO and wild-type littermates at E18 , and dissected mouse cortices from Emx-KO and wild-type littermates at P1 .", "Paired end libraries for 100 nt read length were constructed using the Illumina Tru-seq kit .", "The Emx-KO libraries were strand specific with the second cDNA strand eliminated with the USER enzyme ( Bhatt et al . , 2012 ) .", "Libraries were sequenced on an Illumina HiSeq Genome analyzer in the sequencing core at the Broad Stem Cell Research Center at UCLA .", "Data were analyzed using the standard TopHat , Cufflinks , Cuffmerge , and Cuffdiff pipeline generating 312M and 327M mapped reads from NesKO and wild-type mice respectively at E18 ( Trapnell et al . , 2012 ) .", "At P1 , EmxKO and wild-type samples generated 354M and 317M mapped reads respectively .", "Alternative exon inclusion levels were determined by additional mapping to an exon duo and trio database using Splicetrap ( Wu et al . , 2011 ) .", "Gene Ontology Analysis was performed using DAVID ( Huang da et al . , 2009 ) .", "Mouse primary cortical cultures were prepared from E15-16 embryos and primary hippocampal cultures from E18 embryos as described previously ( Boutz et al . , 2007 ) .", "Briefly , brain tissues were dissected , incubated in 0 . 1% trypsin in Neural Basal medium ( Invitrogen ) , rinsed with Neural Basal containing 5% fetal bovine serum ( FBS ) , and dissociated mechanically .", "Dissociated cells were plated onto poly-L-lysine ( 10μg/ml ) coated dishes with Neural Basal supplemented with B27 and GlutaMax ( Invitrogen ) .", "Cultures were fed with 1/3 volume of fresh Neural Basal/B27/GlutaMax every 3 days .", "All cultures were fixed with 4% paraformaldehyde ( PFA ) in phosphate-buffered saline ( PBS ) for 20 min at room temperature for immunocytochemistry .", "Viability Assays ( Figure 4C ) were carried out using the Life Technologies Live/Dead Cell Viability Kit .", "Briefly , live cells stained with Calcein and dead cells stained with Ethidium dimer were distinguished and counted on multiple microscopic fields to determine the percent of surviving cells at the different time points in culture .", "For the data in Figure 4C , cells from 4 knockout E16-17 embryos derived from two litters , and four wild-type littermate embryos , were plated at 0 . 5 × 106 cells/ml onto coated coverslips in 24-well plates .", "One coverslip from each embryo was stained for viability at each time point , yielding four coverslips for each genotype at each time point ( DIV1 , 4 , 7 , 14 , 18 ) .", "Four regions of each coverslip were counted and summed to yield total cell numbers , with the wild type counts on DIV1 averaged to provide the starting number for determination of ‘percent survival’ at subsequent time points .", "At DIV1 , the four wild-type wells averaged 417 viable cells ( SD = 29 ) and the four knockout wells averaged 442 viable cells ( SD = 30 ) .", "Datasets are submitted to GEO with Accession Number GSE51740 , downloadable at: http://www . ncbi . nlm . nih . gov/geo/query/acc . cgi ?", "acc=GSE51740 ." ] ]

[ "We show that the splicing regulator PTBP2 controls a genetic program essential for neuronal maturation .", "Depletion of PTBP2 in developing mouse cortex leads to degeneration of these tissues over the first three postnatal weeks , a time when the normal cortex expands and develops mature circuits .", "Cultured Ptbp2−/− neurons exhibit the same initial viability as wild type , with proper neurite outgrowth and marker expression .", "However , these mutant cells subsequently fail to mature and die after a week in culture .", "Transcriptome-wide analyses identify many exons that share a pattern of mis-regulation in the mutant brains , where isoforms normally found in adults are precociously expressed in the developing embryo .", "These transcripts encode proteins affecting neurite growth , pre- and post-synaptic assembly , and synaptic transmission .", "Our results define a new genetic regulatory program , where PTBP2 acts to temporarily repress expression of adult protein isoforms until the final maturation of the neuron ." ]

[ "Cells within the developing brain undergo an extended period of maturation .", "A neuronal progenitor cell must first migrate to the proper place within the brain and then develop long extensions that become the axon and dendrites used by the mature neuron to communicate with other cells .", "Finally , the synapses that connect neurons with other neurons must be established .", "Multiple mechanisms are needed to ensure that all the proteins involved in this process are expressed when and where they are needed .", "The production of a protein begins with a region of DNA being transcribed to produce an RNA transcript that consists of segments called exons separated by segments called introns .", "This transcript then undergoes a process called splicing that involves the introns being removed and the exons being joined together to form a messenger RNA molecule that can be translated into protein .", "Specialized RNA binding proteins regulate the splicing process , and most RNA transcripts are subject to a form of splicing called alternative splicing that allows a single gene to express more than one messenger RNA molecule and hence more than one protein product .", "As neuronal progenitor cells in the brain are induced to mature into neurons , many RNA transcripts are seen to change their splicing patterns .", "At the same time , the level of a regulatory RNA binding protein called PTBP1 decreases and the level of a related protein called PTBP2 increases .", "Now Li et al . have studied mutant mice that lack PTBP2 , and have found that structures of the forebrain that normally undergo extensive development after birth instead experience tissue degeneration when PTBP2 is absent .", "Similarly , when neurons lacking PTPB2 are grown in culture , they fail to develop correctly and die .", "Li et al . also found that messenger RNAs from many genes involved in postnatal brain development—affecting processes such as the growth of axons and dendrites and the formation of synapses—exhibit defective alternative splicing in the mutant mice .", "Specifically , protein variants that would normally be expressed only in adult brains were being expressed much earlier .", "By inhibiting the expression of adult forms of proteins until neurons have matured , PTBP2 plays an essential role in controlling the brain’s early development .", "Further work is now required to determine how individual changes in messenger RNA and protein structure controlled by PTBP2 might alter protein function between immature and mature neurons ." ]

[ "Introduction", "Results", "Discussion" ]

[ "evolutionary biology", "computational and systems biology" ]

[ [ "Sewall Wright introduced the concept of fitness landscapes in 1932 ( Wright , 1932 ) , and for decades afterwards it persisted chiefly as a metaphor , due to lack of sufficient data .", "This has changed considerably in recent decades ( de Visser and Krug , 2014; Hartl , 2014; Kondrashov and Kondrashov , 2015; Fragata et al . , 2019 ) .", "There are now a large number of experimental studies that have constructed fitness landscapes for combinatorial sets of mutations relevant to particular phenotypes , such as the resistance of microbial pathogens to antibiotics ( Weinreich et al . , 2006; DePristo et al . , 2007; Marcusson et al . , 2009; Lozovsky et al . , 2009; Brown et al . , 2010; Schenk et al . , 2013; Goulart et al . , 2013; Mira et al . , 2015; Palmer et al . , 2015; Knopp and Andersson , 2018 ) , and the genomic scale of these investigations is rapidly growing ( Wu et al . , 2016; Bank et al . , 2016; Domingo et al . , 2018; Pokusaeva et al . , 2019 ) .", "Mathematical modeling of fitness landscapes has also seen a revival , motivated partly by the need to quantify and interpret the ruggedness of empirical fitness landscapes ( Szendro et al . , 2013; Weinreich et al . , 2013; Neidhart et al . , 2014; Ferretti et al . , 2016; Blanquart and Bataillon , 2016; Crona et al . , 2017; Hwang et al . , 2018; Kaznatcheev , 2019; Crona , 2020 ) .", "Conceptual breakthroughs , such as the notion of sign epistasis ( where a mutation is beneficial in some genetic backgrounds but deleterious in others ) , have shed light on how ruggedness can constrain evolutionary trajectories ( Weinreich et al . , 2005; Poelwijk et al . , 2007; Franke et al . , 2011; Lobkovsky and Koonin , 2012; Zagorski et al . , 2016 ) .", "Despite this progress , a limitation of current studies of fitness landscapes is that they focus mostly on G×G ( gene-gene ) interactions , and little on G×G×E ( where E stands for environment ) interactions , that is on how changes in environment modify gene-gene interactions .", "A few recent studies have begun to address this question ( Flynn et al . , 2013; Taute et al . , 2014; Gorter et al . , 2018; de Vos et al . , 2018 ) .", "In the context of antibiotic resistance , it has been realized that the fitness landscape of resistance genes depends quite strongly on antibiotic concentration ( Mira et al . , 2015; Stiffler et al . , 2015; Ogbunugafor et al . , 2016 ) .", "This is highly relevant to the clinical problem of resistance evolution , since concentration of antibiotics can vary widely in a patient’s body as well as in various non-clinical settings ( Kolpin et al . , 2004; Andersson and Hughes , 2014 ) .", "Controlling the evolution of resistance mutants thus requires an understanding of fitness landscapes as a function of antibiotic concentration .", "Empirical investigations of such scenarios are still limited , and systematic theoretical work on this question is also lacking .", "In the present work , we aim to develop a theory of G×G×E interactions for a specific class of landscapes , with particular focus on applications to antibiotic resistance .", "The key feature of the landscapes we study is that every mutation comes with a tradeoff between adaptation to the two extremes of an environmental parameter .", "For example , it has been known for some time that antibiotic resistance often comes with a fitness cost , such that a bacterium that can tolerate high drug concentrations grows slowly in drug-free conditions ( Andersson and Hughes , 2010; Melnyk et al . , 2015 ) .", "While such tradeoffs are not universal ( Hughes and Andersson , 2017; Durão et al . , 2018 ) , they certainly occur for a large number of mutations and a variety of drugs .", "Tradeoffs can also arise in complex scenarios involving multiple drugs .", "It has been reported in Stiffler et al . , 2015 that certain mutations in TEM-1 β-lactamase are neutral at low ampicillin concentration but deleterious at high concentration , and that a number of the latter mutations also confer resistance to cefotaxime .", "Therefore in a medium with cefotaxime and a moderately high concentration of ampicillin , it is possible that these mutations will be deleterious at low cefotaxime concentrations but beneficial at high cefotaxime concentration .", "Fitness landscapes with adaptational tradeoffs are therefore also of potential relevance to evolution in response to multi-drug combinations .", "Our starting point for investigating fitness landscapes induced by tradeoffs is the knowledge of two phenotypes that are well studied – the drug-free growth rate ( which we call the null-fitness ) and the IC50 ( the drug concentration that reduces growth rate by half ) , which is a measure of antibiotic resistance .", "These two phenotypes correspond to the two extreme regimes of an environmental parameter , that is zero and highly inhibitory antibiotic concentrations .", "The function that describes the growth rate of a bacterium for antibiotic concentrations between these two extremes is called the dose-response curve or the inhibition curve ( Regoes et al . , 2004 ) .", "When tradeoffs are present , the dose-response curves of different mutants must intersect as the concentration is varied ( Gullberg et al . , 2011 ) .", "This is schematically shown in Figure 1 .", "The intersection of dose-response curves of the wild type and the mutant happens at point A , swapping the rank order between the two fitness values .", "The intersection point is known as the minimum selective concentration ( MSC ) , and it defines the lower boundary of the mutant selection window ( MSW ) within which the resistance mutant has a selective advantage relative to the wild type ( Khan et al . , 2017; Alexander and MacLean , 2018 ) .", "When there are several possible mutations and multiple combinatorial mutants , a large number of such intersections occur as the concentration of the antibiotic increases .", "This leads to a succession of different fitness landscapes defined over the space of genotype sequences ( Maynard Smith , 1970; Kauffman and Levin , 1987 ) .", "Whenever the curves of two mutational neighbors ( genotypes that differ by one mutation ) intersect , there can be an alteration in the evolutionary trajectory towards resistance , whereby a forward ( reverse ) mutation now becomes more likely to fix in the population than the corresponding reverse ( forward ) mutation .", "These intersections change the ruggedness of landscapes and the accessibility of fitness maxima .", "In this way a rich and complex structure of selective constraints emerges in the MSW .", "To explore the evolutionary consequences of these constraints , here we construct a theoretical model based on existing empirical studies as well as our own work on ciprofloxacin resistance in E . coli .", "Specifically , we address two fundamental questions:", "( i ) How does the ruggedness of the fitness landscape vary as a function of antibiotic concentration ?", "( ii ) How accessible are the fitness optima as a function of antibiotic concentration ?", "We find that even when the null-fitness and resistance values of the mutations combine in a simple , multiplicative manner , the intersections of the curves produce a highly epistatic landscape at intermediate concentrations of the antibiotic .", "This is an example of a strong G×G×E interaction , where changes in the environmental variable drastically alter the interactions between genes .", "Despite the high ruggedness at intermediate concentrations , however , the topology of the landscapes is systematically different from existing oft-studied random landscape models , such as the House-of-Cards model ( Kauffman and Levin , 1987; Kingman , 1978 ) , the Kauffman NK model ( Kauffman and Weinberger , 1989; Hwang et al . , 2018 ) or the Rough Mt . Fuji model ( Neidhart et al . , 2014 ) .", "For example , most fitness maxima have similar numbers of mutations that depend logarithmically on the antibiotic concentration .", "Importantly , all the fitness maxima remain highly accessible through adaptive paths with sequentially fixing mutations .", "In particular , any fitness maximum ( including the global maximum ) is accessible from the wild type as long as the wild type is viable .", "As a consequence , the evolution of high levels of antibiotic resistance by multiple mutations ( Hughes and Andersson , 2017; Wistrand-Yuen et al . , 2018; Rehman et al . , 2019 ) is much less constrained by the tradeoff-induced epistatic interactions than might have been expected on the basis of existing models ." ], [ "The chief goal of this paper is to develop and explore a mathematical framework to study tradeoff-induced fitness landscapes .", "We consider a total of L mutations , each of which increases antibiotic resistance .", "A fitness landscape is a real-valued function defined on the set of 2L genotypes made up of all combinations of these mutations .", "A genotype can be represented by a binary string of length L , where a 1 ( 0 ) at each position represents the presence ( absence ) of a specific mutation .", "Alternatively , any genotype is uniquely identified as a subset of the L mutations ( the wild type is the null subset , that is the subset with no mutations ) .", "In this paper , unless mentioned otherwise , we define the fitness f as the exponential growth rate of a microbial population .", "The fitness is a function of antibiotic concentration .", "This function has two parameters of particular interest to us – the growth rate at zero concentration , which we refer to as the null-fitness and denote by r , and a measure of resistance such as IC50 which we denote by m .", "Each single mutation is described by the pair ( ri , mi ) , where ri and mi are the null-fitness and resistance values respectively of the ith single mutant .", "We further rescale our units such that for the wild type , r=1 and m=1 .", "We consider mutations that come with a fitness-resistance tradeoff , that is a single mutant has an increased resistance ( mi>1 ) and a reduced null-fitness ( ri<1 ) compared to the wild type .", "To proceed we need to specify two things:", "( i ) how the fitness of the wild type and the mutants depend on antibiotic concentration , and in particular if this dependence exhibits a pattern common to various mutant strains;", "( ii ) how the r and m values of the combinatorial mutants depend on those of the individual mutations .", "To address these issues we take guidance from two empirical observations .", "To understand the evolutionary implications of our model , we first describe how the fitness landscape topography changes with the environmental parameter represented by the antibiotic concentration .", "Next we analyze the properties of mutational pathways leading to highly fit genotypes ." ], [ "Fitness landscapes depend on the environment , and gene-gene-interactions can be modified by the environment .", "Systematic studies of such G×G×E interactions are rare , but they are clearly of relevance to scenarios such as the evolution of antibiotic resistance , where the antibiotic concentration can vary substantially in space and time .", "In this paper we have explored the structure of such landscapes in the presence of tradeoffs between fitness and resistance .", "We summarize the main findings of our work .", "All of these conclusions follow from three basic assumptions that are readily generalizable beyond the context of antimicrobial resistance evolution: the existence of tradeoffs between two marginal phenotypes that govern the adaptation at extreme values of an environmental parameter; the scaling property of the shape of the tradeoff function; and the condition of limited epistasis for the marginal phenotypes .", "How generally these assumptions are valid is a matter of empirical investigation .", "We have shown that they hold for certain cases , and the interesting evolutionary implications of our results indicate that more empirical research in this direction will be useful .", "In the case of antimicrobial resistance , there can be fitness compensatory mutations ( Levin et al . , 2000; Brown et al . , 2010; Durão et al . , 2018 ) that do not exhibit any adaptational tradeoffs .", "These mutations are generally found in a population in the later stages of the evolution of antibiotic resistance , which implies that they emerge in a genetic background of mutations with adaptational tradeoffs .", "An understanding of tradeoff-induced landscapes is therefore a prerequisite for predicting the emergence of compensatory mutations .", "While compensatory mutations are expected to facilitate the evolution of high resistance ( Hughes and Andersson , 2017 ) , our study shows that the acquisition of multiple resistance mutations may readily occur even if compensatory mutations are absent .", "In the formulation of our model we have assumed for convenience that the marginal phenotypes combine multiplicatively , but this assumption is in fact not necessary for all our results .", "As shown in Materials and methods , our key results on accessibility only require the absence of positive epistasis .", "These results therefore hold without exception for the combinatorially complete data set in Table 1 , where epistasis is either absent or negative .", "More generally , our analysis remains valid in the presence of the commonly observed pattern of diminishing returns epistasis among beneficial mutations ( Chou et al . , 2011; Schoustra et al . , 2016; Wünsche et al . , 2017 ) .", "We expect our results to hold approximately even when there is a small degree of epistasis ( positive or negative ) in r and m , but we do not explore that question quantitatively in this paper .", "A strict absence of epistasis , while certainly not universal , can be expected to occur under certain generic circumstances .", "Assuming that we deal with a single antibiotic that has a single target enzyme , we can think of two situations that could lead to a multiplicative behavior of the IC50:", "( i ) Single mutations occur in different genes that affect the concentration of the antibiotic-target enzyme complex through independent mechanisms .", "( ii ) Single mutations occur in the same gene but their effect is multiplicative due to the nature of antibiotic-enzyme molecular interactions .", "An example of scenario", "( i ) would be a combination of mutations in the target gene ( reduction of the binding affinity ) , its promoter ( increase in expression ) , genes regulating the activity of efflux pumps and porins ( decrease in intracellular concentration of the antibiotic ) , or genes controlling the level ( increase in concentration ) or activity of drug-degrading enzymes .", "These mechanisms are ‘orthogonal’ to each other , in the sense that they modify independent pathways within the cell .", "If each of them affects the concentration of the antibiotic-target complex through first-order kinetics , their cumulative effect will be multiplicative in terms of the IC50s of single mutations .", "In the case of ciprofloxacin and E . coli ( Figure 2 and Table 1 ) , we expect mutations in gyrA ( target ) to be orthogonal to mutations in acrR and marR ( efflux pumps ) .", "This is borne out by the observed multiplicativity of the IC50 ( Table 1 ) .", "In contrast , we expect scenario", "( ii ) to apply if the single mutations affect different parts of the antibiotic-enzyme binding site independently .", "This is not the case for two particular mutations in gyrA studied here – S83L and D87N ( see cases of epistasis in Table 1 ) .", "An example for scenario", "( ii ) are the two mutations P21L and A26T in the gene encoding the enzyme dihydrofolate reductase , which increase the resistance to trimethoprim in a multiplicative way in the absence of other mutations ( Palmer et al . , 2015 ) .", "If the antibiotic has more than one target , multiplicativity would not generally hold .", "In particular , topoisomerase IV ( gene parC ) is a secondary target for ciprofloxacin with much weaker affinity than gyrase .", "Therefore , mutations in parC do not contribute to resistance unless there is already a mutation in gyrA .", "As a consequence , in contrast to the mutants listed in Table 1 , combinations containing parC display positive epistasis ( Hughes and Andersson , 2017 ) .", "The co-existence of high ruggedness and high accessibility found in the tradeoff-induced landscapes studied here is counterintuitive , and to the best of our knowledge fitness landscape models with this property have not been described previously .", "The situation is depicted schematically in Figure 8 .", "The first landscape is smooth with a single peak that must be accessible from everywhere else .", "The second landscape is rugged , and each fitness peak is typically accessible from a few genotypes only .", "This is the typical picture of a rugged fitness landscape with limited accessibility , as it would be predicted by simple statistical models such as the HoC , NK or rough Mt . Fuji models ( Szendro et al . , 2013; Neidhart et al . , 2014; Hwang et al . , 2018 ) .", "The landscapes we describe here belong to a third type , where a high number of peaks are accessible from a high number of genotypes , creating overlapping ‘valleys’ from which a population may evolve towards different local fitness maxima .", "Moreover , not only are fitness peaks accessible from all their subset and superset genotypes , but there are many direct paths leading up to each peak .", "This appears contrary to the expectation that in landscapes with high epistasis , accessibility should be facilitated through mutational reversions , that is , indirect paths ( DePristo et al . , 2007; Palmer et al . , 2015; Wu et al . , 2016; Zagorski et al . , 2016 ) .", "We conclude with some possible directions for future work .", "Our model provides a principled framework for predicting how microbial fitness landscapes vary across different antibiotic concentrations .", "This could be exploited to describe situations where the antibiotic concentration varies on a time scale comparable to the evolution of resistance , either due to the degradation of the drug or by an externally imposed treatment protocol ( Marrec and Bitbol , 2018 ) .", "In this context it would be of particular interest to include compensatory mutations that lack the tradeoff between growth and resistance , since such mutations are expected to strongly affect the extent to which resistance can be reversed ( Andersson and Hughes , 2010 ) .", "Significant extension of the theory is required if the drug concentration varies on a faster time scale comparable to the growth time of the microbial population , in which case the concept of a concentration-dependent fitness would need to be reconsidered .", "From the broader perspective of evolutionary systems with adaptational tradeoffs mediated by an environmental parameter , our study makes the important conceptual point that it is impossible to have non-epistatic fitness landscapes for all environments .", "Using the terminology of Gorter et al . , 2016 , the tradeoffs enforce reranking G×E interactions which in turn , as we have shown , induce sign-epistatic G×G interactions at intermediate values of the environmental parameter .", "Notably , this general conclusion does not depend on the scaling property of the tradeoff function .", "It would nevertheless be of great interest to identify instances of scaling for other types of adaptational tradeoffs , in which case the detailed predictions of our model could be applied as well ." ] ]

[ "Fitness effects of mutations depend on environmental parameters .", "For example , mutations that increase fitness of bacteria at high antibiotic concentration often decrease fitness in the absence of antibiotic , exemplifying a tradeoff between adaptation to environmental extremes .", "We develop a mathematical model for fitness landscapes generated by such tradeoffs , based on experiments that determine the antibiotic dose-response curves of Escherichia coli strains , and previous observations on antibiotic resistance mutations .", "Our model generates a succession of landscapes with predictable properties as antibiotic concentration is varied .", "The landscape is nearly smooth at low and high concentrations , but the tradeoff induces a high ruggedness at intermediate antibiotic concentrations .", "Despite this high ruggedness , however , all the fitness maxima in the landscapes are evolutionarily accessible from the wild type .", "This implies that selection for antibiotic resistance in multiple mutational steps is relatively facile despite the complexity of the underlying landscape ." ]

[ "Drug resistant bacteria pose a major threat to public health systems all over the world .", "Darwinian evolution is at the heart of this drug resistance: a mutation that allows bacteria to divide in the presence of a drug appears initially in a single cell .", "This mutation makes this cell and its descendants more likely to survive , so they can end up taking over the population .", "The evolution of resistance can be thought of in terms of ‘bacterial fitness landscapes’ .", "These landscapes visualise the relationship between the mutations present in a population of bacteria and how quickly the bacteria divide or reproduce .", "They are called landscapes because they can be represented as a series of mountains and valleys .", "The peaks of this landscape represent combinations of mutations that give bacteria the greatest chance of dividing ( the greatest fitness ) .", "In a landscape with multiple peaks , some peaks will be higher than others .", "If the landscape is smooth , bacteria can easily acquire mutations for drug resistance .", "However , in a rugged landscape , bacteria may get stuck at sub-optimal peaks , because the mutations that would enable them to reach a higher peak would first lead them to losing fitness .", "Several studies on the evolution of antibiotic resistance exist for specific bacteria and specific drugs , but relatively little is known about the general properties of the underlying fitness landscapes .", "Do these landscapes have features that can help explain the rapid evolution of high levels of resistance ?", "Antibiotic resistance often comes at a cost – more resistant strains of bacteria tend to grow more slowly when the drug is absent .", "To build a model of antibiotic resistance landscapes , Das et al . performed growth experiments on several strains of Escherichia coli exposed to a drug called ciprofloxacin .", "They measured how the rate at which the bacteria divided changed at different antibiotic concentrations , and combined this with the observation about resistant strains growing slower to formulate a mathematical model of antibiotic resistance landscapes .", "The landscapes that resulted were found to be very rugged , but unexpectedly , the bacteria could still evolve to access all fitness peaks .", "This means that landscape ruggedness does not constrain the evolution of resistance .", "Understanding how and when resistance evolves is important both for the design of new drugs and the development of treatment protocols .", "A specific prediction of the model is that resistance evolution in fitness landscapes where resistant strains divide more slowly is reversible .", "This implies that the bacteria could regain their susceptibility to treatment when the drug concentration decreases , but this would depend on the specific bacteria and drug in question .", "More broadly , the model provides a framework for addressing the evolution of resistance in clinical and environmental settings , where drug concentrations vary widely in time and space ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "evolutionary biology", "neuroscience" ]

[ [ "The brain is the anatomical substrate of behavior .", "In turn , the behaviors of a species are closely linked to the ecological context and evolutionary history of that species .", "Large-scale evolutionary modifications in the brain therefore provide essential information about the factors that shape species’ diversification patterns .", "Changes in neurobiological features that directly relate to higher-order cognitive capacities are particularly relevant as they underpin adaptive behaviors such as tool manipulation ( Krützen et al . , 2005; Boesch and Boesch , 1990 ) , flexible problem solving ( Benson-Amram et al . , 2016 ) , planning for the future ( Raby et al . , 2007; Osvath and Osvath , 2008 ) , and sophisticated communication systems ( Janik , 2013 ) .", "Even though it is commonly agreed that instances of intelligent behavior have evolved independently in different lineages of mammals ( Roth and Dicke , 2005; Roth , 2015 ) , it is unclear whether such convergent behavioral abilities arose from modifications of common neural systems or whether lineage-specific contingency has shaped particular brain circuits according to unique socioecological conditions .", "This uncertainty has led to different perspectives on what defines ‘intelligence’ .", "Comparative psychologists describe it as a domain-general problem solving ability that comprises associative-learning .", "Such ‘general intelligence’ has been proposed to equip species with the ability to make mental models of the environment , develop actions based on abstract notions of associations between percepts , and to generate goals from current contexts ( Spearman , 1904; Duncan et al . , 2000; Genovesio et al . , 2014 ) .", "A different view holds that intelligence comprises the aggregate of cognitive modules of special abilities that evolved within a species in response to specific environments ( Barkow et al . , 1995; Cosmides and Tooby , 1992 ) .", "Under this view , intelligence evolved to perform specific computational strategies that are tailored to solve the task demands of ancestrally recurrent adaptive problems ( Cosmides et al . , 2010 ) .", "Here , we examine the mammalian cerebellum to address whether convergent evolution of mammalian cognitive capacities are scaffolded by modifications of this common neural system .", "The cerebellum may be especially informative in uncovering the coevolution of brain structure and cognition for several reasons .", "First , unlike the more commonly investigated cerebral cortex , the cerebellum’s structural , connectional , functional , and developmental anatomy is relatively uniform ( Larsell , 1970 ) and is therefore ideal for the comparison of homologous neural circuits across species ( Smaers , 2014a ) .", "Second , the lateral extension of the cerebellum to form distinct hemispheres arose early in mammalian evolution ( Figure 1 ) , providing the opportunity to investigate the diversification pattern of a newly evolving neural system .", "Third , through its connectional integration with heteromodal association areas in the cerebrum , the lateral cerebellum has been hypothesized to be involved in the generation of domain-general higher-order models of mental activity .", "A central working hypothesis in this context is that the cerebellum imposes a type of cognitive control over information processing that consists of automating sequences of thoughts and actions ( Schmahmann , 1997 ) .", "This cerebellar-type cognitive control may underpin many aspects associated with ‘intelligent’ behavior , such as working memory , executive function , and the development of behavioral learning models ( Strick et al . , 2009 ) .", "Finally , the strong connectional and functional integration between the lateral cerebellar hemisphere and the cerebrum is also evident in developmental modularity .", "Whereas the medial cerebellum develops early , the lateral cerebellum develops later in tandem with cerebral association areas ( Tiemeier et al . , 2010; Altman and Bayer , 1997 ) .", "We investigate the extent to which mammalian lateral cerebellar hemispheres evolved in coordination with the rest of the cerebellum , whether they are correlated with measures of domain-general cognitive performance , and what patterns underlie their evolutionary diversification .", "We primarily focus on the relative measure of lateral to medial cerebellar volume to account for the functional , connectional , and developmental modularity of the cerebellum ( see more details in Materials and Methods ) .", "Volumetric measurements of cerebellar partitions were used because cerebellar volume is a nearly linear function of its number of neurons ( Herculano-Houzel , 2010 ) , and by extension , its relative investment in particular information processing loops ( Herculano-Houzel , 2010 ) ." ], [ "Phylogenetic scaling of lateral to medial cerebellar volume indicates a positive scaling trend ( F = 294 . 6 , p<0 . 001 , λ = 0 . 945 ) with a slope that is higher than unity ( 95% confidence interval: 1 . 167:1 . 478 ) ( Figure 2 ) .", "Residuals were considered as measures of ‘relative lateral to medial cerebellar size’ ( or ‘lateral-medial cerebellar reorganization’ ) .", "The ratio of the observed to the predicted values range from 2 . 3 to 4 . 4 in apes , cetaceans and pinnipeds , from 0 . 6 to 0 . 7 in feliformes , and from 0 . 2 to 0 . 3 in artiodactyls .", "Phylogenetic regression analysis also demonstrated that lateral-medial cerebellar reorganization is a significant predictor of domain-general cognition in primates ( F = 15 . 670 , p=0 . 001 , Figure 2 ) The evolutionary history of lateral-medial cerebellar reorganization was quantified using a Bayesian reversible-jump Ornstein-Uhlenbeck ( ‘OU’ ) approach ( Uyeda and Harmon , 2014 ) ( Figure 3 ) .", "This analysis indicates five shifts in mean value with a posterior probability ( ‘PP’ ) >0 . 8 .", "These regime shifts occurred at the root branches of the apes , the cetartiodactyls , the cetaceans ( note that our sample includes toothed whales only ) , the pinnipeds , and the feliformes ( Figure 3 , Figure 3—figure supplement 1 ) .", "The signal-to-noise ratio of this estimated pattern is 52 . 34 , demonstrating that the analysis has high effect size and high power .", "Phylogenetic analysis of covariance ( Smaers and Rohlf , 2016 ) indicates that these shifts represent significant differences in the intercept of lateral to medial cerebellar scaling ( i . e . grade shifts; Table 1 ) .", "Specifically , apes , toothed whales and pinnipeds are not significantly different from each other , but each ( and as a group ) are significantly different from others .", "Furthermore , feliformes and artiodactyls are not significantly different from each other , but each ( and as a group ) are significantly different from others .", "These results demonstrate that the six regimes identified by OU modelling constitute three significantly different grades ( in order of magnitude of relative lateral to medial cerebellar size ) : apes , toothed whales , and pinnipeds ( grade 1 ) ; rest of the sample ( grade 2 ) ; artiodactyls and feliformes ( grade 3 ) .", "The equality of slopes assumption of analysis of covariance is upheld ( grade 1 versus grade 2: F = 0 . 009 , p=0 . 925; grade 1 versus grade 3: F = 0 . 088 , p=0 . 771; grade 2 versus grade 3: F = 0 . 836 , p=0 . 367 ) .", "The evolutionary history of lateral-medial cerebellar reorganization was also examined by visualizing the evolutionary trait space in an ancestral phenogram ( Figure 3 ) .", "Ancestral states were inferred using a multiple variance Brownian motion ( ‘mvBM’ ) approach ( Smaers et al . , 2016 ) .", "Results using a standard BM and a reversible-jump BM method yielded similar results ( Figure 3—figure supplement 2 ) .", "Lineage-specific amounts of evolutionary change were also estimated using the mvBM approach and compared against a null model of gradual evolution to obtain estimates of how much faster lineages evolve relative to a gradual model of evolution .", "These results are visualized in Figure 3 and presented in full in Figure 3—figure supplement 3 .", "Results using a reversible-jump BM method yielded similar results ( Figure 3—figure supplement 3 ) .", "The same procedures were used to analyze relative cerebellum size ( residuals of total cerebellar volume to the volume of the rest of the brain ) .", "These analyses revealed no regime shifts indicating PP >0 . 8 , and only a single regime shift with PP >0 . 5 ( the ancestral lineage of the zebu ( Bos taurus indicus ) : PP = 0 . 76 ) .", "Two other regimes shifts had 0 . 2 < PP < 0 . 5: the root of musteline carnivorans ( 0 . 38 ) , and cercopithecine primates ( 0 . 29 ) ( Figure 3—figure supplement 1 ) .", "For relative cerebellar size observed/predicted values ranges from 1 . 2 to 1 . 6 for musteline carnivorans , from 0 . 7 to 0 . 9 for cercopithecine primates , and 0 . 5 for the zebu .", "Phylogenetic analysis of covariance ( ‘pANCOVA’ ) confirms that these regimes form significantly different grades ( Supplementary file 1 ) .", "Phylogenetic regression analysis demonstrated that relative cerebellum size is not a significant predictor of domain-general cognition in primates ( lateral-medial: F = 2 . 122 , p=0 . 163 ) .", "The difference in rate of evolution between lateral-medial cerebellar reorganization and relative cerebellum size was tested using a Q-mode approach ( Adams , 2014 ) .", "Rates were found to be significantly different ( p=0 . 002 ) at a ratio of 2 . 9 ( lateral to medial cerebellum σ2 = 0 . 00753 , relative cerebellum size σ2 = 0 . 00261 ) .", "Figure 4 visualizes this rate difference using a standard BM MCMC procedure ( Revell , 2012 ) .", "Similar results were found using mvBM and rjBM procedures .", "We also assessed potential grade shifts in the size of the medial cerebellum relative to the rest of the brain in order to ascertain whether the described convergent trend between apes , toothed whales and pinnipeds may be confounded by different patterns of evolution in the medial cerebellum ( e . g . the medial may be exceptionally small in some clades but not others , confounding the lateral to medial comparison ) .", "Results indicate that apes , toothed whales and pinnipeds are not significantly different in relative medial cerebellum size ( apes versus toothed whales and pinnipeds: F = 1 . 06 , p=0 . 31; toothed whales versus apes and pinnipeds: F = 1 . 11 , p=0 . 30; pinnipeds versus apes and toothed whales: F = 0 . 07 , p=0 . 79 ) .", "Furthermore , analysis of lateral cerebellum size versus rest of brain size yields similar results as the lateral to medial comparison in that apes , toothed whales and pinnipeds are not significantly different from each other , but are different from other mammals ( apes versus toothed whales and pinnipeds: F < 0 . 01 , p=0 . 96; toothed whales versus apes and pinnipeds: F = 1 . 13 , p=0 . 29; pinnipeds versus apes and toothed whales: F = 0 . 56 , p=0 . 45; apes , toothed whales and pinnipeds versus others ( holding constant artiodactyls and feliforms ) : F = 5 . 06 , p=0 . 01 ) .", "These results confirm that the convergent lateral to medial reorganization among these clades is not due to a differential effect on medial and/or lateral cerebellum size , but rather , that it is due to a similarly convergent pattern of lateral to medial reorganization ." ], [ "Further work is needed to expand the detail of the cerebellar delineations , the breadth of the comparative neuroanatomical sample , and the range of behavioral measures on associative learning abilities across mammals .", "Expanding the detail of cerebellar delineations would allow evaluating the extent to which the currently observed macroevolutionary pattern of convergence towards lateral-medial cerebellar reorganization may be driven by different patterns of modularity within the lateral cerebellum across different clades ( e . g . lobule HVII in apes and pinnipeds , lobule HIX in toothed whales ) .", "Further expanding the breadth of the comparative sample and the detail of neurobiological measurements will allow increasing the resolution of evolutionary inference , expanding our understanding of neurobiological modification in relation to different body plans and life styles , and consequently , refining our understanding of the evolutionary pathways that have shaped intelligent behavior in vertebrates .", "Some outstanding questions on species that are not covered by our current sample include the putative expansion of lateral cerebellar hemisphere in bats ( in relation to echolocation and the expansion of the paraflocculus [Larsell , 1970; Larsell and Dow , 1935] ) , and potential differences in lateral cerebellar expansion in baleen versus toothed whales ( baleen whales do not echolocate and may therefore not indicate an expansion of lobule HIX , as observed in toothed whales [Jansen and Jansen , 1969] ) .", "We conclude that a tendency for distantly related mammalian species to converge on lateral-medial cerebellar reorganization plays an important role in explaining cerebellar macroevolution .", "Considering the lateral cerebellum’s hypothesized role in automating higher-order cortical association information processing , this macroevolutionary pattern suggests a tendency for distantly related species to independently acquire ‘cerebellar-like’ associative-learning abilities .", "We propose cerebellar reorganization as a target for broad comparative investigations of neurobiological diversification because it is more reflective of modularity and interconnectivity than overall brain size , and more validly represents homologous functional and neural circuitry than the more traditional focus on overall neocortex ." ], [ "Brain data were taken from MacLeod et al . ( 2003 ) , Smaers et al . ( 2011 ) , and Maseko et al . ( 2012 ) .", "For the anthropoid data , preference was given to data presented in MacLeod et al . ( 2003 ) because it includes more individuals per species .", "Data for anthropoid species not presented in MacLeod et al . ( 2003 ) were then taken from Smaers et al . ( 2011 ) .", "Smaers et al . ( 2011 ) used the same delineation protocol as MacLeod et al . ( 2003 ) , and also used brains processed in the same lab ( Zilles et al . , ( 2011 ) .", "Maseko et al . ( 2012 ) collected additional data using both histological sections ( using similar delineation criteria as MacLeod et al . 2003] and MRI images ( for the elephant and harbor porpoise only ) .", "The comparability between MRI and histological data likely involves a degree of error , although this error was suggested by Maseko et al . ( 2012 ) to be minimal .", "Data are presented in Figure 2—source data 1 .", "Behavioral data were taken from Deaner et al . ( 2006 ) .", "Other data sets of domain-general cognition were considered ( Benson-Amram et al . , 2016; MacLean et al . , 2014 ) , but found to have a limited overlap with the available neuroanatomical data ( ≤7 species ) .", "The phylogeny was adjusted from Faurby and Svenning ( 2015 ) , who used a novel heuristic-hierarchical Bayesian approach for estimating a species-rich ( >4100 species ) phylogeny of mammals .", "In their approach , species with a large amount of sequence data are freely placed in a standard Bayesian MCMC procedure .", "The phylogenetic placements of species with decreasing data quantities are estimated with increasing restrictions on their possible placement .", "Finally , species with no sequence data are placed based on morphological trees or existing taxonomy .", "Additional details can be found in the authors’ full description of their procedure .", "The result of their procedure is a sample of 1000 trees from the final posterior distribution .", "We chose to use the 4160 species tree as this represents the largest possible tree of species all with unambiguous placement in the phylogeny .", "Faurby and Svenning estimated branch lengths on these final trees using a two-step process where some higher-level divergences were manually incorporated from other sources and the remaining branch lengths simulated using the age of the clade and either a Yule or Birth-Death model of evolution .", "Our analysis required a single resolved tree .", "A typical consensus of the 1000 sampled trees would result in negative branch lengths .", "We instead used the maximum clade credibility tree ( MCC ) from the sample , as estimated using TreeAnnotator v2 . 3 . 1 ( Drummond et al . , 2012 ) .", "The resulting tree is presented in Figure 3—source data 1 .", "For the purposes of our analyses , this tree was pruned to contain only those species in our sample .", "To evaluate whether a particular brain structure is enlarged relative to other structures ( or the rest of the brain ) , the standard approach has been to fit a ( phylogenetic ) regression line through a comparative sample and to calculate to what extent predicted values correspond to observed values ( Passingham , 1973 ) .", "The focus of our study lies on the comparison between the lateral and medial cerebellum .", "This measure quantifies changes within the cerebellum between its two major constituent partitions that are functionally , connectionally , and developmentally distinct .", "Whereas the medial ( vermis and paravermis ) cerebellum is involved in basic motor control , proprioception and autonomic functions , the lateral hemispheres are the site of integration for multiple streams of cerebral information processing ( Glickstein et al . , 2011 ) .", "We also ran analyses using an alternative measure that considers the comparison of overall cerebellar size relative to the size of the rest of the brain .", "Although this latter measure is the most commonly used in previous research , it overlooks modularity within the cerebellum .", "Moreover , this measure also does not account for the fact that the cerebellum is highly interconnected with much of the rest of the brain .", "A comparison against the rest of the brain thus performs a statistical control for much of what is neurobiologically relevant .", "We primarily focus on the measure of lateral to medial cerebellum because it represents the cerebellum’s modular organization and is therefore more relevant to understanding the underpinnings of neural information processing ( Passingham and Smaers , 2014 ) .", "To identify the evolutionary dynamics of brain region enlargement we utilize phylogenetic comparative methods that reveal the tempo , mode , and history of trait evolution .", "Using a phylogenetic tree and observed information from contemporary tip taxa , these methods employ statistical and mathematical models of evolution to describe the pattern and rate of trait change along individual branches of a phylogeny .", "As such , these methods infer the temporal origin and rate of evolution of a trait across a phylogenetic landscape .", "The most frequently used statistical model of evolution is standard Brownian motion ( ‘BM’ ) , which assumes that traits change at each unit of time with a mean change of zero and unknown and constant variance ( Cavalli-Sforza and Edwards , 1967; Felsenstein , 1973; Felsenstein , 1985 ) .", "Within Brownian motion , the evolution of a continuous trait ‘X’ along a branch over time increment ‘t’ is quantified as dX ( t ) = σdB ( t ) , where ‘σ’ constitutes the magnitude of undirected , stochastic evolution ( ‘σ2’ is generally presented as the Brownian rate parameter ) and ‘dB ( t ) ’ is Gaussian white noise .", "The standard BM model of evolution is ideally suited not only as a baseline model for hypothesis testing approaches such as least-squares analysis ( ANOVA , ANCOVA , GLS ) , but also as a baseline model for rate analysis .", "The standard BM model is , however , less well suited for estimating the evolutionary history of biological traits as it assumes that the rate of evolution is constant across the sample .", "Therefore , approaches that aim to model the evolutionary history of biological traits commonly incorporate additional parameters to capture possible deviations from the standard gradual mode of evolution assumed by BM .", "Ornstein-Uhlenbeck ( ‘OU’ ) models incorporate stabilizing selection as a constraint and hereby quantify the evolution of a continuous trait ‘X’ as dX ( t ) = α[θ – X ( t ) ]dt + σdB ( t ) where ‘σ’ captures the stochastic evolution of BM , ‘α’ determines the rate of adaptive evolution towards an optimum trait value ‘θ’ ( Hansen , 1997 ) .", "This standard OU model has been modified into multiple-regime OU models allowing optima to vary across the phylogeny ( Butler and King , 2004 ) .", "Such multi-regime OU models allow modelling trait evolution towards different ‘regimes’ that each display a different mean trait value .", "Several methods have been developed that use this modelling approach to model trait diversification by estimating shifts in θ-values along the branches of the phylogeny ( e . g . , Uyeda and Harmon , 2014; Khabbazian et al . , 2016 ) .", "Multi-rate BM approaches expand the standard BM model by including additional rate parameters that capture potential differences in rates among different clades or lineages .", "Venditti et al ( Venditti et al . , 2011; Pagel and Meade , 2013 ) use a reversible-jump algorithm ( ‘rjBM’ ) in a Bayesian MCMC framework to estimate where such potential shifts in rate may have occurred .", "Smaers et al . ( 2016 ) use a heuristic algorithm ( ‘mvBM’: multiple variance BM ) that leverages global and local information to estimate rates of evolution for each lineage in the tree .", "It is clear that these different approaches have different strengths and weaknesses , and should therefore be used within the constraints of what they aim to do .", "OU modelling approaches , for example , are commonly agreed to be a very powerful approach for modelling trait diversification , though recent research has pointed towards some challenges when using such models .", "Specifically , the theoretical properties of the maximum-likelihood estimators for OU parameters can result in non-uniqueness and inaccuracy causing traditional model selection criteria to favor overly complex scenarios ( Lst and Ané , 2014 ) .", "More recent Bayesian ( Uyeda and Harmon , 2014 ) and least-squares ( Khabbazian et al . , 2016 ) procedures , however , have proposed adjustments to traditional procedures that overcome these difficulties .", "Also multi-rate BM models have clear limitations .", "Such approaches are commonly highly parameterized ( Lst and Ané , 2014 ) and therefore less suitable for hypothesis testing ( Smaers and Mongle , 2017 ) .", "Such models are , however , particularly useful for providing best-fit estimates of evolutionary history ( Smaers and Mongle , 2017 ) .", "We modeled changes in mean values along individual branches of the phylogeny using a Bayesian reversible-jump OU procedure ( Uyeda and Harmon , 2014; Uyeda and Eastman , 2014 ) .", "This procedure estimates a best-fit adaptive regime configuration of cerebellar reorganization ( more info in SI ) , whereby ‘regimes’ are defined as a group of lineages with a similar mean value ( θ in the OU model framework ) .", "By using a Bayesian parameter estimation procedure this approach avoids the non-uniqueness of parameter estimation inherent to maximum likelihood procedures ( Lst and Ané , 2014 ) .", "To avoid overfitting this procedure uses a conditional Poisson distribution as a prior on the number of shifts ( ranging from zero to half the number of tips ) .", "Furthermore , this procedure allows the posterior probability ( ‘PP’ ) threshold to call a shift to be adjusted so as to provide more liberal ( PP ≥0 . 2 ) or more conservative ( PP ≥0 . 8 ) estimations .", "A more liberal PP threshold hereby tends to result in high recall rates ( many of the true shifts are detected ) and low precision ( many false positives are detected ) , while a more conservative PP threshold tends to result in low recall rates ( some true shifts are not detected ) and high precision ( few false positives are detected ) .", "Ancestral values were inferred using a multiple variance BM ( ‘mvBM’ ) approach ( Smaers et al . , 2016; Smaers and Mongle , 2018 ) .", "Code to implement mvBM and phylogenetic ANCOVA is available from the 'evomap' R package ( Smaers and Mongle , 2018; copy archived at https://github . com/elifesciences-publications/evomap ) .", "This procedure provides an estimate of evolutionary history that is based on lineage-specific rates of evolution ( visualized in the ancestral phenogram Figure 3b ) .", "This approach has been shown to provide estimates equivalent to standard BM when the trait evolves according to that model , and to outperform it when the trait does not adhere to standard BM by improving the estimation of trait evolution in those location where the evolutionary process deviates from standard BM ( Smaers et al . , 2016; Smaers and Mongle , 2017 ) .", "In Figure 3—figure supplement 2 and 3 we also report results obtained using a reversible-jump BM ( ‘rjBM’ ) method , which is a different multi-rate BM approach ( Venditti et al . , 2011; Pagel and Meade , 2013 ) .", "This different approach provides equivalent results for the analyses presented here .", "Both these methods were used in a Bayesian MCMC framework using 10 million iterations and sampling every 100th iteration , which rendered normal distribution of log likelihood values for all analyses .", "Lineage-specific variation was compared to a baseline expectation given a standard BM model to provide estimates of how much faster evolution in a particular lineage is estimated to be relative to a gradual model .", "The amount of change observed at each branch ( the difference between descendant and ancestral branches as inferred using the mvBM and rjBM approaches ) was compared with a neutral scenario in which all the species in the phylogeny were simulated to evolve at a constant rate ( Gómez-Robles et al . , 2017 ) .", "For these analyses , the original phylogeny was transformed to generations .", "Age at first reproduction as obtained from PanTHERIA database ( Jones et al . , 2009 ) was used as a proxy for generation time .", "When this variable was not available for a given species included in our dataset , the value corresponding to the closest species with known age at first reproduction was used .", "The time-based phylogeny was rescaled to generations by dividing each branch length by the generation time corresponding to their descendant species or descendant inferred node .", "A per-generation rate of evolution was calculated based on available data ( Martins , 1994 ) , and it was later used to simulate evolution over the studied phylogeny at that constant rate ( Polly , 2017; Polly , 2004 ) .", "Simulations were repeated 100 times for each trait and differences between descendant and ancestral values were calculated .", "The average of those differences for each branch were used as the neutral expectation of the amount of change that each branch would have accumulated had all the branches evolved at the same rate .", "The ratio between observed and simulated amounts of change per branch is lower than one for slow-evolving branches and greater than one for fast-evolving branches .", "Because estimation of evolutionary patterns is inherently uncertain we translated the estimated model from the Bayesian reversible-jump OU procedure into a least-squares framework .", "Least-squares analysis allows testing whether the patterning of the extant variation suggested by the evolutionary estimation is significant .", "We hereby used the least-squares solution to phylogenetic analysis of covariance ( pANCOVA ) ( Smaers and Rohlf , 2016 ) to test for differences in slopes and intercepts among the extant values of the estimated regimes .", "This implementation of pANCOVA includes additional indicator variables describing group membership to the standard generalized least-squares procedure ( y=Xb+ϵ ) ( Smaers and Rohlf , 2016 ) .", "This procedure calculates the change associated with the clades of interest in the residual variance simultaneously with the phylogenetic regression parameters , and hereby allows for a direct test of whether a model with multiple grades ( assuming multiple groups with different mean trait values ) provides a significantly better fit to the data than a model with only a single grade ( assuming that no particular group indicates a significantly different mean trait value ) .", "Technical details and examples of implementation are available in Smaers and Rohlf ( 2016 ) .", "Code to implement pANCOVA is available from the ‘evomap’ R package ( Smaers and Mongle , 2018 ) .", "We further include the λ parameter in order to account for the degree of phylogenetic signal in the data ( Pagel , 1997 ) .", "Considering the uncertainties involved in reversible-jump and Ornstein-Uhlenbeck modelling , this step provides a crucial confirmation that the estimated results from the modelling analyses are indeed significant .", "To test for differences in rate among different measures of cerebellar reorganization , we use the procedure proposed by Adams et al ( Adams , 2014; Denton and Adams , 2015; Adams and Otárola-Castillo , 2013 ) .", "This method uses a distance-based approach ( Q-mode ) to quantifying evolutionary rate .", "Q-mode approaches provide estimates of evolutionary rates that are numerically identical to those obtained using covariance-based implementations ( R-mode ) .", "The advantage of the Q-mode approach is that it can be extended to high-dimensional data while maintaining appropriate Type I error and high statistical power for detecting differences in σ2 ( 25 ) .", "This approach assumes a standard BM model of evolution .", "Hypothesis testing is performed by comparing the observed ratio of evolutionary rates with a distribution of possible ratios obtained under the null hypothesis that there is no rate difference between traits .", "Estimating patterns of evolution along individual lineages given comparative trait data and a phylogeny is an inherently uncertain endeavor ( Lst and Ané , 2014 ) .", "Several steps can , however , be taken to confirm the reliability of the estimated patterns ( Smaers et al . , 2017 ) .", "First , when possible results should be translated to least-squares analysis .", "Least-squares analysis allows for hypothesis testing and can hereby confirm or falsify the patterning of the extant variation that is suggested by evolutionary modelling .", "This is particularly true for bivariate allometric analyses .", "The phylogenetic regression ( ‘pGLS’ [Rohlf , 2001] ) and its extensions towards more complex generalized linear models ( e . g . pANCOVA [Smaers and Rohlf , 2016] ) are the most powerful hypothesis testing approaches for comparative data .", "Although least-squares analysis does not allow confirming lineage-specific evolutionary patterns , it is clear that the patterning of the extant variation as suggested by evolutionary modelling analysis is expected to produce significant results when used in least-squares analysis .", "Because observed power is a simple function of the observed P-value in least-squares analysis ( Hoenig and Heisey , 2001 ) , tests that produce significant results can be considered to have high power .", "Second , proxies of effect size can be calculated for evolutionary patterns that have been estimated using OU modelling .", "Cressler et al . ( 2015 ) demonstrated that a signal-to-noise ratio ( ηϕ ) provides a better predictor of power than sample size .", "This ratio compares the minimum difference in mean value among regimes ( multiplied by the strength of directional change among regimes ) with a measure of noise intensity .", "Cressler et al . ( 2015 ) demonstrated that when ηϕ≫1 , high statistical power can be inferred .", "Such measures of effect size are crucial indicators of reliability and can thus be used to build confidence in the accuracy of estimated patterns .", "Third , reliability of the estimated patterns can further be confirmed by testing the same hypothesis using different methods with different model assumptions .", "If the same result is obtained regardless off methods used or models assumed , it can be concluded that the results are reliable .", "We followed these three steps to confirm the patterns estimated by the Bayesian reversible-jump OU procedure .", "We confirmed the statistical significance of differences in intercept among three grades using pANCOVA ( Figure 3 ) , demonstrated that ηϕ≫1 is true for the results presented in Figure 3 , and that the pattern presented in Figure 3 is confirmed using pANCOVA , mvBM ancestral and rate estimation and rjBM ancestral and rate estimation ." ] ]

[ "Given that complex behavior evolved multiple times independently in different lineages , a crucial question is whether these independent evolutionary events coincided with modifications to common neural systems .", "To test this question in mammals , we investigate the lateral cerebellum , a neurobiological system that is novel to mammals , and is associated with higher cognitive functions .", "We map the evolutionary diversification of the mammalian cerebellum and find that relative volumetric changes of the lateral cerebellar hemispheres ( independent of cerebellar size ) are correlated with measures of domain-general cognition in primates , and are characterized by a combination of parallel and convergent shifts towards similar levels of expansion in distantly related mammalian lineages .", "Results suggest that multiple independent evolutionary occurrences of increased behavioral complexity in mammals may at least partly be explained by selection on a common neural system , the cerebellum , which may have been subject to multiple independent neurodevelopmental remodeling events during mammalian evolution ." ]

[ "The brains of mammals consist of the same basic structures , but each of these structures varies from one species to the next .", "A given structure may be larger in one species than another , for example .", "It may contain different numbers or sizes of cells .", "It may even have different connections to other brain regions .", "By comparing individual brain structures between species , we can map how the mammalian brain has evolved .", "Smaers et al . have now done this for the cerebellum , a structure at the back of the brain .", "The mammalian cerebellum consists of three main areas: the vermis , paravermis , and the lateral hemispheres .", "Smaers et al . show that in apes , dolphins and seals , the lateral hemispheres are unusually large relative to the cerebellum as a whole .", "This could indicate that these three groups of animals share a common ancestor with enlarged lateral hemispheres .", "Yet , genetic studies suggest that this is not the case .", "Another possibility is that apes , dolphins and seals independently evolved enlarged lateral hemispheres .", "This may have given rise to a trait that proved beneficial for each of them .", "But what might this be ?", "Studies in people suggest that the lateral hemispheres help to support some forms of learning .", "Apes , dolphins and seals are among only a few species of mammal with the ability to learn new calls and vocalizations .", "The expansion of the lateral cerebellum may therefore have contributed to the evolution of vocal learning , and this may have occurred independently on at least three separate occasions .", "Future work should extend this analysis to other cognitive skills , as well as to other species .", "Bats , for example , would be of particular interest because of their ability to echolocate .", "Finally , the lateral hemispheres consist of several subregions that play different roles in learning and information processing .", "Further experiments should explore whether different subregions have increased in size in different species ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "tools and resources", "neuroscience" ]

[ [ "Data are the essence of what science delivers - to society , to researchers , to engineers , to entrepreneurs .", "These data enable progress , as they provide the basis on which new experiments are designed , new machines are developed , and from which new ideas emerge .", "Independent of the research field , many terabytes of data are produced every year , yet only a small fraction of these data become openly available to other researchers , with even less penetrating the invisible wall between the scientific community and the public ( Mayernik , 2017 ) .", "While research papers report conclusions that are based on data and present summaries and analyses , the underlying data most often remain unavailable , despite their value beyond the original context .", "Whereas this is changing in many fields and the use of open data repositories becomes increasingly mandatory upon publication of a research paper , this is not ubiquitous and older data remain inaccessible in most instances .", "Additionally , merely meeting the data deposition requirement by 'dumping' poorly annotated raw files on an internet platform does not aid transparency or reuse of the data .", "To ensure common standards for data repositories and the datasets to be stored in them , the FAIR principles for data deposition ( Findability , Accessibility , Interoperability , and Reusability ) were developed ( Wilkinson et al . , 2016 ) .", "It is clear from these principles that annotation and rich metadata are essential , if a dataset is supposed to be beneficial to others .", "While this is relatively easily achievable for data such as gene sequences , protein sequences , or numerical datasets , the challenges are much bigger for complex morphological data , physiological observations , or behavioral studies .", "The difficulties result not only from large file sizes of image stacks , high-speed videos , or recorded voltage traces , but also from the heterogeneous data structure often generated by custom designed software or equipment .", "Insect neuroscience is no stranger to these challenges .", "Particularly for research outside the genetically accessible fruit fly Drosophila , no universal data repository exists that allows retrieval of original observations that underlie published articles .", "Research groups worldwide investigate the nervous systems of a wide range of insect species , but mostly operate in isolation of each other .", "Data from these projects are often deposited in local backup facilities of individual institutions and thus remain inaccessible to the community .", "Combined with a lack of interoperability caused by independent choices of data formats this has the potential to severely hamper progress , given that interspecies comparison is one of the existential pillars on which insect neuroscience rests .", "The problem is amplified by the fact that much of the data are both large and heterogeneous ( e . g . 2D and 3D images , models of brain regions , digital neuron reconstructions , immunostaining patterns , electrophysiological recordings , functional imaging data ) .", "A final problem is that depositing well-annotated data takes time and effort , and little incentive is generally given to prioritize this work over acquiring new data or publishing research papers .", "While this is true for all research fields , the complex data in neuroscience requires an extra amount of effort to meet acceptable standards .", "This has made depositing data in a form that is useful to the community a relatively rare event .", "Early efforts were made to develop brain databases for various insect species ( e . g . honeybee [Brandt et al . , 2005] , Manduca sexta [El Jundi et al . , 2009a] , Tribolium castaneum [Dreyer et al . , 2010] , desert locust [Kurylas et al . , 2008] ) , but in those cases , the anticipated interactive platforms for exchange and deposition of anatomical data were short-lived and not used beyond the laboratories that hosted them .", "More recently , several successful databases for anatomical data from insects were developed .", "Most notably , Virtual Fly Brain ( VFB ) ( Osumi-Sutherland et al . , 2014 ) now bundles most efforts in the Drosophila community regarding the deposition of neuroanatomical data - including single-cell morphologies from light microscopy , data from recent connectomics projects ( most notably from Scheffer et al . , 2020 ) , as well as catalogues of GAL4 driver lines , which enable access to specific neurons with genetic methods .", "VFB hosts most content of older independent databases , such as FlyCircuit ( Chiang et al . , 2011 ) and FlyBrain ( Armstrong et al . , 1995 ) and is dedicated to providing smart ways of utilizing and visualizing Drosophila neuroanatomy .", "Similarly , but more focused on data visualization and connectivity modeling , the FruitFlyBrainObservatory allows access to current datasets of single neuron morphologies from Drosophila .", "Another database , founded in 2007 , has grown substantially over recent years: NeuroMorpho . Org ( Ascoli et al . , 2007 ) .", "It provides 3D reconstructed datasets of more than 100 , 000 neurons from across animal species and includes substantial numbers of single-cell data from insects .", "While the latter platform is comparative in nature , it does not offer dedicated tools for comparisons between species or much context for the deposited neuron skeletons .", "In contrast , the data on VFB are much richer and different datasets are tightly linked to each other , allowing , for example , correlation between GAL4 driver lines and electron microscopy based single neuron reconstructions .", "Yet , no comparison to other species is possible or intended via VFB .", "Additionally , no systematic information is provided about the function of the deposited neurons in either database , precluding insights into the structure-function relations that are critically important to understanding the insect brain .", "To address these shortcomings we have developed the Insect Brain Database ( IBdb ) , a cross-species , web-based platform designed for depositing and sharing research data that consist of morphological and functional information from insect brains .", "With an overall modular design , a concept for dual use as depository and data management tool , combined with widely useful visualization tools , this database yields a tool for increasing transparency , accessibility , and interoperability of insect neuroscience data .", "Moreover , the newly developed concepts are not only relevant to insect neuroscience , but to any scientific field that can be linked to a hierarchically organized framework .", "We thus hope that our conceptual design can be adopted by a range of users from across the sciences to simplify data handling and make scientific results in general more transparent ." ], [ "The 'Insect Brain Database' ( IBdb ) can be found on the internet at insectbraindb . org and is freely available to everyone .", "It can be used with most modern web browsers with active Javascript ( tested with Google Chrome , Safari , Firefox ) on computers running any operating system , without the need for any additional plugins .", "A user account can be registered free of charge and is required for users who wish to download data and to contribute content .", "The IBdb is divided into three main hierarchical layers: Species , brain structures , and neuron types .", "Each level is additionally linked to 'experiments' , which is the fourth major organizational layer of the database ( Figure 1A ) .", "At each level , a database entry is represented by profile pages , on which all relevant information is collected ( Figure 1C ) .", "These profile pages are the core of the database .", "They can be reached directly by several search functions , as well as by entry lists .", "As profile pages are embedded in a hierarchical framework of species , brain regions , and cell types , they become automatically associated with metadata .", "For instance , an entry for a pontine neuron of the fan-shaped body ( central body upper division ) in the monarch butterfly would become linked to the brain regions 'fan-shaped body/central body upper division' and its parent region 'central complex' , as well as to 'monarch butterfly' as species .", "The neuron can then be found , for example , by querying the brain regions associated with it , or by exploring the species of interest .", "Species entries contain representative data of an insect species , with the aim of defining that species and the overall layout of its brain .", "Similarly , entries for brain regions and cell types contain representative examples of data that illustrate the respective entity and provide all information to unambiguously define it , essentially providing type-specimen .", "While the definition of species and brain regions are straight forward in the context of this database , neuron types can theoretically be defined in many different ways , based on connectivity , function , developmental origin , morphology or neurochemical identity .", "Given that the database is predominantly organized according to anatomical principles , we have defined a cell type as a collection of individual neurons that are morphologically indistinguishable at the level of light microscopy .", "In many cases , a cell type comprises only a single individual per brain hemisphere , but in cases where multiple identical neurons are present , a cell type is defined as the first level of similarity beyond the individual neuron .", "Higher levels of neuron categories group neurons according to anatomical similarities ( Figure 1—figure supplement 2 ) .", "Contrary to the first three levels , experiment entries contain specific data from individual , defined experiments ( Figure 1B ) .", "As research data can be obtained at the levels of species , brain regions and cell types , experiment entries exist for all three levels and are accessible via the respective profile pages ( Figure 1A ) .", "This distinction between representative and concrete data is important , as for example only one entry for a certain columnar neuron type of the central complex exists in the database , yet , if that single cell type was subject to 30 intracellular recordings , the profile page of that cell type would list 30 experiments , each containing a unique individual neuron with its associated physiology .", "While an anatomical type specimen can easily be defined as the most complete example of any particular neuron’s morphology , the decision of what content to depict as representative functional data is less straight forward .", "The functional information on the profile page should reflect the range of experimental results present in the experiment entries of any particular cell type .", "As those results potentially diverge , for example due to different experimental paradigms used in different research groups , the function section can contain as many entries as needed to capture the available information , without need for consensus .", "With new experiment entries added , this section can evolve over time .", "In the context of this evolving content , it is key to ensure that entries which are cited in published work remain findable in the exact form they existed when they were cited ( Ito , 2010 ) .", "Therefore each entry receives a persistent identifier .", "This identifier ( a 'handle' ) links to a version of the entry that was frozen at the time it was created ( and cited ) .", "Once information is added or removed from that entry , a new handle must be generated , providing a new access link for future citations .", "This system is applied to multiple levels of the database ( experiments , neurons , and species ) and ensures that all information in the database , as well as the interrelations between entries , are truly persistent .", "As locating specific datasets is one of the core functions of a database , we have developed a novel , more intuitive way to find specific neuron data .", "A graphical representation of the insect brain , resembling the overall anatomical outline of all brain regions and highlighting the regions containing neuron data ( Figure 2A ) , makes it easy to search for neurons within single species and across species .", "This graphical interface is generated directly on the database website for each species and is adapted from a generic insect brain , that is a shared ground plan .", "This generic brain is the least detailed fall-back option for any cross-species search and was generated based on the insect brain nomenclature developed by Insect Brain Name Working Group et al . , 2014 .", "It resembles the shared anatomical hierarchy of all brain regions in insect brains .", "Within this hierarchy , the entire brain is divided into 13 super-regions , which consist of individual neuropils .", "The latter can be further divided into sub-regions .", "While all super-regions exist in all species , differences become more pronounced at lower levels of the hierarchy .", "The generic brain therefore largely contains super-regions as well as several highly conserved neuropils ( Figure 2—figure supplement 1 , Figure 2—figure supplement 2 ) .", "As these categories are simply tags of brain region entries used to organize the database , the search interface does not differentiate between them , simplifying the user experience ( Figure 2A ) .", "If more than one species is subject to a query , a schematic brain is generated that displays the commonly shared features of the species involved .", "For both single and multi-species search , when selecting a specific brain region , all neurons in the IBdb that connect to this region become visualized by a dynamically drawn wiring diagram ( Figure 2B ) .", "Filters can be applied to narrow down search results according to neuron polarity , functional class , etc .", "Individual neurons in the wiring diagram can be selected to reveal the neuron's profile page .", "Here , all available information for this cell type is displayed , including links to deposited experiment entries ( Figure 2C ) .", "The schematic display of search results can visualize any neuron in the database , only requiring that a neuron is annotated with respect to the brain regions it innervates .", "A list of search results is additionally made available after each search and offers the possibility to also display experiment entries associated with the found neurons .", "For single species queries , two more modes for visualizing search results are available: the semi-schematic view and the 3D view .", "The semi-schematic view mode emphasizes the natural brain organization on the level of brain regions , while also serving as interface for launching search queries .", "It comprises a full series of automatically generated sections through a segmented 3D brain of a species .", "Each brain region present in that species' 3D brain is shown as an interactive cross section that can be used to query neural connections of that region ( Figure 2D ) .", "If a region is selected , all connected brain areas are highlighted and neurons resulting from this query can be visualized by changing to either the schematic view , the 3D view , or a list view .", "The advantage of the anatomically correct layout of this interface is that a brain region can be queried for a neuron , even if its name is not known to the researcher .", "This is particularly useful for regions with uncommon names that have only recently been introduced to the insect brain naming scheme ( e . g . crepine , superior clamp , etc . , see Insect Brain Name Working Group et al . , 2014 ) .", "Launching a search for neurons in regions with unfamiliar names is made much easier when the search interface resembles the information a researcher has obtained from , for example , confocal images or physical brain slices .", "The semi-schematic mode of the database search function fulfills this demand and bridges the schematic wiring diagram view and the full 3D view .", "The 3D view visualizes search results in an anatomically correct way and shows queried neurons in the context of a species' reference brain ( given that this information was added ) ( Figure 2E ) .", "It displays interactive surface models of that brain together with neuron skeletons obtained from the neuron-type's profile page .", "In the graphical search interface the search parameters are limited to anatomical information defining the neuron's location in the brain ( i . e . likely input and output areas ) .", "In contrast , an additional text-based search function ( ’Expert Search’ ) allows users to query all information deposited on a neuron's profile page .", "Individual search parameters can be logically combined to generate arbitrarily complex searches .", "The results are displayed as a list of neurons , which can be sent to populate a schematic wiring diagram view by a single click .", "Thus , this tool effectively combines complex search with the advantages of an intuitive display of results .", "A different means of locating data on neurons and experiments is achieved via a publication based search function .", "Each neuron and experiment that is associated with a publication becomes automatically part of a dataset linked to this publication .", "By definition , experiments are only part of one publication , while neuron entries can be referenced by many publications .", "In either case , users can locate all data that has contributed to a specific piece of scientific literature .", "Finally , whereas the emphasis of the database search lies on locating cell types , information on brain regions can also be found using identical interfaces .", "The schematic search option allows users to reveal brain region profile pages by selecting schematic neuropil representations .", "The same information can also be obtained by selecting brain regions in the semi-schematic neuropil search interface .", "To maximize the usefulness of the database , we have implemented an integrated 3D viewer to deliver platform independent , high-quality data visualization without any additional software demands .", "Neuropil visibility can be independently switched on and off for each brain region , transparency can be freely adjusted , and colors of neurons can be changed ( Figure 3A ) .", "Neurons can be shown either with diameter information or as simple backbones .", "The built-in screenshot function enables the user to capture any scene displayed in the 3D viewer and produces a high-resolution , publication-ready image with transparent background ( Figure 3B , C ) .", "The IBdb allows users to not only locate neuronal morphologies quickly , but also to combine arbitrary neurons from any single species into a common visualization .", "To achieve this , we have generated a neuron clipboard , in which individual neurons from search results can be stored temporarily ( Figure 3D ) .", "Any subset of cells in the clipboard can be sent to the 3D viewer , as long as all neurons belong to the same species , that is can be displayed using the same reference brain .", "The desired configuration of neurons and neuropils can be generated using the interactive tools of the viewer and the screenshot function can be used to create a high-resolution image to be used for illustration purposes ( e . g . reviews , conference talks , teaching ) .", "Additionally , we have embedded a function to directly compare up to four neurons side by side on screen .", "Any neuron located in the neuron clipboard can be chosen to be included in this comparison .", "The comparison uses the 3D view , the profile image , or the confocal stack located on the respective neurons' profile pages .", "This function is ideally suited to compare homologous neurons from across species to quickly assess differences and shared features of these cells .", "The four-window 3D viewer retains all functions of the normal full screen 3D viewer and thus also allows the capture of high resolution screen shots of each of the neurons being compared ( Figure 3E ) .", "The data in the database are suited for many applications , including more sophisticated ones .", "To provide direct access to all levels of the data in the IBdb we have created an API interface , specifying how to automatically draw data from the database via web-browser based apps .", "Applications produced by third parties that use this function can be embedded directly on the IBdb website , once they are approved by the site administrators .", "Applications envisioned are , for example , quantitative comparisons of both single neuron morphologies and neuropils between species , direct online multi-compartment modeling of neurons deposited in the database , or virtual reality interfaces that allow exploration of anatomical data in a 3D virtual reality environment .", "Over time , we hope that our unified platform will stimulate the insect neuroscience community to generate a collection of online tools to analyze and explore neuroanatomical and physiological data deposited in the IBdb , thereby allowing straight-forward meta-analysis of all raw data deposited in the database .", "As an offline tool , the Natverse package in R already offers the possibility to explore IBdb data ( Bates et al . , 2020 ) .", "All data on the internet is public .", "This also applies to any data publicly available in the IBdb .", "Driven by the requirement to obtain data persistency and implemented by the use of handles , no data can be removed from the database once it is public ( and thus citable ) .", "For all data , the contributors retain ownership and hold the copyright to their data .", "The publishing is performed explicitly by the owners , not by the database administrators , and the license attached to each dataset is a Creative Commons Attribution Non Commercial 4 . 0 International ( CC BY NC 4 . 0 ) .", "Thus , when data in the IBdb are downloaded for reuse , the original work that underlies these data has to be credited together with the IBdb as the source .", "When data are used to generate images with the help of the IBdb , these images are licensed via Creative Commons Attribution 4 . 0 International ( CC BY 4 . 0 ) , that is they can be used in any publication as long as the original data owners and the IBdb are credited .", "Data can be contributed by registered users at all levels of the database , i . e . species , brain regions , neurons , and experiments .", "The process is similar on all levels but requires more expert knowledge the higher in the hierarchy the data reside .", "In the following sections we will briefly illustrate the main principles of how to contribute data ( for full instructions see the Online User Guide ) .", "The database is managed via a group of voluntary curators , a scientific administrator , and a technical administrator .", "Importantly , no single person curates all data in the IBdb , but each species is managed by a specialized curator , who is an expert for that species .", "This distributed curation system ensures that no single person is responsible for too many datasets , and that no curator has to evaluate data outside their area of expertise .", "To additionally reduce the workload for species with many entries , more than one curator can be assigned to any given species .", "The scientific administrator ( the lead author of this publication ) oversees the curators , while technical administration is carried out by the technical administrator ( last author of this publication ) .", "The technical administrator is the only person who has potential access to all data in the database .", "The responsibility of the scientific administrator is to approve new species and to train and support the curators for individual species .", "This training is carried out during a training period during which actions of the curator have to be approved by the scientific administrator before they take effect .", "Once a new curator is sufficiently trained to carry out all tasks independently , the scientific administrator grants full curator privileges .", "The responsibility of each curator is to approve new neuron-type datasets and to re-evaluate major updates of these ( data re-approval ) .", "This process entails checking for formal errors in the submitted data , ensuring that new data do not accidentally duplicate already existing data , and that the quality of the data meets acceptable standards .", "To ensure swift correction of any issues , we have implemented a private communication channel between curator and data owner .", "This was realized through a commenting function that enables the curator to post comments on a neuron page , which are only visible to the data owner .", "The owner can then directly respond to the comments and any issues raised can be resolved .", "While our distributed approach to curation has many advantages , it creates challenges towards ensuring that curation of data across all species and levels is accurate , consistent and complete .", "We have therefore designed multiple tools and features to facilitate effective and consistent data curation .", "Besides formalized training and approval for each new curator , we have provided checklists for both data contributors and curators ( found in the online help menu ) that must be followed to ensure that database entries fulfill predefined standards .", "Yearly meetings among all curators will ensure that these standards are known and can evolve over time .", "If mandatory data is not provided by a data contributor , request for approval of the dataset is automatically blocked , preventing rudimentary datasets to enter the public section of the database .", "Finally , if an entry becomes obsolete , for example when new research data provides conclusive evidence that cell types listed as separate entries belong to the same type , existing entries can be archived .", "This process preserves the persistent handle of the entry , but removes it from public lists and search results in the IBdb .", "This way , references to these datasets remain resolvable , while , at the same time , obsolete data cannot clutter the database .", "In the long run , this function provides the means to keep the database clean without violating the principle of data persistence .", "The definition of what constitutes minimal standards for a neuron or species entry reflects a scientific consensus among curators .", "Rather than imposing these standards onto the field , the IBdb generates the means to develop a common set of rules by providing the platform for continued discussions among curators as well as to reinforce an evolving consensus .", "To enable all users to provide feedback and to discuss topics relevant to other database users , we have added a discussion forum directly to the IBdb website .", "This forum is intended as a means for reporting potential bugs , suggesting new IBdb features , or for discussing scientific content ( methods for data processing or acquisition , requests for literature , staining protocols etc . ) .", "Each database entry has to be explicitly published by the contributor .", "In the process , it is approved by either the database administrator ( species ) , or the species' curator ( neurons ) .", "While this procedure was initially intended only as a quality control measure to prevent incomplete or inaccurate data from compromising the database , we have developed it into a unique feature: the IBdb private mode .", "Before a dataset is made public , it is invisible to all other users , curators and the scientific database administrator .", "The dataset can thus be updated and even deleted .", "This creates the potential of using the IBdb to deposit data while they are being collected or prepared for publication in a research paper , that is , for data management ( Figure 6A ) .", "The API can be used to programmatically integrate the IBdb into any individual data management workflow; a community driven MATLAB implementation for automatic deposition of cell types and experiments is available on GitHub ( https://github . com/zifredder/IBdb-matlab ) .", "To facilitate the use of the IBdb as a data management tool , we have enabled three operational modes of the database site: private , public , and mixed .", "Any user logged in can thus choose to either access only ( own ) private data , only public data , or both .", "The first mode turns the IBdb into a data management site for ongoing research , the second mode is the default mode for viewing publicly available data , and the third mode allows the user to compare their own unpublished data with public data .", "As efficient data management requires researchers from the same laboratory , as well as collaborators , to have access to relevant unpublished data , each user can grant access to their own private datasets ( Figure 7 ) .", "To this end , a user can create a user group and invite other database users to join .", "Datasets can then be added and made visible or editable to all members of the group .", "These data can either comprise individual entries at all levels of the database , or collections of entries defined by common features .", "Finally , users are often reluctant to make datasets available to the public before they are included in a research paper , yet , these data should be available to editors and anonymous reviewers .", "We have therefore created the possibility for 'pre-publishing' database entries ( Figure 6A ) .", "This function allows a user to assign a persistent handle to a dataset ( e . g . a neuron profile page ) without approval by the curator and without making the dataset findable through search or lists in the IBdb .", "Editors and reviewers of the research paper ( anyone in possession of the handle ) then have direct access to the linked pages .", "Once the manuscript is accepted for publication , the user can submit the respective datasets for publication , obtain curator approval , and thus make them findable in the IBdb public mode .", "To avoid having to separately provide numerous independent handles when sharing data , individual entries can be grouped into datasets .", "These receive a unique link that grants access to the entire collection .", "Only public or pre-public data can be included in datasets .", "Public entries of the IBdb are maintained in a dual way; the persistent version is locked and cannot be changed , whereas a second , current version remains visible to the owner and to all members of user groups with appropriate access rights ( Figure 6B ) .", "This current version is fully private and can be freely edited or expanded .", "Importantly , no data that is already part of a public version can be deleted .", "Rather , when for instance a confocal image stack should be replaced by a better one , the old stack can be archived , so that it will not be visible in new versions of the dataset , but will remain present in the database for display of earlier versions .", "Once all required updates of a dataset have been made , the edited version can be re-published and will be assigned a new version of the persistent identifier after re-approval by the assigned curator .", "This new version is now also locked and any further edits will again have to be done in the current version of the dataset .", "This ensures that all data that have been assigned a persistent identifier will remain valid and accessible , while at the same time allowing each entry to evolve .", "The described strategy of publishing and re-publishing and the associated duality of persistent and current versions are implemented at the levels of species , neurons and experiments .", "The IBdb does intentionally not include Drosophila melanogaster as a species .", "This is because huge efforts have been spent developing highly efficient resources for this widely used model system and , as a result , the Virtual Fly Brain ( VFB ) resource has been created ( Osumi-Sutherland et al . , 2014 ) .", "It bundles data from several older Drosophila databases ( e . g . FlyCircuit ) to the most recent connectomics datasets ( Scheffer et al . , 2020 ) .", "Serving as the main repository for anatomical data from the Drosophila brain it has become the main site to locate GAL4 driver lines , single-cell morphologies , and synaptic connectivity data .", "It contains tens of thousands of datasets and is designed to specifically meet the needs of the Drosophila research community .", "By being less specialized , the IBdb has a wider scope .", "We are hosting many species and include both functional and anatomical data .", "We also do not require neuronal anatomies to be registered to a reference brain , if this is not possible for some reason .", "This opens the IBdb up to more diverse data , but as a result cannot provide most of the specialized services that VFB can deliver ( e . g . automatic bridging registrations of 3D data between different reference brains ) .", "Cross-linking the two databases to effectively enable comparing neurons from the insect species deposited in the IBdb with Drosophila , nevertheless , was highly desirable .", "Importantly , both databases have converged on highly similar hierarchical frameworks .", "As the brain nomenclature used by the IBdb and VFB is identical , neuropil identities were mapped across both databases .", "In cases were homology between corresponding neuropils is unclear , regions map to the next higher order brain region ( e . g . Monarch butterfly dorsal bulb maps to the entire Drosophila bulb ) .", "For some neuropils , there are no known counterparts in the fly ( e . g . the posterior optic tubercle ) .", "We use the APIs of both databases to allow direct communication between the IBdb and VFB .", "In principle , when searching the IBdb for neurons , an API mediated query can be automatically sent to VFB and search results are displayed as a list of single neuron entries .", "Each item on the list contains information obtained from VFB and is directly linked to the corresponding entry at VFB .", "This feature is available for the graphic , brain region based search .", "This includes complex multi-neuropil searches , aimed at identifying neurons connecting several brain regions .", "Effectively , this enables a user to launch a query in the IBdb and directly view corresponding neuron data in VFB , including data from recent connectomics efforts ( Scheffer et al . , 2020 ) .", "This seamless interoperability makes maximal use of both complementary resources , without duplicating functionality ." ], [ "Previous and current online databases hosting insect neuroscience data have suffered from several shortcomings: Most severely for old databases , a lack of maintenance often quickly led to outdated file formats , rendering the deposited information no longer compatible for viewing with current web browsers ( anticipated by Ito , 2010 ) .", "Second , while some databases originally allowed interactive viewing of the data , no possibility for contribution of one’s own data existed and data download was limited to very few files , for example Brandt et al . , 2005 , Kurylas et al . , 2008 .", "Usability of larger databases was generally impaired by a layout that often required expert knowledge to be able to launch meaningful database queries or to understand search results ( e . g . FlyCircuit , Invertebrate Brain Platform [now: Comparative Neuroscience Platform] ) .", "This not only applies to old databases , but the restriction to individual species and often highly complex interfaces limit the potential user base to specialists even in cutting edge databases such as VFB or visualization tools such as FruitFlyBrainObservatory .", "Finally , the limitation to purely anatomical data , including in the major current cross-species database NeuroMorpho . org , does not account for one of the key advantages of insect neuroscience: the high level of tractable structure-function relations .", "The IBdb addresses each of these issues .", "Firstly , we have developed the database software to be independent of the operating system and type of web browser used , as well as to not rely on any third party plugins .", "Additionally , we implemented the database as a classic , relational database without experimental data structures ( e . g . intelligent , adaptive search ) , aiming at maximal robustness .", "Having created a conceptionally simple software that uses standard web-technology with standard file-formats makes continued compatibility and technical maintenance comparably simple .", "Second , we have invested substantial effort in making the IBdb intuitive to use and visually attractive to provide a positive user experience .", "The latter factor should not be underestimated in its importance .", "One of the problems encountered in previous databases were user interfaces that were difficult to use , creating an immediate negative experience when attempting to use a site for the first time and therefore reducing the motivation to interact with it .", "As for commercial software , we aspired to generate a user interface that is largely self-explanatory , provides immediate visual feedback when a user action was successful , and which clearly shows what actions can be performed .", "Several years of beta-testing by a multitude of users have streamlined the site to a point at which interacting with the IBdb is both straight forward and fun .", "As the success of the database depends on many users sharing their data , we aimed at making contributing data as intuitive and as easy as searching and visualizing data .", "We have thus simplified the data contribution process to a point where only very limited anatomical knowledge is needed , aiming at enabling physiologists without deep anatomical training to submit data as well .", "Finally , to our knowledge the IBdb is the first database in the field of invertebrate neuroscience that combines functional data with anatomical data , and at multiple levels ranging from entire brains to single neurons .", "This ability , together with the possibility to deposit not only representative data but concrete sets of experiments , provides an opportunity for anatomists to directly interpret their findings in a functional context , as well as allowing physiologists to tether their findings to a coherent anatomical framework that automatically generates context for any functional data .", "The ease of use of the IBdb , combined with housing functional and anatomical data , has the potential to facilitate interactions between expert anatomists and physiologists and thereby strengthen structure-function analysis across the diversity of insect brains .", "The landscape in the insect neuroscience research community has changed dramatically , and most relevant funding bodies are in the process of implementing open data mandates or have already done so ( e . g . European Research Council , National Institutes of Health , Wellcome Trust , etc ) .", "Thus , the initial driving force for data deposition is much larger compared to that surrounding earlier database attempts .", "Yet , why should researchers use the IBdb for meeting these new mandates rather than other available databases ?", "Different from open databases , for example Figshare . org , the IBdb is dedicated to insect neuroscience and thus provides all tools required to manage , annotate , and cross-link the specific data formats generated in this field .", "However , it is not rooted within a single laboratory , nor a single species , thus providing a framework for data from a broad research community .", "The unique possibility for cross-species comparison and the combination of anatomical and functional data additionally broadens the relevance of the IBdb .", "Visualization of anatomical research data is often difficult and particularly complex when involving data from other research groups .", "We have implemented a range of tools enabling the visualization of data in fast , flexible , and effortless ways .", "This saves considerable time compared to other available software tools , in particular for complex 3D neuron data ( e . g . Amira ) .", "The data contributed are also immediately incorporated into the framework of existing data .", "Outside the IBdb these data are distributed across many publications .", "Comparison of one’s own data to any published data would entail contacting authors , obtaining files in unpredictable formats and finding ways to compare them to one’s own work within the software a research group is currently using .", "The IBdb solves these issues and delivers such comparisons within seconds .", "Crucially , these advantages are already present immediately after data upload , prior to publication .", "Via the private mode of the IBdb , individual neurons can already be compared to their counterparts in other species while datasets are being obtained , enabling the user to generate visualizations suitable for conference contributions and publication figures .", "This possibility is to our knowledge unique to the IBdb and provides a major motivation for data contribution .", "Importantly , the dual function of the IBdb as data repository and data management tool eliminates the need to reformat and prepare datasets for publication , a process that is required when submitting datasets to websites dedicated to only data deposition .", "The IBdb therefore provides a streamlined and integrated experience from data acquisition to publication , aimed a minimizing researcher workload .", "This is additionally relevant as formulating a data management plan has also become mandatory for projects funded by most funding organizations .", "By using the IBdb , users in insect neuroscience not only have access to a dedicated data repository , but at the same time have a tool at hand for efficient handling of ongoing research data , making data management plans easy to design and effective .", "This is particularly the case when using the API for accessing the data , as up- and download can be automated and custom designed interfaces can be programmed according to the needs of any particular research group .", "Independent from these aspects , the API of the IBdb offers users the opportunity to expose their published data directly to approved third party applications .", "The bundled availability of data from many research groups generates a possibility of data reuse with a much broader scope than any individual solutions , providing an increased motivation for developers to design workflows that incorporate deposited data .", "High-quality data deposited in the IBdb therefore additionally increases the visibility of the data owners .", "While the data currently deposited in the IBdb represents only a starting point to illustrate the functions and possibilities of our database concept and user interface , the neurons and species present in the IBdb already highlight new biological insights as well as concrete paths towards such insights .", "Most obvious insights can be gained from data deposited in the IBdb that is not published or publishable elsewhere .", "For example , isolated neural morphologies from the Monarch butterfly have been published solely in the IBdb and demonstrated a previously unknown connection between the gall of the lateral complex and the mushroom body lobes ( dp-GA-MB ( L ) neuron , NIN-0000383 ) , thus directly linking the output of the central complex with the mushroom body for the first time in insects .", "The possibility for direct comparison of neuropil and neuron data from many insect species has the potential to identify discrepancies in neuropil definitions across species and pinpoint solutions for revised homologies .", "For example , the main output neurons of the central complex ( PFL or CPU1 neurons ) are most likely homologous across insects and target the dorsal LAL in all species in which this region has been described ( e . g . butterflies , moths , flies , beetles ) , except in locusts , in which they terminate in the ventral LAL .", "This discrepancy suggests that the definition of subregions of the LAL and their borders to surrounding brain regions ( e . g . crepine ) might have to be re-evaluated .", "Along similar lines , the IBdb has generally the potential to expose discrepancies in functional data .", "Work carried out in a research group interested in sensory processing might define the function of a particular cell type very differently than a group focused on motor control , or one focused on neuromodulatory functions , simply because different experimental paradigms are aimed at different aspects of neural function and interpretations of results might be biased toward the underlying hypotheses .", "Such diverging views of neuron function can silently coexist in the literature for long times , but if bundled in side-by-side function entries on the same neuron profile page in the IBdb they become highly obvious .", "Such exposure will facilitate scientific discussion and the emergence of unified ideas of neural function .", "Finally , in any field it is important to consistently define technical terms , agree on key concepts , and have consistent standards regarding what is acceptable data to validate conclusions .", "During the development phase of the IBdb it became clear that the isolated work in many insect model species has led to a wide range of cases in which similar terms have different meanings in different species and where views on what is acceptable proof for , for example , neural function varies .", "The definition of cell type is one such example , where researchers working in different species and brain regions attached substantially different meaning to the term .", "With the unifying approach of the IBdb we had to therefore identify common ground and define cell type in a way that was acceptable to each contributing party .", "Similar issues for other aspects of insect neuroscience research will likely be exposed by the increased levels of communication enabled by the IBdb .", "While resolving these issues will often take time and effort ( and will involve researchers from across the field ) , the main function of the IBdb in this context is not to impose a strict set of rules , but to provide the framework in which inconsistencies can be exposed and resolved , eventually facilitating biological insight .", "The IBdb is designed for long-term accumulation of data by many contributing research groups across the field of insect neuroscience .", "To successfully provide this service , the IBdb has to address several challenges of sustainability .", "These challenges are threefold: First , ensuring technical and financial maintenance of the database infrastructure; second , guaranteed , continued scientific oversight and expert curation , and third , lasting scientific relevance .", "We have therefore taken steps to address each of these points .", "In the light of fast changing web technology , maintenance of high technical standards requires continuous effort and active updating of the database code , which in turn requires financial resources .", "The first issue is covered by an ongoing agreement with the web-developers that built and maintained the database software over the period of the last six years .", "Financially , voluntary contributions from research groups that initiated the database have paid for its creation .", "As the maintenance costs are a small fraction of the development costs , it will be easily possible to run the database within the framework of the existing service agreement for at least the next 5 years without any changes required .", "However , when the data volume increases substantially , the static costs of housing the data will increase accordingly .", "While keeping all public , persistent data available free of charge is mandatory ( given that the IBdb functions as a public data repository ) , maintaining the IBdb as a free data management tool , that is allowing unlimited private data for each user , will likely become unsustainable over time .", "If this becomes a problem , free space in the private section of the database will have to be restricted .", "All space required beyond a certain limit will have to be rented to directly offset the costs for maintaining and administering these data .", "At the same time , research groups involved in creating the IBdb use this platform as their primary tool for management of research data and public data repository .", "The obligation to formulate data management plans and strategies for public deposition of research data , combined with the lack of equally suitable alternative platforms , will ensure that third party funding dedicated to maintaining the database will continuously be available via research funding of the founders of the IBdb .", "To anticipate the slowly growing costs of housing the database due to increasing data volume , we aim at eventually relocating the data from the currently used commercial Amazon cloud platform to an academic server that is provided at minimal costs or free of charge .", "To this end we have ensured that the IBdb does not depend on any core functions of the Amazon cloud storage service , enabling to move the database to a new location with comparably moderate effort .", "The second issue of continued quality control and expert curation is addressed at several levels .", "Firstly , at the level of species , the scientific administrator has the main responsibility to oversee the curation of the species entries by each species’ dedicated group of curators .", "Given the limited number of species that can realistically be included in the IBdb over the coming years ( our estimate is a maximum of several hundred ) , the associated workload for general oversight is limited and manageable by a single person .", "As species curators actively perform research on the species they are responsible for , there is a substantial self interest to maintain high standards to advertise their work and thus facilitate their research .", "At the level of neuron entries , the workload is higher due to larger data volumes .", "Accordingly , the main responsibility for oversight is more distributed and lies with the species’ curators .", "Once datasets exist in the database , updating is generally optional and will only in rare cases be necessary .", "In those cases , the strongest incentive for keeping data up to date lies with the data owners and research group leaders , who also possess the highest expertise for these data .", "Overall , expertise is mostly required to approve new datasets , a process requiring substantial overview over the data available for the relevant species .", "With an increasing number of species , the number of curators will also have to increase .", "This makes curator recruitment and training key requirements both for quality assurance and for enforcing consistent curation strategies .", "For this purpose , regular meetings of all curators will be held and approval of new curators will be carried out only after an extended training period .", "To attract new curators in the future , the IBdb administrators will actively approach researchers in the field of insect neuroanatomy .", "Finally , to identify existing quality issues with deposited data , establish routine workflows , to support curators and contributors at all levels , and to recruit new users , a dedicated , full time database curator position is being created , initially funded by members of the IBdb consortium for a minimum of one year .", "Third , continued scientific relevance of the database will have to be ensured to attract users and contributors long term .", "Most crucially , after introducing the database to the field , the available deposited information has to grow beyond a critical point , at which the IBdb becomes the natural choice for depositing insect neuroscience data .", "We believe that this point has already been reached , as the number of deposited data sets is growing extensively ( Figure 7—figure supplement 1 ) and users without affiliation to the founding consortium have begun to deposit data .", "Nevertheless , attracting more data will remain a key mechanism to increase the attractiveness and acceptance , and , consequently , the relevance of the database in the field .", "As one of the key advantages of our concept is the possibility to deposit all anatomical and functional data from insect neuroscience , irrespective of data format , we have begun to approach authors of published work to enable them to make old data available to the growing IBdb user base .", "While highly useful for classroom teaching of structure-function relations in neural systems , the IBdb has proven to be invaluable to introduce new members of a research team to the basic layout of brains , neurons , and neural circuits in a particular species .", "Using the database serves as an easy ( and fun ) access point to available information on a research species , including key publications , and therefore saves significant effort when writing review papers , PhD thesis introductions , background sections for travel grants , etc .", "While this is true for established researchers as well , it is especially true for younger scientists and students who are new to the field or are at the beginning of their careers .", "Finally , the IBdb provides the possibility for anyone to access original research data in intuitive and attractive ways ( Figure 7 ) .", "This provides opportunities to design teaching assignments for neuroscience students to carry out meta-analyses .", "With access to the data in the IBdb via the application programming interface ( API ) , we have provided the possibility for third parties to develop dedicated teaching tools that provide streamlined methods to use the data for specific classroom exercises .", "Beyond researchers and students , journalists , interested members of the public , or members of funding bodies can also view and explore neuroscience data .", "Ideally , this will contribute to a more transparent understanding of what the output of science is and could spark increased interest in insect neuroscience .", "The framework we have generated with the IBdb is not limited to housing insect brain data .", "Without major modifications it would be equally suited for hosting data from other animal taxa .", "While the intuitive , schematic search engine would not be useful for comparing species that do not share a common basic brain outline ( i . e . a relevant 'generic brain' ) , the text-based expanded search could allow the construction of queries across multiple groups , for example searches according to functional terms .", "We are currently conceptualizing the expansion of the database toward including spiders and envision that crustaceans and other arthropods would be logical next groups .", "The IBdb therefore provides not only a tool for the insect neuroscience community to facilitate data management , data visualization , transparency of results and effective teaching , but it can easily be expanded toward related fields .", "Additionally , it might also serve as a blueprint for how to set up similar databases in unrelated research areas .", "In principle , the strategies used in the IBdb are applicable to any scientific field that can be linked to a hierarchical framework ." ], [ "All web infrastructure is hosted by Amazon web services on servers located in Frankfurt , Germany .", "Data is stored using a PostgreSQL relational database hosted by the Amazon Relational Database Service and files are stored using the Amazon S3 object storage service .", "The servers hosting the website and the local HANDLE system are running in Amazon EC2 containers , which runs Linux .", "Resources communicate using Amazon Virtual Private Cloud .", "The database structure and interaction is managed by a python based Django application .", "User authentication , permissions , and data security are also managed within the Django application .", "A NGINX web server hosts static content and serves as a reverse proxy for dynamic content served by a uWSGI application server hosting the project's Django application .", "Asynchronous tasks are implemented using the Celery distributed task queue and RabbitMQ message broker .", "Data is externally accessible via a web API delivering content in the JSON format to the front-end web application .", "The web API was implemented with Django and the Django REST framework .", "Long-term data persistency is provided to allow users to reference information or profile pages on the site in scientific publications and other external media in a static state , while continuing to allow data to be updated as more information is acquired .", "When a request is made by a user for a persistent copy of a dataset to be created , a copy of the data related to the current state of the dataset is serialized and parsed into JSON .", "A persistent unique identifier is then assigned ( HANDLE ) .", "The JSON data , HANDLE and additional metadata is recorded in a separate table and can no longer be modified .", "All files associated with the persistent dataset are marked as locked in the database and can no longer be modified by the user .", "The recorded state of that dataset can be accessed and viewed on the site using the url associated with the assigned HANDLE .", "The original data copied to create the persistent dataset can be modified without effecting the persistent dataset .", "Additional files may also be added , but will not be reflected in earlier persistent records .", "The front-end of the database is primarily implemented using the Angular web framework in Typescript , HTML and SASS ( CSS extension language ) .", "The Typescript , HTML and SASS are compiled and bundled with the Angular CLI using WebPack to create the distributed application files targeting ECMAScript 2015 capable browsers .", "Graphical consistency is targeted for browsers using Webkit and Gecko-based layout engines adhering to web standards .", "The web based three-dimensional viewer was implemented using Typescript , WebGL and the Three . js three-dimensional graphics framework .", "The two-dimensional schematic view , brain designer and path designer was implemented using Typescript , the Canvas API and Paper . js vector graphics scripting framework .", "User to server communication is protected by the Hyper Text Transfer Protocol Secure ( HTTPS ) .", "User authentication is managed by the Django authentication system .", "Access to data is restricted by object based permissions limited to authorized users through the web interface or API arbitrated by the Django server .", "File downloads of protected content stored on Amazon S3 are accessed using time limited urls , assigned to an authorized user at the time of a download request by the Django server .", "Files are directly downloaded from the S3 storage to the user using the temporary URL .", "The process is seamless to the end user who needs only be logged into the website and click the link associate with the intended file .", "Downloads are logged and accessible to the owner of the data being accessed .", "Files can be uploaded to the S3 object storage only by authorized users with a time limited URL provided by the server .", "The upload is logged and associated with the contributing user .", "Publicly available thumbnail images and other reduced quality images are stored separately in a publicly accessible ( read-only ) S3 bucket .", "Content explicitly designated as public is accessible through the graphical user interface or via the API to any authorized visitor .", "Private content is only accessible to authorized users given permission to access the data .", "The PostgreSQL database is protected by a firewall allowing access only via the Amazon Virtual Private Cloud and is not open to direct access via the web .", "All names for brain areas are in line with previous research .", "The brain regions of the generic insect brain follow the new insect brain nomenclature introduced for Drosophila by Insect Brain Name Working Group et al . , 2014 .", "Accordingly , we have established three hierarchical levels of brain regions: super-regions , neuropils , and sub-regions .", "Super-regions are stereotypical and can be expected to comprise the ground pattern of the brain in all insects ( although some might be reduced in certain species ) .", "The only exception to the Insect Brain Name Working Group et al . , 2014 scheme is the anterior optic tubercle , which we have raised to the level of super-region , given its prominence and distinct nature in most insect species .", "Sub-regions are often specific to individual species and therefore , if such regions were defined , we used the names given to them within the relevant species .", "We did not unify for example names of the mushroom body calyx divisions across species , as this would firstly imply homology where there might not be any , and second , novel naming schemes will have to be developed by the community and not be imposed by a data repository .", "Anticipating that changes to brain names can and will happen in the future , all names , as well as the level of a region within a hierarchy , can be modified .", "Within some neuropils , regular , repeating elements can be found , usually defined as columns and layers .", "We have implemented such a system in the central complex , that is without having to define an array of sub-regions , several strata and orthogonal slices ( following the new brain nomenclature ) can be generated .", "The default number of slices in the generic brain is 16 , assuming that this number is the ancestral state of this region .", "Neuron names follow the conventions within each species , as there is no common naming scheme for insect brain neurons yet in place .", "However , we provide the possibility to define several alternative names for each cell type to allow the parallel use of names .", "This is possible as the identity of a neuron is linked to the persistent ID , and not to the neuron's name .", "Given that we house neurons from multiple species , we add a prefix to the full name of each cell type specifying the species , for example 'am' for Apis mellifera ." ] ]

[ "Insect neuroscience generates vast amounts of highly diverse data , of which only a small fraction are findable , accessible and reusable .", "To promote an open data culture , we have therefore developed the InsectBrainDatabase ( IBdb ) , a free online platform for insect neuroanatomical and functional data .", "The IBdb facilitates biological insight by enabling effective cross-species comparisons , by linking neural structure with function , and by serving as general information hub for insect neuroscience .", "The IBdb allows users to not only effectively locate and visualize data , but to make them widely available for easy , automated reuse via an application programming interface .", "A unique private mode of the database expands the IBdb functionality beyond public data deposition , additionally providing the means for managing , visualizing , and sharing of unpublished data .", "This dual function creates an incentive for data contribution early in data management workflows and eliminates the additional effort normally associated with publicly depositing research data ." ]

[ "Insect neuroscience , like any field in the natural sciences , generates vast amounts of data .", "Currently , only a fraction are publicly available , and even less are reusable .", "This is because insect neuroscience data come in many formats and from many species .", "Some experiments focus on what insect brains look like ( morphology ) , while others focus on how insect brains work ( function ) .", "Some data come in the form of high-speed video , while other data contain voltage traces from individual neurons .", "Sharing is not as simple as uploading the raw files to the internet .", "To get a clear picture of how insect brains work , researchers need a way to cross-reference and connect different experiments .", "But , as it stands , there is no dedicated place for insect neuroscientists to share and explore such a diverse body of work .", "The community needs an open data repository that can link different types of data across many species , and can evolve as more data become available .", "Above all , this repository needs to be easy for researchers to use .", "To meet these specifications , Heinze et al . developed the Insect Brain Database .", "The database organizes data into three categories: species , brain structures , and neuron types .", "Within these categories , each entry has its own profile page .", "These pages bring different experiments together under one heading , allowing researchers to combine and compare data of different types .", "As researchers add more experiments , the profile pages will grow and evolve .", "To make the data easy to navigate , Heinze et al . developed a visual search tool .", "A combination of 2D and 3D images allow users to explore the data by anatomical location , without the need for expert knowledge .", "Researchers also have the option to upload their work in private mode , allowing them to securely share unpublished data .", "The Insect Brain Database brings data together in a way that is accessible not only to researchers , but also to students , and non-scientists .", "It will help researchers to find related work , to reuse existing data , and to build an open data culture .", "This has the potential to drive new discoveries combining research across the whole of the insect neuroscience field ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "developmental biology", "neuroscience" ]

[ [ "The retina is the light-sensitive tissue at the back of the eye that consists of photoreceptor cells , retinal pigment epithelium ( RPE ) , and its basement ( Bruch’s ) membrane .", "Disorder of the retina causes vision loss .", "The primary nutrient source for the retina is the choroid ( Bill et al . , 1980 ) , which is a highly vascularized tissue layer surrounding the retina .", "The choroid consists of three layers: Haller’s layer ( large blood vessel layer ) , Sattler’s layer ( medium size blood vessels ) , and the choriocapillaris ( Hogan et al . , 1971; Nickla and Wallman , 2010 ) .", "The choriocapillaris is a unique anastomosed vascular structure with an extraretinal fenestrated capillary bed that lies in a single plane below Bruch’s membrane .", "In the mouse retina , choroidal development begins from embryonic day ( E ) 13 . 5 ( Marneros et al . , 2005; Saint-Geniez et al . , 2006 ) .", "Vascular endothelial growth factor ( VEGF ) is known to be a central regulator of vascular development during embryogenesis ( Coultas et al . , 2005 ) , and VEGF secreted from RPE cells is indispensable for the vascular development and maintenance of the choroid ( Saint-Geniez et al . , 2009; Le et al . , 2010; Kurihara et al . , 2012 ) .", "VEGF expression is enhanced by a number of transcription factors such as hypoxia-induced factor-1α ( HIF-1α ) , estrogen-related receptor-α ( ERRα ) , and peroxisome proliferator-activated receptor gamma coreceptor 1α ( PGC-1α ) ( Ziello et al . , 2007; Ueta et al . , 2012 ) .", "However , the molecular mechanism of choroidal vascular development and the regulatory mechanism of VEGF secreted from RPE have not been clarified .", "In the present study , we found that aldehyde dehydrogenase one family , member A1 ( Aldh1a1 ) knockout ( Aldh1a1–/– ) mice have hypoplasia in the dorsal region of the choroid .", "Aldh1a1 is an enzyme that synthesizes retinoic acids ( RAs ) and is expressed in the dorsal neural retina from the embryonic stage , not in the RPE and the choroid ( Matt et al . , 2005; Molotkov et al . , 2006; Luo et al . , 2006; Kumar et al . , 2012 ) .", "RAs are essential for biological activities such as reproduction , development , growth , and maintenance ( Kam et al . , 2012; Rhinn and Dollé , 2012; Cunningham and Duester , 2015 ) .", "We analyzed choroidal vascular development in Aldh1a1–/– mice , and demonstrated how Aldh1a1 expressed in the neural retinas in trans enhances VEGF secretion from the RPE to the choroid .", "We also found that , mechanistically , Sox9 expression in RPE is downstream of the signaling pathway mediated by Aldh1a1 in the neural retina ." ], [ "It was previously reported that Aldh1a1 is expressed in the dorsal region of embryonic and adult retinas ( McCaffery et al . , 1991; Fan et al . , 2003; Matt et al . , 2005 ) .", "However , the retinal cell types that express Aldh1a1 have not been precisely identified .", "Therefore , we performed immunohistochemistry and in situ hybridization studies in embryonic and adult mouse retinas .", "At E12 . 5 , Aldh1a1 was localized in the progenitor cells at the dorsal neural retina , and no Aldh1a1 expression was observed in the RPE and choroid complex ( Figure 1A ) .", "Aldh1a3 , an Aldh1 family enzyme that generates RA , was mainly localized in the ventral region of the retina and RPE .", "At E17 . 5 , Aldh1a1 expression was observed in the dorsal region and the ventral edge , but no expression was detected in the RPE/choroid complex ( Figure 1B ) .", "In adult tissue , in situ hybridization showed that Aldh1a1-positive cells were preferentially localized in the middle of the inner nuclear layer ( INL ) cells , and had spread to the dorsal region of the retina ( Figure 1—figure supplement 1A and B ) .", "We also observed Aldh1a1-positive cells at the ventral edge of the adult retina ( Figure 1C ) .", "Immunohistochemistry of sectioned tissues showed Aldh1a1 colocalized with glutamine synthetase ( GS ) , a marker of Müller glia .", "These results indicate that Aldh1a1 is preferentially expressed in the retinal progenitor cells of the dorsal region from the embryonic stage , and in Müller cells in the dorsal region and the ventral edge of the retina in adulthood .", "Next , we analyzed the morphological differences between wild-type ( WT ) and Aldh1a1–/– eyes .", "In adult Aldh1a1–/– mice , the eyeballs displayed a loss of pigmentation in the dorsonasal area ( Figure 1D ) .", "Hematoxylin and eosin ( H and E ) staining of the paraffin sections revealed that pigmentation was lost from the choroidal region , not from RPE cells ( Figure 1E ) .", "The choroidal thickness was significantly less than that of the dorsal and ventral sides of WT eyes and the ventral side of Aldh1a1–/– eyes ( Figure 1F ) .", "There were no obvious morphological differences in the thickness of the neural retina or in the length of the outer segments of photoreceptor cells ( Figure 1G and H ) , as reported previously ( Fan et al . , 2003 ) .", "These findings suggest that the choroidal vessels of Aldh1a1–/– mice are hypoplastic .", "To characterize choroidal vascularization in the Aldh1a1–/– mice , we first immunostained choroidal flat-mounts for endomucin antibody , a specific marker for choriocapillaris , and isolectin B4 , which mainly visualizes choroidal medium-sized/large blood vessels .", "Surprisingly , the Aldh1a1–/– mice exhibited choroidal hypoplasia in the dorsal region of the eyes ( Figure 2A ) , although Aldh1a1 is normally expressed in the neural retina .", "In the dorsal and ventral regions of WT and the ventral region of Aldh1a1–/– eyes , the choriocapillaris was normal , with a dense mesh structure , whereas fewer choroidal vessels with more avascular areas were detected in the dorsal region of Aldh1a1–/– eyes ( Figure 2B and C ) .", "We also performed whole-mount immunohistochemistry for ZO-1 , a tight junction marker of RPE cells ( Figure 2D ) .", "In the dorsal region of Aldh1a1–/– eyes , the vascular density was significantly lower than that in the other regions ( Figure 2E ) , whereas there were no significant morphological differences in the localization of ZO-1 signals and RPE size between WT and Aldh1a1–/– eyes ( Figure 2F ) .", "Taken together , these results demonstrate that Aldh1a1–/– eyes show choroidal hypoplasia without degeneration of the RPE in the dorsal region .", "To investigate further the morphological features of the choroidal vasculature and the neural retina/RPE/Bruch’s membrane complex , sections of WT and Aldh1a1–/– eyes were examined by transmission electron microscopy ( TEM ) .", "The TEM images demonstrated that Aldh1a1–/– eyes exhibited apparently normal morphology of photoreceptor and RPE cells , despite the complete absence of choroidal pigmentation and the presence of a thin dorsal choroid layer ( Figure 3A ) .", "Bruch’s membrane did not differ significantly between WT and Aldh1a1–/– eyes ( Figure 3B ) .", "Remarkably , the choroidal vessels in the hypoplastic dorsal section of Aldh1a1–/– eyes showed fenestrations , a characteristic of capillaries , as did those in the dorsal and ventral regions of WT and the ventral region of Aldh1a1–/– eyes ( Figure 3C ) .", "Taken together , these results suggest that the hypoplastic blood vessels of the Aldh1a1–/– eyes maintain the characteristics of the choriocapillaris , including an intact RPE and Bruch’s membrane .", "Although several reports have suggested that FMS-like tyrosine kinase 1 ( Flt1 , also called VEGF receptor", "1 ) and kinase insert domain protein receptor ( Kdr , also called VEGF receptor", "2 ) are expressed in choroidal vessels ( Zhao and Overbeek , 2001; Witmer et al . , 2003; Cross et al . , 2003; Saint-Geniez et al . , 2006 ) , the blood vessel type-specific receptor expression remains ambiguous .", "Therefore , we examined the regional distribution of Flt1-DsRed and Kdr-EGFP in choroidal vessels in WT and Aldh1a1−/− eyes by using Flt1-BAC-DsRed;Kdr-BAC-EGFP mice ( Matsumoto et al . , 2012 ) .", "In the choroidal flat-mounts from adult WT mice , the choriocapillaris coexpressed both Flt1-DsRed and Kdr-EGFP , while the medium-sized/large choroidal vessels expressed only Flt1-DsRed ( Figure 3—figure supplement 1A–D ) .", "In addition , more Flt1-DsRed appeared to be expressed in the medium-sized/large choroidal vessels than in the choriocapillaris ( Figure 3—figure supplement 1D ) .", "In contrast , in the dorsal region of Aldh1a1–/– eyes , all the blood vessels associated with the hypoplastic choroid coexpressed both Flt1-DsRed and Kdr-EGFP ( Figure 3—figure supplement 1B–D ) .", "Also , the level of expression of Flt1-tdsRed in the Aldh1a1–/– blood vessels appeared lower than that in the medium-sized/large choroidal vessels .", "We next explored the developmental time point at which choroidal hypoplasia appears in Aldh1a1–/– mice .", "Since the formation of the choroidal vascular network starts at approximately E13 . 5 ( Zhao and Overbeek , 2001; Marneros et al . , 2005; Saint-Geniez et al . , 2006 ) , we compared the vascular development in the choroid of the eyes of WT and Aldh1a1–/– mice from E16 . 5 to postnatal ( P ) 28 using a choroidal flat-mounts immunostained with antibodies against endomucin and ETS-related gene ( ERG ) , which are enriched in endothelial cells ( Figure 4A; Figure 4—figure supplement 1 ) .", "In the dorsal region of Aldh1a1–/– eyes , endomucin immunostaining was patchy , indicating that choroidal hypoplasia was already detectable ( Figure 4A and B ) .", "There were significantly fewer endothelial cells in the area than in WT eyes ( Figure 4C ) .", "Based on studies showing that VEGF secreted from RPE cells is necessary for choroidal vascular development ( Sakamoto et al . , 1995; Saint-Geniez et al . , 2009; Le et al . , 2010 ) , we hypothesized that RAs synthesized by Aldh1a1 in the neural retina stimulate the RPE to enhance VEGF secretion to the basal side of the choroid .", "To test this hypothesis , we first evaluated the VEGF level in E17 . 5 and P24 RPE-choroid complexes by ELISA .", "VEGF levels in the Aldh1a1–/– RPE-choroid complex were significantly lower than those in WT eyes at both time points ( Figure 4D ) .", "In addition , in situ hybridization revealed that Vegfa mRNA expression of the E16 . 5 Aldh1a1−/− RPE cells was reduced at the dorsal half where Aldh1a1 is expressed in the WT neural retina ( Figure 4E , shown as arrowheads ) .", "Next , we tested RA-dependent VEGF secretion by the primary human RPE cells .", "We measured VEGF in the culture medium after RA treatment .", "As a result , RAs significantly enhanced VEGF secretion in a dose-dependent manner ( Figure 4F ) .", "Because RA is the active metabolite of vitamin A ( Shams et al . , 1993; Amengual et al . , 2012 ) , we generated vitamin A-deficient ( VAD ) mice by feeding a vitamin A-deficient diet ( Chihara et al . , 2013 ) .", "At P3 , VAD mice showed dorsal choroidal hypoplasia in the flat-mount analysis ( Figure 4G ) .", "In the dorsal region of VAD eyes , the vascular density was significantly lower than that in the other regions such as the dorsal and ventral regions of WT and the ventral region of VAD eyes ( Figure 4H ) .", "Also , RA administration to pregnant Aldh1a1–/– mice by oral gavage from E10 to E16 significantly suppressed the dorsal choroidal hypoplasia in their offspring ( Figure 4I and J ) .", "These results indicate that RA controls dorsal choroidal vascular development and that dorsal choroidal hypoplasia in Aldh1a1–/– mice is causally related to a deficiency in RA synthesis .", "These observations raised the question of whether Aldh1a3 , another RA-producing enzyme in the mouse retina , also affects choroidal vascularization , because Aldh1a3 begins to be expressed in the entire RPE cell layer and in the ventral region of the neural retina from E10 . 5 ( Matt et al . , 2005 ) .", "To test this possibility , we conditionally disrupted Aldh1a3 in the neural retina ( floxed Aldh1a3 mice crossed with Pax6-α-Cre ) , however , in these mice , choriocapillaris development was found to be normal ( Figure 4—figure supplement 2 ) .", "In conclusion , RAs derived from Aldh1a1 but not Aldh1a3 control choroidal vascular development by enhancing VEGF secretion from RPE cells .", "We next investigated the transcription factors that directly enhance VEGF expression in dorsal RPE cells .", "Recently , it was reported that conditional disruption of Pax6 in the RPE at the early embryonic stages resulted in the loss of pigmentation ( Raviv et al . , 2014 ) , and Pax6 conditional knockout mice in which the Vegfa promoter is synergistically transactivated by Pax6 and Sox9 exhibit choroidal hypoplasia ( Cohen et al . , 2016 ) .", "Therefore , we performed immunohistochemistry to detect Pax6 and Sox9 in sections of embryonic WT and Aldh1a1–/– retinas .", "The intensity of Pax6 immunofluorescence in the dorsal Aldh1a1–/– RPE was slightly lower than WT at E12 . 5 and E14 . 5 , but did not show a significant difference ( Figure 5—figure supplement 1A–C ) .", "Next , we measured the developmental expression of Sox9 ( Figure 5A and B ) .", "In the E12 . 5 WT neural retina , Sox9 was predominantly expressed in the dorsal region rather than in the ventral region , and the expression level at the dorsal region became comparable to the ventral region at E14 . 5 .", "In Aldh1a1–/– neural retinas , Sox9 immunofluorescence in the dorsal region was reduced as much as that of the ventral region ( Figure 5A and C ) .", "In the E12 . 5 WT RPE cells , there was no difference in Sox9 immunofluorescence between dorsal and ventral region , and the intensity increased at E14 . 5 .", "In the E12 . 5 Aldh1a1–/– RPE cells , the immunofluorescence was comparable to WT , but was significantly lower than that of E14 . 5 WT ( Figure 5B , D and E ) .", "These densitometry results suggest that Aldh1a1 enhances Sox9 expression in the dorsal neural retina and RPE cells during development .", "To determine whether Sox9 enhances VEGF in RPE cells in an RA-dependent manner , we measured Sox9 and Vegfa mRNA expression in primary RPE cells in response to RA exposure .", "The results showed that both Sox9 and Vegfa mRNAs ( Figure 5F and G ) were enhanced in an RA-dependent manner .", "To examine whether Sox9 regulates Vegfa in RPE cells , we performed overexpression and knockdown experiments .", "Overexpression of Sox9 by transient transfection of a pCAGIG-Sox9 vector resulted in upregulation of Vegfa mRNA ( Figure 5H and I ) .", "In contrast , knockdown by transient transfection of Sox9 siRNA resulted in downregulation of Vegfa mRNA ( Figure 5J and K ) .", "Taken together , these results strongly suggest that Sox9 enhanced by Aldh1a1-mediated RA upregulates Vegfa expression in RPE cells .", "We next explored further whether the Aldh1a1-driven Sox9 expression in the dorsal neural retina and RPE is involved in choroidal vascular development .", "To generate mice with selective deletion of Sox9 in the developing RPE or neural retina , mice with a conditional deletion of Sox9 ( Sox9flox/flox; Kist et al . , 2002 ) were mated with either Tyr-Cre ( RPE-cKO of Sox9; Delmas et al . , 2003 ) or Pax6-α-Cre ( Retina-cKO of Sox9; Marquardt et al . , 2001 ) , respectively .", "In the Cre-reporter assay with R26R-H2B-mCherry mice ( Abe et al . , 2011 ) on an albino background , mCherry expression from E16 . 5 Tyr-Cre mice was observed in all RPE , although a few mCherry-positive cells were found in the neural retina ( Figure 6—figure supplement 1A ) .", "Also , mCherry expression in Pax6-α-Cre mice was restricted to the dorsal and ventral portions of the neural retina as reported previously ( Marquardt et al . , 2001 ) , but no mCherry-positive cells were found in the choroid ( Figure 6—figure supplement 1B ) .", "In RPE-cKO of Sox9 , we found less pigmentation in the dorsal region , and significantly poorer vasculature in the dorsal choroidal area than the other areas ( Figure 6A and B ) , which phenocopied Aldh1a1–/– eyes .", "Interestingly , although Sox9 was disrupted in all RPE cells , the poor vasculature phenotype was restricted to the dorsal region , and did not appear in the ventral region ( Figure 6B ) .", "Conversely , Retina-cKO of Sox9 showed no hypoplasia of the choroidal vasculature or lack of pigmentation ( Figure 6A and B ) , indicating that Sox9 in the neural retina is not responsible for choroidal vascular development .", "Next , we attempted to rescue the choroidal hypoplasia phenotypes imposed by Aldh1a1 deletion by restoring Sox9 signaling using Cre-inducible Sox9-overexpressing mice ( RPE-cOE of Sox9 , Kim et al . , 2011 ) .", "Tyr-Cre-induced overexpression of Sox9 in these mice significantly recovered the choroidal hypoplasia phenotypes in the dorsal region ( Figure 6C and D ) .", "These results indicated that Sox9 expression enhanced by Aldh1a1 in developing RPE cells is critical for normal choroidal vasculature development ." ], [ "The molecular mechanism underlying the development and function of the neural retina has been well studied , but that responsible for the development of the choroidal vessels has not been thoroughly investigated , despite the pathophysiological importance of these structures in human eye disease .", "Here , we could immunohistochemically discriminate choriocapillaris from medium-sized/large blood vessels in Haller’s/Sattler’s layer in mouse eyes and then demonstrated that disruption of Aldh1a1 , which is predominantly localized in the dorsal neural retina , resulted in choroidal hypoplasia in the dorsal portion of the eyes .", "Previous developmental and electrophysiological analyses of the Aldh1a1–/– mouse has shown that the neural retina is apparently normal ( Fan et al . , 2003 ) .", "By broadening the phenotyping scope , we identified vascular hypoplasia in the dorsal choroid of Aldh1a1–/– eyes .", "Aldh1a1 is expressed in dorsal retinal progenitor cells from E10 . 5 ( Matt et al . , 2005 ) , and we observed no Aldh1a1 expression in either RPE cells or choroid in E12 . 5 and E17 . 5 mouse eyes ( Figure 1A and B ) .", "CD31-positive endothelial cells and VEGF expression in mouse eyes can be observed in choroid and primitive RPE cells at E10 . 5 ( Saint-Geniez et al . , 2006 ) , and choroidal vascular development is initiated at E11 . 5 when periocular vessels emerge following formation of the vascular network at around E13 . 5 ( Zhao and Overbeek , 2001; Marneros et al . , 2005; Saint-Geniez et al . , 2006 ) .", "The requirement for RPE-derived VEGF during embryonic development was reported using RPE-specific Vegfa-knockout mice ( Vegfflox/flox;VMD2-Cre mice ) , in which Cre expression can be conditionally induced in RPE cells by doxycycline administration ( Le et al . , 2008 ) .", "Although conditional disruption of Vegfa in RPE cells at E10 or E13 resulted in decreased choroidal vascular density , loss of the RPE-produced VEGF after E15 caused no significant defects in the choroidal vasculature ( Le et al . , 2010 ) .", "Together with our demonstration that choroidal VEGF production is downregulated in Aldh1a1–/– embryos , these data suggest that Aldh1a1 expression during E10 to E13 in trans potentiates VEGF secretion from RPE cells to allow the development of normal choroidal vascularization .", "In addition , it should be noted that choroidal VEGF level was still reduced at P24 in the Aldh1a1–/– mice .", "Considering that severe choriocapillaris vasoconstriction occurs in adult tamoxifen-induced Vegfflox/flox;VMD2-Cre mice ( Kurihara et al . , 2012 ) , it is possible that Aldh1a1 is also responsible for maintenance of the choriocapillaris .", "We also demonstrated that RAs enhanced VEGF secretion from primary RPE cells and that VAD mice showed choroidal hypoplasia in the dorsal region .", "Also , RA administration suppressed the choroidal hypoplasia phenotype in Aldh1a1–/– mice .", "These results strongly suggest that Aldh1a1-mediated RA is responsible for normal choroidal vascular formation by controlling VEGF secretion from RPE cells .", "Moreover , we observed downregulation of Sox9 in the dorsal Aldh1a1–/– eyes .", "RPE-specific disruption of Sox9 replicated the phenotype of Aldh1a1–/– eyes , and Sox9 overexpression in the Aldh1a1–/– RPE cells rescued dorsal choroidal hypoplasia .", "Considering that in primary RPE cells RA exposure enhances both Sox9 and Vegfa expression and that overexpression and knockdown of Sox9 influences Vegfa expression , it is more likely that Aldh1a1-mediated RA production stimulates Sox9 expression in dorsal RPE cells and Sox9 then transactivates the Vegfa promoter .", "Retinoic acid receptor α ( RARα ) and retinoid X receptor α ( RXRα ) are expressed in RPE cells at an early embryonic stage ( Mori et al . , 2001 ) .", "It is plausible that RARα and RXRα enhance Sox9 expression in dorsal RPE cells , although the precise molecular mechanism remains to be investigated .", "We observed interactions between Aldh1a1 , Sox9 , and Vegfa genes; however , the results raised the question of why VAD mice and RPE-cKO of Sox9 show choroidal hypoplasia only in the dorsal region because the reduction of vitamin A and Sox9 also occurs in the ventral region .", "We do not yet have the answer , but one possibility is that ventral choroidal vascular development is governed by a different molecular pathway .", "For example , Aldh1a3 , another RA-producing enzyme in the mouse retina , is expressed in RPE cells from E10 . 5 to E12 . 5 and in the ventral neural retina during embryogenesis ( Matt et al . , 2005 ) , Figure 1B ) .", "However , conditional disruption of Aldh1a3 in the neural retina ( floxed Aldh1a3 mice crossed with Pax6-α-Cre ) did not result in either choroidal hypoplasia or loss of pigmentation in the ventral region ( Figure 4—figure supplement 2 ) , suggesting that Aldh1a3-mediated RAs from the ventral neural retina are unlikely to affect ventral choroidal development .", "In addition , we did not observe upregulation of Sox9 in the ventral RPE cells from E12 . 5 to E14 . 5 ( Figure 5D and E ) , suggesting that Sox9 in the ventral RPE cells is unnecessary for ventral choroidal development .", "Given that mRFP and GFP fluorescent intensity seems different between dorsal and ventral RPEs in Tg-CAG-mRFPfloxed-SOX9-ires-EGFP mice ( Figure 6C ) , the difference might be the result of different developmental or physiological characteristics .", "In summary , we demonstrate a novel role of Aldh1a1 in dorsal choroidal vascular development .", "The results of the present study strongly suggest that RA production resulting from Aldh1a1 expression in the dorsal neural retina upregulates Sox9 expression in the dorsal RPE cells to enhance RPE-derived VEGF secretion ( Figure 6E ) .", "In addition , our results suggest that embryonic RA exposure may regulate future dorsal choroidal vessels in the adult .", "Because vascular hypoplasia could result in hypoxia and impaired nutrient supply , which could affect adjacent RPE and photoreceptor cells , future studies should investigate the degeneration of RPE and photoreceptor cells .", "For example , age-related macular degeneration ( AMD ) is the leading cause of severe vision loss in humans ( de Jong , 2006 ) and is caused by abnormalities of the subfoveal choriocapillaris .", "Aldh1a1−/− and VAD mice would be useful not only to study regional differences in choroidal development and maintenance but also to explore both a risk factor and a potential therapeutic target for AMD , because the RA concentration in the neural retina is affected by various environmental factors such as dietary intake of Vitamin A and light irradiation ." ], [ "All animal experiments were conducted with the approval of the RIKEN Center for Developmental Biology Ethics Committee ( No . AH18-05-23 ) .", "Timed pregnant CD1 and C57BL/6 mice were purchased from the Laboratory for Animal Resources and Genetic Engineering , RIKEN Center for Developmental Biology .", "Aldh1a1–/– mice ( Fan et al . , 2003 ) were purchased from Jackson Laboratory and mated with CD-1 mice to allow visualization of the choroidal vessels .", "Flt1-BAC-DsRed;Kdr-BAC-EGFP mice ( Matsumoto et al . , 2012 ) were mated with CD-1 or albinized Aldh1a1–/– mice .", "To conditionally disrupt Aldh1a3 in neural retinas , Aldh1a3-flox mice ( Dupé et al . , 2003 ) were mated with Pax6-α-Cre mice ( Marquardt et al . , 2001 ) .", "To conditionally disrupt Sox9 in RPE cells and neural retinas , Sox9-flox mice ( Kist et al . , 2002 ) were mated with Tyr-Cre ( Delmas et al . , 2003 ) and Pax6-α-Cre mice , respectively .", "To conditionally overexpress Sox9 in RPE cells , CAG-mRFP1floxed-Sox9-ires-EGFP transgenic mice ( Kim et al . , 2011 ) were mated with Tyr-Cre mice .", "Albinized R26R-H2B-mCherry mice ( Abe et al . , 2011 ) were used for the Cre reporter assay of Tyr-Cre and Pax6-α-Cre mice ( Figure 6—figure supplement 1A and B ) .", "PCR primers used for genotyping are listed in Table 1 .", "VAD diet feeding and RA treatments were performed as described previously ( Chihara et al . , 2013; Fan et al . , 2003 ) .", "Eight-week-old CD-1 mice were fed a vitamin A-deficient ( VAD ) diet ( AIN93G-D13110GC; Research Diets , New Brunswick , USA ) .", "After 16 weeks of feeding this diet , the animals were used as breeding pairs .", "Pregnant mice received the VAD diet until 3 days postpartum .", "All-trans-RA ( Sigma ) was suspended in ethanol ( 5 mg/ml ) and then either diluted in sunflower oil ( 125 μg/ml ) and administered by oral gavage to pregnant females ( 2 mg/kg of body weight ) every 12 hr from E10 to E16 .", "For cryosections , embryos were fixed in 4% paraformaldehyde ( PFA ) , cryoprotected in 30% sucrose in phosphate-buffered saline ( PBS ) overnight at 4°C , embedded in OCT compound ( Tissue Tec; Sakura Fine Technical , Japan ) , and sectioned at 10 μm using a cryostat ( HM560; Thermo Fisher Scientific , Waltham , MA ) .", "Specimens were blocked with horse serum for 1 hr at room temperature and incubated with primary antibodies overnight at 4°C , followed by incubation with secondary antibodies for 1 hr at room temperature .", "For flat-mount immunostaining of the RPE/choroid , the tissues were fixed with 4% PFA at room temperature for 30 min , and washed three times with PBS containing 0 . 5% Triton X-100 ( PBST , Nacalai Tesque , Japan ) , incubated with primary antibodies overnight at 4°C , followed by incubation with secondary antibodies for 1 hr at room temperature ( Zhu et al . , 2012 ) .", "The primary antibodies and dilutions used were as follows: goat anti-Aldh1a1 ( 1:1 , 000; Abcam ) , rabbit anti-Aldh1a3 ( 1:1 , 000; Sigma ) , rat anti-endomucin ( 1:400; Millipore ) , mouse anti-GS ( 1:1 , 000; Millipore ) , FITC-isolectin B4 ( 1:100; Vector Laboratories ) , rabbit anti-ZO-1 ( 1:100; Invitrogen ) , rabbit anti-GFP ( 1:1 , 000; Abcam ) , rabbit anti-ERG ( 1:400; Abcam ) , rabbit anti-Pax6 ( 1:200; BioLegend ) , and rabbit anti-Sox9 ( 1:200; Millipore ) .", "Mice were euthanized and perfused with ice-cold 4% PFA .", "Eyes were fixed with 2% glutaraldehyde in 4% PFA overnight .", "After washing with PBS , the eyes were postfixed with ice-cold 1% OsO4 in 0 . 1 M sodium cacodylate buffer , pH 7 . 3 , for 2 hr .", "The samples were then rinsed with distilled water , stained with 0 . 5% aqueous uranyl acetate for 2 hr or overnight at room temperature , dehydrated with ethanol and propylene oxide , and embedded in Poly/Bed 812 ( Polyscience ) .", "Ultrathin sections were cut , double-stained with uranyl acetate and Reynolds’ lead citrate , and viewed with a JEM 1010 or JEM 1400 transmission electron microscope ( JEOL ) at an accelerating voltage of 100 kV .", "Labeled cells were imaged using an LSM 780 confocal microscope ( Carl Zeiss ) .", "Images were processed using Photoshop CS2 software ( Adobe Systems ) .", "Choroidal vascular density was analyzed using ImageJ software ( NIH ) as described previously ( Le et al . , 2010 ) .", "Fluorescence intensity was quantified using Zen Black software ( version 2012; Carl Zeiss ) according to the manufacturer’s instructions .", "All images shown are representative of three to eight independent experiments .", "In situ hybridizations were performed as described previously ( Acloque et al . , 2008 ) .", "The RPE/choroid was separated from the mouse eye and was homogenized and resolved in 100 μl RIPA buffer containing a protease inhibitor .", "The total VEGFA protein ( pg ) per mg of the extracted RPE/choroid tissue was calculated using ELISA development kits ( R and D Systems , Minneapolis , MN ) ( Ueta et al . , 2012 ) .", "Primary human RPE cells ( Lonza ) were maintained in Dulbecco’s minimal essential medium ( DMEM ) supplemented with 5% fetal bovine serum ( FBS ) without antibiotics .", "Before RA treatment or transfection experiments , the RPE cells were suspended at 2 × 105 cells per well of a 6-well plate and cultured in DMEM supplemented with 5% FBS without antibiotics .", "After overnight culture , the medium was changed to RA-supplemented DMEM without FBS , and the cells were harvested after 24 hr incubation .", "For overexpression , 1 μg of pCAGIG and pCAGIG-Sox9 expression vectors ( Addgene #11159 ) were transfected using Lipofectamine 3000 ( Invitrogen ) according to the manufacturer’s instructions .", "For knockdown , control and human Sox9 siRNAs ( Santa Cruz ) were transfected using siRNA Transfection Reagent ( Santa Cruz ) according to the manufacturer’s instructions .", "After 7 hr incubation , the medium was changed to DMEM without FBS ( for overexpression ) or DMEM plus 10% FBS ( knockdown ) , and cells were harvested after another 24 hr incubation to quantify Sox9 and Vegfa mRNA by reverse transcription–quantitative polymerase chain reaction ( RT-qPCR ) .", "RT–qPCR was performed as described in a previous report ( Sugita et al . , 2015 ) .", "Briefly , Sox9 , Vegfa , and β-actin expression was analyzed in triplicate samples using a LightCycler model 480 ( Roche Diagnostics ) , qPCR MasterMix ( Roche Diagnostics ) , and highly specific Universal ProbeLibrary assays ( Roche Diagnostics ) .", "The tested primers are described in Table 1 , and the Universal Probes used were Probe#61 ( Sox9 and Vegfa ) , and Probe#64 ( β-actin ) .", "Relative mRNA expression was normalized to ΔΔCt of β-actin using relative quantification software ( Roche Diagnostics ) .", "Results were reported as the relative expression of each molecule ( ΔΔCt: control cells = 1 ) .", "All data are presented as the means ± SD .", "JMP Pro version 10 . 0 . 2 ( SAS Institute Inc . ) was used for statistical analysis , and data were analyzed using analysis of variance ( ANOVA ) followed by the Tukey–Kramer multiple-comparison test .", "When only two groups were compared , a two-sided Student’s t-test was used ." ] ]

[ "VEGF secreted from retinal pigment epithelial ( RPE ) cells is responsible for the choroidal vascular development; however , the molecular regulatory mechanism is unclear .", "We found that Aldh1a1–/– mice showed choroidal hypoplasia with insufficient vascularization in the dorsal region , although Aldh1a1 , an enzyme that synthesizes retinoic acids ( RAs ) , is expressed in the dorsal neural retina , not in the RPE/choroid complex .", "The level of VEGF in the RPE/choroid was significantly decreased in Aldh1a1–/– mice , and RA-dependent enhancement of VEGF was observed in primary RPE cells .", "An RA-deficient diet resulted in dorsal choroidal hypoplasia , and simple RA treatment of Aldh1a1–/– pregnant females suppressed choroid hypoplasia in their offspring .", "We also found downregulation of Sox9 in the dorsal neural retina and RPE of Aldh1a1–/– mice and RPE-specific disruption of Sox9 phenocopied Aldh1a1–/– choroidal development .", "These results suggest that RAs produced by Aldh1a1 in the neural retina directs dorsal choroidal vascular development via Sox9 upregulation in the dorsal RPE cells to enhance RPE-derived VEGF secretion ." ]

[ "The retina is the part at the back of our eyes that detects light and sends this information to our brain .", "Within the retina is a layered structure containing the light-sensitive cells , known as the neural retina , and another protective layer of cells called the retinal pigment epithelium .", "A surrounding network of blood vessels , the choroid , keeps the retina healthy by supplying oxygen and nutrients .", "When the choroid does not work properly , eye disease can result .", "A common example is age-related macular degeneration , where blood vessels in the choroid either break down or start growing uncontrollably in the wrong places .", "In both cases , light-sensitive cells are damaged and eventually die .", "This causes vision loss that worsens over time .", "The choroid forms early in life , within the developing embryo .", "The retinal pigment epithelium helps the choroid to develop properly by producing a protein , VEGF , which supports the growth of blood vessels .", "However , it was not clear what signals tell this tissue to start making VEGF in the first place and then to keep making it .", "To address this , Goto et al . looked at eye development in mutant mice that lack an enzyme called Aldh1a1 .", "This enzyme’s role is to make a molecule called retinoic acid , which is known to be vital for many biological processes including the growth and development of embryos .", "Aldh1a1 is not made in the choroid of normal mice , just in the neural retina .", "Yet the choroid in the mutant mice without Aldh1a1 still grew fewer blood vessels than normal .", "Their retinal pigment epithelium also produced less VEGF and had lower levels of a protein called Sox9 , which is known to make the gene for VEGF more active .", "Goto et al . went on to show that simply removing retinoic acid from the diet of normal mice produced the same choroid defect as in the mutant mice with no Aldh1a1 .", "Genetically manipulating otherwise normal mice to decrease the levels of Sox9 in the retinal pigment epithelium had a similar effect .", "In contrast , giving Aldh1a1-deficient mice extra retinoic acid or artificially increasing their levels Sox9 was enough to make the choroid develop normally .", "These experiments showed that retinoic acid produced in the neural retina directs choroid development by making Sox9 more active , which in turn encourages the retinal pigment epithelium to produce VEGF .", "These findings bring new insights into the molecular signals that control choroid development .", "In the future , they may also help scientists to better understand why blood vessels in the choroid become abnormal in eye diseases like age-related macular degeneration ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "plant biology", "microbiology and infectious disease" ]

[ [ "Fungi constitute an evolutionarily and ecologically diverse group of microorganisms .", "Although most species are saprophytic , many are causative agents of disease and include plant pathogens that cause considerable yield losses in agricultural crops worldwide ( Skamnioti and Gurr , 2009; Oliver , 2012 ) .", "To sense the presence of potential pathogens , hosts employ cell surface receptors that detect conserved pathogen-associated molecular patterns ( PAMPs ) to activate immunity ( Medzhitov and Janeway , 1997; Boller and Felix , 2009 ) .", "Chitin , an N-acetyl-D-glucosamine ( GlcNAc ) homopolymer , is the primary structural component of fungal cell walls and is recognized as a PAMP by plant cell surface receptors that contain extracellular lysin motifs ( LysMs ) ( Felix et al . , 1993; Shibuya et al . , 1993; Shibuya et al . , 1996; Kombrink et al . , 2011 ) .", "The first chitin receptor has been cloned from rice ( Oryza sativa ) as the chitin oligosaccharide elicitor-binding protein ( CEBiP; Kaku et al . , 2006 ) that forms a receptor complex in a ligand-dependent manner that furthermore includes the chitin elicitor receptor kinase-1 ( OsCERK1; Shimizu et al . , 2010 ) .", "Similarly , Arabidopsis thaliana CERK1 binds chitin and is required for chitin-triggered immunity ( Miya et al . , 2007; Wan et al . , 2008; Iizasa et al . , 2010; Petutschnig et al . , 2010 ) .", "However , no homologs of rice CEBiP could be implicated in chitin-triggered immunity against fungal infection in Arabidopsis ( Shinya et al . , 2012; Wan et al . , 2012 ) .", "Recently , a crystal structure of the AtCERK1 ectodomain was determined , revealing chitin binding to one of the three AtCERK1 LysMs only ( Protein Data Bank code 4EBZ; Liu et al . , 2012 ) .", "LysM2 of AtCERK1 binds three GlcNAc residues of a longer chitin oligomer in a shallow groove on the surface of the protein , with both ends of the oligomer as well as one half side of each of the three bound GlcNAc residues protruding into the solvent ( Liu et al . , 2012 ) .", "Biochemical experiments suggest that sufficiently long chitin oligomers act as bivalent ligands , leading to ligand-induced AtCERK1 dimerization that is required for immune signaling ( Liu et al . , 2012 ) .", "The evolution of interactions between microbial pathogens and their hosts involves a continuous arms race , in which pathogens secrete effectors to deregulate host immunity ( de Jonge et al . , 2011 ) .", "The leaf mold fungus Cladosporium fulvum abundantly secretes the LysM-containing effector protein Ecp6 ( for extracellular protein 6; Bolton et al . , 2008 ) during colonization of its host tomato .", "Ecp6 acts as a scavenger of chitin fragments and thus prevents recognition of the fungus by host immune receptors ( de Jonge et al . , 2010 ) .", "Intriguingly , Ecp6 homologs occur throughout the fungal kingdom , suggesting a fundamental role of chitin scavenging in fungal pathogenicity ( Bolton et al . , 2008; de Jonge and Thomma , 2009 ) .", "Indeed , LysM effectors from the fungal wheat pathogen Mycosphaerella graminicola and the rice pathogen Magnaporthe oryzae suppress chitin-triggered immunity ( Marshall et al . , 2011; Mentlak et al . , 2012 ) .", "However , the mechanism by which LysM effectors outcompete plant receptors for chitin binding remain unknown thus far .", "LysM-containing proteins are broadly distributed in bacteria , plants , fungi , and animals ( Buist et al . , 2008; Kombrink et al . , 2011 ) .", "Nevertheless , only few tertiary LysM structures have been reported in addition to that of AtCERK1 ( Bateman and Bycroft , 2000; Bielnicki et al . , 2006; Koharudin et al . , 2011; Liu et al . , 2012 ) .", "The canonical three-dimensional LysM domain structure consists of a βααβ-fold , in which two α-helices are packed against one side of a two-stranded antiparallel β-sheet .", "Based on NMR spectroscopy , the loop between the first β-sheet and the first α-helix and the loop between the second α-helix and the second β-sheet were shown to physically interact with chitin oligomers in a 1:1 stoichiometry with an affinity binding constant of up to 21 µM ( Ohnuma et al . , 2008; Koharudin et al . , 2011 ) .", "The AtCERK1 ectodomain binds chitin oligomers with a similar affinity , up to 45 µM for ( GlcNAc ) 8 , as determined by isothermal titration calorimetry ( Liu et al . , 2012 ) .", "Here , we report the crystal structure of Ecp6 to a resolution of 1 . 6 Å , revealing a novel mechanism for chitin binding by LysMs that developed in fungi , involving substrate-induced intrachain dimerization of two LysM domains to form a buried intramolecular chitin-binding groove .", "The composite LysM1–LysM3 binding site shows ultra-high chitin-binding affinity , thus explaining how LysM effectors outcompete plant host receptors for chitin binding ." ], [ "To understand the molecular mechanism of LysM effector chitin scavenging , we pursued a crystal structure of the C . fulvum LysM effector Ecp6 .", "To this end , Ecp6 was heterologously produced in the yeast Pichia pastoris and purified .", "Initial Ecp6 vapor-diffusion crystallization screening consistently yielded intertwined plate-like crystals that grew overnight .", "These crystals were harvested , smashed in stabilizing mother liquid and used for micro-seeding ( Bergfors , 2003 ) .", "In this manner , large Ecp6 protein crystals that belonged to space group P3221 were obtained .", "The structure was determined by single-wavelength anomalous dispersion ( SAD ) and refined to a resolution of 1 . 6 Å with an Rwork and Rfree of 20 . 3% and 22 . 5% , respectively ( Figure 1 and Table 1 ) .", "The structure model comprises nearly the complete mature protein sequence , from amino acid residues 7 to 195 .", "Residues 1–6 and 196–199 of the structure were not defined in the electron density map , suggesting that they occur as flexible tails on both ends of the protein . 10 . 7554/eLife . 00790 . 003Figure 1 . Overall crystal structure of the Cladosporium fulvum LysM effector Ecp6 . ( A ) Crystal structure model of an Ecp6 dimer in which the left monomer is colored orange and the three LysMs of the right monomer are indicated in three shades of blue with the flexible loop between LysM1 and LysM2 in gray .", "The chitin tetramer ( green sticks ) and four disulfide bridges ( yellow sticks ) are indicated .", "Furthermore , in the right monomer , the two ( putative ) chitin-binding loops are shown in red and green for each of the LysMs .", "( B ) Omit map ( 2Fo−Fc; contoured at 1σ above the mean ) with phases calculated omitting ( GlcNAc ) 4 .", "LysMs are colored in three shades of blue as in panel A . ( C ) Interactions between Ecp6 and ( GlcNAc ) 4 .", "Hydrogen bonds are indicated in red , and atoms involved in hydrophobic contacts are represented with transparent surface .", "Only residues forming H-bonds with the chitin are labeled .", "( D ) Clustal-W alignment of the three LysM domains of Ecp6 .", "The distribution of the α-helices ( helices ) and β-sheets ( arrows ) are shown .", "The two chitin-binding sites in LysM1 and LysM3 are indicated with a red line for the first loop between the first β-sheet and the first α-helix and a green line for the second loop between the second α-helix and the second β-sheet , as indicated in the right monomer in panel A . Blue arrows point towards residues targeted for mutagenesis in the three LysM domains . DOI: http://dx . doi . org/10 . 7554/eLife . 00790 . 00310 . 7554/eLife . 00790 . 004Table 1 . Data collection and refinement statisticsDOI: http://dx . doi . org/10 . 7554/eLife . 00790 . 004NativeSADData collection statistics BeamlineBL14 . 1 - BESSYID29 - ESRF Wavelength ( Å ) 0 . 918141 . 70 Space groupP 32 2 1 Cell dimensions a , b , c ( Å ) 57 . 5 , 57 . 5 , 118 . 757 . 9 , 57 . 9 , 119 . 7 Resolution ( Å ) 49 . 80–1 . 59 ( 1 . 68–1 . 59 ) 46 . 24–2 . 10 ( 2 . 21–2 . 1 ) Rsym* ( % ) 5 . 1 ( 44 . 9 ) 6 . 7 ( 40 . 9 ) I/σI†20 . 6 ( 4 . 4 ) 24 . 2 ( 2 . 9 ) Completeness ( % ) 98 . 4 ( 94 . 1 ) 97 . 7 ( 85 . 9 ) Redundancy9 . 2 ( 8 . 2 ) 15 . 4 ( 5 . 6 ) Phasing statistics ( 2 . 5 Å resolution cut-off ) Anomalous completeness ( % ) –96 . 9 ( 81 . 2 ) Anomalous multiplicity–8 . 3 ( 3 . 0 ) Figure of Merit ( FOM ) –0 . 372 Map Skew–0 . 14 Correlation of local R . m . s . density–0 . 82 Correlation Coefficient ( CC ) –0 . 76Refinement statistics Resolution ( Å ) 49 . 8–1 . 631 . 0–2 . 10 No .", "of reflections ( work/free ) 29 , 078/154613 , 072/689 Rwork/Rfree‡ ( % ) 20 . 3 ( 24 . 0 ) /22 . 5 ( 27 . 1 ) 21 . 0 ( 23 . 3 ) /26 . 6 ( 34 . 2 ) No .", "of atoms/average B-factor Protein1392/17 . 61387/34 . 33 Water119/37 . 553/44 . 61 Other99/24 . 3115/39 . 90 R . m . s . deviations bond lengths ( Å ) 0 . 0160 . 018 R . m . s . deviations bond angles ( ° ) 1 . 811 . 94 Ramachandran plot ( % preferred region/% allowed region ) 96 . 74/3 . 2695 . 72/4 . 28The values in the parentheses refer to the highest resolution shell .", "*Rsym = ( ∑│Ihkl−<Ihkl>│ ) / ( ∑ Ihkl ) , where the average intensity <Ihkl> is taken over all symmetry equivalent measurements and Ihkl is the measured intensity for any given reflection .", "†I/σI is the mean reflection intensity divided by the estimated error .", "‡Rwork = ││Fo│−│Fc││/│Fo│ , where Fo and Fc are the observed and calculated structure factor amplitudes , respectively .", "Rfree is equivalent to Rwork but calculated for 5% of the reflections chosen at random and omitted from the refinement process .", "Ecp6 has a tightly packed structure that is stabilized by four disulfide bridges , and two spatially close glycosylation sites ( Asn104 and Asn193 ) were identified .", "The three LysMs of Ecp6 ( LysM1–LysM3 ) share the typical βααβ-fold , with LysM1 being separated by a long and flexible linker from a compact and rigid body formed by LysM2 and LysM3 ( Figure 1A ) .", "Furthermore , the crystal packing revealed the existence of an Ecp6 homodimer that was also observed in gel filtration chromatography during protein purification , with a flat buried surface of 943 Å2 at LysM2 and LysM3 as calculated using PISA ( Protein Interfaces , Surfaces and Assemblies; http://www . ebi . ac . uk/pdbe/prot_int/pistart . html; Figure 1A; Krissinel and Henrick , 2007 ) .", "Unexpectedly , the calculated 2Fo−Fc map showed a well-defined electron density for a bound chitin tetramer ( GlcNAc ) 4 in a large interdomain groove between LysM1 and LysM3 ( ∼16 Å long and ∼11 Å across its widest points; Figure 1B ) , although the protein crystallized in the absence of exogenously added chitin .", "The chitin oligomer was likely co-purified with Ecp6 and derived from the cell wall of the P . pastoris expression system .", "The observation that the chitin oligomer remained adhered to Ecp6 during the protein purification procedure suggests that it is bound to the groove with high affinity .", "Thus , our finding illustrates the remarkable potential of Ecp6 to instantly and strongly bind chitin after secretion by fungal cells , a scavenging activity that is expected to similarly occur after secretion by C . fulvum to prevent chitin oligosaccharides from activating host immune receptors ( de Jonge et al . , 2010 ) .", "The loop between the first β-sheet and the first α-helix and the loop between the second α-helix and the second β-sheet in both LysM1 and LysM3 interact with chitin ( Figure 1 ) , similar to the previously reported chitin binding by single LysM domains including LysM2 of AtCERK1 ( Ohnuma et al . , 2008; Koharudin et al . , 2011; Liu et al . , 2012 ) .", "Evidently , LysM1 and LysM3 of Ecp6 cooperate to bring two chitin-binding regions together , composing a novel type of binding groove in which one chitin tetramer is nearly completely buried and engaged in many noncovalent interactions , including 12 hydrogen bonds ( Figure 1C ) .", "Specifically , the groove is shaped by the amino acids 20GDTLT24 and 46PNLIEL51 of LysM1 and 150GDLFV154 and 176PSKL179 of LysM3 .", "The chitin binding is further strengthened by noncovalent interactions among proximate residues on the complementary surfaces of LysM1 ( N45 and 47NLIE50 ) and LysM3 ( 150GDLFVD155 ) ( Figure 1 ) .", "The concerted action of two LysM domains that compose a single binding groove and the many noncovalent bonds that are involved in the interaction with chitin may collectively provide a mechanistic explanation for high affinity substrate binding , resulting in sequestration by Ecp6 of cell wall chitin from the heterologous host P . pastoris .", "Furthermore , the solute-exposed ends of the chitin tetramer in the structure suggest that longer oligomers can also be bound to the groove , in which case four GlcNAc units of a chitin oligomer make direct contact with the protein while the remaining parts of the molecule protrude into the solvent ( Figure 1 ) .", "To investigate the contribution of the LysM1–LysM3 binding groove to chitin scavenging by Ecp6 , mutants in the chitin-binding site of LysM1 and LysM3 were generated ( Figures 1D and 2A ) , produced in P . pastoris , and tested for the ability to suppress chitin-triggered immunity in tomato cells ( de Jonge et al . , 2010 ) .", "Two residues that are in close contact with the chitin oligomer and present in the loop between the first β-sheet and the first α-helix ( T22 and L152 ) and in the loop between the second α-helix and the second β-sheet ( N47 and S177 ) of LysM1 and LysM3 were chosen to disrupt chitin binding to the LysM1–LysM3 groove ( Figures 1D and 2A ) .", "The selected residues were substituted by relatively large arginine or lysine amino acids to maximize interference with chitin binding .", "Circular dichroism ( CD ) spectra of the mutant proteins were obtained to confirm the correct folding . 10 . 7554/eLife . 00790 . 005Figure 2 . Structural and mutational analysis of chitin binding .", "( A ) Ecp6 monomer in which the residues targeted for mutagenesis are labeled and represented using sticks .", "The chitin oligomer is in green sticks .", "( B ) Structural alignment of the three Ecp6 LysM domains .", "Each of the LysMs are colored in three shades of blue and the chitin tetramer is in green sticks .", "The two chitin-binding loops are shown in red and green for each of the LysMs , as indicated in Figure 1A . DOI: http://dx . doi . org/10 . 7554/eLife . 00790 . 005 Isothermal titration calorimetry ( ITC ) analysis demonstrated that all mutants were able to bind chitin ( Figure 3 ) .", "It was previously reported that Ecp6 protein , carrying three LysM domains , binds chitin with 3:1 stoichiometry ( de Jonge et al . 2010 ) by building on the general observation that LysM domains bind their substrate with 1:1 stoichiometry .", "However , the crystal structure revealed that only LysM2 is available for chitin binding when Ecp6 is produced in P . pastoris , as the LysM1–LysM3 groove is occupied by chitin ( Figure 1 ) .", "Therefore , we integrated the heat measurements using a single binding site model , confirming that P . pastoris-produced Ecp6 binds chitin with 1:1 stoichiometry and a dissociation constant ( kd ) of 4 . 5 µM ( Figure 3A ) .", "The T22R mutant in LysM1 binds chitin with similar thermodynamic values as Ecp6 produced in P . pastoris ( kd = 4 . 69 µM; n = 0 . 75; Figure 3D ) .", "However , chitin binding by the mutants N47K , L152R and S177K did not follow a sigmoidal curve , suggesting that more than one binding event occurs in these mutants ( Figure 3 ) .", "Binding by the N47K mutant could be fitted to a ‘two binding site’ model , revealing that the second binding displayed similar biochemical characteristics ( kd = 5 . 2 µM; n = 0 . 984 ) as Ecp6 produced in P . pastoris ( Figure 3E ) .", "These results likely reflect chitin binding to LysM2 and to the partially disrupted LysM1–LysM3 groove , as only one of these two LysMs was mutagenized in a single mutant . 10 . 7554/eLife . 00790 . 006Figure 3 . Mutants in LysM1 and LysM3 chitin-binding site still bind chitin . Raw data ( upper panels ) and integrated heat measurements ( lower panels ) from isothermal titration calorimetry of ( GlcNAc ) 6 binding to Ecp6 produced in P . pastoris ( A ) and mutants in LysM3 ( B and C ) and in LysM1 ( D and E ) .", "Lines in the lower panel represent best-fit curves for one binding site model .", "ITC control experiments involving chitin ligand into the buffer ( PBS ) , of buffer injection into the buffer , and buffer injection into Ecp6 protein solution are included in panel A . DOI: http://dx . doi . org/10 . 7554/eLife . 00790 . 006 Next , we assessed the ability of the various mutants to suppress chitin-triggered immunity in tomato cells .", "Unfortunately , the two mutants that were generated in LysM1 ( T22R and N47K ) resulted in an autoactive protein that triggered a response of the tomato cells , leading to medium alkalinization already in the absence of chitin ( Figure 4A ) .", "Most probably the induction of medium pH shift by the mutants in LysM1 was due to improper protein folding , as is suggested by CD spectra when compared with wild-type Ecp6 protein ( Figure 4B ) .", "Possibly , these mutant proteins carry P . pastoris chitin with low affinity that is released in the cell suspension , thus activating a pH shift in the absence of exogenously added chitin .", "Consequently , these mutants could not be assessed for their ability to suppress chitin-triggered immunity .", "In contrast , the CD spectra of the two mutants in LysM3 ( L152R and S177K ) were very similar to that of wild-type Ecp6 protein , indicating that they adopt the same secondary structure ( Figure 4D ) .", "As these mutants ( L152R and S177K ) did not trigger a response of the tomato cells in the absence of chitin oligosaccharides , they could be tested for their scavenging ability .", "Interestingly , both mutants still prevented chitohexaose [ ( GlcNAc ) 6]-induced medium alkalinization .", "These results suggest that also LysM2 contributes to the suppression of chitin-triggered immunity by P . pastoris-produced Ecp6 in the cell suspension assay ( Figure 4C ) .", "Indeed , the high sequence and tertiary structure conservation of the three LysM domains of Ecp6 suggests that LysM2 contains a functional chitin-binding site ( Figure 1D; Figure 2B ) .", "To investigate this , conserved residues in the putative chitin-binding site of LysM2 were selected for mutagenesis ( Figures 1D and 2A ) .", "Based on the structural alignment , T95 and D121 were mutagenized because of their localization in the two loops that may be involved in chitin binding by LysM2 .", "P . pastoris-produced mutant proteins ( that contain chitin in the LysM1–LysM3 groove ) were tested for their capacity to suppress chitin-triggered immunity ( de Jonge et al . , 2010 ) .", "Interestingly , mutants T95R and D121K no longer suppressed the chitin oligosaccharide-induced pH shift , revealing that LysM2 contains a functional chitin-binding site that contributes to the suppression of chitin-triggered immunity in the cell suspension assay ( Figure 5A ) .", "Indeed , ITC experiments confirmed that the P . pastoris-produced mutants T95R and D121K were impaired in the binding of exogenously added chitohexaose ( Figure 5C and 5D ) .", "Correct folding of the LysM2 mutants was confirmed by CD spectra that were similar to that of wild-type Ecp6 protein ( Figure 5B ) .", "Collectively , these data confirm that suppression of chitin-triggered immunity in the tomato cell suspension by P . pastoris-produced Ecp6 is established through the µM chitin-binding activity of LysM2 and explains why chitin scavenging by LysM3 mutants was not impaired .", "However , calculation of the equilibrium between Ecp6 protein and chitin oligosaccharide levels at the concentrations that were used , and that were well below the measured dissociation constant of LysM2 , reveals that only a small amount of the available chitin oligosaccharide is bound by this LysM .", "Consequently , the suppression of chitin-triggered immunity by LysM2 is unlikely to work via chitin oligosaccharide sequestration . 10 . 7554/eLife . 00790 . 007Figure 4 . Analysis of the capacity to prevent chitin recognition by Ecp6 mutants on the chitin-binding site of LysM1 ( A–B ) or LysM3 ( C–D ) .", "( A and C )", "The maximum pH shift determined on treatment with 10 nM ( GlcNAc ) 6 and 100 nM wild-type or mutants in LysM1 ( A ) and LysM3 ( B ) after normalization to treatment with ( GlcNAc ) 6 in the absence of Ecp6 protein is represented .", "Bars present averages of at least two replications with standard deviations .", "Significant differences with ( GlcNAc ) 6 treatment are indicated with asterisks and significant differences with Ecp6 treatment are indicated with plusses ( t-test p≤0 . 05 ) .", "( B and D )", "Circular dichroism spectra of the mutants in LysM1 ( B ) and LysM3 ( D ) at 10 µM . DOI: http://dx . doi . org/10 . 7554/eLife . 00790 . 00710 . 7554/eLife . 00790 . 008Figure 5 . LysM2 mutants are impaired in chitin scavenging .", "( A ) Prevention of chitin-triggered medium alkalinization in the tomato cell suspension assay by Ecp6 mutants .", "The maximum pH shift determined on treatment with 10 nM ( GlcNAc ) 6 and 100 nM Ecp6 or the mutants on LysM2 ( T95R and D121K ) after normalization to treatment with ( GlcNAc ) 6 in the absence of Ecp6 is represented .", "Bars present averages of three replicates with standard deviations .", "Significant differences with ( GlcNAc ) 6 treatment are indicated with asterisks , and significant differences with Ecp6 treatment are indicated with plusses ( t-test p≤0 . 05 ) .", "( B ) Circular dichroism spectra of the mutants on LysM2 at 10 µM .", "( C ) T95R and ( D ) D121K mutants on LysM2 are impaired in chitin binding .", "Raw data ( upper panels ) and integrated heat measurements ( lower panels ) from isothermal titration calorimetry of ( GlcNAc ) 6 binding to T95R and D121K mutants produced in P . pastoris . DOI: http://dx . doi . org/10 . 7554/eLife . 00790 . 008 Based on the assumption that we have not assessed the full chitin-binding capacity of the Ecp6 effector protein thus far , but only assessed the µM affinity of the solitary LysM2 , we attempted to obtain chitin-free Ecp6 protein .", "Unfortunately , we did not succeed in recovering functional chitin-free Ecp6 after denaturing with 8 M urea .", "As an alternative strategy , the production of Ecp6 in mammalian ( HEK293 ) cells was pursued .", "A relatively small amount ( 4 mg ) of chitin-free Ecp6 was obtained from these cells .", "We confirmed that the Ecp6 protein produced in mammalian cells was able to suppress chitin-triggered immunity in the tomato cell suspension assay ( Figure 6 ) and subsequently performed substrate affinity measurements ( Figure 7 ) .", "Interestingly , ITC revealed biphasic binding of the chitin hexamer ( GlcNAc ) 6 to chitin-free Ecp6 .", "A first binding phase in which one chitin molecule was bound with ultra-high affinity ( kd = 280 pM; n = 0 . 99 ) occurred , followed by binding of an additional molecule with lower affinity ( kd = 1 . 70 µM; n = 1 . 03 ) ( Figure 7B ) .", "Both binding events displayed 1:1 stoichiometry , demonstrating that the three LysM domains of a single Ecp6 monomer are collectively involved in two binding events ( Figure 7B ) .", "As P . pastoris-produced Ecp6 , in which the LysM1–LysM3 groove is blocked by a P . pastoris chitin fragment , displayed similar characteristics as the second binding event of mammalian cell-produced Ecp6 ( Figure 7A , B ) , we conclude that the µM affinity should be attributed to LysM2 .", "To confirm this hypothesis , we also produced the LysM2 mutant T95R , which only has the LysM1–LysM3 site available for chitin binding , in mammalian cells .", "As this mutant binds a single chitin molecule with high affinity ( kd = 4 . 95 nM ) ( Figure 7C ) , we concluded that the ultra-high chitin-binding affinity displayed by wild-type Ecp6 corresponds to the LysM1–LysM3 binding groove .", "The slightly lower dissociation constant that is measured for the LysM1–LysM3 groove in the HEK293-produced LysM2 mutant when compared with the HEK293-produced wild-type Ecp6 may be attributed to disabled cooperativity that may occur between both binding sites in the wild-type protein .", "We subsequently analyzed the role of the LysM1–LysM3 groove in suppression of chitin-triggered immunity in the tomato cell suspension assay with the HEK293-produced LysM2 mutant T95R .", "As expected , this mutant protein was able to prevent chitin-triggered medium alkalinization ( Figure 6 ) .", "Consequently , the composite LysM1–LysM3 binding site provides a single binding event with ultra-high ( pM ) affinity for chitin binding , the highest chitin-binding affinity described in nature , which is extremely competent to sequester chitin oligosaccharides .", "Through this effector activity , C . fulvum prevents chitin oligosaccharides from activating host immune receptors during infection of tomato . 10 . 7554/eLife . 00790 . 009Figure 6 . Prevention of chitin-triggered medium alkalinization in the tomato cell suspension assay by Ecp6 and T95R produced in HEK293 cells . The maximum pH shift determined on treatment with 10 nM ( GlcNAc ) 6 and 100 nM Ecp6 or T95R mutant after normalization to treatment with ( GlcNAc ) 6 in the absence of Ecp6 is represented .", "Bars present averages of three replicates with standard deviations .", "Significant differences with ( GlcNAc ) 6 treatment are indicated with asterisks , and significant differences with Ecp6 treatment are indicated with plusses ( t-test p≤0 . 05 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 00790 . 00910 . 7554/eLife . 00790 . 010Figure 7 . Ultra-high affinity chitin binding by intrachain LysM dimerization in Ecp6 . Raw data ( upper panels ) and integrated heat measurements ( lower panels ) from isothermal titration calorimetry of ( GlcNAc ) 6 binding to Ecp6 produced in P . pastoris ( A ) and in HEK293 ( B ) and T95R mutant produced in HEK293 ( C ) .", "Lines in the lower panel represent best-fit curves for one ( P . pastoris-produced T95R ) or two ( HEK293-produced ) binding site model . DOI: http://dx . doi . org/10 . 7554/eLife . 00790 . 010" ], [ "Various fungal pathogens secrete LysM effectors to scavenge chitin fragments and thus avoid recognition by host immune receptors that activate immune responses ( Bolton et al . , 2008; de Jonge et al . , 2010; Marshall et al . , 2011; Mentlak et al . , 2012; Kombrink et al . , 2011 ) .", "However , the mechanism used by these LysM effectors to efficiently compete for chitin binding with immune receptors remained unclear .", "Our structural and biochemical analysis of the LysM effector Ecp6 has unveiled a novel mechanism for chitin binding that evolved in fungi , in which the concerted action of two LysM domains results in sequestration of a chitin oligomer with ultra-high affinity .", "The flexible loop between LysM1 and the rigid LysM2–LysM3 body enables the sandwiching of four GlcNAc units of a chitin oligomer in the interface of the LysM1–LysM3 dimer by 12 hydrogen bonds and many other noncovalent interactions , resulting in ultra-high ( pM ) affinity .", "Based on the structure of the binding groove , it is anticipated that the intrachain LysM dimerization is substrate induced rather than pre-formed , as the chitin oligomer is completely buried in between the two LysM domains that are in very close proximity to make contact with the chitin oligomer .", "A pre-formed binding site would require a chitin oligomer to slide along the two LysM domain binding sites , which is practically impossible .", "At minimum , the binding groove has to breathe to let the oligomer enter before the chitin oligomer is bound .", "Furthermore , the noncovalent interactions among proximate residues on the complementary surfaces of LysM1 and LysM3 are likely not sufficient to keep a pre-formed binding groove in a closed conformation .", "More realistically , and considering the long and flexible linker that separates LysM1 from the LysM2–LysM3 body , the binding groove significantly opens in absence of the ligand and closes upon ligand binding , leading to substrate-induced intrachain LysM domain dimerization .", "Longer chitin oligomers may protrude from the Ecp6 protein into the solvent on both ends , which is particularly relevant since these are considered to be the most biologically active in eliciting chitin-triggered immune responses ( Felix et al . , 1993; Liu et al . , 2012 ) .", "Indeed , previously determined affinity constants were similar for chitin tetra- , penta- , hexa- and octamers ( de Jonge et al . , 2010 ) .", "Likely , as soon as Ecp6 is secreted from the plasma membrane of C . fulvum , it will sequester a chitin oligosaccharide that remains firmly attached to the protein .", "The global arrangement of the three LysMs of the Arabidopsis AtCERK1 chitin receptor , all three facing in outward direction on the surface of the protein , clearly prevents the establishment of intrachain LysM dimerization in a similar fashion as observed in Ecp6 ( Figure 8 ) .", "Moreover , only a single of the three AtCERK1 LysM domains is apparently involved in substrate binding , and only a single chitin oligomer is bound by a receptor molecule ( Liu et al . , 2012 ) .", "Consequently , AtCERK1 binds ( GlcNAc ) 6 with a considerably lower affinity ( kd = 44 . 8 µM; Liu et al . , 2012 ) than the LysM1–LysM3 binding groove of Ecp6 ( kd = 280 pM; Figure 7B ) .", "A tomato receptor for chitin has not been identified until now .", "However , it has been described based on radiolabeling assays that tomato cells and microsomal membranes can bind chitin oligomers with binding constants of 1 . 4 nM and 23 nM , respectively ( Baureithel et al . , 1994 ) .", "Also , these affinities are lower than that of the LysM1–LysM3 binding groove of Ecp6 .", "Considering also the high amount of Ecp6 effector protein that is secreted , especially during the initial stages of the infection ( Bolton et al . , 2008 ) , these data collectively explain how C . fulvum , and possibly also other fungal pathogens ( de Jonge and Thomma , 2009; Marshall et al . , 2011; Mentlak et al . , 2012 ) , manage to suppress chitin-triggered immunity during infection of their hosts . 10 . 7554/eLife . 00790 . 011Figure 8 . Spatial distribution of the LysM domains in the Arabidopsis thaliana chitin-binding immune receptor AtCERK1 . In contrast to Ecp6 ( A ) , the global arrangement of the LysMs ( colored in three shades of blue and with chitin-binding loops in red and green ) in the AtCERK1 ectodomain does not allow the formation of an intrachain LysM dimer ( B ) .", "Only one of the three LysMs is reported to bind a chitin oligomer ( Liu et al . , 2012 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 00790 . 011 In Arabidopsis , AtCERK1 receptors have been suggested to bind in tandem to long chitin oligomers , prompting dimerization and activation of immune signaling ( Liu et al . , 2012 ) .", "Thus , considering LysM dimerization as a mechanism for substrate binding , also interchain LysM dimerization may be exploited by nature .", "In this study as well as in previous studies ( de Jonge et al . , 2010 ) , it was found that P . pastoris-produced Ecp6 is able to suppress chitin-triggered immunity of tomato cells although only LysM2 is available for chitin binding .", "Moreover , P . pastoris-produced LysM2 mutants in Ecp6 were impaired in prevention of chitin-triggered alkalinization of tomato cell suspensions ( Figure 5 ) .", "In this respect it is surprising that the affinity that was determined for chitin binding by LysM2 is lower than previously determined affinities for chitin binding by tomato cells ( Baureithel et al . , 1994 ) .", "However , it needs to be noted that different methods have been used to determine these affinities , and that the binding assays were performed under different conditions that furthermore differ from those in planta during the interaction with the pathogen .", "Because the concentrations of P . pastoris-produced Ecp6 ( 100 nM ) and chitin ( 10 nM ) that were used in the alkalinization experiment are much lower than the LysM2 dissociation constant ( 4 . 5 µM ) , P . pastoris-produced Ecp6 will only sequester a small portion of the available chitin oligosaccharides , and the amount of remaining , unbound , chitin oligosaccharides should be sufficient to activate chitin-triggered immunity .", "Thus , the observation that LysM2 is able to suppress chitin-triggered immunity strongly suggests that this LysM suppresses chitin-triggered immunity through another mechanism than through chitin oligosaccharide sequestration .", "Potentially , LysM2 may be involved in perturbation of the activation of chitin-triggered immunity by preventing the immune receptor dimerization that is required for the activation of immune signaling ( Liu et al . , 2012 ) .", "Ecp6 may bind to chitin oligomers that have been bound by host immune receptor monomers , thus physically blocking host immune receptor dimerization .", "These mechanisms may explain how LysM2 of Ecp6 may perturb chitin-triggered immune signaling .", "In conclusion , it appears that the C . fulvum LysM effector Ecp6 is able to suppress chitin-triggered immunity through two mechanisms; efficient chitin oligosaccharide sequestration through a ligand-induced composite LysM1–LysM3 binding groove , and secondly a mechanism exerted by LysM2 that does not involve chitin oligosaccharide sequestration but may involve perturbation of host immune receptor complexes .", "Future studies should reveal whether , and how , perturbation of chitin-triggered immunity by LysM2 occurs in the interaction of C . fulvum with tomato .", "Convergent evolution towards recognition of the same ligand by molecules of taxonomically diverse organisms frequently occurs , as exemplified by the perception of bacterial flagellin by the mammalian and plant immune receptors TLR5 and FLS2 , although these receptors recognize different PAMP motifs within the same ligand ( Felix et al . , 1999; Smith et al . , 2003; Chinchilla et al . , 2006 ) .", "In contrast , our study on chitin perception by LysM proteins shows that convergent evolution has brought about different modes to interact with the same PAMP motif in taxonomically diverse organisms by structurally diverse proteins ( Figure 8 ) ." ], [ "The pGEM-T ( Invitrogen , Carlsbad , CA ) vector containing His6-FLAG tagged Ecp6 ( de Jonge et al . , 2010 ) was used to obtain the mutant constructs by PCR using overlapping primers ( T22R: forward TGACCGCCTCACCTCCATTG and reverse TGAGGCGGTCACCCTTGAC; N47K: forward CCCCAAACTCATCGAGCTCGGCGC and reverse CGATGAGTTTGGGGTTGGCGAG; T95R: forward GAACCCAAAGGCCATCGATGTTGG and reverse GTGAGACGGTCGCCGCTG; D121K: forward GAACCCAAAGGCCATCGATGTTGG and reverse CGATGGCCTTTGGGTTCTCGATCTG; L152R: forward GTGACCGTTTCGTCGATTTGG and reverse ACGAAACGGTCACCGGCC; S177K: AACCCAAAGAAGCTCAAGGTTGGTCAGC and reverse GAGCTTCTTTGGGTTAACGTTGTTG ) that contained the corresponding nucleotide mismatch , followed by digestion of the template DNA by DpnI .", "A pPIC9 vector ( Invitrogen ) containing His6-FLAG tagged Ecp6 was used to express Ecp6 in Pichia pastoris ( de Jonge et al . , 2010 ) .", "Purification was performed using a Ni2+-NTA Superflow column ( Qiagen , Valencia , CA ) .", "Chitin-free Ecp6 was produced deploying HEK293 cells ( Genscript , Piscataway , NJ ) .", "After codon optimization , a signal peptide sequence in Ecp6 gene construct , which already contained a His6 and Flag tag on the N-terminus , was added .", "The recombinant plasmids encoding Ecp6 and the T95R mutant were transiently transfected into 100 ml suspension of HEK293 cell cultures .", "The target protein was captured from the cell culture supernatant by HiTrap chelating HP 5 ml ( GE Healthcare , Milwaukee , WI ) followed by buffer exchange .", "The purified proteins were analyzed using SDS-PAGE and Western blot using the primary antibody Mouse-anti-His mAb .", "Quantification was performed based on the absorbance of the protein solution at 280 nm .", "Medium alkalinization experiments were performed as previously described ( Felix et al . , 1993; de Jonge et al . , 2010 ) .", "Briefly , 2 . 5 ml aliquots of a suspension of tomato cell line Msk8 in 12-well culture plates on a rotary shaker at 200 rpm were allowed to equilibrate for at least 2 hr .", "On addition of chitin oligosaccharides ( Isosep AB , Tullinge , Sweden ) , the pH of the medium was measured , and the maximum increase of the pH ( ΔpHmax ) that occurred within 3–5 min after application of chitin oligosaccharides was calculated .", "As previously noted by others as well , the maximum pH shift obtained after chitin stimulation varied little within an experiment when using the same batch of cells , but varied significantly between different experiments when using different batches of cells ( Felix et al . , 1998 ) .", "Consequently , the maximum pH shift varied between 0 . 06 and 0 . 14 for the different experiments .", "In each experiment , the ΔpHmax was normalized to a ( GlcNAc ) 6 control ( 10 nM ) .", "Prior to addition , mixtures of Ecp6 protein ( 100 nM ) and chitin oligosaccharides ( 10 nM ) were kept at room temperature for at least 10 min while shaking gently .", "All experiments were performed at least three times .", "P . pastoris-produced Ecp6 was further purified by gel filtration chromatography ( Superdex 75; GE Healthcare ) in 20 mM HEPES , pH 7 . 0 , and 50 mM NaCl .", "Large single crystals were only obtained by micro-seeding .", "To this end , intertwined plate-like crystals that grew overnight in the initial Ecp6 vapor-diffusion crystallization screening were harvested , smashed in stabilizing mother liquid , and used for micro-seeding ( Bergfors , 2003 ) , using a reservoir with 200 mM ammonium sulfate , 100 mM sodium acetate , pH 4 . 6 , and 20–30% PEG MME 2000 ( wt/vol ) .", "A SAD experimental dataset was obtained from an I3C ( 5-amino-2 , 4 , 6-triiodoisophthalic acid; Jena Biosciences , Jena , Germany ) soaked Ecp6 crystal at ESRF beamline ID29 ( Grenoble , France ) ( Gabadinho et al . , 2010 ) .", "A native high-resolution dataset was collected at BL14 . 1 of the BESSY II storage ring ( Berlin , Germany ) ( Mueller et al . , 2012 ) .", "Datasets were processed with MOSFLM ( Leslie and Powell , 2007 ) and XDS ( Kabsch , 2010 ) and scaled and merged using SCALA ( Evans , 1997 ) .", "Initial phases were calculated using PHENIX AUTOSOL ( Adams et al . , 2002 ) .", "Automatic model building failed because of the sequence homology between the individual LysM domains and because of the additional density for the ligand .", "Therefore , manual model building in COOT ( Emsley and Cowtan , 2004 ) was used instead .", "The structure model was finally refined to 1 . 6 Å resolution using REFMAC5 ( Vagin et al . , 2004 ) .", "The structure was validated using the wide range of tools offered by the program COOT .", "In addition , structures were validated by the RCSB Protein Data Bank ( PDB ) services as part of the Auto Deposition Input Tool ( ADIT ) process ( Berman et al . , 2000 ) .", "All structural figures were created with PyMOL ( DeLano Scientific/Schroedinger ) .", "Isothermal titration calorimetry ( ITC ) experiments were performed at 25°C following standard procedures using a Microcal VP-ITC calorimeter ( GE-Healthcare ) .", "P . pastoris-produced wild-type Ecp6 ( 30 µM ) and the mutants L152R ( 38 µM ) , S177K ( 30 µM ) , T22R ( 30 µM ) , N47K ( 20 µM ) , T95R ( 30 µM ) , and D121K ( 20 µM ) , containing a chitin tetramer in the LysM1-LysM3 groove , or 8 µM of chitin-free Ecp6 or T95R mutant , produced in HEK293 cells , were titrated with 1 injection of 1 µl , followed by 33 injections of 8 µl of ( GlcNAc ) 6 ( Isosep AB , Tullinge , Sweden ) at 400 µM for P . pastoris-produced Ecp6 , S177K , T22R , and N47K , 200 µM for P . pastoris-produced L152R , 800 µM for P . pastoris-produced T95R , or 200 µM for HEK293 produced-Ecp6 .", "Both ligand and protein were suspended in PBS , pH 7 . 2 .", "Data were analyzed using Origin ( OriginLab ) and fitted to the models describing one ( for P . pastoris-produced protein ) and two types ( for HEK293-produced Ecp6 ) of binding sites .", "Experiments were repeated three times with similar results .", "CD spectra were recorded on Jasco J-715 from 190 to 250 nm at 24°C using a 0 . 1 cm path length cell .", "Proteins were at a final concentration of 10 . 6 µM in water .", "Measurements were recorded at 1 nm wavelength increments at 100 nm/min by using a 1 nm bandwidth , 0 . 25 s response time .", "Final spectra are the average of four replicates .", "For structural data , see Protein Data Bank ID codes 4B8V and 4B9H ." ] ]

[ "While host immune receptors detect pathogen-associated molecular patterns to activate immunity , pathogens attempt to deregulate host immunity through secreted effectors .", "Fungi employ LysM effectors to prevent recognition of cell wall-derived chitin by host immune receptors , although the mechanism to compete for chitin binding remained unclear .", "Structural analysis of the LysM effector Ecp6 of the fungal tomato pathogen Cladosporium fulvum reveals a novel mechanism for chitin binding , mediated by intrachain LysM dimerization , leading to a chitin-binding groove that is deeply buried in the effector protein .", "This composite binding site involves two of the three LysMs of Ecp6 and mediates chitin binding with ultra-high ( pM ) affinity .", "Intriguingly , the remaining singular LysM domain of Ecp6 binds chitin with low micromolar affinity but can nevertheless still perturb chitin-triggered immunity .", "Conceivably , the perturbation by this LysM domain is not established through chitin sequestration but possibly through interference with the host immune receptor complex ." ]

[ "The ability to launch an immune response is not unique to animals .", "Plants have also evolved the ability to detect molecules present on the surface of pathogens such as fungi .", "These molecular signatures are known as pathogen-associated molecular patterns ( PAMPs ) , and they are detected by specialized receptors on the surface of plant cells .", "Chitin , the main structural component of the cell wall in fungi , is one example of a PAMP .", "Many species of plants are able to detect chitin using receptors that contain sequences of amino acids called lysin motifs .", "Previous work in the model plant Arabidopsis has shown that chitin binds to a single lysin motif within each plant receptor .", "However , just as plants have evolved the ability to recognize PAMPs , so fungi have evolved ways to outwit plants .", "They have developed small molecules called effector proteins that bind to PAMPs , in effect hiding them from the plant receptors .", "The tomato fungus Cladosporium fulvum , for example , secretes an effector protein called Ecp6 , which contains lysin motifs just like those in the plant receptors .", "By binding chitin fragments , Ecp6 helps the fungus to avoid detection by its host plant .", "Now , Sánchez-Vallet et al . present the high resolution crystal structure of Ecp6 and reveal the mechanism by which it outcompetes the plant’s own chitin receptors .", "In the presence of chitin , two lysin binding motifs within the Ecp6 protein combine to produce a binding site with ultrahigh affinity for chitin .", "This can outcompete the plant receptors , which use only a single lysin domain to bind the fungal protein .", "As well as providing a molecular explanation for how certain fungi manage to evade the immune response in plants , the work of Sánchez-Vallet et al . offers an unusual example of convergent evolution , in which two evolutionarily distant organisms have evolved the ability to recognize the same molecule through structurally diverse proteins ." ]

[ "Introduction", "Results and discussion", "Materials and methods" ]

[ "evolutionary biology", "short report", "neuroscience" ]

[ [ "The last common ancestor of mice and humans existed around 80 million years ago , sufficient time for divergence in signal-dependent gene regulation ( Villar et al . , 2014 ) .", "However , the conservation or divergence of dynamic signal-dependent programs of gene expression is incompletely understood , particularly in the nervous system .", "A fundamental transcriptional program of this sort is that elicited in neurons by electrical activity , triggered via Ca2+ influx through ligand- and/or voltage-gated Ca2+ channels and activating genes containing Ca2+-responsive transcription factor binding sites in their promoters ( Sheng and Greenberg , 1990; Morgan and Curran , 1991; West et al . , 2001 ) .", "These changes in gene expression are critical to a neuron’s functional response to electrical activity in development and maturity ( Konur and Ghosh , 2005; West and Greenberg , 2011; Bell and Hardingham , 2011 ) , and are totally distinct from the toxic sequelae of excitotoxic Ca2+ influx ( Hardingham and Bading , 2010; Wyllie et al . , 2013 ) .", "For example , rodent studies have shown that activity-dependent gene expression programs direct myriad processes in developing neurons , including neuroprotection , dendritic arborization , and synaptic plasticity ( Konur and Ghosh , 2005; West and Greenberg , 2011; Bell and Hardingham , 2011 ) .", "Moreover , certain neurodevelopmental disorders are associated with defects in activity-dependent transcriptional mechanisms ( West and Greenberg , 2011 ) , making it important to fully understand transcriptional programs that are triggered by electrical activity in developing human neurons .", "Comparing the influence of neuronal electrical activity on mouse-human orthologs , and identifying the basis for any differences , requires a combination of approaches , given the inability to reproducibly study primary human developing neurons .", "Embryonic stem cell ( ESC ) -based technology enables the generation of glutamatergic cortical-patterned neurons from human embryonic stem cells ( hESCCORT-neurons ) of sufficient homogeneity and electrical maturity ( Bilican et al . , 2014; Livesey et al . , 2014 ) to enable the study of activity-dependent gene expression .", "Such responses can then be compared to both primary mouse cortical neurons ( of differing developmental stages ) as well as those derived from mouse ESCs , to distinguish species-specific differences from those dependent on developmental stage or origin ( primary tissue vs . stem cell line ) .", "Moreover , it is in theory possible to study mouse-human ortholog regulation in the same neuron by exploiting the aneuploid Tc1 mouse which carries a freely segregating copy of human chromosome-21 , to identify whether any differences are independent of cellular environment ( i . e . are due to DNA sequence divergence ) .", "A combination of these approaches has been employed to reveal strong conservation of the neuronal activity-dependent transcriptome onto which a significant degree of divergence is overlaid ." ], [ "We generated dissociated glutamatergic cortical-patterned neurons from human embryonic stem cells ( hESCCORT-neurons ) , whose characterization is described fully elsewhere ( Bilican et al . , 2014; Livesey et al . , 2014 ) .", "These cells have a combination of homogeneity and electrical maturity that is hard to achieve with classical human stem cell-derived neurosphere-based preparations , which have a poor dynamic range in terms of gene regulation ( Paşca et al . , 2011 ) compared to primary mouse neuronal preparations ( Spiegel et al . , 2014 ) We studied transcriptional responses to L-type Ca2+ channel activation , an important mediator of activity-dependent gene regulation ( West and Greenberg , 2011; Sheng et al . , 1990; Bito et al . , 1997; Deisseroth et al . , 2003; Wheeler et al . , 2012; Ma et al . , 2014 ) .", "To do this , hESCCORT-neurons were subject to KCl-induced membrane depolarization in the presence of the L-type Ca2+ channel agonist FPL64176 ( KCl/FPL ) , which leads to robust and uniform Ca2+ responses ( [Bilican et al . , 2014] , Figure 1—figure supplement 1a , Figure 1—source data 1 ) similar to rodent neurons ( Hardingham et al . , 1999 ) .", "QPCR analysis revealed robust induction of classical early-response activity-regulated genes ( ARGs ) FOS , FOSL2 ( FRA-2 ) , and FOSB , and neurobiologically important ARGs BDNF , ARC , and NPAS4 ( Figure 1—figure supplement 1b , Figure 1—source data 3 ) whose regulation has hitherto been studied primarily in rodent neurons .", "We then performed RNA-seq analysis of KCl/FPL-induced gene expression changes in hESCCORT-neurons as well as days-in-vitro ( DIV ) 10 mouse primary cortical neurons ( Mus-PRIMCORT-neurons , predominantly excitatory , like Hum-ESCCORT-neurons ) , focusing on a 4h timepoint ( Figure 1a , b , Figure 1—source data 1 ) .", "QPCR validation of fold-induction of selected ARGs revealed a tight correlation with the RNA-seq data ( r > 0 . 99 for Hum-ESCCORT-neurons and mouse neurons respectively , Figure 1—figure supplement 1c , 1d , Figure 1—source data 4 ) , supporting the reliability of the RNA-seq data set .", "Our interspecies comparisons were restricted to 1:1 orthologous pairs whose average expression level met a minimum threshold ( 11 , 302 genes expressed >0 . 5 FPKM on average , in all cell types ) .", "Genes induced >5-fold in both Hum-ESCCORT- and DIV10 Mus-PRIMCORT-neurons according to RNA-seq included neurobiologically important genes such as PER1 , EGR2 , EGR4 and ATF3 ( Figure 1c ) .", "Global comparison of Log2 ( gene fold-change ) in orthologous pairs revealed a correlation between Hum-ESCCORT-neurons and DIV10 Mus-PRIMCORT-neurons ( r = 0 . 480 , 95% CI: 0 . 465 to 0 . 494 , p<0 . 0001 , Figure 1d , Figure 1—source data 1 ) , pointing to a significant , but incomplete , degree of conservation .", "For the 11 , 302 ortholog pairs , we calculated the 'differential regulation index' ( DRI ) , defined as the fold-change ( Hum-ESCCORT-neurons ) divided by the fold-change ( Mus-PRIMCORT-neurons ) .", "The Log2 of the DRIs are normally distributed about zero with a standard deviation of 0 . 68 ( Figure 1—figure supplement 1e , Figure 1—source data 5 ) . 10 . 7554/eLife . 20337 . 003Figure 1 . Conservation and divergence in gene regulation in neurons of human and mouse origin .", "( A , B )", "Analysis of gene expression changes induced by KCl/FPL in Hum-ESCCORT-neurons ( A ) and DIV10 Mus-PRIMCORT-neurons ( B ) .", "Normalised RNA-seq read density ( FPKM ) mapping to each gene in RNA extracted from control vs . KCl/FPL-treated neurons is shown ( n = 3 independent biological replicates ) .", "Genes whose expression was significantly altered by KCl/FPL treatment ( Benjamini-Hochberg-adjusted p-value<0 . 05 , calculated within DESeq2 ) are highlighted in red .", "( C ) Cohort of human:mouse orthologous pairs where both are strongly ( >5-fold ) and significantly ( Benjamini-Hochberg-adjusted p-value<0 . 05 ) induced in Hum-ESCCORT-neurons and DIV10 Mus-PRIMCORT-neurons respectively .", "( D ) Correlation of KCl/FPL-induced fold-change in 11 , 302 ortholog pairs in DIV10 Mus-PRIMCORT-neurons vs . DIV10-Hum-ESCCORT-neurons .", "( E ) Correlation of KCl/FPL-induced fold-change in the same 11 , 302 genes as in ( D ) in DIV10 vs . DIV4 Mus-PRIMCORT-neurons . DOI: http://dx . doi . org/10 . 7554/eLife . 20337 . 00310 . 7554/eLife . 20337 . 004Figure 1—source data 1 . Data set relating to Figure 1a–e . DOI: http://dx . doi . org/10 . 7554/eLife . 20337 . 00410 . 7554/eLife . 20337 . 005Figure 1—source data 2 . Data set relating to Figure 1—figure supplement 1a . DOI: http://dx . doi . org/10 . 7554/eLife . 20337 . 00510 . 7554/eLife . 20337 . 006Figure 1—source data 3 . Data set relating to Figure 1—figure supplement 1b . DOI: http://dx . doi . org/10 . 7554/eLife . 20337 . 00610 . 7554/eLife . 20337 . 007Figure 1—source data 4 . Data set relating to Figure 1—figure supplement 1c–d . DOI: http://dx . doi . org/10 . 7554/eLife . 20337 . 00710 . 7554/eLife . 20337 . 008Figure 1—source data 5 . Data set relating to Figure 1—figure supplement 1e–f . DOI: http://dx . doi . org/10 . 7554/eLife . 20337 . 00810 . 7554/eLife . 20337 . 009Figure 1—figure supplement 1 . Conservation and divergence in gene regulation in neurons of human and mouse origin .", "( A ) Example fluo-3 Ca2+ imaging trace of KCL/FPL-treated Hum-ESCCORT-neurons .", "( B ) qPCR analysis of the fold induction of the indicated genes in Hum-ESCCORT-neurons after 4h of KCl/FPL treatment .", "*p=0 . 0001 , 0 . 0013 , 6E-05 , 0 . 0005 , 0 . 0086 , 0 . 0010 , 2-tailed t-test ( n = 3 ) .", "( C , D )", "A comparison of the magnitude of gene induction of a selection of human ( C ) and mouse ( D ) genes , as assayed by RNA-seq and by qRT-PCR .", "In both cases identical sets of 3 independent replicates were assayed and the mean calculated .", "( E ) For the 11 , 302 orthologous pairs , the 'differential regulation index' ( DRI ) was calculated , defined as the fold-change ( Hum-ESCCORT-neurons ) divided by the fold-change ( DIV10 Mus-PRIMCORT-neurons ) .", "The distribution of the log2 of the DRIs is shown .", "( F ) As for ( E ) except the DRI was calculated for DIV4 vs . DIV10 Mus-PRIMCORT-neurons .", "( G ) Correlation of KCl/FPL-induced fold-change in 11 , 302 ortholog pairs in DIV4 Mus-PRIMCORT-neurons vs . Hum-ESCCORT-neurons . DOI: http://dx . doi . org/10 . 7554/eLife . 20337 . 009 While this imperfect conservation could involve species-specific gene responsiveness , it could be due to other factors:", "( i ) non-developmental equivalency in the human and mouse neuronal preparations combined with a dependency of the responsiveness of certain genes on developmental stage could underlie differences;", "( ii ) if gene-responsiveness were sensitive to the origin of the neurons studied ( embryonic stem cell line ( human ) vs . primary tissue ( mouse ) ) this could also lead to differences , and an erroneous exaggeration of apparent evolutionary divergence .", "To address", "( i ) we compared KCl/FPL-induced gene regulation in DIV10 Mus-PRIMCORT-neurons with that in synaptically immature DIV4 Mus-PRIMCORT-neurons , which revealed a strong correlation ( p<0 . 0001 , r = 0 . 834 , 95% CI: 0 . 833 to 0 . 844 , Figure 1e , Figure 1—source data 1 ) , and a log2 DRI ( DIV4:DIV10 ) distribution curve ( Figure 1—figure supplement 1f , Figure 1—source data 5 ) substantially narrower than that observed in Figure 1—figure supplement 1e .", "Taken together , this suggests that developmental stage does not have a large effect on the activity-responsiveness of genes at this time point and is unlikely to account for the human-mouse differences observed in Figure 1d .", "To address", "( ii ) ( i . e . whether stem cell- vs . primary tissue origin causes differential gene responsiveness ) we generated cortical-patterned neurons from mouse embryonic stem cells ( Mus-ESCCORT-neurons ) using established protocols ( Gaspard et al . , 2008 ) , which exhibit spontaneous firing and synaptic activity ( Figure 2a–d ) , similar to our Hum-ESCCORT-neurons ( Bilican et al . , 2014; Livesey et al . , 2014 ) .", "The Mus-ESCCORT-neurons were subject to the same 4h KCl/FPL stimulation and RNA-seq analysis ( Figure 2—figure supplement 1a ) .", "Same-species comparison of gene fold-change was made between Mus-PRIMCORT-neurons ( DIV4 and DIV10 ) and Mus-ESCCORT-neurons in the 11 , 302 genes compared in Figure 1e , f .", "A strong correlation was observed in gene-responsiveness in Mus-ESCCORT-neurons vs . Mus-PRIMCORT-neurons at either DIV10 or DIV4 ( p<0 . 0001 , r = 0 . 748 ( DIV10 ) , Figure 2e; r = 0 . 788 , ( DIV4 ) , Figure 2f ) , significantly stronger than between Mus-ESCCORT- and Hum-ESCCORT-neurons ( r = 0 . 596 , Figure 2g , see also Figure 2—source data 1 ) .", "Indeed , a comparison of gene fold-change between all biological replicates in all data sets revealed that all mouse neuronal preparations ( primary DIV10 , primary DIV4 , and ESC-derived ) correlated far more strongly with each other than with the human ESCCORT-neurons .", "( Figure 2h ) .", "This suggests that the weaker correlation with the hESCCORT-neurons may indeed be due to some species-specific differences in neuronal gene activity-responsiveness , rather than differences in developmental stage or cellular origin . 10 . 7554/eLife . 20337 . 010Figure 2 . Stem cell origin does not substantially impact on activity-dependent gene responsiveness .", "( A ) Example immunofluorescence pictures of Mus-ESCCORT-neurons stained for neuronal markers Tuj1 ( upper ) and Reelin ( lower ) .", "Note also absence of Nestin staining ( upper ) , a marker of undifferentiated neural precursor cells .", "Scale = 20 µm .", "( B ) Example trace of a burst of action potentials ( APs ) induced in Mus-ESCCORT-neurons by current injection ( see Materials and methods ) .", "( C ) Example trace illustrating spontaneous TTX-sensitive AP firing .", "( D ) Example trace illustrating spontaneous TTX-sensitive EPSCs , as well as TTX-insensitive , CNQX-sensitive miniature EPSCs ( also see inset; scale bar: 20 pA , 5 ms ) .", "Activity returned upon wash out of TTX and CNQX ( not shown ) .", "( E , F )", "Correlation of KCl/FPL-induced fold-change in the same 11 , 302 genes as in Figure 1d , e in Mus-ESCCORT-neurons vs . DIV10 ( E ) or DIV4 ( F ) Mus-PRIMCORT-neurons .", "( G ) Correlation of KCl/FPL-induced fold-change in 11 , 302 ortholog pairs in Mus-ESCCORT-neurons vs . Hum-ESCCORT-neurons .", "( H ) A connection map generated in Cytoscape ( Shannon et al . , 2003 ) illustrating the relative determination coefficients ( R2 ) between the fold-inductions of the 11 , 302 genes studied in each of the three biological replicates of the experiments performed in each of the four different neuronal preparations .", "The thickness of the connecting line and the attractive force of the connecting nodes are both directly proportional to R2 , against a background of constant inter-node repulsion .", "Note that all mouse neurons of differing developmental stage and origin ( primary vs . ES cell ) cluster strongly together , with the Hum-ESCCORT-neuronal replicates clustering away from them .", "( I ) A connection map generated as for ( H ) but illustrating the relative determination coefficients ( R2 ) between the basal expression levels ( FPKM ) of the 11 , 302 genes studied in each of the three biological replicates of the experiments performed in each of the four different neuronal preparations . DOI: http://dx . doi . org/10 . 7554/eLife . 20337 . 01010 . 7554/eLife . 20337 . 011Figure 2—source data 1 . Data set relating to Figure 2e–g . DOI: http://dx . doi . org/10 . 7554/eLife . 20337 . 01110 . 7554/eLife . 20337 . 012Figure 2—source data 2 . Data set relating to Figure 2—figure supplement 1e . DOI: http://dx . doi . org/10 . 7554/eLife . 20337 . 01210 . 7554/eLife . 20337 . 013Figure 2—source data 3 . Data set relating to Figure 2—figure supplement 1b–d . DOI: http://dx . doi . org/10 . 7554/eLife . 20337 . 01310 . 7554/eLife . 20337 . 014Figure 2—figure supplement 1 . Differential gene inducibility is not linked to basal levels of gene expression .", "( A ) Normalised RNA-seq read density ( FPKM ) mapping to each gene in RNA extracted from control vs . KCl/FPL-treated Mus-ESCCORT-neurons is shown ( n = 3 independent biological replicates ) .", "Genes whose expression was significantly altered by KCl/FPL treatment ( Benjamini-Hochberg-adjusted p-value<0 . 05 , calculated within DESeq2 ) are highlighted in red .", "B-D ) Correlation of basal gene expression ( Log2 ( FPKM ) ) across 11 , 302 ortholog pairs in Hum-ESCCORT-neurons vs . DIV4 Mus-PRIMCORT-neurons ( B ) , DIV4 Mus-PRIMCORT-neurons ( C ) and Mus-ESCCORT-neurons ( D ) .", "( E ) For each of the 11 , 302 orthologous pairs , the Log2 ( DRI ) Hum-ESCCORT-vs .", "DIV10 Mus-PRIMCORT-neurons ( i . e . DRIs from Figure 1—figure supplement 1e ) were plotted against the Log2 ( DBEI ) , where DBI ( differential basal expression index ) is the ratio of basal expression in Hum-ESCCORT-vs .", "DIV10 Mus-PRIMCORT-neurons . DOI: http://dx . doi . org/10 . 7554/eLife . 20337 . 014 As a final genome-wide comparison we wanted to determine whether activity-dependent gene responsiveness showed a higher or lower degree of divergence than basal gene expression levels , and whether differences in basal gene expression were any predictor of differential activity-dependent gene responsiveness .", "Comparison of basal expression levels between Hum-ESCCORT-neurons and mouse neurons ( DIV10 Mus-PRIMCORT-neurons , DIV4 Mus-PRIMCORT-neurons , and Mus-ESCCORT-neurons ) revealed correlation coefficients of 0 . 714 , 0 . 711 , and 0 . 710 ( Figure 2—figure supplement 1b–d , Figure 2—source data 3 ) .", "This correlation is substantially stronger than the that observed when comparing gene fold-change after KCl stimulation ( 0 . 480 , 0 . 526 , 0 . 595 , Figures 1d , 2g , Figure 1—figure supplement 1g ) .", "Moreover , we performed a similar clustering analysis as in Figure 2h which illustrates this graphically: i . e . while the basal expression profile of Hum-ESCCORT-neurons does not cluster as closely as the three mouse neuronal populations do to each other , the degree of difference is less than that observed in Figure 2h ( Figure 2i ) .", "Therefore basal neuronal gene expression shows less divergence than the responsiveness of genes to depolarization .", "We also investigated whether gene differential responsiveness to KCl ( DRI ) in human vs . mouse neurons has any relationship with the relative basal expression of that gene in human vs . mouse neurons .", "For each of the 11 , 302 orthologous pairs , we plotted the Log2 ( DRI ) Hum-ESCCORT- vs . DIV10 Mus-PRIMCORT-neurons ( i . e . DRIs from Figure 1—figure supplement 1e ) against the Log2 ( DBEI ) , where DBEI ( differential basal expression index ) is the ratio of basal expression in Hum-ESCCORT- vs . DIV10 Mus-PRIMCORT-neurons ( Figure 2—figure supplement 1e , Figure 2—source data 2 ) .", "As can be seen , there is no link between a gene's relative responsiveness to depolarization in human vs . mouse neurons , and its relative basal expression levels in human vs . mouse neurons .", "Moreover , if we consider the 657 genes where Log2 ( DRI ) >1 , the standard deviation of their respective Log2 ( DBEI ) , 1 . 45 , is similar to the standard deviation of Log2 ( DBEI ) across all 11 , 302 genes ( 1 . 39 ) , suggesting that there is no dramatic change in divergence of basal gene expression regardless of direction , in genes where DRI>1 .", "Thus , evolutionary differences in activity-dependent gene responsiveness are not substantially attributable to differences in basal expression .", "We hypothesized that species-specific gene-responsiveness to neuronal activity could be in part due to evolutionary divergence in genetic sequence , such as the promoters and distal enhancers that mediate activity-dependent changes in gene expression ( West and Greenberg , 2011; Kim et al . , 2010 ) .", "To investigate whether genetic sequence could be sufficient to quantitatively influence species-specific gene inducibility , we utilized neurons cultured from the Tc1 transchromosomic mouse strain which carries a copy of human chromosome 21 ( Hsa21 ) , albeit with approximately 10% deleted ( O'Doherty et al . , 2005; Gribble et al . , 2013 ) .", "Thus , regulation of Hsa21 genes , plus their mouse 1:1 ortholog , could be studied side-by-side in the same cellular environment of a mouse primary neuron .", "We cultured cortical neurons from Tc1 embryos to DIV10 , stimulated ± KCL/FPL , performed RNA-seq , and developed a workflow that distinguished between human and mouse RNA-seq reads , discarding any reads that were ambiguous .", "Once the initial criterion of a perfect sequence match of the RNA-seq read to either Hsa21 or the mouse genome was met , only a further 0 . 055% of reads were discarded due to 100% sequence conservation between species .", "Thus , the expression level of human and mouse genes carried in the Tc1 mouse can be analysed ± stimulation ( Figure 3a ) .", "We first verified that the mouse genes in Tc1 neurons responded similarly to wild-type mouse neurons of the same age ( Figure 3—figure supplement 1a , Figure 3—source data 1 , r = 0 . 900 ) . 10 . 7554/eLife . 20337 . 015Figure 3 . DNA sequence is a contributing factor to species-dependent gene responsiveness to neuronal activity .", "( A ) Analysis of gene expression changes induced by KCl/FPL in mouse Tc1 neurons .", "Mouse genes are shown in grey , human chromosome-21 ( Hsa-21 ) genes in red .", "( B ) The graph concerns 72 orthologous pairs whose human ortholog is on Hsa-21 and carried by the Tc1 mouse strain , and which meets expression cut-off ( 100 reads ) .", "For each pair , the differential responsiveness index ( DRI ) was calculated from the species-separated Tc1 neuron RNA-seq data ( see text ) , and plotted against the DRI calculated from separate neuronal preparations ( Hum-ESCCORT-neurons vs . DIV10 Mus-PRIMCORT-neurons , as was done in Figure 1—figure supplement 1e ) .", "( C ) KCl/FPL-induced fold induction of the human ( red ) and mouse ( blue ) orthologs of Hsa-21 gene ETS2 , analysed side-by-side in Tc1 neurons .", "*p=0 . 035 .", "unpaired t-test; # indicates p=0 . 0464 , 0 . 0026 ( left to right ) vs . unstimulated control ( 4 hr , n = 7; 24 hr , n = 3 ) .", "( D ) Kinetics of KCl/FPL-induced ETS2 induction in Hum-ESCCORT-neurons vs . DIV10 Mus-PRIMCORT-neurons .", "*p=0 . 0017 , 0 . 004 ( left to right ) , unpaired 1-way ANOVA vs . control ( Hum-ESCCORT-neurons: n = 3 ( 2 hr , 8 hr ) , n = 6 ( 4h ) ; DIV10 Mus-PRIMCORT-neurons: n = 4 ( E ) The indicated firefly luciferase reporters based on human ( Hsa ) or mouse ( Mmu ) ETS2 promoters ( See ( Right ) for schematic ) plus a pTK-renilla control were transfected into mouse cortical neurons , treated ± KCl/FPL after which firefly:renilla luciferase ratio was measured , and fold-induction calculated .", "For comparing the effect of TAM67 , a control vector ( encoding β-globin ) was used .", "*p=0 . 0015 , 0 . 0015 , 0 . 0015 , 2-tailed paired t-test ( n = 3 ) .", "( F ) Kinetics of gene regulation of 6 human:mouse ortholog pairs that show quantitative differences in activity-dependent regulation in Hum-ESCCORT-neurons vs . all mouse neuronal preparations from prior RNA-seq analyses .", "Analysis was performed in Hum-ESCCORT-neurons vs . DIV10 Mus-PRIMCORT-neurons ( n = 3 , 4 respectively ) .", "At the timepoints studied , three of these pairs exhibit quantitatively higher induction in human neurons , and three stronger repression in human neurons .", "*p=<0 . 0001 , 0 . 0004 , 0 . 0005 , <0 . 0001 , <0 . 0001 , 0 . 0073 ( top to bottom , 2-way ANOVA , p-value corresponds to the main species effect ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 20337 . 01510 . 7554/eLife . 20337 . 016Figure 3—source data 1 . Data set relating to Figure 3—figure supplement 1a . DOI: http://dx . doi . org/10 . 7554/eLife . 20337 . 01610 . 7554/eLife . 20337 . 017Figure 3—source data 2 . Data set relating to Figure 3b . DOI: http://dx . doi . org/10 . 7554/eLife . 20337 . 01710 . 7554/eLife . 20337 . 018Figure 3—source data 3 . Data set relating to Figure 3c–f . DOI: http://dx . doi . org/10 . 7554/eLife . 20337 . 01810 . 7554/eLife . 20337 . 019Figure 3—figure supplement 1 . Inducibility of mouse genes in wild-type and Tc1 neurons strongly correlate .", "( A ) Correlation of KCl/FPL-induced fold-change in the same 11 , 302 mouse genes analysed in Figures 1 and 2 , in DIV10 Mus-ESCCORT-neurons from wild-type vs . Tc1 primary cortical neurons , showing the expected high correlation . DOI: http://dx . doi . org/10 . 7554/eLife . 20337 . 019 72 orthologous pairs were then focussed on , where the Hsa21 ortholog met an expression cut-off of >100 reads .", "For each pair , we calculated the 'Tc1 DRI' ( fold-change ( human ortholog ) /fold-change ( mouse ortholog ) ) as a measure of whether responsiveness of the orthologs was different or similar , and plotted it against the gene’s DRI calculated from fold-induction in separate human/mouse neuronal preparations: fold-change ( Hum-ESCCORT-neurons ) / fold-change ( DIV10 Mus-PRIMCORT-neurons ) .", "Importantly , we observed a significant correlation between these DRIs ( p<0 . 0001 , r = 0 . 730 , Figure 3b , Figure 3—source data 2 ) , meaning that differences in gene regulation observed in separate human and mouse cortical neuron preparations ( 'separate DRI' ) are broadly recapitulated when the orthologs are studied side-by-side in the same cellular environment ( 'Tc1 DRI' ) .", "This points to the DNA sequence as a contributor to quantitative species-specific gene responsiveness .", "An example of such a gene is ETS2 , the human ortholog of which is induced more strongly and rapidly than the mouse ortholog ( Figure 3c , d , Figure 3—source data 3 ) .", "Interestingly , a region of its proximal promoter contains three human-specific AP-1 sites ( Figure 3e , right ) and was found to confer activity inducibility on a luciferase reporter , unlike the corresponding region of the mouse Ets2 promoter ( Figure 3e , left ) .", "Moreover , expression of inhibitory AP-1 mutant TAM67 ( Brown et al . , 1993 ) inhibited Hum-ETS2-luciferase reporter induction , and mutation of the three AP-1 sites to the corresponding mouse sequence abolished inducibility ( Figure 3e ) .", "Thus , the ETS2 proximal promoter contains functional human-specific AP-1 sites , potentially contributing to the stronger and more rapid induction of the human ortholog in neurons Although the focus of this study is understanding the extent and causes of differences in gene activity-responsiveness in human vs . mouse neurons , it should be noted that qualitative gene up/down regulation is well conserved .", "For example , of the top 100 most strongly induced human genes in Hum-ESCCORT-neurons ( FC>3 . 5-fold , Padj<0 . 05 ) , 85 are also induced in Mus-ESCCORT-neurons ( FC>1 . 5-fold , Padj<0 . 05 ) .", "Of the remaining 15 genes , 6 are induced in DIV4 and/or DIV10 Mus-PRIMCORT-neurons , taking the total to 91/100 .", "Conserved inducibility remains high even at a lower fold-change cut-off: e . g . 409 out of 475 genes induced >2-fold ( Padj<0 . 05 ) in Hum-ESCCORT-neurons are also induced ( FC>1 . 5-fold , Padj<0 . 05 ) in one or more of the mouse neuronal preparations .", "Although we confirmed examples of genes regulated both up and down at 4h in Hum-ESCCORT-neurons but not in mouse neurons , by performing a timecourse in Hum-ESCCORT- and DIV10-Mus-PRIMCORT neurons ( Figure 3f ) , we hesitate to label these as definitively human-specific activity-response genes: there may be time-points , stimulation paradigms , or developmental stages that do expose activity-dependency on mouse neurons .", "Nevertheless , our study points to quantitative differences in activity-dependent gene-responsiveness in developing human neurons , compared to their mouse counterpart , mediated at least in part by DNA sequence divergence .", "It is possible that these differences have a functional impact on the physiological response of human neurons to electrical activity .", "More generally , given that diverse signal dependent transcriptional programs mediate cellular responses to physiological stimuli , as well as the effects ( and side-effects ) of pharmaceutical agents ( Bell and Hardingham , 2011; Gupta et al . , 2012; Bell et al . , 2011; Hardingham and Lipton , 2011; Baxter et al . , 2011 ) , this study points to the value of employing human neurons as a tool to help bridge the translational gap between rodent studies , and human neurophysiology ." ], [ "A detailed description of the derivation of hESCCORT-neurons , including immunohistochemical validation of cortical markers as well as electrophysiological characterization can be found in recent studies from the authors ( Bilican et al . , 2014; Livesey et al . , 2014 ) .", "Briefly , the hESC line H9 was used , and obtained from WiCell ( Madison , WI ) under full ethical / IRB approval of the University of Edinburgh .", "hESCs were maintained on CF-1 irradiated mouse embryonic fibroblasts , neurally converted to anterior-patterned neural precursor cells ( NPCs ) in suspension in chemically defined medium as described ( Stacpoole et al . , 2011 ) , whereupon neural rosettes were mechanically isolated , dissociated and plated for proliferative expansion .", "For differentiation of anterior NPCs , they were plated in default media ( A-DMEM/F12 , 1% P/S , 0 . 5% Glutamax , 0 . 5% N2 , 0 . 2% B27 , 2 μg/mL Heparin ( Sigma ) ) on poly-D-lysine ( Sigma ) , laminin ( Sigma ) , fibronectin ( Sigma ) and matrigel coated coverslips for differentiation and fed twice a week .", "Default media was supplemented with 10 μM forskolin ( Tocris ) in weeks 2 and 3 .", "From week 4 onwards forskolin was removed and default media was supplemented with 5 ng/mL BDNF and 5 ng/mL GDNF .", "Experiments were performed 5 weeks after the differentiation of anterior NPCs was commenced .", "Mouse ES cells ( E14tg2α ) were maintained in GMEM ( Sigma ) supplemented with 2-mercaptoethanol ( Gibco ) , non-essential amino acids ( Gibco ) , glutamine , pyruvate , 10% foetal calf serum ( Gibco ) and 100 units/ml LIF ( made in-house ) on gelatinised tissue culture flasks .", "Monolayer neural differentiation is described elsewhere ( Pollard et al . , 2006 ) and modified here to include cyclopamine .", "Briefly , ES cells were washed to remove all traces of serum and then plated at a density of 1 × 104 cells/cm2 onto gelatin-coated tissue culture plastic in N2B27 serum-free medium .", "N2B27 consists of a 1:1 ratio of DMEM/F12 and Neurobasal media ( Gibco ) supplemented with 0 . 5% modified N2 ( made in house as described ( Pollard et al . , 2006 ) , 1% B27 ( Gibco ) , 2- mercaptoethanol ( Gibco ) , 2 mM L-Glutamine , and 1uM Cyclopamine ( Sigma ) .", "Medium was changed every day .", "Cells were cultured for 9 days under these conditions before transferring to terminal differentiation conditions .", "Terminal differentiation of mouse NPCs into dorsal forebrain ( cortical ) neurons was achieved as described elsewhere ( Gaspard et al . , 2008 ) by withdrawal of cyclopamine from day 9 NPCs dissociated into single cells , with Accutase ( Life technologies ) , and re-plated on Matrigel ( SLS , 354230; 1 in 100 dilution ) , Fibronectin 20 mg/ml ( F2006 – Sigma-Aldrich ) , Laminin 10 mg/ml ( L2020 – Sigma-Aldrich ) coated coverslips at a density of 30 , 000 cells per 0 . 3 cm2 .", "Addition of 5 µM AraC on day 10 , for 24 hr , was used to remove residual proliferating cells .", "Differentiating cortical neurons were maintained for a further 12–14 days , with media changes every 2–3 days , in Advanced DMEM/F12 ( Invitrogen ) containing: 1% N-2 supplement ( Invitrogen ) , 1% B27 supplement ( Invitrogen ) , 1% penicillin/streptomycin ( Invitrogen ) , 0 . 5% GlutaMAX ( Invitrogen ) , and 5 mg/ml heparin ( Sigma ) , supplemented with 1 mM N-acetyl cysteine ( Sigma ) and 10 ng/ml BDNF ( R&D Systems ) and finally processed for RNA-seq and electrophysiological studies .", "For cell culture IHC , cells were fixed in 4% formaldehyde for 20 min at room temperature , washed with PBS and permeabilized with the detergent NP40 ( Life Technologies ) .", "Cells were subsequently incubated in primary antibodies over night at 4°C .", "The next day , cells were washed with PBS and incubated with the appropriate secondary antibody at room temperature for 2 hr .", "Cells were then mounted using the mounting medium Vectashield ( Vector Labs ) .", "Cortical mouse neurons were cultured from either wild-type CD1 mice or Tc1 mice described ( Martel et al . , 2012 ) at a density of between 9–13 × 104 neurons per cm2 from E17 . 5 mice with Neurobasal growth medium supplemented with B27 ( Invitrogen , Paisley , UK ) .", "Stimulations of cultured neurons were done after a culturing period of 9–11 days during which neurons develop a network of processes , express functional NMDA-type and AMPA/kainate-type glutamate receptors , and form synaptic contacts .", "For Tc1 mouse genotyping , DNA was isolated from cerebellum using Qiagen QIAamp DNA Mini Kit following manufacturer’s instructions .", "Genotyping reactions were performed using the following primers: D21S55 Forward =5’-GGT TTG AGG GAA CAC AAA GCT TAA CTC CCA-3’ , reverse =5’-ACA GAG CTA CAG CCT CTG ACA CTA TGA ACT-3’; Myo Forward =5’-TTA CGT CCA TCG TGG TGG ACA GCAT-3’ , reverse =5’- TGG GCT GGG TGT TAG TCT TAT-3’ .", "D21S55 recognizes the Tc1 allele ( product =208 bp ) whereas Myo recognizes both Tc1 and WT alleles ( product =245 bp ) .", "Prior to stimulation , neurons were first placed overnight into a minimal defined medium ( Papadia et al . , 2005 ) containing 10% MEM ( Invitrogen ) , 90% Salt-Glucose-Glycine ( SGG ) medium ( [Bading et al . , 1993]; SGG: 114 mM NaCl , 0 . 219% NaHCO3 , 5 . 292 mM KCl , 1 mM MgCl2 , 2 mM CaCl2 , 10 mM HEPES , 1 mM Glycine , 30 mM Glucose , 0 . 5 mM sodium pyruvate , 0 . 1% Phenol Red; osmolarity 325 mosm/l , [Papadia et al . , 2005] ) .", "KCl/FPL stimulations were performed as described previously ( Hardingham et al . , 1999 , 1997 ) .", "KCl/FPL stimulation involved neurons being exposed to 50 mM KCl by adding 0 . 41 volumes of KCl depolarization solution ( Bading et al . , 1993 ) ( 10 mM HEPES , pH 7 . 2 , 170 mM KCl , 1 mM MgCl2 , 2 mM CaCl2 ) in the presence of 5 µM FPL64176 ( Tocris ) , plus an NMDA receptor antagonist ( MK-801 , 5 µM ) to prevent any excitotoxicity .", "As described recently ( Bilican et al . , 2014 ) , hESCCORT-neurons respond strongly and uniformly to KCl/FPL treatment , with over 80% of hESCCORT-neurons exhibiting a >10-fold increase in [Ca2+] , and the remainder showing at least a 5-fold increase .", "Ca2+ imaging was performed as described ( Hardingham et al . , 1997; Soriano et al . , 2008 ) at 37ºC in aCSF ( 150 mM NaCl , 3 mM KCl , 10 mM HEPES , 2 mM CaCl2 , 1 mM MgCl2 , 1 mM glucose ) .", "Briefly , cells were loaded with 11 µM Fluo-3 AM ( from a stock solution of 2 . 2 mM Fluo-3 dissolved in anhydrous DMSO containing 20% ( w/v ) Pluronic detergent ) for 30 min at 37°C .", "Fluo-3 fluorescence images ( excitation 472 ± 15 nm , emission 520 ± 15 nm ) were taken at one frame per 5 s using a Leica AF6000 LX imaging system , with a DFC350 FX digital camera .", "To calibrate images , Fluo-3 was saturated by adding 50 μM ionomycin to the perfusion chamber ( to obtain Fmax ) and quenched with 10 mM MnCl2 + 50 μM ionomycin to levels corresponding to 100 nM Ca2+ ( Minta et al . , 1989 ) , which was in turn used to calculate Fmin .", "Free Ca2+ concentrations were calculated from fluorescence signal ( F ) according to the equation [Ca2+] = Kd ( F – Fmin ) / ( Fmax – F ) , and expressed as a multiple of the Kd of Fluo-3 ( which is approximately 315 nM ) .", "The whole-cell patch configuration was used to record membrane currents and voltage deflections as previously described ( Bilican et al . , 2014; James et al . , 2014 ) .", "Membrane potential data are corrected for liquid junction potential ( +14 mV ) .", "Current and voltage measurements were typically low-pass filtered online at 2 kHz , digitized at 10 kHz and recorded to computer using the WinEDR V2 7 . 6 Electrophysiology Data Recorder ( J . Dempster , Department of Physiology and Pharmacology , University of Strathclyde , UK ) .", "Neurons were treated with KCl/FPL as described above .", "Cells were lysed and RNA extracted at 4h post-stimulation .", "For both mouse and human-based experiments , three independent biological replicates were performed , and analysed in parallel .", "Three replicates were deemed sufficient based on previous transcriptome studies on activity-dependent gene expression in which we successfully identified differentially regulated genes with this number of replicates ( Papadia et al . , 2008 ) .", "Total RNA was assessed for quality ( Agilent Bionalyzer ) and quantity ( Invitrogen Qubit ) before library preparation .", "Illumina libraries were prepared from 1 µg of total RNA using TruSeq RNA Sample Prep Kit v2 with a 10 cycle enrichment step as per the manufacturer's recommendations .", "Final libraries were pooled in equimolar proportions before Illumina sequencing on a HiSeq 2500 platform using 75 base paired-end reads .", "Raw reads were processed using RTA 1 . 17 . 21 . 3 and Casava 1 . 8 . 2 ( Illumina ) .", "Reads were mapped to the mouse ( mm10 ) and human ( hg38 ) reference genomes using version 2 . 4 . 0i of the STAR RNA-seq aligner ( Dobin et al . , 2013 ) .", "A table of per-gene read counts was generated from the mapped reads with featureCounts version 1 . 4 . 6-p2 ( Liao et al . , 2014 ) , using gene annotations from Ensembl version 82 .", "Differential expression analysis was then performed using DESeq2 ( R package version 1 . 10 . 0 ) ( Love et al . , 2014 ) .", "Raw data will be deposited in the European Nucleotide Archive in advance of publication .", "A region of the human ETS2 promoter ( NM_001256295 -1080 to -19 , or NM_005239 . 5 , positions -1622 to -542 ) was generated by PCR amplification using Stratagene UltraII Fusion HS DNA Polymerase kit and subcloned into pGL4 . 10 .", "All other promoter sequences ( both wild-type and mutant ) were obtained as synthetic clones from Life Technologies ( Geneart ) and then subcloned into pGL4 . 10 .", "This included the corresponding mouse Ets2 promoter sequence ( ENSMUST00000023612 -1055 to -65 , or NM_011809 . 3 -1387 to -397 ) and a mutated form of the human ETS2 promoter with the putative AP-1 sites mutated to the corresponding mouse sequence ( ETS2 ( ΔAP1 ) ) was synthesized .", "The following plasmids have been described previously: pcDNA3-cJun ( 3–122 ) ( TAM-67 ) ( Brown et al . , 1993 ) .", "Firefly luciferase-based reporter gene constructs were transfected ( Lipofectamine 2000 ) into DIV8 mouse cortical neurons as along with a Renilla expression vector ( pTK-RL ) control .", "The number of biological replicates were performed based on previous studies where site-directed mutagenesis was performed on reporter constructs to assess activity-dependent promoter regulation ( Papadia et al . , 2008 ) .", "Neurons were stimulated with KCl/FPL ( where appropriate ) 24 hr after transfection .", "Luciferase assays were performed using the Dual Glo assay kit ( Promega ) with Firefly luciferase-based reporter gene activity normalized to the Renilla control ( pTK-RL plasmid ) in all cases .", "For RNA-seq and qPCR , RNA was isolated using the Roche HP RNA Isolation kit according to manufacturer’s instructions .", "For qPCR , cDNA was synthesized from 1– 3 µg RNA using Roche Transcriptor cDNA Synth kit .", "Briefly , RNA was diluted in RNase-free water and mixed on ice with 1 µl oligo-dT primers ( 50 pmol/µl ) , 2 µl random hexamers ( 600 pmol/µl ) , 4 µl Transcriptor RT Reaction Buffer ( 5x ) , 0 . 5 µl Protector RNase Inhibitor ( 40 U/µl ) , 2 µl Deoxynucleotide Mix ( 10 mM each ) , 0 . 5 µl Transcriptor Reverse Transcriptase and made up to 20 µl with RNAase-free water .", "Reaction mixtures were incubated for 2 min at 25°C , 40 min at 42°C and 5 min at 95°C .", "Resultant cDNA was stored at −20°C .", "qPCR was performed in an Mx3000P QPCR System ( Stratagene ) using Roche FS Universal SYBR Green MasterRox .", "Briefly , cDNA ( equivalent to 6 ng of starting RNA ) was mixed with concentrated master mix ( 2x ) and appropriate forward and reverse primers ( 20 nM ) and made up to a final volume of 15 µl with RNase-free water .", "Technical replicates as well as no template and no RT controls were included in each run .", "Gene expression levels were normalized in all cases to Gapdh housekeeping control .", "Primer sequences used are as follows: PrimerSequenceHuman-ARCF: 5’-CTGAGCCACCTAGAGGAGTACT-3’ .", "R: 5’-AACTCCACCCAGTTCTTCACGG-3’Mouse-ArcF: 5’-GCTGGAAGAAGTCCATCAAGGC-3’ .", "R: 5’-ACCTCTCCAGACGGTAGAAGAC-3’Human-BDNFF: 5’-AGCTGAGCGTGTGTGACAGT-3’ .", "R: 5’-ATGGGATTGCACTTGGTCTC-3’Mouse-BdnfF: 5’-AAAGTCCCGGTATCCAAAGG-3’ .", "R: 5’-CTTATGAATCGCCAGCCAAT-3’Human-CCNHF: 5’-CGATGTCATTCTGCTGAGCTTGC-3’ .", "R: 5’-TCTACCAGGTCGTCATCAGTCC-3’Mouse-CcnhF: 5’-ACTTGCCTGTCACAGTTACTGGA-3’ .", "R: 5’-GAATGACACCGCTCCAGCTTCT-3’Human-ETS2F: 5’-CAACTCCTTTTCAGAGATCAGG-3’ .", "R: 5’-TTTCATCAAGACCCCTACCG-3’Mouse-Ets2F: 5’-TCACGTAAAGGGAGATGTGTCG-3’ .", "R: 5’-TGCTCTGTCTGTGCTTCTGG-3’Human-FOSF: 5’-CTACCACTCACCCGCAGACT-3’ .", "R: 5’-AGGTCCGTGCAGAAGTCCT-3’Mouse-FosF: 5’-CCATGATGTTCTCGGGTTTC-3’ .", "R: 5’-TGGCACTAGAGACGGACAGA-3’Human-FOSBF: 5’-GCCGGGAACGAAATAAACTA-3’ .", "R: 5’-CACCAGCACAAACTCCAGAC-3’Mouse-FosbF: 5’-AGGGAGCTGACAGATCGACTT-3’ .", "R: 5’-CTTCGTAGGGGATCTTGCAG-3’Human-FOSL2F: 5’-ACACCCTGTTTCCTCTCCG-3’ .", "R: 5’-GATGGTGGGGATGAATGCAC-3’Mouse-Fosl2F: 5’-CGGGAACTTTGACACCTCGT-3’ .", "R: 5’-AGGGATGTGAGCGTGGATAG-3’Human-GAPDHF: 5’-CTTCACCACCATGGAGAAGGC-3’ .", "R: 5’-GGCATGGACTGTGGTCATGAG-3’Mouse-GapdhF: 5’-GGGTGTGAACCACGAGAAAT-3’ .", "R: 5’-CCTTCCACAATGCCAAAGTT-3’Human-NPAS4F: 5’-CCTGCATCTACACTCGCAAG-3’ .", "R: 5’-CTCGCTCACACTCTCAGACA-3’Mouse-Npas4F: 5’-AGGGTTTGCTGATGAGTTGC-3’ .", "R: 5’-CCCCTCCACTTCCATCTTC-3’Human-PER1F: 5’-TCAACTGCCTGGACAGCATCCT-3’ .", "R: 5’-TCAGAGGCTGAGGAGGTGGTAT-3’Mouse-Per1F: 5’-GAAACCTCTGGCTGTTCCTACC-3’ .", "R: 5’-AGGCTGAAGAGGCAGTGTAGGA-3’Human-SIK1F: 5’-CTCAGAGAGGGCAGAGGTGA-3’ .", "R: 5’-ATGCATAAACGTCAGCAGCA-3’Mouse-Sik1F: 5’-GTGCCATCCAAACACCTCTG-3’ .", "R: 5’-TGTCTGGAGAGTAAGCGGTA-3’Human-SLC2A3F: 5’-TGCCTTTGGCACTCTCAACCAG-3’ .", "R: 5’-GCCATAGCTCTTCAGACCCAAG-3’Mouse-Slc2a3F: 5’-CCGCTTCTCATCTCCATTGTCC-3’ .", "R: 5’-CCTGCTCCAATCGTGGCATAGA-3’Human-PTDSS1F: 5’- ATGTGATCACCTGGGAGAGG-3’ .", "R: 5’- CCATTGCACAACAGGATGTC-3’Mouse-Ptdss1F: 5’- ACACAGTGCAAGCGTGTAGG-3’ .", "R: 5’-AACCATGCCGTACAGACACA-3’Human-ATP1B1F: 5’-CCGCCAGGATTAACACAGAT-3’ .", "R: 5’-TCCTCGTTCTTTCGGTTCAC-3’Mouse-Atp1b1F: 5’-CAGATTCCCCAGATCCAGAA-3’ .", "R: 5’-CTGCACACCTTCCTCTCTCC-3’Human-KCTD1F: 5’-AGTCGGCCCAATATGTCAAG-3’ .", "R: 5’-CCGATTCTGGATTCAGGGTA-3’Mouse-Kctd1F: 5’-AGTCGGCCCAATATGTCAAG -3’ .", "R: 5’-TTCTGGATTCGGGGTATTTG -3’Human-LMBRD2F: 5’-TTGGGATAGCTGCTGCAAAT-3’ .", "R: 5’-CTCCATGGCATCTTCCAAAT-3’Mouse-Lmbrd2F: 5’-GTGCGGCTTTAGGACTTGAG-3’ .", "R: 5’-CAGCGGCAGTATGAAGACAA-3’Human-TRAFD1F: 5’-AAGGAGGGGAAGATTTTGGA-3’ .", "R: 5’-GCCTGAGCACCTTACCAGAG-3’Mouse-Trafd1F: 5’-AGTCTGTGCCTGAGGCTGAT-3’ .", "R: 5’-GAGAAGGGTTGCAGCTTGTC-3’ Statistical testing of the RNA-seq data is described in that section .", "Other testing involved a 2-tailed paired Student's t-test .", ", or a one- or two-way ANOVA followed by Sidak’s post-hoc test , as indicated in the legends .", "Correlation coefficients were calculated assuming a Gaussian distribution of the data .", "Throughout the manuscript , independent biological replicates are defined as independently performed experiments on material derived from different animals ." ] ]

[ "Evolutionary differences in gene regulation between humans and lower mammalian experimental systems are incompletely understood , a potential translational obstacle that is challenging to surmount in neurons , where primary tissue availability is poor .", "Rodent-based studies show that activity-dependent transcriptional programs mediate myriad functions in neuronal development , but the extent of their conservation in human neurons is unknown .", "We compared activity-dependent transcriptional responses in developing human stem cell-derived cortical neurons with those induced in developing primary- or stem cell-derived mouse cortical neurons .", "While activity-dependent gene-responsiveness showed little dependence on developmental stage or origin ( primary tissue vs . stem cell ) , notable species-dependent differences were observed .", "Moreover , differential species-specific gene ortholog regulation was recapitulated in aneuploid mouse neurons carrying human chromosome-21 , implicating promoter/enhancer sequence divergence as a factor , including human-specific activity-responsive AP-1 sites .", "These findings support the use of human neuronal systems for probing transcriptional responses to physiological stimuli or indeed pharmaceutical agents ." ]

[ "Cells in the brain known as neurons produce electrical activity that allows them to signal to other cells; this in turn allows us to think , feel , move , remember and learn .", "This electrical activity also causes the neurons to increase or decrease the activity of certain genes .", "Whether a gene is controlled by electrical activity depends on the structure of the regions of DNA , called promoters or enhancers , where certain proteins can bind in order to activate the gene .", "Studying how human neurons respond to electrical stimulation has been challenging because they are difficult to obtain and grow in the laboratory .", "Instead , most experiments have been conducted with mouse or rat neurons .", "However , it was not known to what extent the changes seen in gene activity in rodent neurons reflect the changes that occur in their human equivalents .", "Qiu et al . grew human neurons from human embryonic stem cells and compared these cells with the corresponding mouse neurons .", "Overall , the cells from both species generally reacted similarly to simulated electrical stimulation .", "However , some genes responded in significantly different ways in mouse and human neurons .", "For example , a gene called ETS2 was switched on more quickly and strongly in the human neurons than in mouse neurons .", "Further experiments indicated that some of these differences are because the promoter and enhancer regions of the genes have evolved in different ways in mice and humans .", "More research is now needed to test whether the differences in gene activation seen in the mouse and human neurons in response to electrical activity affect how the neurons work .", "It will be equally important to investigate whether neurons from different species respond differently to other factors , such as drugs ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "biochemistry and chemical biology", "neuroscience" ]

[ [ "Circadian clocks are an evolutionarily conserved timekeeping mechanism that allows organisms to anticipate and adapt their behavior , physiology , and biochemistry to predictable changes in their environment .", "Organisms with a circadian period length matched to that of their environment grow faster and have greater reproductive fitness and lifespan than their unsynchronized competitors ( Dodd et al . , 2005; Woelfle et al . , 2004; Ouyang et al . , 1998; Beaver et al . , 2002 ) Circadian timekeeping is a product of a hierarchical system composed of multiple oscillators ( Reppert and Weaver , 2002 ) .", "The suprachiasmatic nuclei ( SCN ) of the anterior hypothalamus represent the top of the hierarchy , responsible for integrating environmental light-dark input and synchronizing timekeeping in peripheral tissues ( Liu et al . , 2007a ) .", "Most tissues possess autonomous cellular oscillators that control rhythmic tissue-specific physiology .", "At the molecular level , the circadian clock is composed of a core transcription-translation feedback loop , in which transcriptional activators aryl hydrocarbon receptor nuclear translocator-like ( ARNTL/BMAL1 ) and circadian locomotor output cycles kaput ( CLOCK ) regulate the transcription of their own repressors , period 1/2/3 ( Per1/2/3 ) and cryptochrome 1/2 ( Cry1/2 ) ( Ko and Takahashi , 2006; Reppert and Weaver , 2002 ) .", "PER and CRY repress their own transcription through the inhibition of the CLOCK/BMAL1 transcriptional complex forming the core feedback loop ( Zhang and Kay , 2010 ) .", "The fidelity of the core clock loop is stabilized by a second interlocking feedback loop composed of retinoic acid receptor-related orphan receptor ( RORα/β/γ ) -dependent transcription of Bmal1 , a process inhibited by the Bmal1 downstream targets Rev-Erbα/β ( Nr1d1/Nr1d2 ) ( Preitner et al . , 2002; Sato et al . , 2004; Liu et al . , 2008; Cho et al . , 2012 ) .", "In addition to the regulation of known clock genes , the CLOCK/BMAL1-complex also regulates the expression of thousands of other genes , which drive rhythmic changes in various tissue-specific physiological and cellular processes ( Zhang et al . , 2014; Perelis et al . , 2015 ) .", "Many clock controlled output genes encode metabolic enzymes leading to diurnal oscillations in carbohydrate flux ( Asher and Schibler , 2011; Bass , 2012; Zhang and Kay , 2010; Bass and Takahashi , 2010 ) .", "Rhythmic carbohydrate catabolism and mitochondrial oxidative phosphorylation , particularly in hepatocytes ( Jacobi et al . , 2015; Peek et al . , 2013 ) , likely contribute to oscillations in reactive oxygen species ( ROS ) ( Pekovic-Vaughan et al . , 2014; Stangherlin and Reddy , 2013; Milev and Reddy , 2015 ) .", "Signaling through oxidative intermediates ( Sies , 2014 ) modulates an array of redox sensitive transcription factors and enzymes to regulate gene expression , metabolic flux , and timekeeping ( Pekovic-Vaughan et al . , 2014; Xu et al . , 2012; Wible and Sutter , 2017 ) .", "Appropriate timing and magnitude of ROS signaling likely facilitates the temporal segregation and transition between incompatible metabolic processes and increases overall metabolic efficiency ( Milev and Reddy , 2015; Jacobi et al . , 2015 ) .", "A key transcription factor , regulated by oxidative signaling , controlling both metabolic ( Mitsuishi et al . , 2012 ) and circadian ( Rey et al . , 2016; Yang et al . , 2014 ) gene expression is NF-E2 related-factor 2 ( NRF2 ) .", "The activity of NRF2 is antagonized by the cytoplasmic repressor kelch like ECH associated protein 1 ( KEAP1 ) .", "KEAP1 destabilizes NRF2 by facilitating its ubiquitinylation and eventual proteasomal degradation .", "Activators of NRF2 signaling relieve NRF2 from KEAP1-mediated repression through the oxidation or covalent modification of KEAP1 cysteine residues .", "Well-studied chemical NRF2 activators include the clinically relevant electrophiles 3H-1 , 2-dithiole-3-thione ( D3T ) and 1-[2-cyano-3 , 12-dioxooleana-1 , 9 ( 11 ) -dien-28-oyl]imidazole ( CDDO-Im ) , and the oxidants tert-butylhydroquinone ( tBHQ ) ( Imhoff and Hansen , 2010 ) and hydrogen peroxide ( H2O2 ) .", "Modification of KEAP1 cysteine residues by these agents results in an increased NRF2 half-life that facilitates nuclear translocation and accumulation ( Wible and Sutter , 2017; Suzuki and Yamamoto , 2015; Kwak et al . , 2004; Saito et al . , 2016 ) .", "The expression of Nrf2 is under the transcriptional regulation of the CLOCK/BMAL1-complex ( Pekovic-Vaughan et al . , 2014; Xu et al . , 2012; Zhang et al . , 2009; Lee et al . , 2013 ) .", "CLOCK/BMAL1-dependent Nrf2 regulation gives rise to diurnal patterns in NRF2 signaling , which underlies the rhythmic expression of antioxidant and metabolic enzymes as well as NADPH reducing equivalents and glutathione biosynthesis ( Xu et al . , 2012 ) .", "Here , we report the effects of NRF2 gain- and loss-of-function on circadian gene expression and rhythmicity , elaborating the coupling of NRF2 and clock and the role of NRF2 to integrate cellular redox status into timekeeping ." ], [ "Microarray data ( GSE99199; Wible et al . , 2018 ) characterizing global gene expression changes in mouse liver in response to D3T suggested a possible alteration in circadian gene expression in response to chemical NRF2-activation ( Figure 1—figure supplement 1 ) .", "To further characterize the effect of D3T on circadian gene expression and to determine whether those effects were dependent on NRF2 , we measured the RNA expression of several circadian genes in the liver of Wt and Nrf2-/- mice treated with three doses of 300 µmol/kg bodyweight D3T ( every other day , with sampling 24 hr following the third dose ) ( Figure 1A ) .", "While E-box and D-box-containing clock genes ( Nr1d1 , Nr1d2 , Dbp , Tef , and Per3 ) were up-regulated in response to D3T in Wt mice , genes that are regulated primarily via RAR-related orphan receptor response elements ( ROREs ) ( Bmal1 , Npas2 , E4bp4 ) were down-regulated , consistent with the network features of the molecular clock ( Ueda et al . , 2005; Liu et al . , 2008; Baggs et al . , 2009 ) .", "The full induction of E-box- and D-box-containing genes ( Per3 , Nr1d1 , Nr1d2 , Dbp , and Tef ) in response to D3T-treatment required the presence of the KEAP1/NRF2 signaling pathway , as their induction was significantly compromised in Nrf2-/- mice .", "Nrf2 deficiency did not alter the down-regulation of Bmal1 , Cry1 , or E4bp4 , suggesting their independence of NRF2 and that the circadian network effect did not transmit to RORE-mediated transcription .", "Next , we evaluated whether the effects of D3T on circadian gene expression observed in vivo manifested in alterations in circadian function in vitro and whether those potential alterations were dependent on NRF2 .", "Chemical activation of NRF2 occurs through either electrophilic or oxidative modification of KEAP1 , antagonizing its repression of NRF2 .", "To assess the effect of each type of KEAP1 modification and subsequent NRF2 activation on the circadian rhythm , we treated Wt and Nrf2-/- mouse embryonic fibroblast ( MEF ) cell lines harboring a Per2 promoter-driven luciferase reporter ( Per2:Luc ) with a single dose of 100 µM D3T ( Figure 1B ) or 50 µM tBHQ ( Figure 1C ) .", "Both treatments resulted in significantly reduced rhythm amplitude , an effect that was not observed in the Nrf2-/- cells .", "As a primary regulator of the antioxidant response pathway , it is likely that an endogenous NRF2 activating signal is H2O2 , generated as a byproduct of metabolic flux .", "To evaluate the potential effect on circadian rhythmicity as a result of the activation of NRF2 by an endogenous oxidative signal , we treated MEFs at the beginning of the cycle with a low concentration ( 100 µM ) of H2O2 ( Figure 1D ) .", "Similar to both D3T and tBHQ treatment , we observed significant NRF2-dependent reductions in rhythm amplitude in the presence of H2O2 .", "As a well-characterized , clinically relevant , NRF2-activating compound , we also evaluated the effect of CDDO-Im on circadian rhythmicity ( Figure 1—figure supplement 2 ) .", "Treatment with CDDO-Im caused significant reductions in both amplitude and period length ( Treatment ) , that were noticeably absent in Nrf2-/- MEFs .", "Because poor circadian oscillations in the presence of CDDO-Im may be a product of chemical toxicity , we replaced the treatment medium in cell lines of both genotypes and continued monitoring rhythmic bioluminescence patterns ( Recovery ) .", "The restoration of normal circadian rhythmicity in the treated Wt cells served as a confirmation of good cell health and a dependency on CDDO-Im for the alterations in the circadian parameters observed .", "In our earlier experiments , we observed inherently low bioluminescence rhythm amplitudes ( Figure 1B–D ) in Nrf2-/- MEFs , indicating that NRF2 may be a required component of the circadian clockwork .", "To test this hypothesis , we determined rhythmic bioluminescence patterns in Wt and Nrf2-/- MEFs .", "In addition to the previously observed reduction in rhythm amplitude , the loss of Nrf2 also resulted in a significant decrease in period length ( Figure 2A , Figure 2—figure supplement 1 ) .", "To confirm that the circadian phenotype observed in these cells was a product of the loss of NRF2 signaling and not an artifact of the established cell lines , we genetically reconstituted Nrf2 expression in the Nrf2-/- MEF cells ( Figure 2B , Figure 2—figure supplement 2 ) .", "The restoration of Nrf2 expression rescued both circadian amplitude and period length relative to the parental Nrf2-/- cell line , but fell short of achieving Wt rhythmicity .", "As a marker of NRF2 activity , we measured increased Nqo1 expression , confirming that exogenous Nrf2 expression yields functional NRF2 protein ( Figure 2C ) .", "Consistent with Nrf2 expression being driven by a constitutive CMV promoter , NRF2 protein expression in the rescued cell line was constitutively elevated at 24 hr and 36 hr post-synchronization .", "This contrasted with circadian NRF2 expression observed in Wt MEFs ( Figure 2D , Figure 2—figure supplement 3 ) .", "To independently validate the circadian phenotype observed in Nrf2-/- MEFs , we established a shRNA-mediated Nrf2 knockdown MEF cell line .", "Nrf2 knockdown led to significant reductions in both rhythm amplitude and period length ( Figure 2E ) , albeit to a lesser extent than what was observed in Nrf2-/- MEFs .", "The efficiency of Nrf2 knockdown was confirmed by measurements of significantly reduced NRF2 protein abundance ( Figure 2F ) .", "Nrf2 and Nqo1 RNA expression was also significantly down-regulated as a result of shNrf2 transduction ( Figure 2G ) .", "Interestingly , Nr1d1 expression was also decreased in response to shNrf2 , consistent with previous reports ( Rey et al . , 2016; Yang et al . , 2014 ) and our results from mouse liver ( Figure 1A ) , suggesting that Nr1d1 may be transcriptionally regulated by NRF2 ( Figure 2G ) .", "Together , these data support the idea that the disrupted circadian rhythmicity observed in Nrf2-/- MEFs is a direct effect of the loss of Nrf2 and implicate a role for NRF2 in the regulation of rhythm amplitude and period length .", "Reduced Nr1d1 expression in shNrf2 cell lines suggested that the mechanistic input of NRF2 into the clockwork may be through the regulation of Nr1d1 expression .", "We determined the temporal abundance patterns of NR1D1 protein as a function of the loss of Nrf2 in an attempt to gain insight as to whether changes in Nr1d1 gene expression had an effect on a rhythmic output .", "In Nrf2-/- MEFs , the peak of NR1D1 protein accumulation was delayed by 4 hr relative to the Wt ( Figure 2H ) .", "Delayed NR1D1 expression , as opposed to the complete loss , in Nrf2-/- cells suggests that Nr1d1 is likely regulated by additional transcription factors beyond NRF2 .", "The improper temporal regulation of NR1D1 in the absence of Nrf2 , however , may contribute to the perturbed circadian rhythmicity observed in MEFs lacking Nrf2 .", "Previous reports regarding the cross-talk between NRF2 and the circadian rhythm have demonstrated that activation of NRF2 in the presence of 6-aminonicotinamide ( Rey et al . , 2016 ) or hyperoxic conditions ( Yang et al . , 2014 ) lead to the transcriptional regulation of Nr1d1 .", "We are currently unaware of any studies evaluating the effect of genetic NRF2 gain-of-function on circadian gene transcription or the circadian rhythm .", "Because genetic gain-of-function and chemical activation have been shown to lead to distinct NRF2-dependent gene responses ( Yates et al . , 2009 ) , we explored the effects of genetic NRF2 activation on circadian rhythmicity and gene expression .", "We established Keap1-/- MEFs harboring a Per2:Luc reporter to determine the circadian phenotype as a function of genetic NRF2 activation .", "Constitutive NRF2 activation in the Keap1-/- background resulted in significant reductions in both amplitude and period length ( Figure 3A ) .", "To confirm our observations in the Keap1-/- MEFs , we overexpressed Nrf2 in Wt MEFs by transducing these cells with a CMV-driven Nrf2 expression construct .", "Overexpression of NRF2 in a Wt background resulted in reduced circadian amplitude and period length in manner similar to the Keap1-/- cell line ( Figure 3B ) .", "The overexpression and enhanced transcriptional activity of NRF2 in this cell line , were validated by measurement of elevated NRF2 protein ( Figure 3C ) and NQO1 protein and RNA expression ( Figure 3C–D ) in the Wt +CMV-Nrf2 cells .", "Moreover , we observed elevated levels of NR1D1 protein ( Figure 3C ) and RNA ( Figure 3D ) in response to Nrf2 overexpression , consistent with NRF2 contributing to Nr1d1 gene expression .", "Interestingly , the circadian phenotypes resulting from the overexpression of NRF2 recapitulated the phenotype of genetically rescued Nrf2-/- MEFs , suggesting that the timing or absolute stoichiometry of NRF2 protein abundance are likely to be important for proper circadian function .", "In addition to genetic gain-of-function , the activation of NRF2 by D3T also had an effect on Nr1d1 expression .", "In response to 100 μM D3T , the expression of both Nqo1 and Nr1d1 were significantly induced ( Figure 3E ) .", "These effects were noticeably absent in D3T-treated Nrf2-/- MEFs .", "We did observe a slight , but significant , induction of Nr1d1 expression in Nrf2-/- MEFs in the absence of D3T-treatment , consistent with the previously reported induction of Nr1d1 under oxidative intracellular conditions ( Yang et al . , 2014 ) , a condition likely to be present in Nrf2-/- MEFs .", "Using a position weight matrix implemented in the JASPAR CORE database ( Mathelier et al . , 2016 ) , we identified putative antioxidant response elements ( AREs ) in the regions surrounding the promoters of Nr1d1 and Nqo1 ( Figure 3—figure supplement 1 , Supplementary files 1–2 ) .", "Chromatin immunoprecipitation of NRF2 in MEFs treated with 100 μM D3T for 24 hr demonstrated direct binding of NRF2 to two of the three ARE elements identified in the Nr1d1 promoter , namely Nr1d1-2 and Nr1d1-3 , and two ARE elements in Nqo1 , namely Nqo1-1 and Nqo1-2 ( Figure 3F ) .", "The binding of NRF2 to the elements Nr1d1-3 and Nqo1-1 are novel findings , while the binding of NRF2 to the element in Nr1d1-2 ( Yang et al . , 2014 ) and Nqo1-1 ( Nioi et al . , 2003 ) confirm previous reports .", "Together these data demonstrate that genetic or pharmacological activation of NRF2 impinges on circadian clock function , which correlates with enhanced occupation of NRF2 on the Nr1d1 promoter and an induction of Nr1d1 expression .", "As the key regulator of an antioxidant stress response , we hypothesized that the activation of NRF2 by appropriately timed endogenous levels of oxidants would cause a reinforcement of the circadian amplitude , similar to what has been observed in plants ( Zhou et al . , 2015 ) .", "To determine the timing of endogenous oxidative signals relative to the circadian-mediated expression of NRF2 , we measured the oxidation state of peroxiredoxin ( PRDX-SO2/3 ) as a biomarker of an oxidized intracellular environment ( Edgar et al . , 2012 ) in synchronized MMH-D3 hepatocytes ( Figure 4A ) .", "Levels of hyperoxidized PRDX were maximal 28 hr post-synchronization ( red arrow ) , which preceded peak NRF2 protein accumulation ( black arrow ) by 12 hr .", "This 12 hr lag phase is likely a product of the kinetics of NRF2 activation .", "NRF2-activating signals inhibit KEAP1-mediated NRF2 turnover , resulting in KEAP1 becoming saturated with bound NRF2 .", "Due to the short NRF2 half-life , a lag time would be expected to allow for the saturation of KEAP1 NRF2-binding sites and the accumulation of newly synthesized active NRF2 following the activation of the pathway ( Wible and Sutter , 2017 ) .", "The timing of oxidative signaling ( Figure 4A ) , however , is consistent with what has been previously observed in mouse liver ( Xu et al . , 2012; Edgar et al . , 2012 ) , indicating that the generation of endogenous oxidative signals occurs at a similar time in vitro relative to Nrf2 expression as it does in vivo .", "To test our hypothesis that appropriately timed oxidative NRF2 activation could lead to circadian amplitude reinforcement , we treated MEFs with 100 μM H2O2 , a concentration likely to be physiologically relevant , at a circadian time corresponding to the endogenous peak of oxidative signaling determined in hepatocytes ( Figure 4B , Figure 4—figure supplement 1 ) .", "Activation of NRF2 in response to an oxidative signal at this time resulted in significantly increased amplitude , without affecting period length .", "A similar effect was also observed in the presence of another NRF2-activating compound D3T ( Figure 4—figure supplement 2 ) , suggesting that amplitude reinforcement is a function of NRF2 activity and not a side-effect of any one particular chemical .", "In addition to reinforcing circadian amplitude , activation of NRF2 by H2O2 at this time also resulted in a subtle advance of the circadian phase , which became more prominent at higher concentrations of H2O2 ( 500 μM ) ( Figure 4—figure supplement 3 ) .", "To confirm that the reinforcement of circadian amplitude in response to oxidative signaling was dependent on NRF2 activation , we treated Wt and Nrf2-/- MEFs with 100 μM H2O2 at a time consistent with the generation of endogenous oxidative signals .", "Treatment with H2O2 , again , resulted in circadian amplitude reinforcement , which was absent in H2O2-treated Nrf2-/- MEFs ( Figure 4C , Figure 4—figure supplement 4 ) .", "Reinforcement of circadian amplitude , following the addition of H2O2 , could be blocked in the presence of an antioxidant ( N-acetylcysteine; NAC ) ( Figure 4D ) , suggesting that oxidation was indeed the relevant signal responsible for the reinforcement of circadian amplitude .", "Interestingly , treatment with NAC alone resulted in significantly decreased rhythm amplitude and period length , suggesting that endogenous oxidative signals ( Sies , 2017 ) may play a role in the maintenance of circadian timekeeping .", "Similar dependence on NRF2 for amplitude reinforcement in response to H2O2 was also observed when the treatment was applied at a different time in the circadian cycle ( Figure 4E ) .", "In contrast to what we observed when applying H2O2 prior to the peak of PER2 expression , addition of H2O2 prior to the trough resulted in a subtle delay of the circadian phase .", "Interestingly , when H2O2 was added to the cultures at the nadir of PER2 expression , the circadian phase appeared to be delayed or possibly reset ( Figure 4F , Figure 4—figure supplement 5 ) .", "This effect was not observed in Nrf2-/- MEFs , indicating that H2O2-induced phase-dependent shifts in circadian phase may occur through an NRF2-dependent mechanism .", "While shifts in circadian phase in response to oxidative signals have been observed previously across multiple cell lines ( Tahara et al . , 2016; Putker et al . , 2018 ) , our data implicates the activation of NRF2 as the likely mechanism through which oxidants signal into the clockwork .", "NRF2-dependent input into the clock effectively couples redox homeostasis and timekeeping yielding a molecular framework to enhance oscillator robustness and shift circadian phase .", "Similar to our observations in MEFs , shRNA-mediated Nrf2 knockdown in hepatocytes caused significant amplitude and period length reductions ( Figure 5A , Figure 5—figure supplement 1 ) .", "Hepatocytes harboring shNrf2 showed a 50% reduction in total NRF2 protein expression and a greater than 90% reduction in NQO1 ( Figure 5B ) .", "These data indicate that a reduction in total NRF2 protein resulted in markedly lower transcriptional activity , which paralleled significant reductions in rhythm amplitude and period length .", "To evaluate the coupling of NRF2 to the clock in vivo , we characterized the rhythmic bioluminescence patterns in liver and lung organotypic slices from two different Nrf2-null PER2::LUC mouse strains ( MYM and YWK ) .", "Loss of Nrf2 in the liver of mice from both strains resulted in a significant alteration in circadian period length ( Figure 5C and E ) .", "Surprisingly , we observed no deleterious effects of Nrf2 loss on circadian rhythmicity in the lung tissue of either strain ( Figure 5D and F ) , despite earlier literature indicating that NRF2 is a circadian output in the lung ( Pekovic-Vaughan et al . , 2014 ) and can be integrated into clock function in that tissue type in response to stress ( Yang et al . , 2014 ) .", "Previous reports have indicated that the loss of Nrf2 had no effect on the locomotor behavioral activity in mice ( Pekovic-Vaughan et al . , 2014 ) .", "In agreement with this observation , we observed no significant alteration in circadian rhythmicity in SCN organotypic slices from the YWK strain of Nrf2-null mice ( Figure 5—figure supplement 2 ) .", "To better understand the mechanistic effects of NRF2 gain-of-function on the circadian clockwork , we established Wt and Nrf2 overexpression MMH-D3 Per2:Luc hepatocyte cell lines .", "Similar to our observations in MEFs , Nrf2 overexpression resulted in decreased rhythm amplitude and period length ( Figure 6A ) .", "Nrf2 overexpression also led to significantly elevated Nqo1 , Cry2 , and Nr1d1 expression ( Figure 6B ) .", "Consistent with the effect of genetic gain-of-function , D3T-induced activation of NRF2 in hepatocytes caused significant increases in Cry2 , Nr1d1 , and Nqo1 RNA expression ( Figure 6C ) , suggesting these genes may be under NRF2 regulation .", "Using a position weight matrix implemented in the JASPAR CORE database ( Mathelier et al . , 2016 ) we identified putative AREs in the promoter regions of each of these genes ( Figure 3—figure supplement 1 , Supplementary files 1–2 ) , supporting the hypothesis that they are indeed regulated by NRF2 .", "Chromatin immunoprecipitation of NRF2 in D3T-treated hepatocytes demonstrated significantly elevated binding of NRF2 to novel enhancer elements in the promoters of Cry2 , Nr1d1 , and Nqo1 ( Figure 6D ) .", "In addition to these novel elements , we also observed D3T-induced enhanced binding of NRF2 to ARE elements previously characterized in Nr1d1 ( Figure 6—figure supplement 1 ) ( Yang et al . , 2014 ) and Nqo1 ( Figure 6—figure supplement 1 ) ( Nioi et al . , 2003 ) .", "Along with these effects on circadian gene expression , transfection of pLV7-Nrf2 into HEK293T cells was able to repress CLOCK/BMAL1-mediated transcription of Per1:Luc and synthetic E-box:Luc reporter constructs in a manner similar to what was observed in response to the transfection of Cry1 ( Figure 6E ) .", "These observations suggested that NRF2 may be physically interacting with the CLOCK/BMAL1 complex , a common property of core clock proteins ( Anafi et al . , 2014 ) , leading to its repression .", "Attempts to characterize protein-protein interactions between NRF2 and any other clock protein using coimmunoprecipitation and two-hybrid assays , however , failed to detect any physical interaction involving NRF2 and another clock protein ( data not shown ) .", "Our data indicating that NRF2 may be a regulator of Cry2 expression , raised the hypothesis that NRF2 may repress CLOCK/BMAL1 transcription indirectly through the regulation of endogenous Cry2 expression .", "In support of this hypothesis , we measured significant increases in both Cry2 and Nqo1 expression in HEK293T cells transfected with pLenti-Nrf2 ( Figure 6F ) .", "To confirm that Cry2 expression could account for a repression in CLOCK/BMAL1 transcription , we measured CLOCK/BMAL1-mediated Per1 reporter-driven bioluminescence in HEK293T cells as a function of Cry1 , Cry2 , or pLenti-Nrf2 expression .", "Expression of either Cry1/2 or Nrf2 resulted in significant repression of CLOCK/BMAL1 transcriptional activity ( Figure 6G ) .", "To evaluate whether the regulation of circadian gene expression in response to NRF2 activation manifests in changes to circadian function , we measured bioluminescence expression patterns in Per2:Luc MMH-D3 hepatocytes in response to increasing doses of D3T .", "Both circadian parameters of rhythm amplitude and period length decreased as a function of D3T dose escalation ( Figure 6H ) , while having no measurable negative effect on cell health ( Figure 6—figure supplement 2 ) .", "Interestingly , the same D3T dose escalation that produced reductions in rhythm amplitude and period length also led to the dose-dependent induction of Nqo1 and Cry2 expression ( Figure 6I ) .", "Together , these data support the concept that NRF2 likely represses CLOCK/BMAL1-mediated transcription indirectly through the regulation of Cry2 and Nr1d1 expression .", "Given the reported E-Box-mediated circadian regulation of Nrf2 ( Pekovic-Vaughan et al . , 2014 ) , these results indicate that NRF2 and clock comprise an interlocking loop ( Figure 6J ) that integrates cellular redox signals into circadian timekeeping ." ], [ "In mammals , the molecular core circadian clock mechanism is a transcription-translation feedback loop .", "Positive transcriptional regulation of the E-box containing genes Per and Cry by CLOCK and BMAL1 is followed by translation of PER and CRY , which heterodimerize and repress their own transcription through the inhibition of the CLOCK/BMAL1-complex .", "Circadian regulation of RORE- and D-box containing genes comprise additional transcription-translation feedback loops interlocked with the core clock mechanism ( Zhang and Kay , 2010; Takahashi , 2017 ) .", "These additional feedback loops stabilize and contribute to clock robustness and also serve as entry points for external feedback .", "Synchronicity between the circadian clock and the environment is maintained by entraining the clock to predictable external cues .", "A predominant entraining signal in peripheral tissues is diurnal metabolic cycles ( Hara et al . , 2001 ) .", "As a natural consequence of oxidative metabolism , perturbations in the ratios of redox coupled cofactors and the production of oxidative signals are thought to be the metabolic inputs into the clockwork .", "Redox rhythms , including oscillations in H2O2 and hyperoxidized peroxiredoxin ( Edgar et al . , 2012 ) , exist across all domains of life and are believed to cross-talk with the molecular clock .", "Parallels between these redox oscillations , metabolic cycles , and circadian gene expression led to the idea that changes in the intracellular redox potential may be the link coupling metabolism to the clock ( Putker and O'Neill , 2016 ) .", "In biochemical assays , increases in the NAD+/NADH ratio , to mimic an oxidized intracellular environment , resulted in reduced CLOCK/BMAL1 DNA-binding affinity ( Rutter et al . , 2001 ) .", "Additionally , the levels of zCry1 and zPer2 were elevated in zebrafish Z3 cells in response to light-induced H2O2 production ( Hirayama et al . , 2007 ) .", "A single H2O2 bolus recapitulated these effects , confirming that H2O2 was indeed a circadian input signal .", "Despite these observations , the direct mechanism of redox , and by extension metabolic , input into the clock has yet to be fully elucidated .", "A previous study reported a NRF2-binding site in the promoter of the clock gene Nr1d1 and its subsequent regulation in response to H2O2 and sulforaphane ( Yang et al . , 2014 ) , a well-characterized NRF2 activator ( Kensler et al . , 2013 ) .", "As a primary sensor of intracellular oxidation , metabolic input into the clock via NRF2-mediated transcription seemed fitting .", "A recent report demonstrated that that genetic or chemical inhibition of the pentose phosphate pathway ( PPP ) altered circadian period length in a NRF2-dependent manner ( Rey et al . , 2016 ) .", "Here , we substantiated these findings , showing that electrophilic or oxidative activation of KEAP1/NRF2 signaling altered clock gene expression and circadian function .", "Expression of E- and D-box-regulated circadian genes , including Per3 , Nr1d1 , Nr1d2 , Dbp , and Tef , were elevated via a NRF2-dependent mechanism in the liver of mice treated with D3T , indicating possible NRF2 input into multiple circadian loops .", "These changes in the levels of RNA paralleled significant reduction of circadian rhythm amplitude in MEFs in the presence of D3T .", "This effect was absent in treated Nrf2-/- cells .", "Oxidative activators of KEAP1/NRF2 signaling , tBHQ and H2O2 , had similar NRF2-dependent effects on rhythm amplitude .", "We further validated the effects on circadian rhythmicity in response to NRF2-activating chemicals using genetic activation of NRF2 .", "Overexpression of NRF2 or deletion of its negative regulator , Keap1 , phenocopied the effects of NRF2-activating chemicals on circadian function , in part through NRF2-mediated Nr1d1 regulation .", "Together , these data support the idea that NRF2 is a critical node linking metabolism to the clock as well as a conduit for the response to changes in intracellular redox status .", "As alterations in rhythm amplitude and period length are considered to be reliable indicators of clock fidelity ( Takahashi et al . , 2008 ) , an important and heretofore unreported observation of our study is the significant alteration in circadian rhythmicity in mouse fibroblasts , hepatocytes , and liver explants lacking Nrf2 .", "Along with the effects of NRF2 gain-of-function on rhythmicity , these observations indicate that the stoichiometry and/or the timing of Nrf2 expression are important for maintaining clock function .", "Marked circadian disruption in the absence of NRF2 supports an endogenous role for NRF2 in timekeeping in certain tissues , as these effects were observed in liver , but not in lung or SCN explants .", "This lack of effect in SCN explants is consistent with a previous report demonstrating that Nrf2-/- mice display Wt locomotor activity patterns ( Pekovic-Vaughan et al . , 2014 ) .", "By characterizing the circadian timing of redox and NRF2 cycling , we showed that intracellular oxidation precedes NRF2 accumulation .", "Activation of NRF2 at a circadian time when the intracellular redox potential is decreasing leads to a NRF2-dependent enhancement of circadian amplitude , a response termed reinforcement ( Zhou et al . , 2015 ) .", "Previous studies have reported that both Keap1 expression and GSH levels are lowered prior to the production of endogenous oxidants and the induction of Nrf2 in mouse liver ( Xu et al . , 2012 ) .", "These results indicate that NRF2 expression is rapidly increasing at a time when intracellular reducing capacity is at its lowest .", "Decreasing intracellular redox potential promotes the accumulation of oxidants and a reduction in intracellular pH . It is therefore plausible that the simultaneous decreases in redox potential and intracellular pH lead to the oxidative modification of KEAP1 cysteine residues and the activation of NRF2 .", "In light of these previous findings , our results showing that oxidative activation of NRF2 at this time reinforces the circadian clock to increase oscillator amplitude is consistent with tight coupling of cellular redox status , NRF2 activation , and clock function .", "The NRF2-dependent reinforcement of circadian amplitude is nearly identical to that attributed to the Arabidopsis NPR1 ( non-expressor of pathogenesis-related gene 1 ) , a redox-sensitive modulator of clock gene expression .", "When salicylic acid is produced and activates NPR1 at an appropriate time , NPR1 transcriptionally regulates gene expression in the morning and evening plant circadian loops to reinforce the circadian amplitude .", "In addition to reinforcing circadian amplitude , NPR1 also segregates the initiation of cellular defense and immunity pathways to the morning phase of the cycle ( Zhou et al . , 2015 ) .", "This provides a mechanism to maximize energy expenditure during the night phase to support plant growth and overall fitness ( Nozue et al . , 2007; Dodd et al . , 2005 ) .", "As an analog of NPR1 , NRF2 may contribute to a similar temporal segregation of energy utilization pathways in animals .", "In addition to amplitude reinforcement , our data indicate that physiological levels of H2O2 may cause phase-dependent circadian phase-resetting .", "Similar findings have been reported recently in multiple mammalian cell lines demonstrating that redox signals and the intracellular redox environment can elicit shifts in circadian phase and regulate rhythm amplitude ( Putker et al . , 2018 ) .", "These findings , in combination with our observations that both Cry2 and Nr1d1 are transcriptionally regulated by NRF2 , suggest a plausible mechanism through which endogenous oxidative signals directly input into the circadian clockwork to link metabolism and timekeeping .", "Thus , while acute changes in redox balance may act as nonessential auxiliary timekeepers ( Putker et al . , 2018 ) , proper alignment of the intracellular redox and circadian phases appears to enhance circadian function , implying the existence of a direct and reciprocal coupling between the redox state and the circadian mechanism .", "We believe that NRF2 is the mechanistic conduit interconnecting these two processes .", "Activation of NRF2 is emerging as a key regulator of metabolism through the regulation of the PPP and by directing glutamine breakdown into glutathione biosynthetic pathways ( Mitsuishi et al . , 2012; Hayes and Dinkova-Kostova , 2014 ) .", "NRF2-dependent regulation of the PPP suggests that NRF2 may be a contributor in the daily transition between catabolic and anabolic metabolism .", "NRF2 feedback into the circadian clockwork could be an effective mechanism to facilitate this transition by integrating metabolic output and intracellular redox homeostasis into rhythmicity and carbohydrate flux .", "Similarities between NPR1 and NRF2 in their regulation of antioxidant response and metabolic pathways , and their common integration into the circadian function of their respective organisms , indicate that the interconnectedness of these pathways is likely a product of convergent evolution .", "We propose that the coordinated integration of metabolism , redox homeostasis , and the circadian clock in mammals through NRF2 is a mechanism to align energy production and utilization to the environmental light-dark cycle , as has been observed in plants ( Dodd et al . , 2005 ) .", "Proper integration of redox signals into the clock requires the activation of NRF2 by levels of oxidants low enough to affect signaling without inducing damage .", "A recent study seeking to link metabolic redox oscillations to the clock through NRF2 relied on PPP inhibition to reduce NADPH production , increasing the NADP+/NADPH ratio ( Rey et al . , 2016 ) .", "However , data from this study shows that these conditions created a persistently elevated oxidizing environment , as evidenced by the hyperoxidation of PRDX , leading to the activation of a NRF2-mediated stress response and a disruption in circadian function .", "Similar disruptions in clock function in response to oxidative stress were observed in the lungs of newborn mice exposed to 95% O2 ( Yang et al . , 2014 ) .", "This hyperoxic exposure altered the timing and magnitude of E-box containing gene expression in vivo and NRF2 regulation of Nr1d1 in cells .", "Interestingly , we observed unaltered circadian rhythmicity in lung explants from two independent strains of Nrf2-null mice , suggesting that NRF2 signaling is not required for normal circadian function in the lung .", "Nonetheless , the reports showing that NRF2 alters circadian Nr1d1 expression and the disruption of cellular clock function by persistent oxidative activation of NRF2 ( Yang et al . , 2014 ) indicates that ROS-activated NRF2 signals into the clock mechanism .", "Thus , it appears that NRF2 and the stress-response it regulates are at the top of a molecular hierarchy whereby the resolution of oxidative stress likely takes precedence over the preservation of timekeeping .", "However , because the circadian clock is reset daily by environmental and systemic zetigebers , stress-induced disruption of circadian function is likely to be transient and unlikely to manifest in deleterious effects on cellular or tissue health .", "In hepatocytes , where NRF2 and the clock appear to be tightly coupled , we found that activated NRF2-bound specific enhancer regions of the core clock repressor gene Cry2 , increased Cry2 expression , and repressed CLOCK/BMAL1-regulated E-box transcription .", "Moreover , activation of NRF2 in these cells also resulted in dose-dependent decreases in rhythm amplitude and period length , with associated dose-dependent increases in Cry2 .", "Circadian amplitude and period length are largely dependent on CRY repression of CLOCK/BMAL1-mediated E-box gene transcription , placing the regulation of Cry at the heart of the clock mechanism ( Sato et al . , 2006; Kume et al . , 1999; Reppert and Weaver , 2002; Griffin et al . , 1999 ) .", "Despite its role in the clockwork , little is known about the transcriptional regulation of Cry2 .", "Our characterization of a NRF2-binding site in the Cry2 promoter , in addition to its induction , supports the idea that NRF2-dependent regulation of Cry2 is a mechanism to relay timing information from the redox oscillator into the molecular clock .", "Interestingly , circadian regulation of metabolism also occurs via this same mechanism .", "Beyond being NRF2-dependent E-box repressors , both CRY2 and NR1D1 are also important regulators of nuclear hormone receptors , governing lipid , glucose , and xenobiotic metabolism ( Zhang et al . , 2015; Kriebs et al . , 2017 ) .", "Additional NRF2-dependent regulation of the production of reducing equivalents through the PPP ( Mitsuishi et al . , 2012 ) and heme levels through heme oxygenase one expression ( Ishii et al . , 2000 ) , which serve as cofactors for CRY2 and NR1D1 , respectively , further integrate metabolism and the clock mechanism and highlight the interconnectedness of these oscillators .", "While the principle that metabolism regulates the clock through oscillations in the redox balance is generally accepted ( Asher and Schibler , 2011 ) , the molecular mechanisms through which the redox state is integrated into the clockwork is just emerging .", "As an E-box-regulated circadian output ( Pekovic-Vaughan et al . , 2014 ) , the data presented here suggest that NRF2 likely represses its own transcription through the regulation of Cry2 and subsequent CRY2-mediated repression of CLOCK/BMAL1 , thus forming a redox-responsive transcription-translation feedback loop interlocked with the core molecular circadian mechanism .", "From these observations , NRF2 appears to be a key mechanistic link between circadian oscillations in redox balance and clock gene expression rhythmns .", "Reciprocal integration of redox potential and the clock through NRF2-dependent regulation reinforces the efficiency of both the circadian and metabolic oscillators , contributing to enhanced organismal fitness ." ], [ "CDDO-Im was obtained from Dr . Michael B . Sporn at Dartmouth Medical School .", "D3T and H2O2 were purchased from LKT Laboratories ( St . Paul , MN ) and Sigma ( St . Louis , MO ) , respectively .", "The integrity and concentration of hydrogen peroxide was verified prior to each experiment by spectroscopic detection at 240 nm , using an extinction coefficient of 43 . 6 M−1 cm−1 ( Noble and Gibson , 1970 ) .", "Vehicle treatments for all experiments contained either water or DMSO as indicated in the figure legends .", "The concentration of DMSO never exceeded 0 . 05% .", "All experiments were approved by the University of Memphis Institutional Animal Care and Use Committee and carried out in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the U . S . National Institutes of Health .", "Male mice ranging from 3 to 6 months of age were used for all experiments .", "Animals were housed in a temperature and humidity controlled room with a 12:12 hr light:dark cycle .", "Food and water were provided ad libitum .", "Nrf2-/- mice were developed by Dr . Yuet Wai Kan ( YWK strain ) ( Chan et al . , 1996 ) and Dr . Masayuki Yamamoto ( MYM strain ) ( Itoh et al . , 1997 ) and obtained from Jackson Laboratories ( Bar Harbor , ME ) or from Dr . Thomas Kensler at the University of Pittsburgh , respectively .", "PER2::LUC transgenic mice , originally generated by Dr . Joe Takahashi ( Yoo et al . , 2004 ) , were obtained from Jackson Laboratories .", "Wt and Nrf2-/- PER2::LUC mouse strains were created by crossing Wt C57BL/6J and Nrf2-/- C57BL/6J mice with PER2:LUC transgenic mice .", "Offspring heterozygous for both alleles were crossed and homozygous littermates were used for each experiment .", "All genotypes were confirmed using PCR amplification of tail genomic DNA ( Supplementary file 3 ) .", "For gene expression studies , animals were treated by gavage with vehicle [10% Cremophor EL ( Sigma ) , 10% dimethyl sulfoxide , 80% phosphate-buffered saline] or 300 µmol/kg bw D3T every other day for 5 days .", "Mice were euthanized 24 hr following the final treatment .", "Liver tissue was excised and snap frozen .", "Stable Per2:Luc reporter MEF cell lines were created by infecting MEFs of each genotype with lentiviral particles ( Ramanathan et al . , 2012 ) carrying a vector expressing a Per2-driven luciferase reporter construct , as described ( Liu et al . , 2008; Liu et al . , 2007b ) .", "Reporter cell lines were selected by the addition of 0 . 01 mg/mL blasticidin added to the growth medium .", "Genotypes were confirmed by PCR ( Supplementary file 3 ) .", "All cultures were checked and confirmed to be free from mycoplasma using the MycoSensor PCR Kit ( Agilent , Santa Clara , CA ) .", "All MEF cell lines were maintained in DMEM high glucose ( Fisher Scientific , Hampton , NH , 10–013-CV ) medium supplemented with 10% fetal bovine serum and 1x penicillin/streptomycin ( complete growth medium ) .", "2 μg/mL puromycin was added to the growth medium for all MEF cell lines transduced with shRNA or Nrf2 overexpression lentiviral vectors .", "Cells were cultured to confluence on collagen-coated 35 mm cell culture plates .", "At confluence , the growth medium was removed , the cells were washed once with PBS , and freshly made Recording Medium ( Yoo et al . , 2004; Yamazaki and Takahashi , 2005 ) containing 2% FBS , 2% B27 ( Thermo Fisher , Waltham , MA ) , and 1 mM luciferin was added to the cultures .", "Plates were sealed with a vacuum grease coated cover slip and loaded into a Lumicycle 32 ( Actimetrics , Wilmette , IL ) housed in a non-humidified 37°C CO2-buffered incubator for real-time bioluminescence recording .", "The addition of Recording Medium was sufficient to synchronize the cell population producing coherent luminescence signals .", "For assays evaluating the effects of D3T , tBHQ , H2O2 , and CDDO-Im , each treatment was made in Recording Medium and added to the plates at the time they were loaded into the Lumicycle .", "For treatment-induced amplitude reinforcement assays , plates were removed from the Lumicycle at the indicated time , H2O2 or D3T was added to the existing medium at the times indicated , the plates were resealed with cover slips , and the dishes were loaded back into the Lumicycle .", "We did not observe measurable deviations in bioluminescence independent of treatment following this procedure .", "MMH-D3 Per2:Luc reporter cell lines were provided by Dr . Andrew Liu at the University of Memphis and confirmed to be free from mycoplasma using the MycoSensor PCR Kit ( Agilent ) .", "MMH-D3 hepatocytes were cultured in RPMI1640 medium ( Fisher Scientific , 11875 ) supplemented with 10% fetal bovine serum , 1x penicillin/streptomycin , 10 µg/ml insulin , 55 ng/ml epidermal growth factor , and 16 ng/ml insulin-like growth factor-II to confluence in collagen-coated 35 mm cell culture plates .", "At confluence , the hepatocytes were differentiated by adding 2% ( v/v ) DMSO to the growth medium .", "Differentiation medium was refreshed every other day for 5 days .", "Following differentiation , cells were washed with PBS and serum-free growth medium containing 200 nM dexamethasone was added for synchronization .", "Cells were incubated in dexamethasone-containing medium for 2 hr in a 37°C humidified and CO2-buffered cell culture incubator .", "Following synchronization , cells were washed once with PBS , and freshly made Recording Medium containing 2% FBS , 2% B27 , and 1 mM luciferin was added .", "Plates were sealed with vacuum-coated coverslips and loaded into a Lumicycle 32 for real-time bioluminescence recording .", "For high-throughput luminescence studies , MMH-D3 hepatocytes were cultured in collagen-coated 96-well plates , differentiated , and synchronized as described above .", "Bioluminescence was recorded using a Synergy 2 SL microplate reader ( Bio Tek , Winooski , VT ) , as previously described ( Ramanathan et al . , 2012 ) .", "Liver , lung , and SCN organotypic slices were prepared as previously described ( Liu et al . , 2007b ) .", "Briefly , Wt and Nrf2-/- mice harboring at least one copy of the PER2::LUC transgene were euthanized and dissected to remove the liver , lung , and SCN .", "The liver and lung were washed in ice cold PBS and sliced by hand on ice with a razor blade in approximately 2 mm x 2 mm square pieces .", "Liver and lung slices were washed again with Recording Medium and placed directly into 35 mm culture dishes .", "The SCN sections were washed with ice cold HBSS and sliced using a vibrating microtome .", "SCN slices were then washed with Recording Medium and cultured on Millipore membrane inserts ( Merck Millipore , Billerica , MA , PICM0RG50 ) , in 35 mm cell culture plates .", "Recording Medium ( 900 μL ) was added to dishes containing each tissue slice .", "Dishes were sealed with a vacuum grease-coated cover slip and loaded into a Lumicycle for bioluminescence recordings .", "Bioluminescence data were analyzed using the LumiCycle Analysis software ( Actimetrics , Version 2 . 31 ) .", "Baseline subtraction was performed to detrend traces using a 24 hr moving average .", "Baseline subtracted bioluminescence data from cells and tissues were least-square fit to a damped sine wave from which the amplitude and period were determined as described previously ( Ramanathan et al . , 2014 ) .", "Amplitude and period were determined from data collected between days ~ 1 . 5 and 5 , with the exception of data shown in Figure 4B–F in which amplitude and period were determined following the addition of treatment until the end of the recording .", "All fittings of the data had a goodness of fit >85% for cells and >80% for tissues .", "For cell and tissue slice cultures , any culture that did not produce sustained rhythmic bioluminescence as defined by the inability to fit a sine wave with >80% goodness of fit to the baseline subtracted data was deemed to be an outlier and removed from the analysis .", "Due to transient spikes in luminescence immediately following a change of media , the first 24 hr of recording data was excluded from period length and amplitude determination .", "shNrf2 Per2:Luc MEF cell lines were generated using infectious lentiviral particles .", "Infectious lentiviral particles containing shRNA constructs targeting Nrf2 or an empty vector backbone were purchased from Sigma ( St . Louis , MO , SHC001V , SHCLNV-NM_010902:TRCN0000012128 , Supplementary file 4 ) .", "MEFs were infected at an MOI = 3 , following the manufacturer’s protocol .", "Briefly , viral particles plus polybrene ( 8 µg/ml ) were added to the complete cell culture growth medium 1 day after seeding the MEFs .", "Cells were incubated in viral containing medium overnight in a 37 °C cell culture incubator .", "The next day , the cells were washed with PBS and the medium was changed to complete growth medium without viral particles .", "Two days post-infection , cells were selected with complete growth medium containing 2 µg/ml puromycin .", "Puromycin-containing medium was replaced every other day for 4 days .", "By the fifth day the non-infected control cell population was no longer viable .", "Generation of shNrf2 knockdown MMH-D3 hepatocytes was performed following a similar protocol using viral particles containing either a nonspecific sequence ( Scramble ) or Nrf2 shRNA target sequences ( Supplementary file 4 ) .", "Nrf2 overexpression or genetic rescue cell lines were generated following published protocols ( Ramanathan et al . , 2014; Ramanathan et al . , 2012 ) .", "An Nrf2 lentiviral expression vector was created by amplifying Nrf2 cDNA using primers to install a 5’-CACC leader sequence on the amplicon ( Supplementary file 3 ) .", "The Nrf2 PCR product was then cloned into pENTR/D-TOPO following the manufacturer’s instructions ( Invitrogen , Carlsbad , CA , K2400 ) .", "The resulting pENTR-Nrf2 vector and a pENTR-CMV promoter vector were moved into a pLV7-Puro destination vector ( Liu et al . , 2008 ) through a multisite Gateway recombination reaction to generate the pLV7-CMV-Nrf2 lentiviral expression vector .", "Viral particles were generated following standard protocols in 293 T cells , as described previously ( Tiscornia et al . , 2006 ) with the exception that the transfection of 293T was performed using Lipofectamine 2000 ( Invitrogen ) in place of the calcium phosphate method described .", "Viral titers were determined by p24 ELISA assay using the Lenti-X p24 Rapid Titer Kit ( Clontech , Mountain View , CA , 632200 ) .", "Viral titers of >107 were normally achieved .", "Cells were infected at an MOI = 3–5 and selected with 2 µg/ml puromycin .", "To isolate RNA from mouse liver approximately 500 mg of tissue was homogenized in 1 mL RNA STAT-60 ( Tel-Test , Friendswood , TX ) .", "RNA was isolated from cells by adding RNA STAT-60 directly to the cell culture plate .", "In both instances , RNA was extracted from the STAT-60 solution by the addition of chloroform:isoamyl alcohol ( 24:1 ) .", "Extracted RNA was dissolved in water , reprecipitated using sodium acetate and isopropanol , washed with 75% ethanol , and quantified using a Nanodrop .", "For qPCR measurement of RNA transcripts , 1 µg of RNA was reverse transcribed to cDNA .", "6 ng of cDNA was mixed with forward and reverse primers for the intended gene target ( Supplementary file 5 ) and ABsolute Blue SYBR Green qPCR master mix ( Thermo Fisher Scientific ) .", "Gene expression measurements were normalized to the expression of the endogenous reference gene , Gapdh , which not affected by any of the treatments or genetic manipulations .", "Cells grown to 100% confluence were washed with ice cold PBS and harvested by scraping in cold PBS .", "Cell pellets were lysed in RIPA buffer [25 mM Tris pH 7 . 4 , 150 mM NaCl , 0 . 1% SDS , 0 . 5% sodium deoxycholate , 1% Triton X-100 , and freshly added Protease Inhibitor Cocktail ( Sigma ) , 1 mM phenylmethylsulfonyl fluoride , and 1 mM Na3VO4] .", "Lysates were cleared by centrifugation at 13 , 000 g , 10 min , 4°C .", "Total protein was quantified by Micro BCA Assay ( Thermo Fisher ) .", "50 µg of total protein was resolved on 10% SDS polyacrylamide gels .", "For all western assays , protein was transferred to PVDF membranes .", "Antibodies used are as follows: rabbit anti-NRF2 ( Santa Cruz , SC-722; Proteintech , 16396 ) ; rabbit anti-NQO1 ( Abcam , AB80588 ) ; rabbit anti-NR1D1 ( Cell Signaling , 13418 ) .", "Following incubation with primary antibody , blots were incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies for 1 hr at room-temperature .", "Clarity ( Bio-Rad , Hercules , CA ) substrate was used for chemiluminescent detection using a ChemiDoc ( Bio-Rad ) .", "Wt and Nrf2-/- Per2:Luc MEFs were cultured to confluence in complete growth medium .", "Cells were washed once with PBS and changed into serum-free growth medium containing 200 nM dexamethasone and incubated for 2 hr in a standard cell culture incubator for synchronization .", "Following synchronization , cells were washed once with PBS and complete growth medium was added ( t = 0 ) .", "Cells were harvested at the indicated times post-synchronization as described above .", "MMH-D3 hepatocytes were cultured , differentiated , and synchronized following the protocol described for the bioluminescence assays .", "Following synchronization , Recording Medium was added to begin the timecourse .", "Samples were harvested every 4 hr for 52 hr .", "For all timecourse assays , 50 μg of total protein extract was resolved on 10% polyacrylamide gels and transferred to PVDF membranes .", "Membranes were incubated with rabbit anti-PRDX-SO2/3 ( Thermo LF-PA0004 ) ; rabbit anti-PER2 ( Proteintech , 20359 ) ; rabbit anti-NRF2 ( Santa Cruz , SC-722 ) ; and rabbit anti-NR1D1 ( Cell Signaling , 13418 ) .", "ChIP-PCR was performed as described previously ( Sutter et al . , 2009 ) .", "Lysates from 2 × 150 mm dishes were sonicated in 1 ml of lysis buffer ( 1% SDS , 10 mM EDTA , 50 mM Tris-HCl , ph 8 ) using the Covaris S220 ( 35 min , 147 Watts , 200 cycle/burst , duty factor of 5 ) and tubes containing an AFA fiber ( Covaris , Woburn , MA ) .", "NRF2 was immunoprecipitated from the lysate using rabbit anti-NRF2 antibodies ( Diagenode , Denville , NJ , C15410242-100; Santa Cruz , SC-722 ) .", "Genomic DNA sequence 3 . 5 kb upstream and 1 kb downstream of the Cry2 , Nr1d1 , and Nqo1 transcriptional start sites were analyzed for putative ARE elements using a position weight matrix implemented in JASPAR ( Mathelier et al . , 2016 ) .", "ARE sequences characterized by JASPAR in the mouse genome reference GRCm38 . p4 are located in Supplementary file 2 .", "Primers were designed ( Supplementary file 1 ) to flank the putative ARE sequences with a relative profile score of 79% or better .", "HEK293T cells were grown in DMEM high-glucose ( Fisher Scientific ) medium supplemented with 10% fetal bovine serum and 1x penicillin/streptomycin .", "HEK293T cells seeded in six well plates were transfected with 25 ng/well pLenti-Nrf2 ( OriGene , Rockville , MD , MR226717L1 ) using Lipofectamine 2000 following the manufacturer’s protocol and incubated overnight at 37°C in a CO2-buffered incubator .", "Following overnight incubation , the transfection medium was replaced with fresh growth medium .", "48 hr post-transfection untransfected and transfected cells were harvested in RNA STAT-60 .", "RNA was extracted , reverse transcribed , and used in a qPCR reaction to amplify either Cry2 or Nqo1 as described above .", "For in vitro transcription repression assays , approximately 15 , 000 cells/well were seeded in a 384-well plate .", "Cells in every well were transfected with 12 . 5 ng of either pGL3-P ( Per1 ) -dLuc or pGL3-3xEbox-P ( SV40 ) -dLuc reporter constructs , as indicated , and 2 . 5 ng of a phRL-SV40 plasmid expressing Renilla luciferase using Lipofectamine 2000 following the manufacturer’s protocol .", "25 ng of each of the following expression plasmids were cotransfected as indicated: pLV7-P ( CMV ) -Nrf2 ( Figure 6E ) , pLenti-Nrf2 ( Figure 6G ) , pLV7-P ( CMV ) -Bmal1 , pLV7-P ( CMV ) -Clock , pCMV10-P ( CMV ) -Cry1 , or pCMV10-P ( CMV ) -Cry2 .", "Empty pCMV10 vector was used to normalize the total amount of DNA/well to 90 ng .", "48 hr post-transfection , cells were harvested and assayed with the Dual-Glo Luciferase Assay System ( Promega , Madison , WI ) .", "Firefly luminescence was normalized to Renilla luminescence as an internal control for transfection efficiency ." ] ]

[ "Diurnal oscillation of intracellular redox potential is known to couple metabolism with the circadian clock , yet the responsible mechanisms are not well understood .", "We show here that chemical activation of NRF2 modifies circadian gene expression and rhythmicity , with phenotypes similar to genetic NRF2 activation .", "Loss of Nrf2 function in mouse fibroblasts , hepatocytes and liver also altered circadian rhythms , suggesting that NRF2 stoichiometry and/or timing of expression are important to timekeeping in some cells .", "Consistent with this concept , activation of NRF2 at a circadian time corresponding to the peak generation of endogenous oxidative signals resulted in NRF2-dependent reinforcement of circadian amplitude .", "In hepatocytes , activated NRF2 bound specific enhancer regions of the core clock repressor gene Cry2 , increased Cry2 expression and repressed CLOCK/BMAL1-regulated E-box transcription .", "Together these data indicate that NRF2 and clock comprise an interlocking loop that integrates cellular redox signals into tissue-specific circadian timekeeping ." ]

[ "Like many other animals , our behavior often follows a familiar pattern each day .", "We tend to wake up with the morning sunlight and start by eating breakfast to satisfy our hunger .", "Then , at night , most of us sleep , and our bodies use chemical building blocks from the day's meals to replenish and repair our tissues .", "As such , its not just our behavior that shows a daily cycle .", "The countless chemical reactions that keep us alive , collectively referred to as our metabolism , also change over the course of each day .", "Daily patterns of activity , known as circadian rhythms , can be seen even at the level of individual cells .", "Each cell in the body has its own molecular clock that works by rhythmically cycling the levels of different molecules .", "Proteins called CLOCK and BMAL1 trigger the production of proteins PER and CRY .", "As levels of PER and CRY rise , they interfere with CLOCK and BMAL1 , essentially switching off their own production .", "Then , levels of PER and CRY fall and the cycle starts again .", "Thus , these molecules rise and fall throughout the day to drive circadian rhythms , similar to how a pendulum swings back and forth to keep a clock ticking .", "Metabolism and circadian rhythms are clearly linked , but it is not well understood how this works .", "Now , Wible , Ramanathan et al . identify a protein called NRF2 as an important bridge between the molecular clock and metabolism .", "NRF2 is a activated by hydrogen peroxide , a byproduct of cell metabolism , and when activated this protein grabs hold of DNA to increase the activity of specific genes .", "A combination of experiments revealed that mouse liver cells need NRF2 to maintain a normal circadian rhythm , and a closer inspection of the liver cells revealed that NRF2 specifically attaches to part of the gene for a clock protein called CRY2 .", "This enhances the production of this protein , which in turn , switches CLOCK and BMAL1 off .", "In this way , NRF2 links metabolism signals to the ticking of the circadian clock .", "Previous research has shown a link between shift work , which disrupt these rhythms , and metabolic diseases like obesity and diabetes .", "Understanding more about the underlying molecular biology that links metabolism to circadian rhythms could help scientists to find new ways to improve public health ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "structural biology and molecular biophysics", "microbiology and infectious disease" ]

[ [ "Human immunodeficiency virus ( HIV ) replication depends on the coordinated expression of viral proteins from fully spliced , partially spliced , and unspliced forms of its RNA genome .", "Only fully spliced RNAs encoding viral regulatory proteins initially reach the cytoplasm for translation ( Feinberg et al . , 1986 ) .", "Later in the life cycle , the regulatory protein Rev directs the nuclear export of unspliced and partially spliced viral RNAs by engaging the Crm1 nuclear export pathway typically used to transport host protein cargoes and small RNAs ( Sodroski et al . , 1986; Emerman et al . , 1989; Felber et al . , 1989; Kohler and Hurt , 2007; McCloskey et al . , 2012 ) .", "Rev coopts Crm1 to export viral RNAs by bridging the interaction between Crm1 and the Rev Response Element ( RRE ) , a structured RNA located within an intron of unspliced and singly-spliced viral RNAs ( Figure 1A ) .", "The amino terminal portion of Rev recognizes the RRE through an arginine rich motif flanked by hydrophobic domains that facilitate Rev oligomerization on the RRE ( Malim and Cullen , 1991 ) .", "The Rev nuclear export sequence ( NES ) , located near the carboxy terminus , recruits Crm1 in cooperation with Ran bound to GTP ( RanGTP ) to elicit nuclear export of viral RNAs ( Fischer et al . , 1995 ) . 10 . 7554/eLife . 04121 . 003Figure 1 . A dimer of Crm1-RanGTP binds the Rev-RRE complex .", "( A ) Rev assembles on the Rev Response Element ( RRE ) ( step 1 ) and presents nuclear export sequences ( NESs ) for Crm1 to recognize in conjunction with RanGTP ( step 2 ) .", "The assembled particle passes through the nuclear pore complex ( step 3 ) to the cytoplasm where Ran GTP hydrolysis stimulates the dissociation of Rev-RRE from Crm1 .", "( B ) The gel shows the protein composition from affinity purifications of GB1-Rev incubated in the presence ( + ) or absence ( − ) of the RRE , Crm1 , and Ran .", "T or D denotes Ran exchanged with GTPγS or GDP , respectively .", "RevM10 is a mutation within the NES of Leu78Asp and Glu79Leu ( Malim et al . , 1989 ) .", "The gel is representative of three independent experiments .", "( C–D )", "Reconstituted Rev-RRE particles preferentially bind two copies of Crm1 and RanGTP .", "( C ) Size-exclusion chromatograms of Rev-RRE with increasing molar ratios of Crm1 and RanQ69L show saturation at a molar ratio of two Crm1 per RRE .", "Excess Crm1 eluted as free monomers ( 9 . 49 ml fraction ) or in a small population of larger complexes ( 6 . 76 and 7 . 02 ml fractions ) .", "Molecular masses were determined using multi-angle laser light scattering after size-exclusion chromatography and have a technical error of three percent .", "( D ) The composition of peak fractions was analyzed by SDS-PAGE for proteins and by Urea-PAGE for RNA .", "These results are representative of two independent experiments .", "( E–F )", "Negative-stain electron micrographs show two copies of Crm1 in complexes reconstituted with Rev-RRE and RanGTP .", "Representative fields and class averages of 7196 nuclear export particles ( E ) and 6445 Crm1 molecules alone ( F ) are shown . DOI: http://dx . doi . org/10 . 7554/eLife . 04121 . 00310 . 7554/eLife . 04121 . 004Figure 1—figure supplement 1 . Assessment of the stoichiometry of Crm1 , RanGTP , Rev , and the RRE .", "( A ) Size-exclusion chromatograms of the reconstituted complex ( top ) , Rev-RRE ( second from top ) , Crm1 ( third from top ) , or RanGTP ( bottom ) with indicated molecular masses ( MW ) determined by multi-angle laser light scattering after size exclusion chromatography .", "The RRE has a mass of 80 . 172 kD and each Rev subunit has a mass of 13 . 179 kD , so the total 159 kD mass of the complex corresponds to six Rev subunits per RRE .", "( B–C )", "Determination of stoichiometry based on protein standards .", "( B ) Size-exclusion chromatograms from Figure 2E of human or murine Crm1 assembled with Rev-RRE and RanGTP with fractions used for determining stoichiometry marked with an asterisk .", "( C ) An SDS-PAGE gel stained with Sypro-Ruby showing a dilution series of protein standards , which were quantified by amino acid analysis , and indicated fractions from the chromatograms in panel B . ( D ) Quantification of ( C ) and the calculated stoichiometry of the complex .", "The RRE concentration was determined by UV spectrometry and used to determine the molar ratios for proteins .", "These data indicate that this reconstitution had slightly sub-stoichiometric amounts of Crm1 and RanGTPand six subunits of Rev bound to the RRE , with a small population having slightly more Rev bound . DOI: http://dx . doi . org/10 . 7554/eLife . 04121 . 004 A set of structures detail the architecture of the RRE ( Fang et al . , 2013 ) , Rev binding to a single sites in the RRE ( Battiste et al . , 1996 ) , Rev oligomerization ( Daugherty et al . , 2010b; DiMattia et al . , 2010 ) , and the Crm1-NES interaction ( Güttler et al . , 2010 ) , but the organization of the entire export complex has remained unknown .", "Without a complete structure , it is unclear , for example , how HIV modulates Rev-RRE activity through mutations in the RRE that change the RNA fold yet minimally affect Rev binding ( Legiewicz et al . , 2008; Sloan et al . , 2013 ) or how subtle amino acid substitutions between nearly identical orthologs of Crm1 restrict HIV replication in different species ( Nagai-Fukataki et al . , 2011; Sherer et al . , 2011; Elinav et al . , 2012 ) .", "Here we report the overall architecture of the Rev-RRE/Crm1-RanGTP export complex using single-particle electron microscopy ( EM ) .", "Unexpectedly , Rev-RRE forms a complex with an ordered Crm1 dimer , unlike previously characterized cargoes that bind a Crm1 monomer ( Dong et al . , 2009 ) .", "Residues unique to simian primates form a dimerization interface between Crm1 monomers that is seen as a crystal contact in the structure of human Crm1 ( Dong et al . , 2009 ) .", "It appears that RRE-directed remodeling of the Rev oligomer ( Jayaraman et al . , 2014 ) may organize the Rev NESs to enhance avidity for the Crm1 dimer and help tune its export activity .", "It will be interesting to determine if some cellular protein-RNA complexes may also utilize Crm1 dimerization to tune nuclear export ." ], [ "In order to define the arrangement of HIV RNA export complexes for structural studies , we first determined the biochemical requirements for complex formation using recombinant Crm1 , Ran , Rev tagged at the amino-terminus with a GB1 domain ( GB1-Rev ) , and a 245 nt portion of RRE RNA transcribed in vitro .", "Crm1 co-purified with GB1-Rev in the presence of both RanGTP and the RRE ( Figure 1B , lane 11 ) , but showed only a weak interaction with RanGDP or without the RRE ( compare lane 11 to lanes 12 and 9 ) .", "The interaction between Crm1 and Rev requires a functional NES , as GB1-Rev bearing a dominant-negative M10 mutation in the NES , Leu78Asp and Glu79Leu ( Malim et al . , 1989 ) , did not bind Crm1 under any condition ( lanes 17–18 and 21–24 ) .", "These results mirror in vivo and in vitro experiments showing that Rev uses an intact NES to bridge the interaction between the RRE and Crm1 utilizing RanGTP ( Fischer et al . , 1995; Fornerod et al . , 1997; Askjaer et al . , 1998 ) .", "Furthermore , our data directly show for the first time that the RRE enhances the Rev-Crm1 interaction , supporting the hypothesis that the Rev NES must be recognized in the proper Rev-RRE context to function ( Askjaer et al . , 1999 ) .", "Previously , we demonstrated that the RRE assembles six subunits of Rev into discrete , asymmetric complexes with a total mass of 160 kD ( Daugherty et al . , 2010a ) .", "We used size-exclusion chromatography to further determine if these Rev-RRE complexes can associate with Crm1 and RanQ69L , a mutant that stabilizes the GTP-bound form of Ran ( Bischoff et al . , 1994 ) .", "When increasing amounts of Crm1-RanQ69L were added to a fixed concentration of Rev-RRE , all four components eluted in a 462 kD fraction ( Figure 1C , D ) , corresponding to two copies of Crm1 and RanQ69L per Rev-RRE complex ( Figure 1—figure supplement 1 ) .", "In fact , negatively stained electron micrographs of complexes from this fraction clearly show two Crm1 molecules per single particle , compared to images of Crm1 monomers alone ( Figure 1E , F ) .", "A Crm1 dimer explains the need for at least two NESs in the Rev oligomer to function ( Hoffmann et al . , 2012 ) , and it may also be important for efficiently transporting the RRE through the nuclear pore complex since the number of receptors required for nuclear transport scales with the size of the cargo ( Ribbeck and Görlich , 2002 ) .", "A Crm1 monomer is thought to use a protein adaptor to export relatively small , structured RNAs ( McCloskey et al . , 2012 ) .", "In contrast , the RRE is almost twice the size of host RNA cargoes , is embedded in a much larger mRNA , and is bound to the Rev oligomer .", "Previous structures of Crm1 bound to host cargo proteins have shown monomeric binding , so we sought to understand how Rev-RRE organizes a Crm1 dimer .", "We utilized single-particle negative-stain EM and a random conical tilt method ( Radermacher , 1988 ) to generate a 3D reconstruction of the Crm1-RanGTP/Rev-RRE complex ( Figure 2—figure supplement 1 ) .", "We recorded two images of the same sample area , one tilted to 60° and one at 0° , and assigned particles from the 0° images into classes .", "We then calculated 3D reconstructions for each class using corresponding particle images from the 60° tilted specimen .", "All reconstructions produced the same structure but in different orientations , permitting us to align and then average the classes to generate a final structure of the complex at ∼25 Å resolution .", "In this reconstruction , we observed two connected bowl-shaped densities , reminiscent of the known Crm1 morphology ( Dolker et al . , 2013 ) , arranged with twofold symmetry .", "To place Crm1 into the reconstruction , we initially fit the crystal structure of two murine Crm1-RanGTP monomers ( Güttler et al . , 2010 ) into the EM density ( Figure 2—figure supplement 2 ) , even though our complex was assembled with the human receptor , because RanGTP is absent in the human Crm1 crystal structure ( Dong et al . , 2009 ) and is known to bind on the interior of Crm1 and stabilize a strained conformation that opens the hydrophobic NES binding cleft ( Monecke et al . , 2009 , 2013 ) .", "Remarkably , seven residues specific to simian primates that lie outside the NES-binding site and are known to be important for Rev function and HIV biogenesis ( Nagai-Fukataki et al . , 2011; Sherer et al . , 2011; Elinav et al . , 2012 ) were symmetrically apposed in this arrangement of the Crm1 dimer .", "This fitting suggested that the species-specific residues may form a protein–protein interface between the Crm1 monomers , and indeed , we discovered that this same interface formed a crystal contact between human Crm1 monomers in the crystal structure bound to Snurportin1 ( Dong et al . , 2009 ) .", "We further found that a Crm1 dimer held together by this symmetric interface unambiguously fit as a rigid body into the EM map ( Figure 2A ) . 10 . 7554/eLife . 04121 . 005Figure 2 . Species-specific residues define a Crm1 dimerization surface that enhances Rev-RRE binding .", "( A ) A dimer of human Crm1 extracted from a unit cell of a Crm1-Snurportin1 complex that lacks RanGTP ( PDB 3GB8 ) was fit into the EM reconstruction ( grey envelope ) of Crm1 and RanGTP bound to Rev-RRE ( Figure 2—figure supplement 2 ) .", "Residues that differ between murine and human Crm1 that enhance Rev-RRE activity ( Sherer et al . , 2011; Elinav et al . , 2012 ) are shown in gold and form an interface between two Crm1 monomers ( detailed in panel B and Figure 2—figure supplement 3 ) .", "Two NES binding sites poised to engage two Rev NES peptides are shown in cyan .", "( C–D )", "Human Crm1 recognizes Rev-RRE with higher affinity and increased cooperativity compared to its murine ortholog , shown by gel mobility shift assays with radiolabeled RRE , a molar excess of Rev , and increasing concentrations of Crm1-RanGTP .", "The Hill equation or a version of the McGhee-von Hippel model ( Epstein , 1978 ) was fit to the quantified data from three independent experiments ( R2 > 0 . 99 for all models compared to average values ) as shown in ( D ) and described in ‘Materials and methods’ .", "Asterisks denote *p < 0 . 05 and **p < 0 . 01 from one-tailed t-tests .", "A schematic model of the complex is shown to highlight the physical meaning of the McGhee-von Hippel parameters .", "( E ) Size-exclusion chromatograms of Rev-RRE complexes assembled with murine Crm1 or human Crm1 show that murine Crm1 elutes with a larger apparent mass , presumably due to a larger hydrodynamic radius in the absence of Crm1-Crm1 interactions .", "( F ) Class averages of Rev-RRE complexes bound to a dimer of human ( 426 particles ) or murine ( 617 particles ) Crm1 from negatively-stained micrographs of particles from ( E ) .", "Murine Crm1 particles showed a wider range of sizes than the human particles , with most being larger , and the murine particles showed different features , such as an hourglass shape with fewer details in the center of the particle than human Crm1 particles .", "Scale bars show 10 nm . DOI: http://dx . doi . org/10 . 7554/eLife . 04121 . 00510 . 7554/eLife . 04121 . 006Figure 2—figure supplement 1 . Random conical tilt reconstruction and refinement .", "( A ) Representative fields from a random conical tilt pair of particles assembled with Rev-RRE , Crm1 , and RanGTP .", "( B ) Class averages from untilted particles used for random conical tilt .", "Particle numbers are inset in each image .", "Classes six and seven were observed with lower concentrations of sample and resemble partially disassembled complexes .", "Scale bars show 10 nm .", "( C ) Volumes from each class after random conical tilt refinement show similar features among all classes suggesting the classes are different views of the same particle .", "The volume from the final reconstruction is shown in ( D ) .", "( E ) Fourier shell correlation curves for each random conical tilt class and the final reconstruction .", "The boost in Fourier shell correlation when C2 symmetry is applied during refinement confirms the symmetric arrangement of bowl-like densities .", "( F ) Angular distribution from projection matching shows no major gap in Euler space . DOI: http://dx . doi . org/10 . 7554/eLife . 04121 . 00610 . 7554/eLife . 04121 . 007Figure 2—figure supplement 2 . Comparison of fitting murine Crm1 or human Crm1 crystal structures in the EM reconstruction .", "( A ) Two murine Crm1-RanGTP ( PDB 3NC0 ) monomers individually fit well ( Correlation ( R ) = 0 . 7526 or 0 . 7393 ) into two bowl-like densities from the reconstruction of Crm1-RanGTP bound to Rev-RRE .", "The final fit places residues different between murine and human Crm1 ( gold ) to potentially form an interface between two Crm1 monomers .", "( B ) From the fit of murine Crm1 , we inferred that the same residues might form a crystal contact between Crm1 monomers in the crystal structure of human Crm1 bound to Snurportin-1 ( PDB 3GB8 ) and this , indeed , was observed .", "The fit of the human Crm1 dimer structure is shown ( R = 0 . 8227 ) .", "Although the crystal structures of murine Crm1 either have two Crm1 molecules in the asymmetric unit or other dimers in the unit cell , these alternate dimers use different crystal contacts to build the unit cell and do not have the same conformation as the human Crm1 dimer ( data not shown ) .", "( C ) A hybrid model of the Crm1 dimer bound to RanGTP .", "The human Crm1 interface was used to orient two copies of murine Crm1 bound to RanGTP by aligning murine Crm1 with the human Crm1 interface , which shows little deviation between Cα atoms .", "The final model fits well ( R = 0 . 9047 ) into the EM map and was used to search for Rev-RRE density shown in Figure 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 04121 . 00710 . 7554/eLife . 04121 . 008Figure 2—figure supplement 3 . A comparison of species-specific residues at the interface of the Crm1 dimer . Species-specific residues form many interactions in the human Crm1 dimer interface ( A ) but few at the analogous murine positions ( B ) .", "Aligning the Cα atoms from murine and human Crm1 at the dimer interface ( B ) shows steric clashes between side chains near Thr411 in murine Crm1 interface and between the side chains of His352 and Thr487 that would prevent murine Crm1 dimerization .", "( C ) Swapping residues between human and murine Crm1 in the dimer interface alters virus production .", "A summary of data from references ( Sherer et al . , 2011 ) and ( Elinav et al . , 2012 ) testing the effect of murine or human residues on virus-like particle production or infectivity of HIV or FIV vectors shows the biological relevance of the Crm1 interface .", "Sherer et al . substituted human residues into murine Crm1 to determine which residues enhanced virus-like particle production ( Sherer et al . , 2011 ) .", "Elinav et al . substituted mouse residues into human Crm1 and scored for loss of function ( Elinav et al . , 2012 ) .", "In both studies , the gain in activity denoted by ‘h’ is a substitution of all human interface residues into murine Crm1 and vice versa for the loss of activity denoted by ‘m’ .", "Data were normalized according to the following equations: Gain of Function = ( mutant activity−murine Crm1 activity ) / ( human Crm1 activity−murine Crm1 activity ) and Loss of Function = Gain of function−1 . DOI: http://dx . doi . org/10 . 7554/eLife . 04121 . 008 This Crm1 dimer interface was not previously noted because Crm1 was known to bind the host Snurportin1 cargo as a monomer and was seen as a monomer in the asymmetric unit of the crystal .", "Subsequent studies interpreted species-specific residues in the context of the Crm1 monomer structure ( Nagai-Fukataki et al . , 2011; Sherer et al . , 2011; Elinav et al . , 2012 ) .", "Based on steric considerations from the structure of a Rev oligomer , our lab previously proposed a ‘jellyfish’ model in which a Rev hexamer with dangling unstructured NES peptides could accommodate no more than two Crm1 subunits ( Daugherty et al . , 2010b ) .", "As described below , our data now suggest that Crm1 forms an intimate dimer with Rev-RRE bridging across the interface , permitting us to propose a more comprehensive model of the export complex .", "A detailed examination of the Crm1 dimer reveals an extensive interface that buries a total surface area of 2170 Å2 in the crystal structure ( Figure 2B ) and corresponds to functionally important residues ( Figure 2—figure supplement 3 ) .", "Central to the interface are symmetric hydrogen bonds between Arg474 and Glu478 across the dimer ( 2 . 9 Å donor to acceptor; Figure 2B ) and , indeed , substituting these residues decreases Rev function ( Elinav et al . , 2012 ) .", "The surrounding interactions are largely hydrophobic: Phe414 fills a cavity between Glu478 and His481 from the opposite subunit while Pro411 , Met412 , and Met 348 interact with the apposing Val484 .", "Consistently , swapping murine residues for Pro411 , Met412 , and Phe414 results in the largest reduction in activity ( Sherer et al . , 2011; Elinav et al . , 2012 ) .", "The ends of the interface near Thr346 utilize Glu345 to form a salt bridge with Lys531 ( 2 . 8 Å ) and hydrogen bond with Gln530 ( 2 . 8 Å ) on the other monomer .", "The sum of these interactions enables human Crm1 to form an ordered dimer interface upon Rev-RRE binding .", "To assess the importance of this interface for Rev-RRE recognition , we compared the binding of murine and human Crm1 to Rev-RRE complexes using gel mobility shift assays ( Figure 2C ) , which were quantified and fit with a standard Hill binding model ( Figure 2D ) .", "The apparent binding constant for human Crm1 to Rev-RRE was slightly , yet significantly ( p < 0 . 01 ) , higher than for murine Crm1 ( K1/2 = 297 ± 30 nM vs 450 ± 43 nM ) , and the human protein achieved higher maximal saturation ( M = 0 . 96 ± 0 . 010 vs 0 . 90 ± 0 . 016 , p < 0 . 01 ) .", "An interesting but puzzling difference also was observed in the cooperativity term , as measured by the Hill coefficient ( h = 2 . 49 ± 0 . 02 vs 1 . 96 ± 0 . 40 , p < 0 . 05 ) .", "For human Crm1 , the Hill coefficient exceeded the dimeric stoichiometry of the complex , which—while unusual—is not unprecedented ( Atkinson , 1966 ) .", "As an initial interpretation , we considered that the Hill coefficient of two with murine Crm1 reflected the stoichiometry of the complex while the higher value with human Crm1 might also include an additional component of cooperativity resulting from the arrangement of the Crm1 dimer .", "Overall , the result of these small differences between the human and murine proteins is comparable to the fourfold enhancement in nuclear export activity seen when human Crm1 is expressed in murine cells ( Sherer et al . , 2011; Elinav et al . , 2012 ) .", "Because fitting to the Hill model yielded somewhat atypical parameters , we explored whether an alternative model , which describes how large ligands bind to a linear array of sites ( McGhee and von Hippel , 1974 ) , might better explain the data .", "This model has typically been used to analyze protein binding to DNA sites , which may resemble how Crm1 binds to the array of NES peptides in the Rev-RRE complex .", "According to this model , the affinity between the ligand and each binding site ( Kd ) would describe Crm1 binding to one Rev NES , the number of binding sites that a large ligand occludes ( n ) would reflect the number of Rev sites Crm1 obscures due to its large size , and cooperativity between ligands ( ω ) would be reflected by ω > 1 , indicating an interaction between Crm1 monomers ( Figure 2D ) .", "In this model , human and murine Crm1 both have similar affinities for an individual NES ( Kd ≈ 6–8 µM ) but human Crm1 shows more than 2-fold higher cooperativity ( ω = 27 . 8 ± 3 . 4 vs 12 . 1 ± 3 . 8 , p < 0 . 01 ) .", "It is common for ω values to be quite large if complexes have extensive protein–protein interfaces , such as the binding of RecA filaments to DNA , with ω ≈ 50 ( Menetski and Kowalczykowski , 1985 ) .", "The relative ω values for Crm1 suggest a tighter interaction between the human Crm1 monomers , but the absolute value of ω > 1 for murine Crm1 still suggests some interaction between monomers .", "Indeed , murine Crm1 complexes with Rev-RRE elute from size-exclusion columns with an even larger apparent size than with human Crm1 ( Figure 2E ) and display a different , elongated , hour-glass shape in class averages of negatively-stained particles ( Figure 2F ) , suggesting that these differences in the dimer complexes may reflect observed differences in Rev-RRE binding and export activities .", "Both human and murine Crm1 occlude three binding sites ( Figure 2D ) , consistent with the stoichiometry of a Rev hexamer harboring six NESs binding to only a dimer of Crm1 ( Daugherty et al . , 2010b ) .", "These binding analyses also suggest that the Crm1 dimer interface helps promote cooperative assembly of the complex .", "The stabilization of the Rev-RRE export complex imparted by the human Crm1 dimer interface may explain how human Crm1 enhances oligomerization of a Rev-like protein from Human T-cell Leukemia Virus-1 ( Hakata et al . , 2003 ) or why it enhances the nuclear export activity of Rev for other lentiviruses , such as feline immunodeficiency virus ( Elinav et al . , 2012 ) .", "The micromolar affinities for Crm1 binding to an individual NES calculated with the McGhee-von Hippel model are consistent with previous reports ( Paraskeva et al . , 1999; Güttler et al . , 2010 ) and underscore the importance of the RRE for scaffolding the Rev oligomer to promote the interaction with Crm1 .", "For example , murine Crm1 , with apparently weak or no interaction between Crm1 monomers , still binds the Rev-RRE complex with an order of magnitude greater affinity than to a single NES ( K1/2 = 450 nM vs Kd = 5910 nM ) .", "This result may indicate that the increased local concentration of NESs in the Rev-RRE complex enhances Crm1 binding and is consistent with our observations in Figure 1B where the RRE was required for Rev to strongly bind Crm1 .", "Furthermore , a recent study has shown that increasing the local concentration of NESs in the Rev-RRE complex by appending additional NESs to Rev overcomes the defect in murine Crm1 ( Aligeti et al . , 2014 ) .", "Overall , the combined contributions from the RRE scaffolding the Rev oligomer and the interaction between Crm1 monomers cooperatively assemble the whole complex for nuclear export and may strongly influence the disassembly of the particle in the cytoplasm to regulate downstream steps for translating viral proteins and virion assembly ( Yedavalli et al . , 2004; Nagai-Fukataki et al . , 2011; Elinav et al . , 2012 ) .", "To reveal how the Rev-RRE particle docks onto the export complex , we fit a dimer of Crm1 with bound RanGTP ( Figure 2—figure supplement", "2 ) into the EM map and then searched for extra density .", "Such extra density , which sits on one side of the Crm1 dimer between the two NES binding sites , is expected to correspond to bound Rev-RRE ( Figure 3A , B ) .", "The location of this density sharply contrasts with how a host cargo , Snurportin1 , binds to monomeric Crm1 , where the main docking surface is on the opposite side of Crm1 ( Figure 3C ) .", "Yet , the observed density is too small to fully accommodate a Rev hexamer bound to RNA , likely reflecting the poor visualization of RNA in uranyl-stained images as well as conformational heterogeneity ( Figure 3—figure supplement", "1 ) arising from the disordered carboxy-terminus of Rev where the NES is located ( Daugherty et al . , 2010 ) . 10 . 7554/eLife . 04121 . 009Figure 3 . Rev-RRE binds between NES binding sites on the Crm1 dimer .", "( A–B )", "A model of the Crm1-RanGTP dimer displays extra density ( red ) on one side of the structure that locates the Rev-RRE complex between the Rev NESs bound to each Crm1 ( green ) .", "The Crm1-RanGTP dimer model was built by aligning two murine Crm1-RanGTP complexes onto the human Crm1 dimer .", "( C ) A rotated view of the structure showing the location of Snurportin1 bound to a Crm1 monomer on the opposite side of the protein .", "( D ) Class averages of particles containing Rev tagged with GB1 or RRE tagged with streptavidin , and cryo-EM of the complex without any tags , confirm the location of Rev-RRE .", "Raw micrographs , class averages , and particle numbers are shown in Figure 3—figure supplement 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 04121 . 00910 . 7554/eLife . 04121 . 010Figure 3—figure supplement 1 . Heterogeneity within class averages reveals Rev-RRE density . Particles collected from an independent data set were aligned to references from random conical tilt class averages ( A ) , and 1150 particles from one class were sub-classified using a reference-free alignment ( B ) .", "Some of the sub-classes further reveal extra densities ( arrows ) at the Rev-RRE location .", "Scale bars show 10 nm . DOI: http://dx . doi . org/10 . 7554/eLife . 04121 . 01010 . 7554/eLife . 04121 . 011Figure 3—figure supplement 2 . Localization of Rev-RRE density . Representative fields and class averages from negative stain ( A–B ) or cryo-electron micrographs ( C ) show extra density from Rev-RRE when Rev is tagged with GB1 ( A , 794 particles ) or the RRE tagged with Streptavidin ( B , 383 particles ) .", "Extra density from Rev-RRE also is apparent in cryo-EM images of complexes that do not have any additional tag ( C , 3262 particles ) , where the contrast is generated from the particles themselves rather than from uranyl ions that typically obscure nucleic acids .", "Scale bars show 10 nm . DOI: http://dx . doi . org/10 . 7554/eLife . 04121 . 011 To be certain of the locations of Rev and the RRE in the particle , we compared class averages of the complex to those formed either with GB1-tagged Rev or with streptavidin-tagged RRE ( Figure 3D ) .", "Despite heterogeneity arising from flexible linkers to the tags and from different conformations within each class , additional density was plainly visible in both tagged complexes ( Figure 3—figure supplement", "2 ) with Rev and the RRE unambiguously positioned on one side of the Crm1 dimer .", "Class averages from cryo-electron micrographs , where RNA staining is not an issue , also show extra density in the same location ( Figure 3D ) .", "Together these data indicate that the positioning of NES sites constrains Rev-RRE to flexibly dock on one side of the Crm1 dimer ." ], [ "This work provides the first glimpse of an assembled HIV nuclear export complex , involving an essential HIV-host interaction between the Rev-RRE complex and Crm1 .", "Most surprisingly , we revealed the existence of a Crm1 dimer , whose details could be inferred from crystal contacts observed in an existing Crm1 structure ( Dong et al . , 2009 ) .", "The ability to assemble these complexes in vitro provides a good starting point to investigate how additional host proteins may work with Crm1 during viral RNA trafficking .", "The Rev-RRE particle is clearly positioned at the Crm1 dimer interface although the extent of the interface between Rev-RRE and Crm1 is unclear beyond the Rev NES interaction because there was insufficient resolution to precisely place Rev-RRE into the reconstructed complex .", "In addition to poor staining of the RNA , heterogeneity of the Crm1/Rev-RRE interaction contributes to the lack of distinct density for Rev-RRE and presumably results from multiple arrangements of the complex as well as peptide flexibility surrounding the Rev NESs .", "Furthermore , models of Rev oligomers using combinations of Rev dimer structures free or bound to RNA suggest that a range of oligomer conformations can interface with the Crm1 dimer ( Jayaraman et al . , 2014 ) .", "Determining which conformations are present in the entire export complex will require higher resolution structures .", "Because a portion of the RRE stabilizes an alternate conformation of the Rev dimer ( Jayaraman et al . , 2014 ) and the whole RRE directs Rev oligomerization ( Pond et al . , 2009; Fang et al . , 2013; Bai et al . , 2014 ) , we anticipate that the RNA modulates the conformation of the Rev oligomer and consequently its interaction with the Crm1 dimer .", "Indeed , we observe that the RRE enhances the interaction with Crm1 , probably reflecting the high local concentration of Rev NESs promoted by the RRE scaffold .", "If the RRE stabilizes distinct conformations of the Rev oligomer ( Jayaraman et al . , 2014 ) , then the volume occupied by the Rev NES in those different states could influence the affinity for the Crm1 dimer .", "Such a mechanism could explain how HIV can evolve to fine-tune the levels of nuclear export through changes in the RRE during the course of infection within a single host ( Legiewicz et al . , 2008; Sloan et al . , 2013 ) .", "It is clear that the Crm1 dimer architecture is functionally important for HIV replication since mutations in Crm1 that decrease Rev-dependent export and restrict viral replication correspond to residues that form the interface between Crm1 monomers ( Nagai-Fukataki et al . , 2011; Sherer et al . , 2011; Elinav et al . , 2012 ) .", "The difference between murine and human Crm1 amplifies into a fourfold defect in nuclear export and a similar defect in translation of viral proteins ( Figure 4 ) .", "Overall , these subtle differences in binding lead to a 12-fold increase in virion production when human Crm1 is expressed in mouse cells ( Sherer et al . , 2011 ) , and even these values may underestimate the differences in an endogenous context since in these experiments human Crm1 is overexpressed in cells already containing murine Crm1 . 10 . 7554/eLife . 04121 . 012Figure 4 . Cooperative assembly of a nuclear export complex with human Crm1 enhances nuclear export and virion production . Human Crm1 ( top ) associates with Rev-RRE with higher affinity than murine Crm1 ( bottom ) due to species-specific residues that form the dimer interface ( gold ) .", "This increased association amplifies into even larger differences in nuclear export activity and virion assembly between the two Crm1s . DOI: http://dx . doi . org/10 . 7554/eLife . 04121 . 012 The significance of the Crm1 dimer for Rev-RRE function is underscored by a comparison of Crm1 orthologs ( Sherer et al . , 2011 ) .", "This work proposed an intriguing evolutionary path for Crm1 starting from an ancestral sequence that lacked the full set of residues that we now know form the Crm1 dimer interface .", "The simian primate lineage might have acquired mutations that stabilize the interface , whereas the murine lineage might have accrued mutations that destabilize the interface .", "Even though mouse Crm1 lacks the residues to fully stabilize the Crm1 dimer , the Rev-RRE complex can still recruit two Crm1 subunits , which presumably accounts for its partial function in Rev-mediated export .", "Thus , Rev oligomerization scaffolded by the RRE may have initially allowed ancestral viruses to recruit two Crm1 subunits even if an ancestral form of Crm1 did not have a dimeric interface .", "The oligomerization of Rev-like proteins in other retroviruses may similarly facilitate binding to existing Crm1 orthologs that lack the dimeric interface .", "The selective pressures that fixed the dimer interface in the simian primate lineage are unknown , but its evolution has certainly provided an advantage for HIV replication .", "The Crm1 surface bound by Rev-RRE is clearly distinct from where the host cargo Snurportin1 binds ( Figure 3C ) and could reflect a virus-specific interface and potential therapeutic target .", "However , it seems more likely that the Crm1 dimer plays some role in host cell biology .", "Previous studies have implicated the same species-specific Crm1 residues as important for binding to RanBP3 , a protein that enhances interactions of Crm1 with RanGTP and some NESs , although it is not known if a Crm1 dimer is involved ( Hakata et al . , 2003 ) .", "Alternatively , Crm1 dimerization may modulate levels of nuclear export for host mRNAs .", "Crm1 mediates export of a small set of mRNAs via proteins bound near their 3′ ends ( Brennan et al . , 2000; Yang et al . , 2001; Culjkovic et al . , 2006; Prechtel et al . , 2006 ) .", "One of these proteins , HuR , even forms an oligomeric complex at its 3′ RNA element ( Fialcowitz-White et al . , 2007 ) .", "Perhaps analogous to the Rev , the HuR oligomer may also bind a Crm1 dimer .", "Moreover , the positioning of cis-acting elements within a cellular mRNA may define a topology for RNA-binding proteins to recruit a Crm1 dimer without homooligomeric assemblies of proteins .", "Such a mechanism would allow Crm1-mediated export to integrate different protein signals to tune the levels of nuclear export for a given mRNA ." ], [ "All plasmids used in this study are listed in Supplementary file 1 .", "Crm1 and Ran were cloned between the NdeI and XhoI sites in a pET19b vector ( EMD Millipore , Darmstadt , Germany ) that encodes an amino-terminal deca-histidine tag followed by a tobacco etch virus ( TEV ) protease site .", "Genes were amplified from their source vectors by polymerase chain reaction with phusion polymerase ( New England Biolabs ( NEB ) , Ipswich , MA ) using forward and reverse primers ( Integrated DNA technologies ( IDT ) , Coralville , IA , listed in Supplementary file 2 ) containing NdeI or XhoI restriction sites , respectively .", "PCR products and the target vector were digested with restriction enzymes ( NEB ) , purified ( Qiagen , Venlo , Netherlands ) , ligated ( Roche , Basel , Switzerland ) , and then transformed into E . coli DH5α .", "Plasmids from positive transformants were isolated ( Qiagen ) , screened by restriction digests , and sequenced ( University of California - Berkeley DNA Sequencing Facility , Berkeley , CA ) .", "Mutations were introduced using a single primer method ( Makarova et al . , 2000 ) .", "Briefly , DNA was synthesized using mutation-bearing primers ( IDT ) using Pfu Turbo ( Agilent , Santa Clara , CA ) , purified , DpnI digested , and transformed into E . coli DH5α .", "Plasmids from transformants were isolated and sequenced .", "RNA was transcribed by T7 polymerase from a linearized plasmid and then purified by Urea-PAGE ( Daugherty et al . , 2008 ) .", "A single-stranded segment found to be a cloning byproduct at the 5′-end of the RRE was removed from the original template by site-directed mutagenesis .", "Pure RNA pellets were resuspended in buffer A [1 mM HEPES-KOH , pH 7 . 5 @ 20°C] and stored at −20°C .", "RNA was annealed by heating to 95°C and cooling to 30°C in an aluminum heating block placed at room temperature .", "RNA was quantified by UV spectroscopy using a 260 nm extinction coefficient of 2 . 5 × 106 M-1cm−1 for the RRE .", "Purified RNA was treated with calf intestinal phosphatase according to the manufacturer ( NEB ) .", "RNA was purified by phenol-chloroform extraction , ethanol precipitated in 300 mM NaCH3CO2 , washed with 70% ice-cold ethanol , and resuspended in buffer A . RNA was labeled for ≥1 hr at 37°C in buffer B [50 mM imidazole-HCl , pH 6 . 4 @ 20°C , 10 mM MgCl2 , 5 mM DTT] with 10 Units of Polynucleotide Kinase ( NEB ) by adding either γ-32P-ATP ( Perkin Elmer , 3000 Ci/mmol ) or ATPγS ( Sigma-Aldrich , St . Louis , MO ) to final concentrations of 450 nM and 1 mM , respectively .", "For radiolabeling , RNA was purified by phenol chloroform extraction , diluted in buffer A , and desalted with PD Spintrap G-25 column ( GE Healthcare Lifesciences , Piscataway , NJ ) according to manufacturer’s instructions .", "For biotin labeling , RNA was purified by adding NH4CH3CO2 to a final concentration of 2 . 5 M and centrifuging at >20 , 000×g and 4°C for 1 hr to pellet the protein .", "RNA in the supernatant was ethanol precipitated and resuspended in buffer A . Maleimido-biotin was added at a final concentration of 100 µM , incubated at room temperature for 4 hr , and quenched with an excess of 2-mercaptoethanol .", "The reaction was diluted in buffer A , desalted as above , and ethanol precipitated twice in NaCH3CO2 in order to get rid of excess label .", "The washed RNA pellet was resuspended in buffer A . After labeling , RNAs were annealed as above and evaluated on Urea-PAGE to ensure no degradation .", "Radiolabeled RNA was quantified by scintillation counting , aliquoted , and then stored at −20°C .", "Proteins were expressed in E . coli BL21 ( DE3 ) .", "Bacteria were grown in Luria–Bertani Miller Formula ( LB ) media with noted supplements by diluting overnight cultures 50-fold into 1 l of media and by shaking at 200 rpm in baffled Fernbach flasks to the indicated OD600 .", "Expression was induced by adding isopropyl-β-D-thiogalactopyranoside ( IPTG ) to the indicated final concentration .", "Bacteria were harvested by centrifugation at 4500×g and 4°C for 20 min .", "Pellets were frozen in N2 ( l ) and stored at −80°C .", "Rev was expressed as previously described ( Daugherty et al . , 2008 ) .", "LB media and agar plates were supplemented after autoclaving with 1% ( wt/vol ) glucose , 100 µg/ml Ampicillin , and 34 µg/ml chlorampenicol .", "Crm1 expression vectors were co-transformed with pLysS-Rosetta2 ( EMD Millipore ) , and colonies were selected on LB agar plates .", "Bacteria were grown for protein expression with chloramphenicol reduced to 17 µg/ml at 37°C until OD600 = 1 . 0–1 . 2 .", "Cultures were shifted to 20°C for 20 min , induced by adding IPTG to 200 µM , and grown overnight .", "Bacteria were grown in LB with 100 µg/ml Ampicillin at 37°C to OD600 = 0 . 6-0 . 8 , shifted to 18°C for 20 min , induced with 500 µM IPTG , and grown overnight .", "Each gram of bacterial pellet was resuspended in 10 ml of buffer C [50 mM Tris–HCl , pH 8 . 0 @ 20°C; 2 M NaCl; 2 mM 2-mercaptoethanol; 10 mM imidazole; 0 . 1% ( vol/vol ) Tween-20] .", "Proteases were inhibited by adding phenylmethanesulfonylfluoride ( PMSF ) to a final concentration of 1 mM and one complete , Mini , EDTA-free protease inhibitor cocktail tablet ( Roche ) was added for every 5 g of bacteria .", "Bacteria were lysed with an Emulsiflex-C3 homogenizer ( Avestin , Ottawa , Canada ) , and the lysate was cleared by centrifugation at 30 , 000×g and 4°C for 30 min .", "The supernatant was loaded on Ni-NTA Agarose resin ( Qiagen ) with 1 ml of resin for every 2 . 5 g of bacterial pellet .", "The resin was washed extensively with buffer C and then buffer D [125 mM K2HPO4-KH2PO4 , pH 7 . 5 @ 20°C; 250 mM NaCl; 2 mM 2-mercaptoethanol; 10% ( vol/vol ) glycerol; 0–500 mM imidazole] .", "A step-wise increase in imidazole concentration eluted proteins from the resin .", "Pure fractions were combined , supplemented with TEV protease at a 1:20 molar ratio , and dialyzed overnight in a 3500 Da MWCO Slide-A-Lyzer Cassette ( Thermo Scientific , Waltham , MA ) against buffer D without imidazole .", "Dialyzed protein was passed over Ni-NTA resin .", "The flow-through was collected and filtered through a 0 . 22 µm syringe filter .", "The protein was then loaded onto a Mono-S 5/50 GL column ( GE Healthcare Lifesciences ) , washed , and eluted with a linear gradient of buffer D into buffer E [500 mM K2HPO4-KH2PO4 , pH 7 . 5 @ 20°C; 1 M NaCl , 2 mM 2-mercaptoethanol; 10% ( vol/vol ) glycerol] .", "After analysis by SDS-PAGE , pure fractions were pooled , frozen in N2 ( l ) and stored at −80°C .", "Rev concentration was determined by UV spectroscopy using 280 nm extinction coefficients of 20 , 000 M-1cm−1 for GB1-Rev and 8480 M-1cm−1 for Rev . Crm1 was resuspended in 4 ml of buffer F [50 mM Tris–HCl , pH 7 . 5 @ 4°C; 600 mM NaCl; 2 mM Mg ( CH3CO2 ) 2; 2 mM 2-mercaptoethanol; 10-200 mM imidazole; 20% ( vol/vol ) glycerol] containing 0 . 1% ( v/v ) Tween-20 and 5% glycerol for each gram of bacterial pellet .", "A protease inhibitor tablet was added for every 40 g of bacteria with PMSF .", "Lysis was as for Rev . Contaminating nucleic acids were removed from the supernatant of the clarified lysate by adding polyethylenimine to 0 . 2% ( wt/vol ) and cleared by centrifugation at 20–25 , 000×g and 4°C for 30 min .", "Ni-NTA chromatography was as for Rev with with 1 ml of resin for every 20 g of bacterial pellet and washes and elutions were in buffer F . TEV protease was added to pooled fractions in a 1:20 molar ratio and incubated at 4°C overnight .", "Protein was concentrated with a 10 kD MWCO Amicon Ultra Free Centrifugal Concentrator ( EMD Millipore ) and filtered through a 0 . 22 µm syringe filter .", "Crm1 was further purified by size-exclusion chromatography with buffer F containing 10% glycerol and 20 mM imidazole on a Superdex 200 , HiLoad 16/60 column ( GE Healthcare Lifesciences ) followed in tandem by a HisTrap Fast Flow , 5 ml column ( GE Healthcare Lifesciences ) .", "Fractions were analyzed by SDS-PAGE .", "Pure fractions were pooled , concentrated , frozen in N2 ( l ) , and stored at −80°C .", "Prior to use Crm1 was exchanged into buffer G [50 mM HEPES-KOH , pH 7 . 5 @ 20°C , 50 mM KCl , 2 mM Mg ( CH3CO2 ) 2 , 2 mM 2-mercaptoethanol , 2% ( vol/vol ) glycerol] using a 7 kD MWCO , Zeba Spin column ( Thermo Scientific ) , and the concentration was determined using Bradford assays with a bovine serum albumin ( BSA ) standard ( Thermo Scientific ) .", "Ran was purified in a similar manner as Rev with the following modifications: Lysis , Ni-NTA purification , and dialysis were performed with buffer H [64 mM Tris–HCl , pH 7 . 5 @ 4°C , 400 mM NaCl , 5 mM MgCl2 , 2 mM 2-mercaptoethanol , 0–250 mM Imidazole] .", "Prior to cation exchange , protein was concentrated with a 3 kD MWCO Amicon Ultra Free Centrifugal Concentrator ( EMD Millipore ) , nucleotides were removed by adding EDTA to a final concentration of 10 mM , and exchanged into buffer I [25 mM MES-HCl , pH 6 . 5 @ 4°C , 0 . 020–1 M KCl , 1 mM EDTA , 4 mM 2-mercaptoethanol , 10% ( vol/vol ) glycerol] using a PD-10 column ( GE Healthcare Lifesciences ) .", "Any precipitate was removed by centrifugation at 20 , 000×g followed by filtration through a 0 . 22 µm syringe filter .", "Cation exchange was performed with buffer I and eluted with a linear gradient of KCl in buffer I . Prior to use , Ran nucleotides were exchanged into Ran by adding nucleotide and MgCl2 to final concentrations of 1 mM and 5 mM , respectively , and then incubating at room temperature for 30 min .", "Excess nucleotide was removed by exchanging the protein into buffer G using a 7 kD MWCO , Zeba Spin Column ( Thermo Scientific ) .", "Protein concentration was determined with a BSA standard as for Crm1 .", "Ran was exchanged with GTPγS or GDP as described above .", "Rev was diluted with buffer G containing 30 mM imidazole , and Crm1 and Ran were exchanged into the same buffer in order to reduce non-specific interactions with the resin . 1 . 6 µM GB1-Rev was mixed with 200 nM RRE , 400 nM Crm1 , and/or 800 nM Ran in 50 µL .", "Reactions were incubated with 50 µl of Ni-NTA agarose resin for 2 hr at 25°C .", "The supernatant was removed and the beads were washed three times with 500 µl of buffer .", "Proteins were eluted by rotating the resin for 5 hr with 100 µl of assembly buffer containing 750 mM imidazole .", "Eluates were separated on a 4–12% Novex Bis-Tris Gel in SDS-MES buffer ( Life Technologies , Waltham , MA ) and stained using SYPRO Ruby ( Lonza , Basel , Switzerland ) .", "In these gels , only Rev and Crm1 are easily visible because Rev and Ran migrate with the same apparent mass .", "Rev-RRE complexes were assembled with a 10-fold excess of Rev added to the RRE .", "Crm1 and RanQ69L were then added to Rev-RRE and diluted to 70 µl .", "The final concentration of the Rev-RRE complex was 4 µM for stoichiometric titrations .", "At these concentrations , Rev-RRE completely assembles with Crm1 and RanQ69L but disassembles through dilution on the column , as supported by native gel electrophoresis ( Figure 2B ) .", "The assembly reactions were incubated at 37°C , filtered through a 0 . 22 µm centrifugal filter ( EMD Millipore ) , and then kept on ice .", "A Shodex KW-804 ( Showa Denko America , New York , NY ) column equilibrated in buffer G was loaded with 50 µl of assembly reaction .", "Complexes were separated at a flow rate of 0 . 35 ml/min , and the 12 . 5 ml column volume was collected in 135 µl fractions .", "Fractions were analyzed by SDS-PAGE as for in vitro pulldowns .", "Molecular masses were determined using the program ASTRA from MALLS and refractometry ( Wyatt Technology Corp , Santa Barbara , CA ) measurements following size-exclusion chromatography .", "For these experiments , the concentration of Rev-RRE was increased to 8 µM for accurate mass determination .", "The masses of Crm1 and RanQ69L were determined at 20 µM and 50 µM , respectively .", "Because the technical error of measuring the mass of the nuclear export particle was nearly the same as a Rev monomer , we validated the stoichiometry by densitometry of SDS-PAGE gels stained with Sypro-Ruby scanned on a Typhoon ( GE Healthcare Lifesciences ) and quantified with ImageQuant .", "Concentrations for standards of each protein were determined by amino-acid analysis ( UC-Davis Proteomics Facility , Davis , CA ) .", "Crm1 and Ran were exchanged into buffer J [50 mM HEPES-KOH , pH 7 . 5 @ 20°C , 100 mM KCl , 2 mM Mg ( CH3CO2 ) 2 , 0 . 5 mM EDTA , 2 mM 2-mercaptoethanol , 12% ( vol/vol ) glycerol] and protein concentrations were determined by quantitative SDS-PAGE .", "Stocks of Rev and the RRE were diluted in buffer J and then combined at 80 nM and 2000 cpm/µl ( ∼0 . 5 nM ) , respectively .", "Crm1 and RanGTP were serially diluted in buffer J containing 100 µg/ml BSA , 10 µM yeast tRNA , and 200 nM methionine .", "6 µl of Crm1-RanGTP was added to 6 µl of Rev-RRE and assembled at 37°C for 15 min . 10 µl of the assembly reaction was loaded onto a 5% acrylamide ( Protogel , 37 . 5 acrylamide to 1 bisacrylamide ) native gel in buffer K [25 mM HEPES-25 mM imidazole , pH 7 . 4 @ 20°C] and continuously running at ∼10 V/cm and 20°C .", "The gel was dried onto filter paper , exposed to a phosphor-screen overnight , and imaged on a Typhoon .", "Band intensities ( I ) were quantified using ImageQuant and normalized with the equation , Inorm=1− ( IjRev−RREIjTotal ) • ( IoTotalIoRev−RRE ) where Ij is the intensity at the indicated concentration of Crm1-RanGTP and Io is the initial intensity of Rev-RRE without Crm1-RanGTP .", "A modified Hill equation , Inorm=M•ChCh+ K1/2hwhere K1/2 is the apparent binding constant , h is the Hill coefficient , M is the maximum instensity , and C is the concentration of Crm1 and RanQ69L , was fit to each binding experiment using sum-of-squares minimization implemented with the Solver tool in Microsoft Excel .", "A version of the McGhee-von Hippel binding model ( McGhee and von Hippel , 1974 ) derived for a finite lattice ( Epstein , 1978 ) was also used to fit the data with a sum-of-squares minimization ( Kowalczykowski et al . , 1986 ) .", "The model is described by the equation , θ=nL• ( ∑k=0L/n∑j=0k−1k•PM ( k , j ) • ( Kd−1C ) kωj∑k=0L/n∑j=0k−1PM ( k , j ) • ( Kd−1C ) kωj ) Where , PL ( k , j ) = ( L−nk+1 ) !", "( k−1 ) !", "( L−nk−k+j+1 ) !", "( k−j ) !", "j !", "( k−j−1 ) !", "θ is the fraction bound and is assumed to be the same as Inorm , L is the maximal number of binding sites , Kd is the dissociation constant for Crm1 binding to a Rev NES , n is the number of sites occluded by Crm1 binding , ω is the cooperative interaction between Crm1 monomers , C is the concentration of Crm1/Ran , k is the number of Crm1 monomers bound to Rev-RRE , j is the number of times Crm1 monomers are adjacent to each other , and Pl ( k , j ) is number of combinations for k monomers binding to Rev-RRE with j adjacencies on a lattice of length L . In order to obtain reliable fits , L was set to an arbitrarily large number of 100 binding sites , which effectively describes an infinitely long lattice ( McGhee and von Hippel , 1974; Epstein , 1978 ) .", "Such a lattice can serve as a valid model for even a small number of binding sites ( Epstein , 1978; Kowalczykowski et al . , 1986 ) .", "Because L is constant and the parameters k and j are determined from L and n , the only free parameters that vary are K , n , and ω .", "We initiated the parameter search by setting Kd = 1 µM , ω = 1 , and n = 3 . We additionally tested n = 1 , 2 , 3 , or", "4 . n = 3 provided the most consistent fits with the best correlation , as measured by R2 , between the model and each of the titration curves .", "2 . 5 µl of 100–400 nM samples were adsorbed to glow-discharged , carbon-coated , 400 mesh grids and stained with 0 . 75% uranyl formate as previously described ( Booth et al . , 2011 ) .", "For random conical tilt , 200 mesh grids were used .", "A substoichiometric amount of streptavidin was added to biotinylated RNA immediately before adsorbing onto grids .", "Images were acquired at 1 . 5 µm defocus for untilted images and ∼2 . 0 µm for tilted images using low dose procedures on a T12 microscope ( FEI , Eindhoven , Netherlands ) operating at 120 kV and recorded on a 4096 x 4096 CCD camera ( Ultrascan 4000 , Gatan , Pleasanton , CA ) with a 52k magnification corresponding to a pixel size of 2 . 02 Å .", "Tilt pairs were manually collected at 60° and then 0° angles .", "2 µl of sample was applied to 1 . 2/1 . 3 µm Quantifoild Grids ( Electron Microscopy Sciences , Hatfield , PA ) , blotted , and frozen in liquid ethane with a Vitrobot ( FEI ) .", "Images were recorded with a 2–4 . 5 µm defocus range using low dose procedures on a TF20 microscope ( FEI ) operating at 200 kV and recorded on a 8192 × 8192 CMOS camera ( TVIPS , Gauting , Germany ) at 62k magnification corresponding to a pixel size of 1 . 29 Å .", "Negative-stain images were binned twofold for a pixel size of 4 . 04 Å .", "Cryo-images were binned fourfold for a final pixel size of 5 . 17 Å .", "The defocus astigmatism , tilt angle , and tilt axis were determined using CTFFind and CTFTilt ( Mindell and Grigorieff , 2003 ) .", "Tilted images were rotated to align the tilt axis with untilted images .", "Particles were manually selected using Samviewer ( Liao et al . , 2013 ) .", "The tilt angle for each pair was further refined based on particle positions .", "Particles were boxed from images , combined into a single stack , normalized , and contrast transfer function ( CTF ) corrected .", "For cryo images , the contrast was inverted to have white particles against a black background as required for SPIDER ( Shaikh et al . , 2008 ) .", "Particles from untilted negative-stain and cryo-images were subjected to reference free classification followed by multiple rounds of correspondence analysis , k-means classification , and multi-reference alignment using SPIDER ( Shaikh et al . , 2008; Liao et al . , 2013 ) .", "For each class average of untilted images , we calculated a random conical tilt ( RCT ) reconstruction from tilted particles with angular parameters from the in plane rotation of class averages and from the tilt angle determined during particle picking .", "This initial RCT reconstruction was first refined for translational alignment of individual particles .", "Images of untilted particles were then added to the reconstructions followed by translational alignment .", "All refinements were performed using FREALIGN ( Grigorieff , 2007 ) in a frequency-limited range of 240–38 Å .", "Volumes were low-pass filtered after each cycle of refinement using the FCREF option .", "When we compared RCT reconstructions using UCSF Chimera , we concluded the first five classes were different views of the same structure and the remaining two classes were substoichiometric particles ( Figure 2—figure supplement 1 ) .", "Untitled and tilted particles from the first five classes were merged into a single dataset for further projection matching refinement .", "The RCT reconstruction from Class02 was used as an initial model to determine Euler angles and in-plane shifts of all particles .", "In the subsequent iterative refinement , the frequency was limited between 240 Å and 34 Å .", "The final resolution was estimated to be 25 Å ( FSC = 0 . 143 ) to 30 Å ( FSC = 0 . 5 ) ( Figure 2—figure supplement 1 ) .", "The Euler angle distribution of all particles suggests there is no major gap in Euler space .", "A b-factor of −1000 Å2 with a cosine-edge , low-pass filter to 25 Å and a 2-pixel width was applied to the final volume using b-factor ( N . Grigorieff ) .", "We also tested applying C2 symmetry during the refinement and reconstruction .", "Symmetry improves the resolution of the overall reconstruction but weakens the features from Rev-RRE .", "We , therefore , only interpreted the volume with no symmetry applied .", "All molecular modeling was performed using UCSF Chimera ( Goddard et al . , 2007 ) .", "Crystal structures were initially fit by eye into the EM density and further optimized using the ‘Fit in Map’ function in Chimera to optimize the correlation between the crystal structure filtered to 25 Å resolution and the EM map .", "In order to find the difference density for Rev-RRE , the hybrid model of the Crm1 dimer crystal structure ( Figure 2—figure supplement 2 ) filtered to 25 Å resolution was used to mask out Crm1 and Ran density in the EM reconstruction using the ‘mask’ command in Chimera ." ] ]

[ "The HIV Rev protein routes viral RNAs containing the Rev Response Element ( RRE ) through the Crm1 nuclear export pathway to the cytoplasm where viral proteins are expressed and genomic RNA is delivered to assembling virions .", "The RRE assembles a Rev oligomer that displays nuclear export sequences ( NESs ) for recognition by the Crm1-RanGTP nuclear receptor complex .", "Here we provide the first view of an assembled HIV-host nuclear export complex using single-particle electron microscopy .", "Unexpectedly , Crm1 forms a dimer with an extensive interface that enhances association with Rev-RRE and poises NES binding sites to interact with a Rev oligomer .", "The interface between Crm1 monomers explains differences between Crm1 orthologs that alter nuclear export and determine cellular tropism for viral replication .", "The arrangement of the export complex identifies a novel binding surface to possibly target an HIV inhibitor and may point to a broader role for Crm1 dimerization in regulating host gene expression ." ]

[ "To be able to multiply , viruses first have to infect a host cell and then hijack the host's molecular machinery to make viral proteins .", "The first stage of this process takes place in the nucleus of the host cell and involves the DNA being transcribed to make RNA molecules .", "These RNA molecules must then be exported from the nucleus to the cytoplasm , where the proteins are made .", "For RNA molecules that have been transcribed from the cell's own DNA , this export process happens automatically .", "However , the export of viral RNA molecules requires help from the virus .", "In the case of HIV-1 , the virus supplies a protein called Rev , which binds to a region on the viral RNA molecules called the Rev Response Element .", "The Rev protein then binds to a group of host proteins called the Crm1 export complex to send the viral RNA molecules to the cytoplasm .", "Although the 3D structures of the individual components have been worked out , it is not known how the viral RNA molecule , the Rev protein and the Crm1 proteins all fit together to make a complex .", "Booth et al . have used a technique called single-particle electron microscopy to produce a 3D structure of the whole complex .", "It shows that this complex forms with two Crm1 proteins contacting each other as they bind to the Rev protein that is already bound to the RNA molecule .", "It also reveals a new surface of the complex that had not been previously predicted to exist .", "In parallel work from the same laboratory , Jayaraman et al . , 2014 .", "have used a different technique to reveal a highly-detailed 3D structure of Rev molecules binding to the Rev Response Element .", "Both structures shed new light on how the HIV-1 virus is able to multiply in its host , which may aid future efforts to develop new treatments for the disease ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "neuroscience" ]

[ [ "Understanding the neural basis of developmental disorders such as congenital prosopagnosia ( CP ) remains a challenge from both a basic science and a translational perspective as there are no obvious identifiable deficits on conventional anatomical MR brain scans .", "Furthermore , many studies show that CP individuals evince normal fMRI activation in the 'core' face-related posterior patches of the brain ( Hasson et al . , 2003; Avidan et al . , 2005 , 2014; Avidan and Behrmann , 2009 ) ( but see von Kriegstein et al . , 2008; Dinkelacker et al . , 2011; Furl et al . , 2011 ) .", "In contrast , more sensitive methods that have been used to map structural changes in CP relative to controls , such as diffusion tensor imaging ( DTI ) have revealed a reduction in long-range white matter tracts connecting the ‘core’ face-related posterior patches and the anterior temporal lobe face patch ( ATL ) in CP ( Thomas et al . , 2009; for related papers , see Grossi et al . , 2014; Scherf et al . , 2014 ) .", "Other studies have also reported local structural and functional atypicalities in the vicinity of face-selective regions ( Gomez et al . , 2015; Song et al . , 2015; Lohse et al . , 2016 ) .", "Using standard functional connectivity ( FC ) analysis , which measures the temporal correlations across different brain areas within an individual , we have previously documented abnormal deviations from the control pattern in the connectivity patterns between the 'core' and 'extended' nodes of the face system ( Avidan and Behrmann , 2014 ) .", "The pattern of FC within each individual , as utilized in previous studies , is a combination of stimulus-induced correlations , intrinsic neural fluctuations , and correlations induced by non-neuronal artifacts ( such as head motion , respiration ) .", "Separating these factors is challenging within the framework of standard FC , given the strength of the intrinsic neural fluctuations .", "Hence , group differences in FC may not be sufficiently robust to be detected following whole brain statistical correction , and , therefore , are less suitable for mapping large-scale changes in network topology .", "In contrast with these previous studies that have examined the neural profile of CP based on a subset of brain regions and their connectivity , here , we adopt an innovative , large-scale network approach .", "The primary goal of this approach is to elucidate the functional brain topology in individuals with CP ( and matched controls ) as a means of examining alterations in neural circuitry .", "Because there is consensus that multiple regions ( face ‘patches’ ) are implicated in normal face recognition in humans ( Haxby et al . , 2000; Pyles et al . , 2013; Weiner and Grill-Spector , 2013 ) and in non-human primates ( Tsao et al . , 2006; Hung et al . , 2015; Moeller et al . , 2017 ) , elucidating alterations in the topology of this distributed cortical circuit is of great interest .", "To that end , in the present study , we have used a novel method , termed ‘inter-subject functional correlation’ ( ISFC ) , which is designed to isolate stimulus-locked functional responses , by correlating the response profile across the brains of multiple participants ( Simony et al . , 2016 ) .", "Importantly , intrinsic neural dynamics during rest and during task conditions that are not related to the pattern of activation evoked by stimulation , as well as non-neuronal artifacts ( e . g . , respiratory rate , motion ) , can only influence the pattern of correlations within each individual brain , but cannot induce correlations between subjects .", "In contrast , neural processes that are locked to the structure of the stimulus can be correlated across brains .", "Thus , the ISFC method allows us to track stimulus-locked brain responses within the high-level visual network in control subjects and to contrast these correlation patterns with the patterns uncovered in CP individuals .", "Such an approach enables us to explore possible alterations in connectivity across large swaths of the cortex in an assumption-free manner rather than focusing on a predetermined subset of regions and connections ." ], [ "To detect stimulus-locked changes in functional responses to faces in CP relative to controls , we used the inter-subject functional correlation ( ISFC; see Materials and methods ) , which calculates the inter-regional correlations in the brains of different individuals who viewed the same stimuli ( Simony et al . , 2016 ) .", "Simony et al . ( 2016 ) demonstrated that the ISFC method substantially increases the signal-to-noise ( SNR ) ratio in detecting shared stimulus-locked network correlation patterns by filtering out idiosyncratic intrinsic neural dynamics and non-neuronal artifacts ( e . g . , respiratory rate; motion ) that can influence FC patterns within a brain but that are uncorrelated across the brains of different participants .", "Capitalizing on the high SNR of the ISFC procedure permits the construction of a fine-grained functional brain network even with a relatively small sample size as is the case in the present study and potentially in other situations of relatively rare disorders ( see Materials and methods ) .", "We calculated the ISFC within the CP and the control groups using the BOLD signal elicited in response to faces and buildings ( see below ) .", "For comparison , we also ran the same analysis using a standard FC procedure .", "In the FC analysis , the response profile in each node was correlated with the response profile of all other nodes within an individual .", "The analysis was repeated for each individual in each group and statistical significance for each edge was determined using a t-test followed by FDR correction .", "We return to these results below .", "ISFC is similar in logic to FC , with one critical difference: instead of correlating the response profile within the brain of each individual , we calculated the correlation patterns across brains .", "For each experimental group ( controls and CPs ) , the correlation between the response profile of each subject and the mean of the remaining 9 subjects was calculated ( see ISFC procedure in Materials and methods ) .", "The average non-thresholded networks for each group are presented in Figure 1a , b for visualization purposes .", "As is evident , the raw mean networks over the ISFC of each group are visually similar .", "To evaluate whether there are any statistical differences in the network structure across the two groups , the networks were directly compared using a permutation test ( see Materials and methods for details ) .", "This analysis resulted in a difference network in which the edges indicate the significant difference between the two groups ( either controls>CPs or CPs>controls ) .", "Non-significant edges were eliminated ( see details of statistical analysis in Materials and methods ISFC Formulation section ) .", "For the following analysis , we applied a stringent statistical criteria .", "This approach is followed by a second , more descriptive network analysis which captures the overall difference in the pattern of ISFC across the two groups ( see Network analysis section ) .", "The controls>CPs difference network revealed that control participants , but not CP , exhibited an overall , non-selective increase in ISFC patterns from nodes in the vicinity of the ATL ( see Table 2 and Network analysis section ) .", "In contrast , the opposite analysis of CPs>controls revealed a significant edge between nodes in the vicinity of the LOC ( see Figure 1c , d ) .", "To examine whether the neuronal ISFC of these significant edges is related to behavioral performance , the mean raw ISFC value for each group was correlated with the famous faces questionnaire and CFMT scores of each participant ( see Materials and methods for details ) .", "We hypothesized that higher ISFC values in the network edges located in posterior visual areas ( e . g . LOC ) should suffice for face representations that are largely driven by the immediate visual input and do not necessarily enable recognition ( see Gainotti , 2007 , 2013; Fox et al . , 2008 and the Discussion section ) .", "We focused the analysis on the single , significant posterior edge that was obtained from the contrast of the CPs>controls and examined the raw ISFC value of this edge within the CP and control groups .", "Moreover , based on previous studies ( Furl et al . , 2011 ) , we set to examine whether the two behavioral measures are redundant and consequently , whether there is a latent behavioral measure which can account for both .", "In accordance with this study , we found a significant correlation between the CFMT and famous faces behavioral measures r ( 18 ) =0 . 71 , p=0 . 0003 .", "Accordingly , the 2 measures were factorized using PCA ( Furl et al . , 2011 ) , and the first principal component which accounted for 85 . 9% of the variance served as our new face recognition behavioral score .", "The correlation between the face behavioral score was r ( 18 ) = 0 . 9 , p<0 . 0001 and r ( 18 ) = 0 . 94 , p<0 . 0001 for CFMT and famous faces questionnaire respectively .", "Subsequently , a multivariate regression was conducted to examine if ISFC and group predicted the face recognition behavioral score .", "This was done for the significant ISFC edges obtained from both the CP >Controls and the Controls > CP contrasts .", "As for the CP >controls contrast , overall a significant effect was found for both the group and the ISFC edge and the independent variables indeed explained a significant amount of the variance of the face recognition behavioral score ( R2 = 0 . 83 , R2Adjusted = 0 . 81 , F ( 2 , 17 ) = 42 . 67 , p<0 . 0001 ) .", "Moreover , the group significantly predicted face recognition behavioral score ( β = −6 . 08 , p<0 . 0001 , t = −6 . 83 , stde = 0 . 87 ) , as did the ISFC value ( β = 4 . 89 , p=0 . 017 , t = 2 . 63 , stde = 1 . 85 ) .", "The intercept term was not statistically significant ( α = 0 . 93 , p=0 . 089 , t = 1 . 8 , stde = 0 . 52 ) .", "Hence , ISFC score was positively associated with behavioral face performance ( see Figure 2a ) .", "When conducting the same procedure but examining the edges obtained from the controls>CPs contrast , the ISFC edge and the independent variables also explained a significant amount of the variance of the face recognition behavioral score ( R2 = 0 . 77 , R2Adjusted = 0 . 74 , F ( 2 , 17 ) = 29 . 19 , p<0 . 0001 ) .", "Nevertheless , only the group effect coefficient was significant ( β = −5 . 23 , p=0 . 005 , t = −3 . 22 , stde = 1 . 62 ) , while the ISFC coefficient was not ( β = −5 . 12 , p=0 . 44 , t = −0 . 79 , stde = 6 . 48 ) .", "The intercept term was not significant , but showed a strong trend ( α = 3 . 17 , p=0 . 052 , t = 2 . 09 , stde = 1 . 51 ) .", "Thus , indicating the lack of a linear relation between the behavioral measure and ISFC in this contrast . 10 . 7554/eLife . 25069 . 004Figure 2 . The relation between the neuronal measures and the face recognition behavioral score . ISFC and FC values of the significant CPs > controls edges and the face recognition behavioral score .", "( a ) ISFC regression – group , as well as ISFC value significantly predict face recognition behavioral score ( group: β = −6 . 08 , p<0 . 0001 , t = −6 . 83 , std = 0 . 87; ISFC value: β = 4 . 89 , p=0 . 017 , t = 2 . 63 , std = 1 . 85 ) .", "( b ) FC regression - group significantly predict face recognition behavioral score while the FC value does not ( group: β = −4 . 99 , p<0 . 0001 , t = −4 . 68 , stde = 1 . 06; FC value: β = 0 . 95 , p=0 . 31 , t = 1 . 04 , stde = 0 . 91 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 25069 . 004 In the previous section , we used the stringent FDR correction .", "To further understand and quantify the networks obtained from comparing CPs and controls , we used a complementary approach in which the non-thresholded positive t-values matrix connectivity patterns were compared to various topological and topographical attributes , while using correspondingly appropriate weighted graph measures for each contrast ( CP>control and control>CP ) .", "These group differences were further quantified for each contrast using a measure of 'node strength' , which quantifies the sum of positive edge weights connected to a node ( Rubinov and Sporns , 2010 ) .", "First , we assessed the selectivity of the differences in the ATL connectivity .", "The ISFC of the edges connecting the ATL to both face and non-face selective nodes located throughout the visual cortex was higher in controls>CPs compared to the opposite contrast ( see specific pattern of ATL ISFC in Table 2 ) ( Figure 1e , f ) .", "The strength scores of all nodes in the network were ranked in a descending order , and , of interest , the three nodes located in the ATL were ranked as the top 3 for the control subjects ( see Figure 1e , f and Table 3a ) .", "Note that the ATL served as a hub connecting both face and non-face selective nodes ( see Table 2 ) .", "The node rankings confirm the centrality of the ATL in the face network of controls in contrast with that of CP , whose top 3 rankings include the right inferior temporal gyrus and the left lateral occipital cortex .", "The network obtained from the CPs>controls comparison revealed that , at a low-edge threshold , significant edges were located throughout the visual cortex , but , as the threshold was increased , edges from anterior regions were eliminated and the remaining significant edges were located only in posterior parts of the visual cortex ( Figure 3 ) .", "To further quantify this effect , the Y coordinates of the 3D MNI space were ranked in an ascending order and binned into 10–21 equally sized bins measuring distance parallel to the posterior-anterior commissure axis ( the maximal number of bins was chosen with a constraint that each bin contained at least one node ) .", "The significance level of the posterior to anterior pattern was then quantified using the Spearman correlation between the Y coordinate bins and the nodes' strength for CPs>controls and controls>CPs contrasts separately .", "The results indicate that the higher Y coordinates ( more anterior regions along the anterior posterior axis ) were associated with higher positive strength values for the controls>CPs contrast across all bin divisions ( rs = 0 . 52 to 0 . 8 , p=0 . 004 to 0 . 053 ) .", "Note that division into 14 bins resulted in the lowest correlation ( rs = 0 . 52 ) , which was only marginally significant ( p=0 . 053 ) .", "Contrarily , the CPs>controls contrast revealed an opposite result such that more posterior regions had higher node strengths across all bin divisions ( rs = −0 . 67 to −0 . 83 , p=0 . 0003 to 0 . 01 ) .", "These correlations validate the result of greater posterior ISFC in CPs versus controls . 10 . 7554/eLife . 25069 . 005Figure 3 . ISFC correlation between strength and node location along posterior-anterior axis . Linear regression fit with a 95% confidence interval band between the strength measure of the nodes and the Y coordinate ascending rank order of each node binned into 21 equally sized bins for", "( a ) controls > CPs ( rs = 0 . 58 , p=0 . 005 ) and", "( b ) CPs > controls ( rs = −0 . 70 , p=0 . 0003 ) .", "The x-axis of the graph denotes the Y coordinates of the 3D MNI space ranked in ascending order , the y-axis of the graph marks the mean strength value of nodes per bin .", "As is evident , the higher the y-coordinate ( more anterior ) , the higher the strength value for controls > CPs and the lower the value for CPs > controls . DOI: http://dx . doi . org/10 . 7554/eLife . 25069 . 005 Additionally , this analysis revealed face-specific dominance , such that the nodes that had the highest degree were face-selective .", "Specifically , higher ISFC patterns in the control group , compared to the CP group , were associated with face selective nodes .", "For this comparison , eight out of the top ten nodes ( 80% ) were face-related ( ranked in descending order by degree score ) ( Table 3a ) , compared to an overall face node base rate of 35% ( number of face nodes divided by overall number of nodes ) .", "The difference between the face-selective node rate in the controls > CPs contrast and the overall face nodes base rate was statistically significant , χ² ( 1 , n = 20 ) =5 . 05 , p=0 . 02 .", "Moreover , 9 out of these 10 nodes , consisted of voxels originating from the same seed ( FFA or LOC ) .", "In the remaining node , voxels consisted of 90% of one tag ( either FFA or LOC ) and 10% of the other tag .", "Thus , the face network of the controls was associated with a higher number of face-tagged nodes compared with the face network of the CPs .", "When comparing the opposite contrast ( CPs>controls ) , no statistically significant difference was found in the face-selectivity of the nodes χ² ( 1 , n = 20 ) =1 . 25 , p=n .", "s .", "In support of these findings , 7 out of the 10 nodes included a substantial majority of LOC tagged voxels ( >75% ) .", "As for the remaining 3 , they were more heterogeneous and contained a mixture of voxels with all 3 possible taggings .", "In fact , four of the top ten nodes , of which only one was face specific , belonged to the lateral occipital cortex ( one in the left hemisphere and three in the right ) and six nodes belonged to the adjacent right inferior temporal cortex .", "We next compared the results obtained using ISFC to the results obtained using standard functional connectivity ( FC ) analysis .", "In accordance with our prediction , when comparing the controls to CPs , we observed the expected greater connectivity between the ATL and posterior regions .", "This effect was evident in a single edge following the application of the FDR multiple comparisons procedure ( see Figure 4a , controls>CPs ) .", "Similar to the ISFC , the opposite contrast of CPs>controls revealed hyperconnectivity in posterior visual regions with a main hub in the vicinity of the LOC and inferior temporal cortex .", "Critically , this effect was also evident following FDR correction ( see Figure 4b , CPs>controls ) . 10 . 7554/eLife . 25069 . 006Figure 4 . Networks obtained using the FC approach . The top schematic denotes the intra-subject functional correlation , which calculates the inter-regional correlations within the brains of single individuals .", "( a ) The FC difference network of controls>CPs .", "This comparison reflects the difference between the FC correlation coefficient values of the controls compared to the group of CP individuals .", "The maps are presented following the application of the FDR correction ( q < 0 . 05 ) .", "( b ) Difference networks obtained from the comparison of FC in CPs>controls . DOI: http://dx . doi . org/10 . 7554/eLife . 25069 . 006 The multiple regression analysis described above was repeated .", "Specifically , we used the edges obtained from the FC as opposed to the ISFC and group independent variables to predict the face recognition behavioral score .", "This was done for the significant FC edges obtained from both the CP>Controls and the Controls>CP contrasts ( Figure 2b ) .", "The regression model with FC and the group independent variables explained a significant amount of the variance of the face recognition behavioral score for both CPs>controls ( R2 = 0 . 78 , R2Adjusted = 0 . 75 , F ( 2 , 17 ) = 30 . 17 , p<0 . 0001 ) and controls>CPs ( R2 = 0 . 78 , R2Adjusted = 0 . 75 , F ( 2 , 17 ) =30 . 78 , p<0 . 0001 ) .", "Nevertheless , for both the CPs>Controls and Controls>CPs only the group effect coefficient was significant ( β = −4 . 99 , p<0 . 0001 , t = −4 . 68 , stde = 1 . 06 ) and ( β = −5 . 23 , p<0 . 0001 , t = −4 . 52 , stde = 1 . 15 ) respectively , while the FC coefficient was not ( β = 0 . 95 , p=0 . 31 , t = 1 . 04 , stde = 0 . 91 ) and ( β = −1 . 34 , p=0 . 25 , t = −1 . 16 , stde = 1 . 15 ) respectively .", "Moreover , the intercept terms were statistically significant ( α = 2 . 2 , p<0 . 0001 , t = 5 . 34 , stde = 0 . 41 ) and ( α = 2 . 77 , p=0 . 002 , t = 3 . 71 , stde = 0 . 74 ) for the CP>Controls and Controls>CPs contrasts respectively .", "Thus , no direct link was found between the face recognition behavioral score and FC connectivity .", "Hence , in accordance with ( Simony et al . , 2016 ) only the ISFC , but not standard FC could be linked to the behavioral measures thus capturing an additional important aspect of the data .", "The stimulus locked response patterns among nodes in the posterior occipital cortex were highly reliable among the CP group , as measured by ISFC .", "Such stimulus locked reliable activation suggests that CP individuals process visual information in a unique , but systematic fashion , which is not observed in the control group .", "Such positive effect rules out the possibility that the differences in network topology across the groups emerge from a global reduction in SNR within the CP subjects .", "Rather , this finding indicates that the stimulus-locked activity patterns are reliable in both groups , but that the network topology differed across the two groups ." ], [ "Perhaps the most striking difference in the topology of the face network in the controls versus CPs concerns the differences in the connectivity to the anterior temporal lobe ( ATL ) .", "The ATL is implicated in the integration of person-specific information ( Kriegeskorte et al . , 2007; Simmons et al . , 2010; Nestor et al . , 2011; Yang et al . , 2016 ) and familiar people recognition ( Gainotti et al . , 2003; Gainotti , 2007 ) .", "Specifically , damage to the left ATL is more associated with impaired representation of semantic information , while damage to the right ATL impedes the visual recognition of familiar faces ( Gainotti , 2007 ) .", "Based on both functional activation data , functional connectivity data ( during task and at rest ) and structural volumetric and connectivity findings , we have argued previously that an abnormality in the anterior temporal lobe and its connectivity may play a critical role in the neural basis of CP ( Behrmann et al . , 2007; Avidan and Behrmann , 2009; Avidan et al . , 2014; Thomas et al . , 2009 ) .", "The abnormality of the ATL is evident in the current findings as well , and the analysis , conducted in an assumption-free fashion revealed that the ATL was the most important hub that distinguished the network topology of the CPs and controls .", "Together , these findings point to abnormal structure , function and connectivity of this region in CP individuals ( but see Gomez et al . , 2015; Song et al . , 2015; Lohse et al . , 2016 ) .", "Our findings indicate that the impaired ISFC of the ATL may result in hyper-ISFC of the more posterior nodes , as in the right inferior temporal gyrus and the LOC , in CP compared to controls .", "These alterations in topology offer a possible account for the face recognition deficits exhibited by CP individuals ( Avidan et al . , 2011; Tanzer et al . , 2013; Yang et al . , 2016 ) .", "For example , the posterior hyper-connectivity in CP , in tandem with the residual , weak , connectivity with anterior regions may allow for some structural encoding of face stimuli derived from the immediate visual input ( see Behrmann et al . , 2005 ) for relevant behavioral findings ) .", "Although insufficient for facial recognition and individuation of identity , this rudimentary processing may partially compensate for the failure to utilize the ATL and its connectivity ( Yang et al . , 2016 ) .", "Furthermore , in the current study , CP subjects exhibited greater LOC-related ISFC compared to normal subjects .", "Consistent with this finding , it has been demonstrated that individuals with autism spectrum disorder ( ASD ) who have selective deficits in face recognition show greater activation of the LOC ( Schultz et al . , 2000; Hubl et al . , 2003 ) .", "Numerous studies have shown that the LOC is associated primarily with object perception ( Malach et al . , 1995; Grill-Spector et al . , 2001; Freud et al . , 2015 ) and plays a major role in the processing of inverted faces , perhaps through object-like and feature-based processing ( Rosenthal et al . , 2016; Yovel and Kanwisher , 2005; Pitcher et al . , 2011; Matsuyoshi et al . , 2015 ) .", "Together , these findings offer a possible account for the fact that most CP individuals , despite their severe deficit in recognizing the identity of individual faces , are typically able to detect the presence of a face in a scene .", "Specifically , it is possible that the network edges located in posterior visual areas ( e . g . LOC ) allow for the computation of face representations that are largely driven by the immediate visual input .", "This information may be relied on disproportionately by the CP individuals and perhaps serves as partial compensation for the failure to represent person-selective information which , in the normal brain is supported by the anterior temporal cortex and its connectivity ( Gainotti , 2007 , 2013; Fox et al . , 2008 ) .", "The current investigation does not allow us to infer causality , and thus , it remains to be determined whether the disconnection between the anterior and the posterior regions are the cause or the effect of the altered network organization .", "A final related , but unresolved issue , is whether the impairment in CP represents the lower end of the continuum of the normal distribution of face processing abilities , or whether the face recognition deficit in CP reflects a separate phenomenon and , hence , a distinct disorder .", "This important issue is outside the scope of the present study and further imaging and behavioral research is warranted in order to address it .", "To the best of our knowledge , these data provide the first demonstration of wide-scale network topological differences in individuals with CP .", "Utilizing the ISFC approach , the results validated previous atypical ATL connectivity findings in CP and enabled us to gain further insight into the altered network-wise configuration of individuals with CP .", "We propose that investigations of the topology of other neurodevelopmental disorders might benefit from the analytic approach developed here and that insights into their underlying neural mechanisms might also be gained ( Steinbrink et al . , 2008; Odegard et al . , 2009 ) ." ], [ "A standard measure which is often used in fMRI studies for characterizing the edges in a functional network is the correlation coefficient between pairs of nodes identified within each subject ( Smith et al . , 2011 ) .", "This approach was applied in the present study as well .", "However , despite its popularity , a major limitation of this measure , which captures within-subject synchronous activity , often referred to as functional connectivity , is that spontaneous intrinsic neuronal activity cannot be reliably separated from the evoked activity associated with the task ( Greicius et al . , 2003; Fox and Raichle , 2007; Simony et al . , 2016 ) .", "Additionally , non-neuronal artifacts such as respiratory rate and motion might also affect the results , usually by decreasing the signal to noise ratio ( SNR ) .", "A possible partial solution to these limitations , formulated to increase the network inference SNR ( Simony et al . , 2016 ) , relies on network construction using inter-subject functional correlation ( ISFC ) rather than exploiting the more widely used within-subject functional correlations .", "Briefly , for each pair of preselected regions of interest ( ROIs ) or nodes , correlation coefficients are computed between each participant and the mean signal of the remaining group without this particular participant ( leave-one-out ) , and then correlations are averaged across all pairwise iterations .", "As the correlations are calculated between different participants , any intrinsic activity and artifacts , which are not correlated between participants , are cancelled out and , consequently , only the task-induced activity of interest serves as the basis of the correlations , resulting in an improved SNR .", "The present study is conducted on individuals with CP , a relatively uncommon disorder and , hence , sample size is inherently limited , especially when contrasting the group size against the large number of nodes and edges ( i . e . , the number of variables outweighs the number of samples ) .", "This situation is typical in investigations of special populations and so we used a version of the inter-subject functional correlation ( ISFC ) ( Hasson et al . , 2009; Simony et al . , 2016 ) to increase the SNR .", "ISFC , is composed of the following steps: First , each experimental group ( e . g . controls and CPs ) which is composed of 10 subjects is divided into a group of 9 subjects and one remaining subject , and the raw signal for each node is averaged across all participants in the 9 subjects group .", "Second , for each experimental group , correlation coefficients are calculated between each pair of independent nodes , such that each pair of nodes is composed of one node from the individual subject and one node from the mean of the remaining nine subjects .", "The correlation coefficients are transformed by Fisher z-transformation .", "This process is repeated for all subjects in each experimental group such that , in each iteration , one of the subjects is left out .", "At the end of the process , each subject in each experimental group has all correlation coefficient values between all its nodes and the mean of all the other 9 subjects .", "An independent-samples t-test is conducted for each pair of nodes to compare its ISFC in CP and control .", "The positive and negative t-values reflect the controls>CPs and CPs>controls contrast respectively .", "Next , to determine statistical significance and assess the null distribution , the subjects are randomly divided into 2 groups of 10 and the same procedure is performed on permuted groups 10000 times .", "This results in a null distribution for each edge .", "Finally , an empirical p-value is calculated for controls>CPs and for CPs>controls as the number of times that each empirical t-value was smaller compared to the null distribution of each edge .", "The p-values are corrected for multiple comparisons using the false discovery rate [qFDR<0 . 005; ( Benjamini and Hochberg , 1995 ) ] procedure .", "Participants were scanned in a 3T Philips Ingenia scanner equipped with a standard head coil , located at the Soroka Medical Center , Beer Sheva , Israel .", "fMRI BOLD contrast was acquired using the gradient-echo echo-planner imaging sequence with parallel acquisition ( SENSE: factor 2 . 8 ) .", "Specific scanning parameters were as follows: whole brain coverage 35 slices , transverse orientation , 3 mm thickness , no gap , TR = 2000 ms , TE = 35 ms , flip angle = 90° , FOV = 256 × 256 and matrix size 96 × 96 .", "High-resolution anatomical volumes were acquired with a T1-weighted 3D pulse sequence ( 1 × 1 × 1 mm3 , 170 slices ) .", "Stimuli were presented using the E-prime 2 . 0 software ( Psychology Software Tools , Inc . , Pittsburgh , PA , USA ) and projected onto an LCD screen located in the back of the scanner bore behind the subject’s head .", "Participants viewed the stimuli through a tilted mirror mounted above their eyes on the head coil .", "A standard blocked-design localizer experiment was used to define face and non-face selective regions .", "Stimuli were presented in 10 s blocks of famous faces , unfamiliar faces , buildings , daily objects , or scrambled objects ( 1 image was presented twice as part of the task ) interleaved by 6 s rest periods .", "Within each block there were 9 images .", "Each image was presented for 800 ms followed by 200 ms inter-stimulus interval and participants performed a two alternative forced choice task ( see detailed description of the protocol in [Avidan et al . , 2014] ) .", "The data from these participants were used to identify the nodes or regions to be used in the analysis of the CP individuals ." ] ]

[ "Using a novel , fMRI-based inter-subject functional correlation ( ISFC ) approach , which isolates stimulus-locked inter-regional correlation patterns , we compared the cortical topology of the neural circuit for face processing in participants with an impairment in face recognition , congenital prosopagnosia ( CP ) , and matched controls .", "Whereas the anterior temporal lobe served as the major network hub for face processing in controls , this was not the case for the CPs .", "Instead , this group evinced hyper-connectivity in posterior regions of the visual cortex , mostly associated with the lateral occipital and the inferior temporal cortices .", "Moreover , the extent of this hyper-connectivity was correlated with the face recognition deficit .", "These results offer new insights into the perturbed cortical topology in CP , which may serve as the underlying neural basis of the behavioral deficits typical of this disorder .", "The approach adopted here has the potential to uncover altered topologies in other neurodevelopmental disorders , as well ." ]

[ "Human babies prefer to look at faces and pictures of faces over any other object or pattern .", "A recent study found that even fetuses in the womb will turn their heads towards dots of light shone through the mother’s skin if the dots broadly resemble a face .", "Brain imaging studies show that face recognition depends on the coordinated activity of multiple brain regions .", "A core set of areas towards the back of the brain processes the visual features of faces , while regions elsewhere process more variable features such as emotional expressions .", "Around 2% of people are born with difficulties in recognizing faces , a condition known as congenital prosopagnosia .", "These individuals have no obvious anatomical abnormalities in the brain , and brain scans reveal normal activity in core regions of the face processing network .", "So why do these people have difficulty with face recognition ?", "One possibility is that the condition reflects differences in the number of connections ( or “connectivity” ) between brain regions within the face processing network .", "To test this idea , Rosenthal et al . compared connectivity in individuals with congenital prosopagnosia with that in healthy volunteers .", "In the healthy volunteers , an area of the network called the anterior temporal cortex was highly connected to many other face processing regions: that is , it acted as a face processing hub .", "In individuals with congenital prosopagnosia , this hub-like connectivity was missing .", "Instead , a number of core regions involved in processing the basic visual features of faces , were more highly connected to one another .", "The greater this “hyperconnectivity” , the better the individual’s face processing abilities .", "The findings of Rosenthal et al . pave the way for developing imaging-based tools to diagnose congenital prosopagnosia .", "The same approach could then be used to investigate the basis of other neurodevelopmental disorders that are thought to involve abnormal communication within brain networks , such as developmental dyslexia ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "immunology and inflammation" ]

[ [ "T lymphocytes responding to antigen undergo rapid proliferation as they differentiate to produce effector populations .", "T cells also undergo large-scale , dynamic transcriptional remodelling during differentiation in response to immune activation ( Sen et al . , 2016; Crompton et al . , 2016; Gray et al . , 2014; Zhang et al . , 2014 ) .", "Immune activation of T lymphocytes requires that these cells adapt their metabolic programs to support rapid clonal expansion and cell differentiation .", "For example , activated T cells accelerate glucose metabolism to fuel oxidative phosphorylation , glycolysis and the production of UDP-GlcNAc to allow critical intracellular protein O-GlcNAcylations ( Donnelly and Finlay , 2015; Swamy et al . , 2016 ) .", "Activated T cells also dramatically upregulate lipid production programs to meet the demand for membrane biosynthesis associated with growth and proliferation ( Kidani et al . , 2013 ) .", "T cells also need a supply of glucose , leucine and arginine to sustain the activity of the serine/threonine kinase complex Mammalian Target of Rapamycin Complex 1 ( mTORC1 ) ( Finlay et al . , 2012; Nicklin et al . , 2009; Wang et al . , 2015 ) , a critical kinase that regulates the differentiation and migratory capacity of effector T cells ( Delgoffe et al . , 2011; Sinclair et al . , 2008 ) ; serine is required for biosynthesis of purine nucleotides needed for T cell proliferation ( Ma et al . , 2017 ) .", "It is thus increasingly recognised that ensuring that T cells have a sufficient supply of metabolic substrates for key biological processes is critical for T cell participation in adaptive immune responses .", "Hence understanding how the supply of these substrates is controlled is essential .", "In this context , it is evident that protein and nucleotide methylations are essential for T cell differentiation: Histone and DNA methylations are key epigenetic modifications that control the accessibility of DNA to the transcriptional machinery ( Allis and Jenuwein , 2016 ) .", "The methylation of RNA is also critical for cell function: mRNA cap methylation controls mRNA binding to the eukaryotic translation initiation factor 4E ( eIF4E ) to regulate translation initiation ( Aregger and Cowling , 2017; Gonatopoulos-Pournatzis and Cowling , 2014; Varshney et al . , 2018 ) ; the methylation of adenosine , ‘m6A’ , in RNA controls multiple processes including translation , splicing and stability of mRNA ( Dominissini et al . , 2012; Louloupi et al . , 2018; Li et al . , 2017; Mauer et al . , 2017 ) .", "Furthermore , there is an increasing awareness that protein arginine methylation is critical for T lymphocyte activation ( Inoue et al . , 2018 ) .", "How do T cells ‘manage’ the increased demand for methyl donors as they respond to immune activation ?", "In this context , methionine is an essential amino acid in mammals for de novo protein synthesis but it is also required for the production of S-adenosylmethionine ( SAM ) , the universal methyl donor for DNA , RNA and protein methyltransferases .", "The unanswered question then is how is methionine metabolism and the production of methyl donors controlled in T cells ?", "There are many studies showing the importance of protein , RNA , DNA and histone methyltransferases in T cells ( Inoue et al . , 2018; Li et al . , 2017; Lee et al . , 2001; Thomas et al . , 2012; Yang et al . , 2015; Karantanos et al . , 2016; Gray et al . , 2017; Sellars et al . , 2015; DuPage et al . , 2015; Geoghegan et al . , 2015; Zhang et al . , 2014; Tumes et al . , 2013; Gamper et al . , 2009 ) .", "However , critical facts about methionine metabolism are unknown; do naïve T cells express all the key enzymes for methionine metabolism to provide methyl donors for methyltransferases ?", "Is there any immune regulation of flux through the methionine cycle ?", "It is also unknown how T cells control intracellular methionine availability in terms of the balance between the production of methionine from intracellular recycling ( salvage pathways ) versus external sources .", "How dependent is T cell activation on the external methionine supply ?", "What are the relevant methionine transporters in T lymphocytes and is methionine metabolism coupled to T cell activation and differentiation into effector cells ?", "The present study uses high resolution mass spectrometry and metabolic labelling strategies to address these fundamental questions .", "The data identify and quantify expression of the key enzymes of the methionine cycle in T cells and document how these are regulated by immune activation .", "Key observations are that methionine flux through the methionine cycle is controlled by the T cell antigen receptor and that a constant sustained external supply of methionine is necessary for T cell immune activation to sustain protein biogenesis , histone methylation and RNA methylation .", "We identify a dominant rate limiting step for the production of methyl donors for nucleotide and protein methylations in immune activated T cells is methionine transport across the T cell membrane .", "Antigen receptor regulation of methionine transport across the cell membrane is thus a key step that licenses the use of methionine for fundamental cellular process that drive T lymphocyte proliferation and differentiation ." ], [ "To investigate whether methionine availability in the external environment is important for T cells , we initially assessed the impact of methionine deprivation on the immune activation of CD4+ T cells .", "Flow cytometric forward and side scatter analysis of CD4+ T cells activated in the absence of methionine revealed that these cells do not undergo normal cell growth/blastogenesis ( Figure 1a ) .", "TCR-activated CD4+ T cells deprived of methionine are smaller than CD4+ T cells activated in the presence of methionine ( Figure 1a ) , and fail to proliferate ( Figure 1b ) .", "However activation markers such as CD69 are expressed normally ( Figure 1c ) .", "Methionine levels in RPMI tissue culture media are 100 μM .", "Serum methionine availability has been shown to range from 3 to 30 μM ( Mentch et al . , 2015 ) , consequently we used levels of methionine spanning this range to further explore the importance of extracellular methionine availability for CD4+ T cell differentiation .", "In these experiments CD4+ T cells were activated by triggering TCR complexes and CD28 and cultured in Interleukin 2 ( IL2 ) and IL12 to differentiate into Th1 cells that produce high levels of interferon gamma ( IFNγ ) .", "We assessed the impact of restricting methionine availability on CD4+ T cell differentiation by activating the cells in media with different levels of methionine .", "The data in Figure 1d show that the frequency of IFNγ producing CD4+ T cells is dependent upon extracellular methionine availability ( Figure 1d ) .", "A 2-fold reduction in methionine from 10 μM to 5 μM thus had a striking impact on the frequency of IFNγ producing CD4+ T cells that could develop under Th1 polarising conditions ( Figure 1e ) and also controlled the amount of IFNγ produced per cell ( Figure 1f ) .", "These initial experiments show that methionine availability is rate limiting for T cell activation and differentiation .", "However , another question is how important is sustained methionine supply for T cell function ?", "To address this question T cells were activated through TCR/CD28 in a saturating concentration of external methionine ( 100 μM ) for 20 hr prior to culturing in reduced concentrations of methionine for a further 2 hr .", "We then used quantitative single cell assays to assess the impact of acute restriction of methionine availability for protein synthesis , RNA and DNA synthesis in immune activated T cells .", "Protein synthesis was assayed using a single cell assay that quantifies the incorporation of an analogue of puromycin into nascent protein chains in the ribosome; total RNA and DNA synthesis were measured by monitoring incorporation of the nucleoside analogue , 5-ethynyl uridine ( EU ) or a modified thymidine analogue ( EdU ) respectively .", "The data show that T cells activated in the presence of 100 μM methionine have high rates of protein , RNA and DNA synthesis ( Figure 1g–i ) .", "However , limiting extracellular methionine availability for 2 hr strikingly impacted on the ability of the cells to maintain these key processes .", "The EC50 , that is the concentration of methionine required for half maximal effect was in these TCR/CD28 activated CD4+ cells was 1 . 39 μM for protein synthesis; 2 . 94 μM for RNA synthesis and 12 . 62 μM for the frequency of cells undergoing DNA synthesis ( Figure 1j–l ) .", "Similar experiments were done with Th1 effector cells .", "These were differentiated for 5 days in 100 μM methionine and then maintained for five hours in methionine limited media .", "The data show that the ability of Th1 cells to sustain RNA , protein and DNA synthesis is also dependent on sustained methionine supply ( Figure 1m–o ) .", "Methionine is the predominant ‘start’ amino acid used to initiate polypeptide synthesis during mRNA translation .", "Figure 2a shows naïve CD4+ T cells have almost undetectable incorporation of extracellular 3H-methionine into protein , however incorporation of 3H-methionine into protein is greatly increased upon activation through the TCR ( Figure 2a ) .", "One explanation for the environmental methionine requirement for T cells is that it fuels protein synthesis .", "However methionine fuels other essential metabolic pathways , consequently we used mass spectrometry to explore methionine metabolism in CD4+ T cells stimulated via the T cell antigen receptor/CD28 complex .", "In particular , the methionine cycle which is initiated when methionine is converted into S-adenosylmethionine ( SAM ) in an ATP-consuming reaction and catalysed by methionine adenosyltransferase ( MAT2A ) .", "Methyltransferases then transfer the methyl group from SAM to yield S-adenosylhomocysteine ( SAH ) and a methylated substrate .", "SAH is swiftly converted into homocysteine ( HCy ) by S-adenosylhomocysteine hydrolase ( AHCY , also known as SAHH ) .", "The T cell metabolomics data show that SAM levels remain relatively constant between TCR stimulated and naïve CD4+ T cells ( Figure 2b ) .", "However , TCR activated cells show an increase in the generation of S-adenosylhomocysteine ( SAH ) and HCy ( Figure 2b ) .", "This increased production of SAH and HCy demonstrates that triggering the TCR drives increased flow through the methionine cycle .", "HCy has two potential metabolic fates , that is , it can be converted to cystathionine , or recycled back into methionine via subsequent enzymatic reactions through the de novo pathway .", "In the de novo pathway , methionine synthase ( MTR ) and the cofactor vitamin B12 perform the rate-limiting step of incorporating methyl groups derived from folate metabolism and HCy to produce methionine .", "SAM can also be utilised for polyamine synthesis , providing spermine and spermidine and yielding 5-methylthioadenosine ( MTA ) .", "The sulphur of MTA can be recycled back into methionine using the salvage pathway , the first step of which is catalysed by MTA phosphorylase ( MTAP ) to generate 5-methylthioribose-1-phosphate ( reviewed in Mentch and Locasale , 2016; Albers , 2009 ) .", "In this context , further evidence that TCR triggering drives the methionine cycle is provided by the data showing that activated CD4+ T cells accumulate 5-methylthioadenosine ( MTA ) and Methyl-5-thio-5-D-ribose 1-phosphate; metabolites produced downstream of polyamine synthesis .", "Activated CD4+ T cells also had increased levels of cystathionine and 2-oxobutanoate ( C4H6O3 ) ; the latter is produced when cystathionine gamma-lyase converts cystathionine to cysteine ( Figure 2b ) .", "One function for the methionine metabolite , S-adenosylmethionine ( SAM ) , is as a methyl donor for methylation modification reactions .", "The increased production of S-adenosylhomocysteine ( SAH ) in TCR activated cells argues that SAM is being used as a methyl donor during T cell activation .", "To explore links between TCR triggering and methylation pathways we first explored if activated T cells showed increased protein methylation by monitoring the methylation status of histone H3 at both activating ( lysine 4; H3K4 ) and inhibitory ( lysine 27; H3K27 ) sites .", "To investigate the methylation status of H3K4 and H3K27 in CD4+ T cells we used flow cytometry with antibodies that recognise trimethylation on H3K4 and H3K27 , respectively .", "These experiments show that trimethylation ( me3 ) of both H3K4 and H3K27 in CD4+ T cells is increased upon in vitro TCR- stimulation ( Figure 2c ) .", "Figure 2d shows the ratio of staining of total H3 , H3K27me3 and H3K4me3 of TCR-stimulated , compared with non-stimulated CD4+ T cells ( Figure 2d ) .", "We also addressed whether a similar increase in H3K27me3 and H3K4me3 occurs in vivo .", "Accordingly , CD4+ T cells from OT2 TCR transgenic mice were adoptively transferred into normal hosts prior to immunisation with the cognate antigen , ovalbumin .", "Analysis of the H3K27me3 and H3K4me3 staining shows that H3 trimethylation on K27 and K4 increases upon immune activation in vivo ( Figure 2e , f ) .", "SAM is also used as a substrate for RNA methyltransferases , producing methylated RNA and SAH .", "To investigate if there are changes in RNA methylation during T cell activation we designed experiments where CD4+ T cells were activated in methionine-free media supplemented with 3H-methionine where the radiolabel is attached to the methyl group .", "This allowed us to purify RNA from the activated cells and quantified the incorporation of the 3H-methyl group into RNA .", "The data show that the 3H-radiolabel is incorporated into RNA after 6 hr of TCR/CD28 stimulation , and this is further increased after 18 hr ( Figure 2g ) .", "A key control in this experiment is to ensure that there is no protein contamination in the RNA extractions .", "For this we did a parallel quantification of the incorporation of 3H-methionine and 3H-phenylalanine: the latter would only be present into cellular proteins .", "The data show there was no protein contamination of these RNA preparations as judged by absence of any detectable 3H-phenylalanine label , compared with high levels of 3H-phenylalanine incorporated into protein ( Figure 2h ) .", "How dependent are these protein and nucleotide methylation reactions on extracellular methionine ?", "Figure 2i shows that SAH levels in TCR activated CD4+ T cells and Th1 cells are dependent upon extracellular methionine supply which argues that sustained methionine availability is required to produce methyl donors for protein and nucleotide methylation ( Figure 2i ) .", "In this context , the data in Figure 2j show that the increase in H3K4 and H3K27 trimethylation in CD4+ T cells in response to TCR activation is regulated by extracellular methionine availability ( Figure 2j ) .", "We also examined the importance of extracellular methionine for RNA methylation in T cells .", "One of the most abundant mammalian RNA modifications is methylation of the N6 adenosine ( m6A ) regulated by the METTL3 methyltransferase ( Liu et al . , 2014a ) .", "This RNA methylation is important for mRNA stability and in T cells has been shown to be essential for normal T cell function ( Li et al . , 2017 ) .", "The data in Figure 2k show that the percentage of total m6A methylation in effector Th1 cells decreases as extracellular methionine is decreased ( Figure 2k ) .", "Another RNA post-translational methyl modification found in RNA is 5-methylcytosine ( m5C ) , the data in Figure 2l show that the amount of m5C in mRNA from effector Th1 cells is dependent upon extracellular methionine ( Figure 2l ) .", "Collectively , these data demonstrate that extracellular methionine is not only directed into protein synthesis in T cells but also into the methionine cycle to generate methyl donors for histone and RNA methylation reactions .", "Furthermore , the sustained supply of extracellular methionine is necessary for these processes .", "The elevated levels of SAH and HCy and the increased levels of RNA and histone methylation demonstrate that immune activated T cells increase metabolic flow through the methionine cycle .", "To explore the molecular basis for the increases in methionine metabolism in activated T cells we used quantitative , mass spectrometry-based proteomics to interrogate the expression and abundance levels of methionine cycle enzymes in naïve , TCR activated and effector CD4+ T cells .", "Critical enzymes that regulate the metabolic flow through the two arms of the methionine cycle , the de novo and salvage pathways , are shown in Figure 3a , b .", "We initially looked at the expression levels of these key proteins in naïve T cells by comparing their relative abundance against the backdrop of the total protein landscape .", "The data show that naïve CD4+ T cells express the key methionine cycle enzymes MAT2A , AHCY and MTR ( Figure 3c , d and e ) .", "Interestingly , both MAT2A and AHCY are highly abundant proteins in the naïve cell proteomic landscape ( Figure 3c and", "d ) .", "Similarly , SRM/SMS and MTAP , enzymes which use SAM for polyamine synthesis and subsequent methionine salvage , are abundantly expressed in naïve T cells ( Figure 3f and g ) .", "mtnA-D , enzymes involved in the final steps of methionine salvage , are also expressed at levels higher than the ‘average’ protein is expressed in a T cell ( Figure 3h ) .", "The relative abundance , as indicated by their concentration , of these proteins within the total proteomic landscape is consistently maintained at high levels upon activation and differentiation of CD4+ T cells ( Figure 3i ) .", "The data in Figure 2 show that immune activation of T cells drives increased RNA methylation .", "We therefore interrogated the proteomic data to quantify expression of essential RNA methyltransferases in naïve and immune activated T cells .", "mRNA methyl cap formation marks RNAs for further processing , nuclear export and translation initiation ( Gonatopoulos-Pournatzis and Cowling , 2014 ) ; the m6A RNA methylation regulates mRNA stability ( Geula et al . , 2015 ) .", "Key methyltransferases for RNA cap methylation are RNMT , the RNA ( guanine-7- ) methyltransferase ( Cowling , 2009 ) , and the cap methyl transferases CMTR1 or CMTR2 ( Inesta-Vaquera and Cowling , 2017 ) .", "The proteomics data show that RNMT and CMTR1 are abundantly expressed in naïve and at similar levels in TCR activated CD4+ T cells , whereas CMTR2 is not expressed at high levels in any population ( Figure 4a ) .", "The data show that naïve T cells also express equimolar levels of the METTL3/METTL14 m6A methyltransferase complex ( Figure 4b ) ( Liu et al . , 2014b ) .", "Another frequent RNA modification is cytosine −5 methylation ( m5C ) .", "The proteomics data show that naïve T cells express high levels of NSUN2 , the methyltransferase responsible for RNA m5C modification , these high levels are maintained after TCR activation and through differentiation ( Figure 4c ) ( Yang et al . , 2017 ) .", "The high levels of expression of the RNA methyl transferases in naïve T cells is in contrast to the pattern of expression of the histone and DNA methyl transferases .", "The expression of the DNA methyl transferases DNMT1 and DNMT3a is markedly higher in immune activated CD4+ T cells than in naïve T cells ( Figure 4d ) .", "Moreover , activated CD4+ T cells dramatically increase expression of UHRF1 , which is required for DNMT1 recruitment and activity ( Nishiyama et al . , 2016 ) .", "The expression of the polycomb repressive complex 2 ( PRC2 ) , which is responsible for histone 3 lysine 27 methylation ( H3K27 ) , is also highly increased upon T cell activation and in differentiated effector T cells compared with naïve T cells ( Figure 4e , f ) as is expression of other key histone methyltransferases , responsible for histone methylations including H3K4 , K9 and K36 and H4K20 methylation ( Bochyńska et al . , 2018; Mozzetta et al . , 2015; Wagner and Carpenter , 2012 ) ( Figure 4g ) .", "One striking observation in the current study was that even short periods of methionine deprivation cause a loss of RNA and histone methylations .", "A key question then , is whether the dependence of these cells on extracellular methionine for histone and RNA methylation reflects that methionine is needed to sustain expression of the methionine cycle enzymes and/or RNA and histone methyltransferases .", "To address this question , we used mass spectrometry to assess the impact of acute methionine deprivation on the proteome of effector Th1 CD4+ T cells .", "In these experiments , Th1 CD4+ T cells were switched into media containing 1μM or 100μM methionine for 5 hr prior to processing for single-shot proteomics from which we identified 4’400 proteins .", "The data shows that the protein expression profile did not significantly vary upon acute methionine deprivation , with a very high correlation coefficient between the two conditions of 0 . 98 ( Figure 5a ) .", "Proteins that showed significant changes in expression ( increased or decreased ) are listed in Figure 5b .", "With regard to the methionine cycle specifically , these experiments found that acute short term methionine deprivation of Th1 cells had no impact on expression of key enzymes including; MAT2A , AHCY , SRM/SMS , MTAP and mtnA ( Figure 5c–g ) .", "The expression of RNA , DNA and histone methyl transferases was also not changed by short-term methionine restriction ( Figure 5h–j ) .", "The need for T cells to sustain supply of methionine to maintain the methionine cycle is thus explained by the need for methionine to produce methyl donors .", "How much does methionine deprivation impact on other metabolic programs in T cells ?", "In this context , the serine/threonine kinase mTORC1 and the transcription factor c-myc can be described as hubs of metabolic regulation in T cells .", "Appropriate mTORC1 activity and c-myc expression are critical for correct effector T cell responses ( Xu et al . , 2012; Zeng and Chi , 2017; Wang et al . , 2011 ) .", "In both T cells and NK cells , expression of c-myc protein is highly sensitive to amino acid availability and expression of amino acid transporters ( Sinclair et al . , 2013; Loftus et al . , 2018 ) .", "To explore whether c-myc expression was dependent upon extracellular methionine availability , we used a mouse model where a fusion protein of GFP-Myc is expressed under the control of the endogenous Myc promoter ( GFP-MycKI ) ( Huang et al . , 2008; Preston et al . , 2015 ) .", "GFP-MycKI CD4+ T cells increase expression of the activation marker CD69 in response to TCR/CD28 stimulation in both the presence ( 100μM ) and absence of methionine .", "The data show that the induction of GFP-Myc expression in response to TCR/CD28 activation is blunted in the absence of methionine ( Figure 6a , b ) .", "It has also recently been demonstrated that methionine availability regulates mTORC1 activity through metabolite SAM binding to SAMTOR; SAM- SAMTOR then associates with and inhibits GATOR1 , resulting in lysosomal recruitment and activation of mTORC1 ( Gu et al . , 2017; Valvezan and Manning , 2019 ) .", "To investigate whether mTORC1 activity in T cells is sensitive to extracellular methionine availability , effector CD4+ Th1 cells were switched to methionine free conditions and the impact of this on mTORC1 activity in Th1 cells was assessed .", "The data show mTORC1 activity in T cells was partially sensitive to methionine deprivation , though the impact of methionine loss was not equivalent to total amino acid deprivation or rapamycin treatment ( Figure 6c , d ) .", "Treatment with SAM ( 50μM ) modestly restores mTORC1 activity in the absence of methionine ( Figure 6e , f ) .", "These data show that methionine availability and the production of SAM can feed into control of mTORC1 activity .", "The impact of methione deprivation on c-myc expression and mTORC1 activity highlights the diversity of cellular responses in T cells that are able to sense the external methionine environment .", "The proteomic data reveal that increased flux through the methionine cycle and increases in RNA methylation are not explained by changes in the abundance of the key enzymes of the methionine cycle or the RNA methyl transferases .", "However , the sustained availability of extracellular methionine is critical which raises the possibility that the rate limiting step for methionine metabolism in activated T cells is the rate of methionine transport .", "To investigate the methionine transport capacity of naïve versus effector T cell populations , we compared radiolabeled methionine uptake in naïve and activated T lymphocytes .", "Naïve CD4+ T cells show very little/near undetectable uptake of 3H-labeled methionine ( Figure 7a ) whereas methionine transport was readily detected in CD4+ T cells activated with CD3/CD28 crosslinking antibodies and in effector Th1 cells ( Figure 7a ) .", "Figure 7b show that the high levels of methionine transport in Th1 cells is dependent on sustained signalling via the IL2 receptor .", "Hence , either the removal of IL2 , or the exposure of cells to limiting IL2 concentrations , results in a decrease of 3H-methionine uptake .", "Together , these data demonstrate that naïve CD4+ T lymphocytes greatly increase methionine uptake in response to antigenic stimulation .", "These changes in methionine transport could reflect changes in either the expression or activity of T cell methionine transporters .", "Mammalian methionine transporters include the System ASC ( alanine-serine-cysteine preferring ) transporter SLC1A5; the System L transporters SLC7A5 and SLC7A8; the System y + L transporters SLC7A6 and SLC7A7; and the System A transporters SLC38A1 and SLC38A2 ( Utsunomiya-Tate et al . , 1996; Baird et al . , 2009; Bröer and Palacín , 2011; Kanai et al . , 1998; Nii et al . , 2001; Napolitano et al . , 2015 ) .", "Interrogation of the naïve , TCR and effector T cell proteomic data identified several candidate methionine transporters in TCR stimulated CD4+ T cells and effector Th1s; notably SLC1A5 , SLC7A5 , SLC7A6 and SLC38A2 ( SNAT2 ) ( Figure 7c ) .", "The candidate methionine transporters that were detected in activated T cells have different expression levels: the most abundant candidates were SLC7A5 and SLC1A5 ( ASCT2 ) .", "SLC7A6 , and SLC38A2 ( SNAT2 ) are both expressed at far lower levels than SLC7A5 .", "The proteomic data moreover reveal the basis for the failure of naïve T cells to transport methionine: they have a very low copy number of any candidate methionine transporter ( Figure 7c ) .", "Hence in contrast to methionine cycle enzymes which are abundantly expressed in naïve and effector CD4+ T cells , expression of methionine transporters is restricted to TCR activated and effector T cells .", "One way to address which methionine transporter dominates in activated T cells is to use selective pharmacological approaches that would distinguish the different candidates .", "For example , 2-aminobicyclo- ( 2 , 2 , 1 ) -heptane-2-carboxylic acid ( BCH ) is a competitive blocker for System L transporters ( Verrey et al . , 2004 ) ; MeAIB competitively blocks SLC38A2 ( Mackenzie and Erickson , 2004 ) .", "Alanine has a high affinity for SLC1A5 ( Kanai and Hediger , 2004 ) ; lysine is transported preferentially by SLC7A6 .", "Accordingly , alanine and lysine competition of methionine transport can be used as evidence for involvement of SLC1A5 or SLC7A6 respectively ( Verrey et al . , 2004 ) .", "Furthermore , SLC1A5 , SLC38A2 and SLC7A6 are all dependent on sodium to mediate methionine transport ( Kanai and Hediger , 2004; Mackenzie and Erickson , 2004; Verrey et al . , 2004 ) .", "Accordingly we assessed the biochemical properties of the methionine transporters in activated T cells .", "The data show that 3H-methionine uptake in CD4+ effector Th1 cells is blocked by BCH .", "The data also show that competition by either Alanine , Lysine or MeAIB which would block System A mediated uptake has little impact on methionine transport in activated CD4+ T cells ( Figure 7d ) .", "Furthermore , 3H-methionine uptake in effector CD4+ T cells is sodium independent ( Figure 7e ) .", "This contrasts with glutamine uptake in the same effector cells , which is not affected by BCH and is sodium dependent ( Figure 7e , f ) .", "Collectively these data establish that methionine transport in activated T cells is via System L transporters .", "Further evidence for this conclusion is shown in Figure 6g: the cellular uptake of the System L substrate 3H-phenylalanine is competitively inhibited by methionine ( Figure 7h ) ; 3H-methionine incorporation into protein in activated T cells is blocked by the System L transport inhibitor BCH ( Figure 7h ) ; TCR induced production of methylated RNA is prevented in the presence of BCH ( Figure 7i ) ; BCH treatment reduces SAH levels in TCR activated CD4+ T cells ( Figure 7j ) ; m6A mRNA methylation is reduced in the presence of BCH ( Figure 7k ) .", "Collectively these data show that methionine delivery mediated via System L amino acid transporters is the rate limiting step for the methionine cycle in T cells and rate limiting for protein synthesis and RNA methylation .", "The only System L candidate identified in the proteomics was SLC7A5 and this was also very abundant ( Figure 7c ) .", "To test the involvement of SLC7A5 directly we examined the methionine transport capacity of T cells lacking SLC7A5 expression .", "Figure 7l shows that SLC7A5 null CD4+ T cells do not increase methionine uptake in response to activation with CD3/CD28 antibodies .", "SLC7A5 null CD4+ T cells T cells also show a striking decrease in their ability to increase levels of the methionine metabolite SAH in response to TCR/CD28 stimulation ( Figure 7m ) .", "This is consistent with SLC7A5 expression being required for the increased flux of methionine through the methionine cycle in response to TCR activation and differentiation ." ], [ "The present study explores how T cells control a fundamental metabolic pathway and shows that antigen receptor triggering of T cells initiates a cycle of methionine metabolism that generates the methyl donors required for RNA and histone methylation .", "We establish that a critical , rate limiting step to fuel protein synthesis and the methionine cycle in T cells is antigen receptor and cytokine regulation of methionine transport across the cell membrane .", "Naïve T lymphocytes express high levels of all the key methionine cycle enzymes but cannot transport sufficient methionine to provide the substrates for these enzymes or to fuel protein synthesis .", "The methionine cycle and protein synthesis are thus only initiated in T cells when antigen receptor engagement signals the expression of methionine transporters that support high rates of methionine transport .", "This provides some new understanding regarding mechanisms that ensure the immunological specificity of effector T cell differentiation .", "Only T cells that respond to antigen to upregulate methionine transport will be able to fuel protein synthesis and supply the methyl donors that permit the dynamic nucleotide methylations and epigenetic reprogramming that drives T cell differentiation .", "Activated T cells express multiple candidate mammalian methionine transporters , therefore we have used a combination of pharmacological and genetic strategies and pinpoint that the sodium independent System L amino acid transporter , SLC7A5 is the dominant methionine transporter in activated T cells .", "Previous studies have shown that SLC7A5 is important for T cell differentiation in vitro and in vivo ( Sinclair et al . , 2013 ) and proposed that this reflected the role of SLC7A5 as the key leucine transporter in activated T cells and its subsequent role in regulating the activity of the leucine sensing kinase mammalian target of rapamycin complex 1 ( mTORC1 ) .", "The present data provide additional novel information as to why SLC7A5 is critically important for T cells; it is required to transport methionine .", "The present data further highlight that the importance of SLC7A5 for T cell biology reflects that it is a common transporter for multiple essential amino acids .", "The dependence of T cell activation , differentiation and proliferation on extracellular methionine was striking and indicates that T cells cannot rely on methionine salvage pathways or autophagy of intracellular cargo to meet their demands for methionine .", "One reason methionine supply is so important for immune T cells is to fuel de novo protein synthesis .", "For example , we show that the expression of c-myc , a critical transcription factor for T cells , is controlled by external methionine availability and this is a simple reflection of the fact that proteins with a short half life like c-myc will be dependent on sustained availability of amino acids .", "In this latter context , it has been shown that in vivo activated CD8+ T cells exhibit dynamic control of translation as they differentiate from naïve ( low translation rates ) into effector ( high translation rates ) and memory ( low translation rates ) cells ( Araki et al . , 2017 ) .", "The present data highlight that the most critical rate limiting step for mRNA translation is the rate of methionine transport via the System L transporter SLC7A5 because it is this transporter that determines intracellular methionine bioavailability .", "We have previously shown that SLC7A5 expression is restricted to TCR activated effector cells and low in naïve and memory T cells ( Sinclair et al . , 2013; Preston et al . , 2015 ) .", "The dynamic changes in mRNA translation observed by Araki et al . ( 2017 ) could thus reflect changes in the methionine transport capacity of naïve versus effector versus memory T cells .", "One other insight from present data was how much T cells depend on an external methionine supply and the controlled expression of methionine transporters to sustain the production of S-adenosylmethionine ( SAM ) , the methyl donor for DNA , RNA and protein methyltransferases .", "Methionine availability and SAM production also modulate mTORC1 activity in T cells analogous to the recently described methionine/SAM sensing pathways that regulate mTORC1 in HEK 293 T cells ( Gu et al . , 2017 ) .", "The supply of methyl donors for protein and nucleotide methylations is critical for many cellular processes .", "For example , mRNA cap methylation controls mRNA binding and translation ( Gonatopoulos-Pournatzis and Cowling , 2014 ) ; the methylation of adenosine , ‘m6A’ , in mRNA controls mRNA stability and is important for T cell homeostasis ( Li et al . , 2017 ) .", "The data herein give new insights namely that engagement of the TCR induces RNA methylation and it is the ability of TCR triggering to control methionine transport to fuel the methionine cycle that allows the TCR to control RNA methylation .", "In this context the TCR does not control expression of the RNA methyltransferases; these are already present in naive T cells .", "The generation of methyl donors by the methionine cycle is also necessary for DNA methylations which are known to be critical for the transcriptional reprogramming that controls T cell differentiation with both increased and repressed transcription of multiple genes , coordinated by changes in DNA methylations ( Crompton et al . , 2016; Youngblood et al . , 2017; Thomas et al . , 2012; Hashimoto et al . , 2013; Makar and Wilson , 2004; Ladle et al . , 2016 ) .", "Regulated changes in histone methylation are also critical for T cell ( Henning et al . , 2018 ) .", "Our quantitative proteomics analysis revealed that expression of histone methylation complexes is low in naïve T cells and increased upon T cell activation .", "The ability of immune activated T cells to control histone methylations requires the co-ordination of the expression of methionine transporters to supply the methionine cycle to make methyl donors as well as to supply methioinine for ‘building’ the expression of the key methyltransferase complexes .", "This is in contrast to the control of RNA methylation where methyltransferases responsible for RNA modifications are present in naïve T cells and poised awaiting substrate availability .", "The present work highlights that a full understanding of epigenetic control of cell phenotypes and the importance of RNA methylations requires knowledge of the rate limiting processes that control the methionine cycle under physiological conditions .", "Hence identification of methionine transporters and understanding the molecular details of how methionine transporter expression is regulated is important to understand how cells control the supply of methyl donors to support key biological processes .", "In this respect the importance of methionine bio-availability to other cell lineages is now recognized eg for liver cells and embryonic stem cells ( Tang et al . , 2017; Shiraki et al . , 2014; Mentch et al . , 2015 ) .", "However , these studies do not explore or discuss the possibility that methionine availability to cells is determined by regulated expression of methionine transporters .", "There is only a limited understanding of the identity of the relevant methionine transporters in different cell types and very little understanding of the signals that control expression of methionine transporters in different tissues .", "Finally , the importance of regulated changes in methionine transport for T cell activation predict that changes in dietary methionine , as seen by Mentch et al . ( 2015 ) , could impact on T cell function .", "Extracellular methionine availability in plasma is thus dependent upon dietary intake and dietary methionine restriction has been shown to alter histone methylation in the liver ( Mentch and Locasale , 2016; Mentch et al . , 2015 ) .", "Whether a T cell would encounter limiting methionine concentrations is unknown , but it is a possibility .", "In particular it is possible that T cells may have limited access to methionine if they are positioned in tissues where other cells compete for nutrients .", "For example in a tumour microenvironment .", "The present data indicate that any study of how dietary methionine impacts any cell will need to consider the impact of restricting methionine on a range of intracellular signaling and metabolic pathways , RNA modifications and mRNA stability and not only consider the availability of methionine for protein synthesis ." ], [ "C57BL/6 ( wild-type , WT ) , Cd4-Cre::Slc7a5fl/fl , GFP-MycKI and OT2 TCR transgenic mice were bred and maintained in the WTB/RUTG , University of Dundee in compliance with UK Home Office Animals ( Scientific Procedures ) Act 1986 guidelines .", "To activate primary T cells , lymph nodes were removed and disaggregated .", "Cells were cultured in RPMI 1640 containing L-glutamine ( Invitrogen , ThermoFisher Scientific , UK ) , 10% FBS ( Gibco , ThermoFisher Scientific , UK ) , 50 μM β-mercaptoethanol ( β-ME , Sigma-Aldrich , UK ) and penicillin/streptomycin ( Gibco ) .", "Cells were stimulated with 1 μg/ml of the CD3 monoclonal antibody ( 2C11 ) and 2 μg/ml anti-CD28 ( 37 . 51; ebiosciences , ThermoFisher Scientific , UK ) in the presence of cytokines IL12 ( 10 ng/ml; RnD Systems , UK ) and 20 ng/ml IL2 ( 20 ng/ml; Proleukin , Novartis , UK ) .", "To generate Th1s , murine CD8+ T cells were depleted from lymph node preparations using CD8 depletion kit ( EasySep , STEMCELL Technologies , UK ) .", "The resulting mix of CD4+ T cells and APC were cultured at 3 × 105 cells/ml for 5 days in the presence of anti-CD3 ( 2 μg/ml ) and anti-CD28 ( 3 μg/ml ) and cytokines IL12 ( 10 ng/ml ) and 20 ng/ml IL2 ( 20 ng/ml ) .", "For proteomics samples , naïve CD4+ T cells were isolated from lymph node and sorted gated on CD4+ , CD62Lhigh and CD44low .", "Live TCR activated CD4+ cells and Th1 cells were sorted for CD4+ expression and DAP1 exclusion .", "Cells were then cultured in 100 μM or 1 μM methionine for a further 5 hr before processing for single-shot proteomics analysis .", "Where indicated , methionine free RPMI ( ThermoFisher ) was supplemented with 10% dialysed FBS ( ThermoFisher ) and reconstituted with L-methionine ( Sigma ) .", "Cells were incubated at 37 ˚C with 5% CO2 throughout .", "For in vivo activation and proliferation , OT2 ( CD45 . 1 ) lymph node cells were injected into C57/Bl6 ( CD45 . 2 ) hosts .", "After 24 hr , mice were immunised i . p . with 4-Hydroxy-3-nitrophenylacetyl hapten conjugated to ovalbumin ( NP-OVA; 100 μg; BioSearch technologies , UK ) adsorbed to alum ( Pierce , UK ) .", "Spleens were harvested and transferred cells were identified and analysed at D3 after activation .", "For cell surface staining , antibodies conjugated to FITC , PE , APC , AlexaFluor 647 , APC-efluor780 , AlexaFluor 700 , PerCPCy5 . 5 , Brilliant Violet 421 and 605 were obtained from either BD Biosciences , eBioscience or Biolegend .", "Fc receptors were blocked using Fc Block ( BD Biosciences ) .", "Antibody clones used were: CD4 ( RM4-5 ) , TCRβ ( H57-597 ) , Vα2 ( B20 . 1 ) , CD62L ( MEL-14 ) , CD44 ( IM7 ) , CD45 . 1 ( A20 ) , CD45 . 2 ( 104 ) , CD69 ( H1 . 2F3 ) .", "Cells were fixed using 1% paraformaldehyde .", "Standard intracellular cytokine staining protocols were followed for INFγ ( clone XMG1 . 2; Biolegend ) staining .", "For intracellular histone methylation staining or phospho-S6 staining , cells were permeabilised post fixation by incubation with 90% ( v/v ) methanol at −20°C for at least 30 min .", "Following permeabilisation , cells were washed twice and incubated with antibody against Tri-methyl-Histone H3 ( Lys 27; clone C36B11 ) , Tri-methyl-Histone H3 ( Lys 4; clone C42D8 ) , Histone H3 ( clone D1H2 XP ) or phospho-S6 Ser235/236 ( # 2211 ) with anti-rabbit Alexa 647 ( # 4414 ) secondary ( Cell Signaling Technology ) .", "Cells were then washed and resupended in 0 . 5% FBS ( v/v ) in PBS for acquisition .", "For flow cytometry assays for protein , RNA and DNA synthesis; cells were treated with either 20 μM O-propargyl-puromycin ( OPP , Jena Bioscience ) for 10 min , Click-iT EdU ( 10 μM; Thermo Fisher ) for 45 mins or Click-iT EU ( 2 mM; Thermo Fisher ) for 30 mins .", "The incorporation of the analogues into newly synthesized protein , RNA and DNA was measured by labelling with Alexa 647-azide ( Invitrogen ) using a standard Click-IT chemistry reaction ( Thermo Fisher ) .", "As negative controls , cyclohexamide ( 100 μg/ml; Sigma; 30mins ) and Actinomycin D ( 5 μg/ml; Sigma; 45 min ) were added to stop protein and RNA synthesis , respectively .", "Data were acquired on a LSR Fortessa II with DIVA software or a FACSVerse flow cytometer with FACSuite software ( BD Biosciences ) and analyzed using FlowJo software ( TreeStar , version 9 and 10 ) .", "Gating strategies are shown in Supplemental data .", "S-adenosyl-homocysteine ( SAH ) levels were measured using a competitive ELISA ( Axis Homocysteine EIA , FHCY 100 , Axis-Shield ) as recommended , omitting the primary enzymatic conversion of homocysteine to SAH step .", "Briefly , nutrient uptake was carried out using 1 × 106 cells resuspended in 0 . 4 ml uptake medium .", "Each uptake for a biological replicate is performed in triplicate .", "Methionine uptake was carried out in HBSS ( ThermoFisher Scientific ) containing [3H] L-methionine ( 1 μci/ml ) and a final extracellular L-methionine concentration of 0 . 5 μm .", "Similarly , phenylalanine uptake was performed using [3H] L-phenylalanine ( 0 . 5 μci/ml ) and a final extracellular L-leucine concentration of 0 . 5 μm .", "2 min uptake assays were carried out layered over 0 . 5 ml of 1:1 silicone oil ( Dow Corning 550 ( BDH silicone products; specific density , 1 . 07 g/ml ) :dibutyl phthalate ( Fluka ) .", "Where indicated , [14C] L-glutamine ( 0 . 1 μci/ml ) was added to assay glutamine uptake simultaneously .", "Cells were pelleted below the oil , the aqueous supernatant solution , followed by the silicon oil/dibutyl phthalate mixture was aspirated , and the cell pellet underneath resuspended in 200 μl NaOH ( 1M ) and β-radioactivity measured by liquid scintillation counting in a Beckman LS 6500 Multi-Purpose Scintillation Counter ( Beckman Coulter ) .", "Where indicated , 5 mM BCH , L-Alanine , L-Lysine , L-Leucine , L-Methionine or MeAIB were used respectively to quench radiolabeled ligand uptake .", "The sodium free buffer was TMACl as described in Baird et al . ( 2009 ) .", "Data is expressed as molecules radiotracer per cell per minute .", "[3H] L-methionine , [3H] L-phenylalanine and [14C] L-glutamine were obtained from Perkin Elmer .", "All other chemicals were obtained from Sigma .", "IL2/12 maintained effector Th1 cells were cultured with 3H-methionine for 6 hr .", "Protein from 5 × 106 cells was precipitated with 0 . 5 ml 10% trichloroacetic acid ( TCA ) , for 15mins at room temperature .", "The protein pellet was washed ( x3 ) with cold acetone , and acetone was allowed to evaporate .", "RNA was isolated from 5 × 106 cells with RNeasy minikit ( Qiagen ) , and quantified by NanoDrop ( ThermoFisher ) .", "Samples were resuspended in scintillation fluid ( Optiphase HiSafe 3 , PerkinElmer ) and 3H radioactivity in TCA precipitated protein , or RNA was measured by liquid scintillation counting in a Beckman LS 6500 Multi-Purpose Scintillation Counter ( Beckman Coulter ) .", "m6A and m5C RNA methylation m6A and m5C RNA methylation was quantified using the fluorometric EpiQuik m6A or 5-mC RNA Methylation quantification Kit ( Epigentek ) respectively , following RNA isolation from 5 × 106 cells using RNAEasy minikit ( Qiagen ) .", "Statistical analyses were performed using Prism 4 . 00 , GraphPad Software , or Sigma Plot ( Systat ) .", "A Shapiro-Wilk test for normality was performed to determine suitable tests for parametric or non-parametric populations .", "F-tests were performed to determine equal variance of populations , otherwise tests assuming unequal variance were performed .", "Multiple comparisons in one-way ANOVA analyses were corrected for using the Holm-Sidak method .", "Where indicated , EC50 and R2values were calculated with least squares fit , no constraints applied .", "All used tests are stated in the respective figure legends , statistical tests used for proteomics analysis are stated in the Proteomics Materials and methods section ." ] ]

[ "Immune activated T lymphocytes modulate the activity of key metabolic pathways to support the transcriptional reprograming and reshaping of cell proteomes that permits effector T cell differentiation .", "The present study uses high resolution mass spectrometry and metabolic labelling to explore how murine T cells control the methionine cycle to produce methyl donors for protein and nucleotide methylations .", "We show that antigen receptor engagement controls flux through the methionine cycle and RNA and histone methylations .", "We establish that the main rate limiting step for protein synthesis and the methionine cycle is control of methionine transporter expression .", "Only T cells that respond to antigen to upregulate and sustain methionine transport are supplied with methyl donors that permit the dynamic nucleotide methylations and epigenetic reprogramming that drives T cell differentiation .", "These data highlight how the regulation of methionine transport licenses use of methionine for multiple fundamental processes that drive T lymphocyte proliferation and differentiation ." ]

[ "White blood cells known as T cells are an essential part of the immune system .", "If these cells do not work properly the immune system falls down , leading to disease and eventually death .", "T cells have receptors on their surface that can detect molecules that do not belong in the body and that may indicate an infection or cancer .", "When one of these foreign molecules is detected , the T cell will activate and transform to become better equipped to protect the body .", "These two processes known as activation and differentiation involve extensive changes within the T cell .", "Many of these changes rely on the addition of a small chemical tag onto molecules such as DNA , RNA or proteins .", "The tag , a methyl group , is most often obtained by breaking down a chemical called methionine , one of the building blocks of proteins .", "Like all other animals , humans cannot make methionine , and so we must instead obtain it through our diet or recycle it from existing proteins .", "This raised some questions: does the availability of methionine limit the activity of T cells ?", "And , if so , is it the uptake or breakdown of methionine that has the biggest effect ?", "Sinclair et al . answered these questions by studying T cells from mice .", "First , the T cells were activated , the proteins from those cells were then collected and quantified using a technique called high resolution mass spectrometry .", "In further experiments , the uptake and use of a radioactively labeled version of methionine was followed in activated T cells .", "Using these approaches , Sinclair et al . showed T cell activation increased the cells demand for methionine , and that T cells need a steady supply of methionine to remain activated .", "The main limiting factor in this process was the speed at which the cell could make the transport systems it needs to collect methionine from its surroundings .", "These findings could prove useful in developing treatments for diseases associated with uncontrollable T cells , such as leukaemia and certain autoimmune diseases .", "Such treatments could , for example , involve restricting the transport of methionine into T cells through drugs , or potentially via the diet ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "developmental biology", "neuroscience" ]

[ [ "MicroRNAs ( miRNAs ) are a class of small regulatory non-coding RNAs that participate in various biological functions by repressing gene expression post-transcriptionally ( Ambros , 2004; Bartel , 2004; He and Hannon , 2004; Kim , 2005 ) .", "Several mouse knockout ( KO ) studies of Dicer , the pivotal RNase for generating mature miRNAs , have revealed that global depletion of these small regulatory RNAs leads to obvious defects in embryonic development , from cell differentiation to animal survival , underlining the functional importance of miRNAs during embryonic development ( Bernstein et al . , 2003; Chen et al . , 2011; Harfe et al . , 2005; Kanellopoulou et al . , 2005; Tung et al . , 2015 ) .", "However , loss-of-function studies on most individual miRNAs have revealed minor or no overt developmental or behavioral phenotypes in multiple organisms , partly reflecting the functional redundancy of miRNAs due to their polycistronic and paralogous features ( Alvarez-Saavedra and Horvitz , 2010; Miska et al . , 2007; Olive et al . , 2015; Park et al . , 2012 ) .", "Moreover , a single miRNA can target multiple mRNAs , and a targeted mRNA can also be bound by different miRNAs at its 3ʹ untranslated region ( 3ʹ UTR ) with differential stoichiometric affinities , representing a reciprocal one-to-multiple regulatory mechanism ( Bartel , 2009; Krek et al . , 2005; Loh et al . , 2011 ) .", "These multiple interactions with differential affinities and stoichiometries add complexity to functional redundancy , rendering it exceedingly difficult to study the function of individual miRNAs .", "Hence , considering the extensiveness of miRNA regulatory networks as well as their buffering effects , unequivocal functional assessments of a miRNA can only be established upon complete removal of all associated redundant miRNAs and pathways .", "Fortunately , the advent of CRISPR-Cas9-mediated technology has made it more feasible to study miRNA function in mouse models ( Li et al . , 2017 ) .", "Many miRNAs are clustered with redundant paralogs in the genome .", "One well-characterized example of a polycistronic miRNA family is MiR34/449 , which comprises six homologous miRNAs ( MiR34a , 34b , 34c , 449a , 449b , and 449c ) located at three different genomic loci—Mir34a , Mir34b/c , and Mir449a/b/c ( Mir449 ) —that encode evolutionary-conserved sequences among vertebrates ( Olive et al . , 2015; Song et al . , 2014 ) .", "In this study , we use ‘Mir34’ and ‘MiR34’ to indicate the mouse gene and the mature form of miRNA , respectively ( Desvignes et al . , 2015 ) .", "The MiR34a , MiR34b , and MiR34c ( MiR34 ) miRNAs appear to be direct targets of p53 , and they have been shown to be involved in p53-mediated apoptosis in cancer contexts ( Bommer et al . , 2007; Corney et al . , 2007; He et al . , 2007; Raver-Shapira et al . , 2007 ) .", "Whereas Mir34a and Mir34bc are not located within other genes , the Mir449 cluster is embedded in the second intron of a host gene , Cdc20b ( cell division cycle 20 homologue B ) , and similar expression patterns of MiR449 and Cdc20b in mouse testis support that they may be subjected to the same mechanism of transcriptional regulation ( Bao et al . , 2012 ) .", "Previous studies have demonstrated that the p53-mediated MiR34 and E2F1-mediated MiR449 pathways both target pro-survival genes to regulate cell fate determination ( Lizé et al . , 2011; Lizé et al . , 2010 ) .", "Apart from this functional redundancy in the context of DNA damage , an additional role for MiR449 in regulating multiciliogenesis by suppressing the Notch pathway has been demonstrated ( Lizé et al . , 2011; Marcet et al . , 2011 ) .", "Mature MiR34a has been shown to be ubiquitously expressed in a variety of organs , whereas MiR34bc and MiR449 exhibit a cell-type-specific expression pattern , predominantly in ciliated cells , such as in respiratory and reproductive tissues , as well as in the ependymal cells enriched with cilia responsible for cerebrospinal fluid flow within the CNS ( Bao et al . , 2012; Kasai et al . , 2016; Olive et al . , 2015 ) .", "These reports highlight that MiR34/449 might have a functional significance in ciliogenesis ( Chevalier et al . , 2015; Concepcion et al . , 2012; Otto et al . , 2017; Song et al . , 2014; Wu et al . , 2014 ) .", "Due to their shared and redundant seed sequences , MiR34/449 displays compensatory expression upon single KO in a context-dependent manner , emphasizing the necessity to enact combinatorial KO of all Mir34/449 alleles ( Bao et al . , 2012; Choi et al . , 2017; Concepcion et al . , 2012; Wu et al . , 2014 ) .", "Based on three studies of Mir34bc and Mir449 double KO ( DKO ) mice , as well as Mir34/449 triple KO ( TKO ) mice , the most prominent phenotype arising from MiR34/449 ablation is the impairment of motile ciliogenesis , which results in respiratory dysfunction , male/female infertility , and postnatal lethality ( Otto et al . , 2017; Song et al . , 2014; Wu et al . , 2014; Yuan et al . , 2019 ) .", "Given that the MiR34/449 family is strongly expressed in the CNS , it is rather surprising that their function there has been largely unexplored , with only a potential role for MiR34/449 in regulating cortex development having been revealed in embryos ( Fededa et al . , 2016 ) .", "In fact , information on the postnatal and adult functions of MiR34/449 has been lacking , perhaps because Mir34/449 TKO mice exhibit a low survival rate , so acquiring sufficient numbers of adult Mir34/449 TKO mice for behavioral assay is arduous .", "We previously uncovered that MiR34/449 exhibits a dynamic expression pattern during spinal cord development ( Li et al . , 2017 ) , so we hypothesized that MiR34/449 might exert an as yet uncharacterized function in the spinal cord .", "In this study , we tested that hypothesis for embryonic , postnatal and adult mice .", "The spinal cord is one of the most important executive centers for body movement , conveying somatosensory information and integrating motor commands emanating from spinal cord per se or the brain ( Arber , 2017; Arber and Costa , 2018 ) .", "The sophisticated processing and integration of sensory and motor circuits relies on proper organization of multiple neuron types in the spinal cord that possess related anatomical functions ( Osseward and Pfaff , 2019 ) .", "Interneurons ( INs ) that reside in the dorsal horn of the spinal cord terminate in exteroceptive sensory fibers and mediate sensory processing .", "The ventral horn contains both motor neurons ( MNs ) and various INs , which are critical for motor function and for generating the rhythmicity of motor behaviors ( Dasen , 2018; Garcia-Campmany et al . , 2010 ) .", "The hallmarks of neuron populations in the intermediate region of the dorso-ventral spinal cord have just emerged following identification of specific markers ( Dobrott et al . , 2019 ) .", "This region serves as a relay station that receives multiple convergent inputs from proprioceptive , exteroceptive , and corticospinal neurons , which connect monosynaptically to MNs ( Levine et al . , 2014; Tripodi et al . , 2011 ) .", "Activation of these premotor neurons , denoted motor synergy encoders ( MSE ) , induces multiple motor pools that contribute to complex movements ( Levine et al . , 2014; Osseward and Pfaff , 2019 ) .", "These sensory feedback pathways are indispensable for proper formation of spinal circuits and for motor execution , and their perturbation can result in defects of limb position , or imprecise or stiff movement ( Gatto et al . , 2019; Koch et al . , 2018; Osseward and Pfaff , 2019 ) .", "Among MSE , Satb1/2on neurons , a sub-population of lamina V/VI and III sensory relay neurons , represent a family of typical intermediate INs that control proper circuit development in a core sensory-to-motor spinal network .", "Conditional ablation of Satb2 from the intermediate spinal cord results in a hyperflexion phenotype upon triggering the limb withdrawal reflex ( Hilde et al . , 2016 ) .", "Although expression of Satb2 is quite dynamic during spinal development , how generation of Satb2on INs controls precise sensory-to-motor output during development remains unknown .", "It is well documented that miRNAs are critical regulators during embryonic spinal cord development , playing roles in progenitor patterning , subtype specification , functional maintenance , and neural regeneration ( Chen et al . , 2011; Chen and Wichterle , 2012; Chen and Chen , 2019; Kye and Gonçalves , 2014; Tung et al . , 2015; Wu et al . , 2012; Zhu et al . , 2013 ) .", "Furthermore , emerging evidence also demonstrates that miRNAs are not only required for early development but that they also display critical postnatal or adult-specific functions ( Sun and Lai , 2013 ) .", "Notably , loss of Dicer from late-stage post-mitotic spinal MNs using ChAT-Cre results in development of a spinal muscular atrophy ( SMA ) -like phenotype in a mouse model ( Haramati et al . , 2010 ) .", "SMA is an autosomal recessive MN disease causing early childhood death .", "Furthermore , mice in which Dicer has been knocked out from proprioceptive sensory neurons display impaired maintenance of monosynaptic sensory-motor circuits in the spinal cord ( Imai et al . , 2016 ) .", "However , it remains unclear if any miRNA participates in IN development and maintenance , or if miRNAs participate at neural circuit and physiological levels .", "Given that MiR34/449 expression varies dynamically during spinal cord development , we were prompted to test if this miRNA family is important for MN and IN development , as well as in the operation of sensory-motor circuits in the spinal cord .", "To address these possibilities , we generated Mir34bc/449 DKO and Mir34/449 TKO mouse models , and then systematically investigated their phenotypes relating to CNS development .", "First , we uncovered low survival of postnatal and adult Mir34bc/449 DKO and Mir34/449 TKO mice .", "However , we applied several behavioral tests to assay spinal functions in survivors and observed several unusual phenotypes , including blepharoptosis , abnormal posture with back-arching , hind tiptoe walking , jumpy movement , and stimulant-induced hypersensitivity .", "Although motor systems of Mir34bc/449 DKO or Mir34/449 TKO mice only mildly compromised , we found that the MiR34/449 mutant mice displayed a more hypersensitive threshold in response to thermally induced pain stimulation .", "Mechanistically , we observed that the number of Satb1/2-expressing INs increased in the spinal cords of Mir34/449 KO mice .", "This Satb1/2 increment is potentially targeted by MiR34/449 via direct binding in the spinal cord .", "Our findings demonstrate that MiR34/449 depletion results in a change to a spinal IN population that might contribute to altered sensory-motor responses , thereby revealing an important role for MiR34/449 in regulating the sensory-motor circuitry ." ], [ "In previous studies , we identified a series of specific developmental stage enriched miRNAs during spinal cord development by taking advantage of an embryonic stem cell ( ESC ) differentiation approach ( Chen et al . , 2011; Wichterle et al . , 2002 ) .", "The MiR34/449 family particularly drew our attention as NanoString miRNA profiling revealed a very dynamic and distinct expression pattern , even they have the same seed sequence ( Li et al . , 2017; Figure 1A ) .", "Despite other studies having illustrated the expression patterns and functions of MiR34/449 in lung tissues and germ cells ( Concepcion et al . , 2012; Otto et al . , 2017; Song et al . , 2014; Wu et al . , 2014 ) , it remained enigmatic if these miRNAs are expressed in embryonic and adult neural tissues and what were their potential functions in the CNS .", "To address this knowledge gap , we systematically compared expression of MiR34/449 among the CNS ( neocortex , cerebellum , and spinal cord ) , peripheral neural tissues ( trigeminal ganglion and dorsal root ganglion ) , and non-neural tissues ( heart , liver , and muscle ) at an adult stage ( postnatal day P50; Figure 1B~1F ) by quantitative RT-PCR ( RT-qPCR ) .", "We found MiR449b-5p to be undetectable in most tissues .", "Generally , the guide strands of the remaining five MiR34/449 members were enriched in neural tissues relative to non-neural tissues ( Figure 1B~1F ) .", "Although MiR34a-5p is expressed in a variety of organs , it was much more abundant in neural than non-neural tissues .", "Notably , MiR34a-5p exhibited the highest expression level in the spinal cord ( Figure 1B ) .", "This greater neural tissue enrichment was also apparent for MiR34b-5p , MiR34c-5p , MiR449a-5p , and MiR449c-5p ( Figure 1C~1F ) .", "This pattern of neural tissue enrichment prompted us to scrutinize expression profiles of MiR34/449 for embryonic ( embryonic day E11 . 5~E17 . 5 ) to postnatal ( P1~P28 ) stages by RT-qPCR ( Figure 1G~1K ) .", "Generally , MiR34a-5p expression increased gradually across postnatal stages , with MiR34b-5p and MiR34c-5p also displaying a propensity for postnatal enrichment ( Figure 1G~1I ) .", "Conversely , MiR449a-5p and MiR449c-5p were enriched at embryonic stages , manifesting much lower expression levels at postnatal stages when compared to MiR34a/b/c ( Figure 1J and K ) .", "To further determine the spatial distribution of MiR34/449 expression in mouse spinal cord , we assessed in situ hybridization of MiR34a-5p , representing the most abundantly expressed member of the MiR34/449 family in postnatal spinal cords ( Figure 1L and M ) .", "To label neurons in the spinal cord or adjacent regions , we performed immunostainings using pan-neuronal ( NeuN ) and mature MN ( ChAT ) markers after in situ hybridization .", "For both embryonic and postnatal stages , we found that MiR34a-5p is expressed in most spinal cord neurons ( Figure 1M ) , including ventral MNs ( Figure 1N ) and most dorsal INs ( Figure 1O ) .", "These RT-qPCR and in situ hybridization results demonstrate that MiR34/449 members are strongly expressed in the CNS , most prominently in the spinal cord , albeit with a very dynamic expression pattern during spinal cord development .", "Among the MiR34/449 members , MiR34a-5p exhibits the strongest expression in the spinal cord , with low embryonic-stage expression and increasing levels as postnatal stages progress .", "Motivated by our above-described findings , we hypothesized that the MiR34/449 miRNAs may play critical roles in the developing spinal cord .", "As MiR34/449 members display extensive homology and a given miRNA family typically manifests dominant cell-type-specific expression ( Olive et al . , 2015 ) , we generated a Mir34/449 TKO mouse line in which all six family members were completely deleted from the Mir34a , Mir34bc , and Mir449abc loci ( Figure 2A ) .", "To do that , previously generated Mir34afloxed and Mir34bc-/- mice ( Concepcion et al . , 2012 ) were first interbred with germ line Cre mice ( E2a-Cre ) to acquire Mir34a-/-;Mir34bc-/- DKO mice .", "Consistent with previous studies ( Bao et al . , 2012; Concepcion et al . , 2012 ) , we did not observe an overt phenotype in this DKO mouse line ( data not shown ) .", "Subsequently , we generated Mir449-/- mice via a CRISPR-Cas9 approach with the intention of interbreeding it with our Mir34a-/-;Mir34bc-/- DKO mice to acquire the TKO ( Mir34a-/-;Mir34bc-/-;Mir449-/- ) line ( Figure 1—figure supplement 1A ) ( see Materials and methods for details ) .", "Given that the gene encoding the Mir449 cluster is embedded in the second intron of a host gene , Cdc20b , we designed two single-guide RNAs ( sgRNAs ) flanking the Mir449 cluster in exon 2 and exon 3 of Cdc20b ( Figure 1—figure supplement 1A ) , and then confirmed complete deletion of Mir449 by sequencing and genotyping ( Figure 1—figure supplement 1B and C ) .", "RT-qPCR further confirmed that expression of MiR449 had been abolished ( Figure 1—figure supplement 1D ) .", "Although we recorded elevated Cdc20b mRNA levels specifically in testis and lung of our MiR449-deficient mouse model ( Figure 1—figure supplement 1E ) , we did not observe overt defects in survival or fertility for either male or female mice .", "Moreover , we noticed compensatory expression of MiR34bc and MiR449 expression in the spinal cords of Mir34a-/-;Mir34bc-/- and Mir34a-/-;Mir449-/- compound KO mice ( Figure 1—figure supplement 1F~H ) , supporting the desideratum to generate complete MiR34/449 KO mice to assess the functions of that miRNA family in the spinal cord .", "To achieve this , mice harboring one wild-type ( WT ) allele of the Mir34/449 family were then intercrossed to generate Mir34/449 TKO or Mir34bc/449 DKO mice ( Figure 2A ) .", "Consistent with previously reported phenotypes for these lines , both Mir34/449 TKO and Mir34bc/449 DKO mice exhibited partial postnatal lethality and growth retardation with reduced body weight ( Figure 2B and C; Fededa et al . , 2016; Song et al . , 2014; Wu et al . , 2014 ) .", "Therefore , we used either Mir34/449 TKO or Mir34bc/449 DKO mice for further analyses and other genetic combinations were employed as littermate controls ( Ctrl ) ( Figure 2A and Supplementary file 1f ) .", "Unexpectedly , both the Mir34bc/449 DKO and Mir34/449 TKO mice presented two waves of postnatal mortality: a first wave occurred after the first week ( P7~P14 ) ( Song et al . , 2014 ) , with a second wave between P21 and P28 ( Figure 2B ) .", "Approximately 50% of Mir34bc/449 DKO and Mir34/449 TKO mice died within the first 2 weeks of birth , with an additional ~40% died within 4 weeks ( Figure 2B ) .", "Consequently , only ~10% of mice survived to adulthood with less obvious growth retardation or abnormalities .", "This pattern of mortality prompted us to further investigate several of the late postnatal ( P14~P28 ) phenotypes .", "For instance , we found that Mir34bc/449 DKO and Mir34/449 TKO mice presented significantly reduced body weights relative to respective controls ( Figure 2C ) , and they manifested several unusual abnormalities , including blepharoptosis ( ptosis , drooping eyelids ) or microphthalmia ( Figure 2—figure supplement 1A and B ) , abnormal posture ( back-arching; Figure 2D ) , and uncoordinated hind tiptoe walking and jumpy behavior , which resemble neurological deficits related to sensory-motor function defects ( Imai et al . , 2016; Figure 2—videos 1 and 2 ) .", "Given that MiR34/449 miRNAs are highly expressed in the spinal cord ( Figure 1 ) and our MiR34/449 mutant lines displayed apparent neurological disorders and peculiar moving patterns ( Figure 2—videos 1 and 2 ) , we subsequently focused on identifying the functions of MiR34/449 miRNAs in mouse spinal cord .", "As both spinal INs and MNs exhibited strong MiR34/449 expression ( Figure 1N and O ) , we first explored if MN differentiation is affected upon MiR34/449 depletion .", "Detailed examination of the ventral spinal progenitor domain by immunostaining with a battery of cross-repressive transcription factor hallmarks ( Chen and Chen , 2019 ) did not uncover obvious progenitor domain changes in MiR34/449 mutant embryos at embryonic day E10 . 5 ( Figure 3—figure supplement 1A ) .", "Next , we investigated if MN differentiation was abrogated by Mir34/449 locus deletion .", "We crossed our Mir34/449 TKO mice with the MN reporter Hb9::GFP mouse line to facilitate MN identification .", "The number and distribution of post-mitotic MNs were comparable between MiR34/449-deficient and Ctrl embryos ( Figure 3—figure supplement 1B ) .", "As our previous study showed that specific motor columns are preferentially affected and motor pools are eroded in conditional MN-Dicer KO mice ( Chen and Wichterle , 2012 ) , we assessed if MN subtype is affected in the Mir34bc/449 DKO and Mir34/449 TKO mice .", "To do that , we conducted immunostaining with the following known markers to identify each MN column type in specific spinal segments: MMC ( medial motor column; Lhx3on ) , LMC", "( m ) ( medial division of lateral motor column; FoxP1on , Isl1on ) , LMC", "( l ) ( lateral division of lateral motor column; FoxP1on , Isl1off ) , HMC ( hypaxial motor column; Lhx3off , Isl1on ) , and PGC ( preganglionic motor column; FoxP1on , pSmadon ) .", "However , MN subtype identity did not appear to be altered in MiR34/449-deficient embryos at E12 . 5 ( Figure 3—figure supplement 1C and D ) .", "Thus , MiR34/449 miRNAs do not play an overt role in MN development .", "Despite MN molecular identity not being affected by MiR34/449 deficiency , this does not necessarily mean that motor function at physiological or behavior levels is not disrupted .", "Therefore , we tested basal motor activity and locomotor coordination of mutant mouse lines by means of open field , rotarod , and treadmill tests ( Figure 3 and Figure 3—figure supplement 2 ) .", "We also conducted these assays on age-matched WT mice to assist evaluations of the phenotypic variance caused by interbreeding complex genetic KO mice .", "Our results show that both Mir34bc/449 DKO and Mir34/449 TKO mice exhibited slightly yet significantly reduced locomotor activity relative to WT and Ctrl mice ( Figure 3B ) , while their locomotor coordination appeared normal assayed by the rotarod ( Figure 3C and D ) .", "Notably , we recorded obvious and spontaneous jumpy movements in the home cages of Mir34bc/449 DKO and Mir34/449 TKO mice , as well as in the open arena ( occurrence ~35%; Figure 2—video 2 ) .", "Ctrl mice with single or double combination KO with other MiR34/449 members also manifested this behavior with a milder extent , whereas the aged-matched WT hardly showed this phenomenon .", "Subsequently , we performed treadmill to assay fine-scale kinematic study of limb coordination and gait patterns ( Herbin et al . , 2007; Leblond et al . , 2003 ) .", "Both Mir34bc/449 DKO and Mir34/449 TKO mice showed comparable locomotion patterns relative to WT and Ctrl mice in terms of running speed ( limb moving speed ) ( Figure 3E ) and temporal compositions during the step cycle ( Figure 3F and G ) .", "Moreover , phase analyses of inter-limb coordination , including alternating homologous and homolateral limbs as well as synchronous diagonal limbs , were all normal ( relative to WT and Ctrl ) among MiR34/449 mutant mice during treadmill walking ( Figure 3H ) , indicating that the ventral motor circuitry remained intact in the absence of MiR34/449 .", "Finally , at the molecular level , immunostaining of ChATon MNs in postnatal spinal cords further revealed that MiR34/449-deficient mice had similar numbers of MNs compared to Ctrl ( Figure 3—figure supplement 1E and F ) .", "Based on these findings , we suggest that the overall motor function appeared hyper-jumpy while most motor outputs were largely preserved upon loss of MiR34/449 miRNAs .", "Since MiR34/449 also displayed strong expression in dorsal INs ( Figure 1O ) , we examined if it is required to transduce pain modalities using several behavioral paradigms ( Figure 4A; Deuis et al . , 2017; Gregory et al . , 2013 ) .", "We first performed a von Frey test on MiR34/449 mutants , Ctrl and WT mice to measure their responses to mechanical stimulation , and observed that the response threshold in nociceptive withdrawal behavior was comparable among WT , Ctrl , and MiR34/449 mutant lines ( Figure 4B ) .", "Next , we measured responses to heat stimulation by means of a tail flick assay ( representing a spinal reflex ) , whereby the tail withdrawal response is evaluated upon applying a heat stimulus to a mouse’s tail ( Figure 4C; Chapman et al . , 1985; Irwin et al . , 1951 ) .", "We found that MiR34/449 mutant mice displayed a significantly reduced latency in the tail flick test relative to both Ctrl and WT mice ( Figure 4C ) , indicative of increased sensitivity to heat stimulation .", "We then performed a hot-plate experiment whereby a heat stimulus is applied to the hind paws of mice , as this assay is considered to involve more complex supraspinal pathways ( Giglio et al . , 2006 ) .", "We observed a minor ( but not statistically significant ) decrease in hindlimb response latency among MiR34/449 mutant mice when compared to WT and Ctrl mice ( Figure 4D ) .", "Thus , we suggest that the major function of MiR34/449 in the spinal cord relates to spinal reflex circuits .", "Thereafter , we sought to identify the primary targets of MiR34/449 that might be involved in the prominent defects of spinal reflex circuits upon MiR34/449 deletions , first we catalogued in silico-predicted targets of MiR34/449 and performed gene ontology analysis using the DAVID online database ( Huang et al . , 2009; Tung et al . , 2015 ) .", "Interestingly , ‘synapse organization’ , ‘neurogenesis’ , ‘transcription factor regulation’ , and ‘regulation of ion transport’ were the most prominent MiR34/449-mediated pathways arising from that analysis ( Figure 5A ) .", "To establish which genes could explain the spinal reflex phenotype , we further filtered our predicted targets according to ( 1 ) whether they manifested both miRNA ( MiR34/449-5p ) /miRNA* ( MiR34a-3p and MiR34b/c-3p ) targeting sites and ( 2 ) exhibited strong IN expression in postnatal spinal cords ( Figure 5B; Lai et al . , 2016; Osseward and Pfaff , 2019 ) .", "Based on those criteria , we identified potential candidate targets of MiR34/449 that were predicted to participate in IN specification or maturation , including multiple genes previously characterized as being important in INs , that is , Satb1 , Satb2 ( Figure 5B and Supplementary file 1g ) .", "To determine if Satb1/2 are direct targets of MiR34/449 , we constructed luciferase reporters containing the full-length 3ʹ UTR of those genes that harbor predicted MiR34/449 target sites ( Figure 5—figure supplement 1 ) .", "The miRNA target-predicting algorithms identified potential binding sites for MiR34/449-5p and MiR34/449-3p in the 3ʹ UTR of these genes ( Figure 5—figure supplement 1 ) .", "Co-transfection of the luciferase construct with three MiR34/449 expression vectors resulted in a reduction in luciferase activity for the WT 3ʹ UTRs of Satb1/2 constructs ( Figure 5C and D ) , whereas a construct harboring the mutated targeting sequences was completely insensitive to miRNA-mediated silencing ( Figure 5C and D ) .", "These findings indicate that MiR34/449 targets the 3ʹ UTRs of Satb1 and Satb2 .", "Notably , a previous study characterized the cellular identity of MSE neurons and found that Satb1/2 are expressed in medial lamina V/VI and lamina III ( Levine et al . , 2014 ) .", "Unlike for WT and control littermates at P14 , we observed a significantly increased number of Satb1on and Satb2on INs in the MiR34/449 mutant spinal cords ( Figure 5E~5G ) .", "Moreover , we also observed a drastic increase in the number of hybrid cells expressing both Satb1 and Satb2 in the MiR34/449 mutant spinal cords at P14 compared to WT and Ctrl groups ( Figure 5E and H ) .", "Thus , we have shown that Satb1 and Satb2 are directly regulated by MiR34/449 miRNAs in the spinal cord .", "To further explore MiR34/449 and Satb1/2 expression in vivo , we conducted in situ hybridization of MiR34a-5p/MiR34c-5p and compared their expression patterns to that of Satb1/2 .", "Notably , we identified many Satb1/2on INs that co-expressed MiR34a-5p or MiR34c-5p in the dorsal/intermediate regions of the spinal cord at P14 , suggesting potentially direct regulation of Satb1/2 by MiR34/449 in spinal INs ( Figure 5—figure supplement 2 ) .", "Together , these findings indicate that MiR34/449 may control optimal development of Satb1/2on INs in the spinal cord to fine-tune sensory-motor circuit outputs .", "While the function of Satb1 in the spinal interneurons is not elucidated yet , a previous study revealed that Satb2 expression is important for establishing appropriate local connectivity in spinal circuits ( Hilde et al . , 2016 ) , highlighting the possibility that MiR34/449-regulated Satb2 expression might be involved in IN development and consequently contribute to sensory-motor circuit outputs .", "To test this possibility , we first examined if IN development is affected upon MiR34/449 abrogation .", "At E11 . 5 , there were no apparent differences in IN progenitors in the dorsal or ventral horns—as revealed by molecular markers Lbx1 ( dI4 ~6 ) , Lhx1/5 ( dI2 , 4 , 6~V1 ) , Isl1 ( dI3 ) , and Tlx3 ( dI3 , 5 ) for the dorsal horn , or Foxd3 ( dI2 , V1 ) , Chx10 ( V2a ) , and Lhx3 ( V2 ) for the ventral horn , respectively—indicating that MiR34/449 is not required for most IN development ( Figure 5—figure supplement 3A~K ) .", "At E13 . 5 ( the earliest time-point at which we could detect dorsal Satb2 INs ) , there was no significant difference in numbers of Satb2on INs in MiR34/449 mutant spinal cords relative to WT and Ctrl ( Figure 5—figure supplement 3L and M ) .", "Although comparable numbers of Satb2on INs persisted until E16 . 5 ( data not shown ) , there was a dramatic increase of Satb2on INs relative to WT and Ctrl at P14 in the MiR34/449 mutant spinal cords throughout the spinal cord segments ( Figure 5E and F and Figure 5—figure supplement 4 ) .", "This outcome indicates that optimal Satb2on IN number and maintenance is particularly sensitive to MiR34/449 expression in the postnatal spinal cord .", "Collectively , we suggest that MiR34/449 miRNAs control optimal development of Satb2-expressing INs in the spinal cord , particularly at the postnatal stage , enabling this miRNA family to fine-tune and maintain sensory-motor circuit outputs .", "Given that the position of the Satb2on INs is highly associated with the connectivity of the sensory afferents ( Hilde et al . , 2016; Levine et al . , 2014 ) , we next tested if the position of the Satb1on or Satb2on INs is affected in the absence of the MiR34/449 at P14 .", "However , neither Satb1 or Satb2 expressing IN positions were obviously affected ( Figure 5—figure supplement 5 ) , reflecting the different phenotype manifestation between MiR34/449 mutants and Satb2 KO mice ( Hilde et al . , 2016; Levine et al . , 2014 ) .", "Based on previous study ( Hilde et al . , 2016 ) , the molecular identity of the spinal cord is altered upon loss of Satb2 , particularly with regard to Ctip2- and Pax2-expressing IN subpopulations at the embryonic stage .", "Thus , we further examined if the increased Satb2on INs in the MiR34/449 mutant spinal cords display an altered complement of subpopulations .", "Notably , we observed an increase in the number of Satb2on;Ctip2on INs in the MiR34/449 mutant spinal cords ( Figure 6A and B ) , yet only a subtle ( but not statistically significant ) change in the Satb2on;Pax2on IN subpopulation was found ( Figure 6A and C ) .", "As the individual Ctip2on and Pax2on cell populations were of comparable size , this outcome further indicates that the Satb2on;Ctip2on IN subpopulation had preferentially increased , with a concomitant decrease in the Satb2on;Pax2on subpopulation , in the Mir34/449 TKO spinal cords relative to those of WT and control littermates ( Figure 6D and G ) .", "Further spatial distribution analyses revealed no significant change in the distribution of the increased number of Satb2on;Ctip2on , yet these cells tended to exhibit a more condensed pattern in the intermediate region of the MiR34/449 mutant spinal cord ( Figure 6E and F ) .", "Given all of these findings , we suggest that MiR34/449 controls optimal development of Satb2-expressing INs in the spinal cord , and that loss of MiR34/449 leads to a change in the molecular identity of Satb2on IN subtypes .", "Our results reflect a potential role for these miRNAs in maintaining precise cell identity in the postnatal spinal cord , which is critical for refining sensory-motor circuit outputs ( Figure 7 ) ." ], [ "The roles of miRNAs during spinal MN development have been relatively well illustrated .", "Specifically , we have previously shown that MiR17-3p participates in the Olig2/Irx3 bistable loop to carve out the pMN/p2 boundary , and MiR27 operates in the Hoxa5/Hoxc8 regulatory circuit to control the timing of Hoxa5 protein expression to exert brachial MN pool identity ( Chen et al . , 2011; Li et al . , 2017 ) .", "However , despite a series of elegant experiments demonstrating spinal IN identity at molecular and physiological levels ( Levine et al . , 2014 ) , the functions of miRNAs in spinal INs and their relationships to the transcription factor-mediated loop have remained puzzling .", "Here , we have demonstrated that the MiR34/449 family directly regulates Satb1/2 expression at the late differentiation stage , thereby potentially altering sensory-motor circuits given that Satb1/2on INs act as a hub located in the intermediate part of the spinal cord .", "As Satb1/2on INs receive inputs from multiple streams of sensory information and relay their outputs to motor command layers of the spinal cord ( Hilde et al . , 2016 ) , it is conceivable that our Mir34/449 KO mice exhibited hypersensitivity to these sensory inputs , accounting for the peculiar walking patterns we observed .", "Of the IN types in the dorsal spinal cord , why does Satb1/2 expression and the number of Satb1/2on INs need to be precisely controlled by MiR34/449 ?", "Perhaps motor behaviors such as walking or pain-induced limb withdrawal depend on integration of multimodal sensory circuits to elicit appropriate responsive muscle contractions .", "Moreover , Satb1/2on INs are particularly critical in interpreting multiple sensory streams and coordinating them into singular behavioral outputs , with loss of Satb2 leading to impairments of IN cellular position , molecular profile , and pre- and post-synaptic connectivity ( Hilde et al . , 2016 ) .", "The complex behavioral repertoire of animals is regulated by a set of motor synergy encoder ( MSE ) neurons , in which both Satb1 and Satb2 are prominent molecular regulators ( Levine et al . , 2014 ) .", "In contrast to Satb2 , the function of Satb1on INs is not yet completely understood ( Hilde et al . , 2016 ) .", "Although Satb1 and Satb2 are close homologs , they may regulate genes via convergent and divergent pathways .", "In this study , we have also uncovered that Satb1 and Satb2 are expressed largely exclusively in spinal INs .", "Upon MiR34/449 loss-of-function , both Satb1 and Satb2 are aberrantly upregulated , with a consequent increase in Satb1/2 co-expressing cells .", "This scenario highlights the potential involvement of miRNAs in the control of mediating intricate balance of the MSE neurons formation , as well as the critical involvement of miRNAs in maintaining spinal neural circuits at the postnatal stage ( Imai et al . , 2016 ) .", "We found that expression of Satb2 is aberrantly upregulated in our MiR34/449 mutant mice .", "Moreover , we revealed the Satb2on;Ctip2on subpopulation change upon MiR34/449 deletion which enhancement may result in the aberrant connection in the spinal circuitry .", "Therefore , similar to miRNA-mediated establishment of MN subtype identity ( Tung et al . , 2015 ) , we suggest that a Satb1/2-MiR34/449 regulatory axis might be critical to exert and fine-tune the precise level of Satb1/2 expression and to refine Satb1/2on IN number for receipt of multimodal sensory inputs .", "This latter is pivotal for animals to react appropriately to diverse sensory stimuli ( Figure 7 ) .", "In cortical neurons , Satb2 inhibits Ctip2 activity to define two major classes of projection neurons ( Alcamo et al . , 2008; Britanova et al . , 2008 ) .", "However , some neurons in perinatal mice manifest a subpopulation of Satb2/Ctip2 co-expressing cortical neurons ( Harb et al . , 2016 ) .", "Interestingly , postnatal Satb2on spinal INs also co-express Ctip2 at the medial region .", "While the genetic network regulating this Satb2/Ctip2 subpopulation in the cortex and spinal cord remains enigmatic , it is clear that the considerable diversity of projection neurons and spinal INs in mammals cannot simply be attributable to Satb2 inhibitory activity .", "Consequently , the regulatory mechanism underlying Satb2on;Ctip2on neuron subtypes has yet to be identified .", "In this study , we have unveiled that numbers of Satb2on;Ctip2on spinal INs increase upon Mir34/449 KO in mice , raising the possibility that one major function of MiR34/449 is to refine the optimal MSE neurons for precise sensory-motor outputs .", "Given the prominent activity-dependent and tuning role of miRNAs ( Chen and Chen , 2019; Sim et al . , 2014 ) , it is tantalizing to test in the future if MiR34/449 also establishes the balance of Satb2on;Ctip2on INs among cortical projection neurons .", "Given that MiR34/449 also targets a series of synaptic genes ( Figure 5A ) , it warrants further investigation if MiR34/449 also engages in the pre- and post-synaptic connectivity by Satb1/2on INs to support input convergence .", "Importantly , other IN genes such as Rorα and Rorβ are potential targets of MiR34/449 ( Figure 5B ) , so whether MiR34/449 miRNAs regulate a battery of spinal INs and if disruption of those pathways account for diverse neurological disorders are interesting topics for further study .", "Given that Dicer is required to maintain monosynaptic sensory-motor circuits in the spinal cord ( Imai et al . , 2016 ) , it is conceivable to hypothesize that other miRNA candidates might be involved in establishing spinal IN subtype identity and regulating sensory-motor connectivity in the spinal cord .", "Experiments to elucidate the full spectrum of miRNAs involved in these mechanisms will provide deep insights into spinal circuit formation and control .", "A series of studies have revealed that removal of individual miRNA paralogs alone results in partial penetrance of phenotypic deficits .", "For instance , Mir196 TKO gives rise to homeotic patterning defects , whereas single/compound Mir196 KO mice exhibited similar defects to varying degrees ( Wong et al . , 2015 ) .", "In agreement with previous studies ( Bao et al . , 2012; Concepcion et al . , 2012; Song et al . , 2014 ) , we did not observe an overt phenotype in our Mir34 or Mir449 single KO lines relative to Ctrl mice .", "However , a hypersensitivity stress test revealed that our Ctrl mice harboring at least one Mir34/449 allele exhibited trivial but statistically non-significant hypersensitivity in response to a heat stimulus ( Figure 4C ) .", "Hence , it was important that we also subjected age-matched WT mice from an identical genetic background to our assays for comparative analyses ( Andolina et al . , 2018; Andolina et al . , 2016; Otto et al . , 2017; Wu et al . , 2014 ) .", "To obtain reasonable numbers of Mir34bc/449 DKO and Mir34/449 TKO mice for our experiments , we intercrossed the mice possessing one WT Mir34/449 allele ( Figure 2A; see Materials and methods ) .", "Therefore , our molecular and behavioral characterizations reflect comparisons between Mir34bc/449 DKO or Mir34/449 TKO mice and both WT and Ctrl lines , with the genetically-matched Ctrl mice displaying complex heterogeneity and possessing at least one and up to at most five alleles targeted for deletion in KO lines ( Simpson et al . , 1997 ) .", "Our observations provide evidence for two roles of miRNAs during development: ( 1 ) miRNAs can function redundantly to control or fine-tune critical developmental steps , and ( 2 ) overt phenotypes upon disrupting only some miRNA family members might only be evident at physiological levels when stress conditions are encountered ( Bartel , 2018; Chen and Chen , 2019 ) .", "Why is it that Mir34bc/449 DKO mice displayed a seemingly similar defective phenotype to Mir34/449 TKO mice , whereas the Mir34a/449 DKO mice are largely normal ?", "Given that MiR34a seems to be expressed at high levels ubiquitously in almost all cell types , including spinal neurons ( Concepcion et al . , 2012; Otto et al . , 2017; Song et al . , 2014; Wu et al . , 2014 ) , this is a puzzling scenario .", "It is generally accepted that pre-microRNA is processed through Dicer to generate a miRNA duplex , consisting of miRNA and miRNA* strands ( also termed guide and passenger strands , respectively ) .", "Although most studies assume that miRNA* has no regulatory activity , some evidence exists to suggest that the abundance , functionality , and physiological relevance of miRNA* are underestimated ( Okamura et al . , 2008 ) .", "Our previous study showed that both MiR17-5p ( guide strand ) and MiR17-3p ( passenger strand , i . e . MiR17* ) are equally functionally important in precursor MN patterning and MN differentiation , having the same or different targets ( Chen et al . , 2011 ) .", "Interestingly , in the current study , we found that MiR34b-3p ( MiR34b* ) is significantly expressed in the developing spinal cord ( data not shown ) , raising the possibility that MiR34b-3p might also be functionally relevant , even though its seed sequence is not conserved when compared to that of MiR34a-3p ( MiR34a* ) .", "We focused on Satb1 and Satb2 in this study as it is a shared target of both MiR34a-5p ( seed , GGCAGUG ) and MiR34b/c-3p ( seed , AUCACUA ) ( Figure 5—figure supplement 1A ) .", "Accordingly , we argue that one plausible reason why Mir34bc/449 DKO mice exhibit a similar lethality phenotype to Mir34/449 TKO mice , whereas Mir34a/449 DKO mice are relatively normal , might reflect an unappreciated function of MiR34b/c-3p .", "We are now in the process of dissecting the possible convergent and divergent targets of the MiR34/449 miRNA family and their implications for embryonic development by examining individual Mir34/449 KO embryonic stem cell lines in our possession .", "The two most prominent and documented roles of MiR34/449 miRNAs are their tumor suppressor function in cancer cells ( He et al . , 2007 ) and their regulation of motile cilia in multiciliated cells ( MCCs ) during development ( Song et al . , 2014 ) .", "From a tumor cell perspective , numerous cell- and animal-based studies have explored the tumor-suppressive function of MiR34a and proposed that restoring MiR34a expression could serve as a potential therapeutic anti-cancer strategy .", "An advantage of such MiR34a-based therapy is the possibility of simultaneously repressing multiple oncogenic and immune evasion pathways ( Di Martino et al . , 2012 ) .", "Moreover , MiR34a overexpression does not seem to be excessively toxic to normal cells in vitro or in vivo ( Raver-Shapira et al . , 2007; Tazawa et al . , 2007 ) .", "In terms of development , MiR34/449 is known to regulate ciliogenesis in MCCs that have hundreds of motile cilia projecting from their apical surfaces .", "The critical role of MiR34/449 in regulating MCCs is evolutionary conserved .", "Ciliation defects caused by MiR34/449 deficiency have been reported for MCCs of the murine airway and fallopian tube , the embryonic epidermis of Xenopus , and the multiciliated pronephros and nasal pits of zebrafish embryos ( Chevalier et al . , 2015 ) .", "In addition to Satb2-mediated hypersensitivity to sensory stimuli in our MiR34/449 mutant mice , we uncovered a series of neurological disorders—particularly staggering , ataxia , and seizure-like behaviors—that could not be fully accounted for by aberrant upregulation of Satb2 in the spinal cord .", "Ependymal cells in the nervous system extend their motile cilia into the brain ventricles and contribute to cerebrospinal fluid ( CSF ) flow , a process important for normal brain function and adult neurogenesis ( Silva-Vargas et al . , 2016 ) .", "A previous study has shown that MiR449 miRNAs express in the early development of choroid plexus , supporting the putative roles of MiR34/449 in the CSF production ( Redshaw et al . , 2009 ) .", "It is not yet clear if some of the neurological disorders we uncovered in this study reflect a defect in cilial motility or CSF secretion in the spinal cord .", "Given the prominent and conserved roles of MiR34/449 miRNAs in motile cilia , it will be tempting to analyze the relevance of ependymal cilia motility for CSF circulation and brain ventricle morphogenesis in conditional neural Mir34/449 KO mice in the future .", "In addition , the observed neurological defects in Mir34/449 mutant mice may also have arisen from proprioceptive impairment since previous study has revealed that Satb2on INs reside in the terminal region of proprioceptive afferents ( Hilde et al . , 2016 ) .", "As one set of measures of proprioceptive function , the spatial parameters of stride length we measured for the treadmill assay presented a trivial but statistically significant difference in MiR34/449-deficient mice relative to WT ( Figure 3—figure supplement 2E ) , implying a potential involvement of the sensory connectivity function of MiR34/449 in the spinal cord .", "Although we have reported an increase in the number and dense positioning of the Satb2on;Ctip2on IN subpopulation upon MiR34/449 abrogation , further assessments are required to validate a link between the proprioceptive afferents displaying this molecular change in IN identity and the peculiar mouse behaviors we observed .", "Taken together , our findings reveal that MiR34/449 miRNAs have important yet underappreciated roles in regulating sensory-motor circuit outputs by refining IN numbers .", "As MiR34 has already undergone clinical trials as a potential gene therapy for cancer and we have uncovered several neurological functions for the MiR34/449 family , we envision that MiR34/449 miRNAs might serve as targets for treatments of neurodegenerative disease in the future ." ], [ "To generate the Mir449-/- mice , we applied the CRISPR-Cas9 system using a published protocol ( Li et al . , 2017 ) .", "We designed two single-guide RNAs ( sgRNAs ) targeting to the Mir449 locus , as depicted in Figure 1—figure supplement 1 .", "The sequences of two designed sgRNAs ( Supplementary file 1a ) , together with the T7 promoter and trans-activating CRISPR RNA ( tracrRNA ) , were concatenated during plasmid synthesis .", "The two Mir449 sgRNAs and the Cas9 mRNA were microinjected simultaneously into fertilized embryos of C57BL/6J mice .", "Mir449+/- mice were obtained and genotyped , with the deleted sites verified by Sanger sequencing , followed by intercross mating to acquire Mir449-/- mice ( Figure 1—figure supplement 1D-F ) .", "Mir34afl/fl ( Mir34atm1 . 2Aven/J , Stock #018545 ) , E2a::Cre ( B6 . FVB-Tg ( EIIa-cre ) C5379Lmgd/J , Stock #003724 ) , and Mir34bc-/- ( B6 . Cg-Mirc21tm1 . 1Aven/J , Stock #018546 ) mice were imported from Jackson Laboratory .", "Mir34a-/- mice were generated by crossing Mir34afl/fl mice with E2a::Cre , resulting in germ-line deletion of loxP-flanked Mir34a followed by depletion of E2a::Cre .", "All individual KO mice were further crossed with each other to obtain Mir34bc/449 DKO and Mir34/449 TKO mice .", "The mutant mouse lines were maintained and bred by the following intercross matings: Mir34a-/-;Mir34bc-/-;Mir449+/- , Mir34a-/-;Mir34bc+/-;Mir449-/- , Mir34bc-/-;Mir449+/- , and Mir34bc+/-;Mir449-/- , as shown in Figure 2A .", "All age-matched littermates from the above matings served as a control ( Ctrl ) group for all experiments , unless otherwise specified .", "To analyze survival and body weight of newborn Mir34bc/449 DKO , Mir34/449 TKO , and Ctrl mice , we monitored the health of these mice every day and determined their body weight every two days for the first 21 days .", "The primers used for all genotyping are listed in Supplementary file 1b .", "All mice generated and used for this study had a C57BL/6J background .", "The smallest sample size that would still give a significant difference was employed , in accordance with 3Rs principles .", "No animals were involved in previous unrelated experimental procedures .", "The influence of mouse sex was not evaluated in this study .", "All the live animals were maintained in a specific-pathogen-free ( SPF ) animal facility , approved and overseen by IACUC Academia Sinica .", "Mice were sacrificed under deep anesthesia by 20 mg/mL Avertin ( 2 , 2 , 2-Tribromoethanol , Sigma ) with a dosage based on mouse body weight .", "Then , cardiac perfusion of cold PBS was performed before collecting tissues and placing in Trizol ( Thermo Scientific ) for RNA extraction .", "A subsequent perfusion was performed with freshly prepared 4% paraformaldehyde ( PFA ) in PBS , followed by whole spinal cord dissection , for immunostaining or in situ hybridization .", "Spinal cords were sucrose-cryoprotected and embedded in FSC 22 frozen section media ( Leica ) , before being cut into 25 μm cryostat sections as previously described ( Tung et al . , 2019 ) .", "For embryo analyses , pregnant mice underwent cervical dislocation , and the embryos were dissected from the sacs .", "After removing the head and internal organs , the embryos were immersed in 4% PFA for 1 ~ 2 hr at 4°C , followed by a PBS wash .", "The same procedures described above for cryosectioning were used for the embryo samples , except that 20 μm cryostat sections were collected .", "For all experimental procedures , we used diethylpyrocarbonate ( DEPC ) -treated water or PBS for washing steps or reagent preparation .", "Spinal cord cryosections were initially treated with 10 μg/mL proteinase K ( Invitrogen ) , followed by acetylation in acetic anhydride/triethanolamine , and then fixed again with 4% PFA .", "Next , sections were pre-hybridized in hybridization solution [50% formamide , 5 X SSC , 0 . 5 mg/mL yeast tRNA ( Ambion ) , 5 X Denhardt’s solution ( Fisher ) , 0 . 5 mg/ml salmon sperm DNA ( Thermo Fisher Scientific ) , 0 . 02% Roche blocking reagent] at room temperature for 2 ~ 4 hr , followed by hybridization with each miRNA probe overnight at 55°C .", "After post-hybridization washes in 2 X SSC and then 0 . 2 X SSC at 55°C , the in situ hybridization signals were detected using the NBT/BCIP ( Roche ) system according to the manufacturer’s instructions .", "After termination of color development , slides were subjected to immunostaining as described below .", "Slides were mounted in Aqua-Poly/Mount and analyzed with a Zeiss LSM 780 confocal microscope .", "The 5’ FITC-labeled LNA MiR34a-5p probe ( ACAACCAGCTAAGACACTGCCA ) was purchased from Exiqon .", "To detect MiR34c-5p , spinal cord samples were dissected and fixed with 4% PFA , as described previously ( Tung et al . , 2019; Yen et al . , 2018 ) .", "The cryostat sections were processed using miRNAscope technology ( Advanced Cell Diagnostics , ACD ) according to the manufacturer’s instructions .", "The probe to detect mmu-MiR34c-5p was customized from 896831-S1 ( ACD ) .", "Total RNA was isolated using the Quick-RNA MiniPrep kit ( Zymo Research ) .", "For mRNA analysis , 50–300 ng of total RNA from each sample was reverse-transcribed with Superscript III ( Thermo Scientific ) .", "One-tenth of the reverse transcription reaction was used for subsequent quantitative real-time PCRs on a LightCycler480 Real-Time PCR instrument ( Roche ) using SYBR Green PCR mix ( Roche ) for each gene of interest .", "GAPDH served as an internal reference for normalization of amplification efficiency .", "PCR primers were designed using the online database Primer3 and subjected to Basic Local Alignment Search Tool ( BLAST; NCBI ) analysis to avoid annealing to non-specific sequences during amplification .", "The sequences of the primers used are listed in Supplementary file 1c .", "For miRNA expression analyses , 100–200 ng of total RNA from each sample was reverse transcribed with a miRNA-specific primer from TaqMan MicroRNA Assays ( Life Technology ) .", "The following assays were used: MiR34a-5p ( Assay ID: 000426 ) , MiR34b-5p ( Assay ID: 002617 ) , MiR34c-5p ( Assay ID: 000428 ) , MiR449a-5p ( Assay ID: 001030 ) , MiR449c-5p ( Assay ID: 001667 ) .", "A ubiquitous MiR16 ( Assay ID: 000391 ) was used as the endogenous control for normalization .", "Each quantitative real-time PCR was performed in duplicate per sample , with at least three different experimental samples .", "Immunostaining was performed on cryostat sections as previously described ( Tung et al . , 2019; Yen et al . , 2018 ) .", "Commercially available primary antibodies used in this study are listed in Key Resources Table .", "Alexa488- , Cy3- and Cy5-conjugated secondary antibodies were obtained from either Invitrogen or Jackson Immunoresearch and used at 1:1000 dilutions .", "Images were acquired by using a Zeiss LSM710 or LSM780 confocal microscope .", "Locomotor activity was measured by the open field , rotarod , and treadmill tests , whereas nociceptive sensory responses were evaluated by the mechanically stimulated von Frey test and heat-stimulated tail flick and hot-plate tests .", "For the Mir34bc/449 DKO and Mir34/449 TKO mice , we selected surviving mutant mice as well as littermate Ctrl for the behavioral analyses .", "Age-matched WT mice from the same C57BL/6J background were also used for experimental comparisons .", "Both sexes of adult mice ( ~4 months-old ) were used in this study .", "The experimenters conducting all behavioral assays were blind to mouse genotypes .", "Three mouse DNA fragments encompassing the Mir34a , Mir34bc , and Mir449abc clusters ( 700 bp , ~1 . 2 Kb , and ~2 Kb in size , respectively ) were amplified from mouse genomic DNA by using 2X PCR Dye Master Mix II ( ADPMX02D-100 ) or Phusion High-Fidelity DNA Polymerase ( F-530L; Thermo Fisher Scientific ) , and cloned into the pENTR/D-TOPO vector ( K2400-20; Life Technology ) according to the manufacturer’s instructions .", "The primers used for TOPO cloning are listed in Supplementary file 1d .", "We then performed GATEWAY recombination ( 11791019; Thermo Fisher Scientific ) to transfer mmu-Mir34a , mmu-Mir34bc , or mmu-Mir449abc into a Dest plasmid , p2Loxa ( Iacovino et al . , 2011 ) , under a PGK promoter .", "To identify putative MiR34/449-regulated genes , we used the online database TargetScan ( Release 7 . 1 or 7 . 2 , http://www . targetscan . org/mmu_72/ ) .", "To narrow down selected candidates , we conducted gene ontology analysis via Database for Annotation , Visualization , and Integrated Discovery ( DAVID ) ( Huang et al . , 2009 ) .", "Functional clustering annotated from DAVID , expressed as gene enrichment , is presented in this study ( Tung et al . , 2015 ) .", "The WT 3’UTR sequence containing four putative MiR34/449-binding sites of the Satb2 gene was amplified from mouse genomic DNA by PCR using 2X PCR Dye Master Mix II ( ADPMX02D-100 ) or Phusion High-Fidelity DNA Polymerase ( F-530L; Thermo Fisher Scientific ) .", "The purified PCR products were then inserted into the psiCHECK-2 vector ( C8021; Promega ) at XhoI and NotI restriction sites by using T4 DNA ligase ( M0202S; NEB ) .", "The mutated versions ( Mut ) of the Satb2 3’UTR were cloned by designing primers that resulted in the complementary sequence of the predicted binding site mismatching with the MiR34/449 seed sequence .", "Each fragment possessing a mutated binding site was initially amplified by PCR , followed by overlapping extension PCR , and the four mutated binding sites within the Satb2 3’UTR were then cloned into the psiCHECK-2 vector described above .", "The primers used for the WT and Mut Satb2 3’UTR reporter are listed in Supplementary file 1e .", "HEK293T cells were plated at a density of 5 × 104 per well ( 24-well plate ) , expanded for 16 ~ 20 hr , and co-transfected with a mixture of 60 ng of WT or Mut reporter and 2 μg of either Mir34a , Mir34bc , Mir449abc , or control plasmids using 2 μL of PLUS Reagent and 2 μL of Lipofectamine LTX Reagent ( A12621; Invitrogen ) .", "After 24 hr , cells were lysed and processed for luciferase assay using the Dual-Luciferase Reporter Assay System ( E1910; Promega ) according to the manufacturer’s instructions .", "We measured luciferase activity using a 20/20 n luminometer ( Turner Biosystems ) .", "To normalize transfection efficiency , luciferase activity was calculated as the ratio of firefly to Renilla luciferase activity , and the relative luciferase activity was further expressed as the ratio of measured luciferase activity to the control ( Chen et al . , 2011; Tung et al . , 2015 ) .", "HEK293T cells were cultured in DMEM ( Gibco ) supplemented with 2 mM L-Glutamine ( Invitrogen ) , 1% Penicillin/Streptomycin ( Invitrogen ) , and 10% FBS ( Gibco ) at 37°C in a humidified incubator with 5% CO2 .", "All cell lines used in this study are subjected to regular mycoplasma tests .", "The positioning of Satb2on INs in cervical/brachial spinal segments of P14 mice was analyzed in Imaris 9 . 5 . 1 ( Bitplane ) using the ‘Spots’ function .", "Cartesian coordinates were constructed for each image , with the midpoint of the central canal defined as position ( 0 , 0 ) .", "The position of each individual neuron was exported from Imaris as a .", "csv file and visualized using a custom R script ( http://www . r-project . org ) , with the dorso-ventral and medio-lateral density distributions shown alongside the respective figures .", "To estimate the probability density of Satb2on INs in the transverse spinal cord , we computed two-dimensional kernel density estimations using the ‘kde2d’ function from the ‘MASS’ library in R . Estimates were displayed as contour plots of density values in the 30th–90th percentiles ( at intervals of 10% ) , as described previously ( Bikoff et al . , 2016; Paixão et al . , 2019; Sweeney et al . , 2018; Tripodi et al . , 2011 ) .", "Results are expressed as mean ± SD ( standard deviation ) .", "Each experiment was repeated at least three times to confirm the findings unless otherwise specified .", "Statistical analysis was performed in Graph Pad Prism 6 . 0 ( GraphPad Software ) .", "Multiple groups were analyzed by one-way or two-way analysis of variance ( ANOVA ) with Tukey’s multiple comparisons test as indicated in the figure legends .", "Two groups were analyzed by Student's t-test .", "p-Values of less than 0 . 05 were considered significant .", "Kaplan-Meier curves were used to analyze mouse survival , with statistically significant differences assessed by the log-rank test ." ] ]

[ "Although the function of microRNAs ( miRNAs ) during embryonic development has been intensively studied in recent years , their postnatal physiological functions remain largely unexplored due to inherent difficulties with the presence of redundant paralogs of the same seed .", "Thus , it is particularly challenging to uncover miRNA functions at neural circuit level since animal behaviors would need to be assessed upon complete loss of miRNA family functions .", "Here , we focused on the neural functions of MiR34/449 that manifests a dynamic expression pattern in the spinal cord from embryonic to postnatal stages .", "Our behavioral assays reveal that the loss of MiR34/449 miRNAs perturb thermally induced pain response thresholds and compromised delicate motor output in mice .", "Mechanistically , MiR34/449 directly target Satb1 and Satb2 to fine-tune the precise number of a sub-population of motor synergy encoder ( MSE ) neurons .", "Thus , MiR34/449 fine-tunes optimal development of Satb1/2on interneurons in the spinal cord , thereby refining explicit sensory-to-motor circuit outputs ." ]

[ "The spinal cord is an information superhighway that connects the body with the brain .", "There , circuits of neurons process information from the brain before sending commands to muscles to generate movement .", "Each spinal cord circuit contains many types of neurons , whose identity is defined by the set of genes that are active or ‘expressed’ in each cell .", "When a gene is turned on , its DNA sequence is copied to produce a messenger RNA ( mRNA ) , a type of molecule that the cell then uses as a template to produce a protein .", "MicroRNAs ( or miRNAs ) , on the other hand , are tiny RNA molecules that help to regulate gene expression by binding to and ‘deactivating’ specific mRNAs , stopping them from being used to make proteins .", "Mammalian cells contain thousands of types of microRNAs , many of which have unknown roles: this includes MiR34/449 , a group of six microRNAs found mainly within the nervous system .", "By using genetic technology to delete this family from the mouse genome , Chang et al . now show that MiR34/449 has a key role in regulating spinal cord circuits .", "The first clue came from discovering that mice without the MiR34/449 family had unusual posture and a tendency to walk on tiptoe .", "The animals were also more sensitive to heat , flicking their tails away from a heat source more readily than control mice .", "At a finer level , the spinal cords of the mutants contained greater numbers of cells in which two genes , Satb1 and Satb2 , were turned on .", "Compared to their counterparts in control mice , the Satb1/2-positive neurons also showed differences in the rest of the genes they expressed .", "In essence , these neurons had a different genetic profile in MiR34/449 mutant mice , therefore disrupting the neural circuit they belong to .", "Based on these findings , Chang et al . propose that in wild-type mice , the MiR34/449 family fine-tunes the expression of Satb1/2 in the spinal cord during development .", "In doing so , it regulates the formation of the spinal cord circuits that help to control movement .", "More generally , these results provide clues about how miRNAs help to determine cell identities; further studies could then examine whether other miRNAs contribute to the development and maintenance of neuronal circuits ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "neuroscience" ]

[ [ "The molecular mechanism of how Ca2+ influx accelerates synaptic vesicle ( SV ) release remains at the heart of understanding synapse action in the normal brain and under disease conditions ( Jahn and Fasshauer , 2012 ) .", "In most synapses , SV release evoked by Ca2+ influx consists of a fast synchronous phase that occurs on a millisecond timescale and a slow asynchronous phase that occurs with some delay and persists for tens or hundreds of milliseconds .", "Additionally , all synapses manifest spontaneous release , corresponding to stochastic fusion of individual SVs .", "The key proteins mediating SV release include soluble Nethylmaleimide–sensitive factor attachment protein receptor ( SNARE ) proteins , Sec1/Munc18 ( SM ) proteins , UNC-13/Munc13s , synaptotagmins and complexins ( Wojcik and Brose , 2007; Südhof and Rothman , 2009 ) .", "The presynaptic active zone is enriched with Ca2+ channels ( Meinrenken et al . , 2002 ) and cytomatrix proteins that organize the action of presynaptic release through multi-domain protein interaction network ( Schoch and Gundelfinger , 2006; Südhof , 2012b ) .", "The mechanisms for why some SVs release rapidly and others slowly in response to membrane depolarization have been primarily attributed to the heterogeneity in their intrinsic Ca2+ sensitivities , such that a low-affinity Ca2+ sensor promotes the fast phase , while a high-affinity Ca2+ sensor supports the slow phase ( Südhof , 2012a ) .", "Yet , numerous studies have suggested that the distance between SVs and Ca2+ entry sites is also a critical determinant for release kinetics and release probability ( Neher and Sakaba , 2008; Hoppa et al . , 2012 ) .", "For example , in the calyx of Held , a giant synapse in the brainstem , it was shown that SVs involved in the slow phase of evoked release triggered by prolonged depolarization are as sensitive to Ca2+ as those in the fast phase , and can undergo rapid release upon uniform elevation of intracellular Ca2+ ( Wadel et al . , 2007 ) .", "A recent study also showed that presynaptic over-expression of an auxiliary α2δ subunit of voltage-gated calcium channels ( VGCCs ) led to a dramatic increase in release probability even though the total Ca2+ influx was reduced .", "It was suggested that the α2δ subunit may promote a closer spatial correlation between sites of Ca2+ influx and vesicle release ( Hoppa et al . , 2012 ) .", "Nonetheless , the mechanism for distance mediated regulation of SV release remains poorly understood .", "The UNC-13/Munc13 family of proteins are conserved core components of the presynaptic active zone , and are essential for both evoked and spontaneous SV release ( Augustin et al . , 1999; Richmond et al . , 1999 ) .", "UNC-13/Munc13 proteins contain multiple protein interaction domains , and have been linked to nearly all aspects of presynaptic release .", "Common to all protein isoforms are a diacylglycerol-binding C1 domain followed by a MUN domain including the MHD ( Munc13 homology domain ) flanked by a C2B and a C2C domain ( Figure 1A ) .", "The MUN domain is structurally similar to the vesicle tethering factors of the CATCHR ( Complex Associated with Tethering Containing Helical rods ) family ( Li et al . , 2011 ) , and is necessary for vesicle priming ( Basu et al . , 2005; Madison et al . , 2005; Stevens et al . , 2005 ) through binding to SNARE and Munc18 ( Betz et al . , 1997; Ma et al . , 2011 ) .", "The N-terminal regions of UNC-13/Munc13 isoforms are divergent in amino acid sequences , and have been hypothesized to contribute to the distinct properties of SV exocytosis in different types of synapses ( Augustin et al . , 2001; Rosenmund et al . , 2002 ) .", "Of direct relevance , a non-calcium binding C2A domain resides at the N-terminus of the major isoforms , which include Munc13–1 , ubMunc13–2 and C . elegans UNC-13 long isoform .", "This C2A domain can homodimerize ( Lu et al . , 2006 ) , and heterodimerize with the zinc finger domain of the active zone protein RIM ( Betz et al . , 2001; Dulubova et al . , 2005 ) .", "RIM can tether presynaptic Ca2+ channels to the active zone , and lack of RIM reduces Ca2+ channel density and Ca2+ influx at active zones ( Han et al . , 2011; Kaeser et al . , 2011; Muller et al . , 2012 ) .", "Recently , an elegant study has demonstrated a mechanism in which homodimerization by the C2A domain keeps ubMunc13–2 under priming-inhibitory state , while heteromeric binding between the C2A domain of ubMunc13–2 and the zinc finger of RIM relieves the inhibition to promote SV priming ( Deng et al . , 2011 ) .", "However , a direct test for C2A domain’s function in vivo is not yet shown . 10 . 7554/eLife . 01180 . 003Figure 1 . The C2A domain of UNC-13L regulates the release probability of evoked synaptic vesicle release .", "( A ) Illustration of UNC-13 long and short isoforms , and location of unc-13 mutations .", "* marks possible initiation methionines downstream of n2609 mutation .", "The purple domain is the calmodulin binding site ( CaM ) .", "( B ) Bright field images of adult animals from wild type , unc-13 ( s69 ) , unc-13 ( e1091 ) and unc-13 ( n2609 ) .", "Scale bar: 0 . 5 mm . ( C and D ) .", "Average recording traces of eEPSCs in animals of genotype indicated .", "( E ) .", "Summary of peak amplitudes of eEPSCs from genotypes shown in C and D . ( F and G ) .", "Average recording traces ( F ) and summary of transferred charges ( G ) of 0 . 5 M hypertonic sucrose solution induced vesicle release in animals of genotype indicated .", "The number of animals analyzed is indicated for each genotype .", "Error bars in E and G indicate SEM .", "Statistics , one way ANOVA .", "***p<0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 01180 . 00310 . 7554/eLife . 01180 . 004Figure 1—figure supplement 1 . Alignment of C2A domains among UNC-13/Munc13 isoforms . Alignment of C2A domains among worm UNC-13L , Rat Munc13-1 and Rat ubMunc13-2 .", "Residues that are identical are shown on a black background , and residues that are similar are shaded .", "The C2A domain of worm UNC-13 shows 50% identity to Rat Munc13-1 and 49% identity to Rat ubMunc13-2 , respectively .", "* marks possible initiation methionines downstream of n2609 mutation . DOI: http://dx . doi . org/10 . 7554/eLife . 01180 . 00410 . 7554/eLife . 01180 . 005Figure 1—figure supplement 2 . Transcripts of unc-13 ( n2609 ) .", "Transcripts of unc-13 long and short isoforms were detected by RT-PCR .", "The long isoform contains exon 1–13 and exon 15–31 .", "The short isoform contains exon 14–31 .", "The primers of RT-PCR1 are designed to be localized in exon 2 and exon 5 .", "The primers of RT-PCR2 are localized in exon 2 and exon 9 .", "The primers of RT-PCR3 are localized in exon 6 and exon 9 .", "The primers of RT-PCR4 are localized in exon 14 and exon 17 .", "The primers of RT-PCR5 are localized in exon 26 and exon 27 . DOI: http://dx . doi . org/10 . 7554/eLife . 01180 . 00510 . 7554/eLife . 01180 . 006Figure 1—figure supplement 3 . The effects of loss of the C2A domain on locomotion speeds .", "( A ) Schematics of single copy insertion ( Si ) for full length UNC-13L and UNC-13LC2A- lacking the C2A domain , driven by pan-neuronal promoter Prgef-1 .", "( B ) The locomotion speeds in wild type and unc-13 ( n2609 ) with or without OP50 bacteria , respectively .", "The locomotion speeds in wild type , unc-13 ( s69 ) ;Si ( UNC-13L ) and unc-13 ( s69 ) ;Si ( UNC-13LC2A− ) without OP50 bacteria .", "The number of animals analyzed is indicated for each genotype .", "Error bars indicate SEM .", "Statistics , two-tailed Student's t test for comparison between N2 and unc-13 ( n2609 ) and one way ANOVA among N2 and MosSCI rescue lines .", "***p<0 . 001; **p<0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 01180 . 00610 . 7554/eLife . 01180 . 007Figure 1—figure supplement 4 . Ratios of mean charge transfers during eEPSC and during sucrose application and the rescue effects of overexpression of UNC-13L and UNC-13LC2A− in unc-13 ( s69 ) .", "( A ) .", "Summary of ratios of mean charge transfers during eEPSCs within 50 ms and during sucrose applications within 5 s after triggers for each given genotype .", "( B ) .", "Overexpression of UNC-13LC2A− did not fully rescue the eEPSC amplitude in unc-13 ( s69 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 01180 . 007 In C . elegans the unc-13 locus produces two main isoforms that differ at the N-terminal region ( Figure 1A ) ( Kohn et al . , 2000 ) .", "The abundant long isoform UNC-13L closely resembles Munc13–1 and ubMunc13–2 .", "Previous studies using genetic mutations that eliminate function of all isoforms or UNC-13L demonstrate an essential role of UNC-13L in neurotransmitter release ( Richmond et al . , 1999 ) .", "A recent study reveals that UNC-13L is involved in both fast and slow release of SVs , while the short isoform UNC-13S is required for the slow release ( Hu et al . , 2013 ) .", "Here , we identified a unique unc-13 mutant that specifically deletes the C2A domain of UNC-13L .", "Exploiting this mutant , we show that the C2A domain regulates the release probability of SVs , likely through positioning UNC-13L to the active zone .", "The C2A domain also has a specific role in spontaneous release .", "Loss of the C2A domain of UNC-13L blocks the enhanced spontaneous release caused by loss of complexin .", "Furthermore , using the genetically encoded photosensitizer miniSOG ( mini singlet oxygen generator ) , we find that acute ablation of the active-zone specific UNC-13L results in a strong inhibition of spontaneous release and of the fast phase of evoked release , while ablation of a non-active-zone variant of UNC-13L alters primarily the slow phase of evoked release .", "These observations support an idea that spontaneous release and the fast phase of evoked release may use a common pool of SVs .", "We also show that reducing SV release by eliminating the function of UNC-13L C2A domain ameliorates behavioral deficits in a C . elegans model for epileptic seizure .", "Together , these data demonstrate that the distance between UNC-13/Munc13 to the Ca2+ entry site plays a critical role in SV release probability and release kinetics ." ], [ "The unc-13 locus contains 31 exons , and produces a major long isoform UNC-13L of 1816 amino acid residues and a short isoform UNC-13S that lacks the N-terminal 607 amino acid residues of UNC-13L and has a different N-terminal domain , through the use of different promoters and alternative splicing ( Figure 1A , Figure 1—figure supplement", "2 ) ( Kohn et al . , 2000 ) .", "We isolated the unc-13 ( n2609 ) mutation in a genetic screen for suppression of the convulsive behavior caused by a gain-of-function mutation in the neuronal acetylcholine receptor acr-2 ( Jospin et al . , 2009 ) ( see ‘Materials and methods’ and below ) .", "unc-13 ( n2609 ) changes glutamine 46 to a stop codon within the C2A domain of UNC-13L ( Figure 1A ) .", "The C2A domain shares 50% identity with rat Munc13–1 and 49% identity with rat ubMunc-13–2 , respectively ( Figure 1—figure supplement 1 ) .", "The key amino acids for homodimerization and for heterodimerization with RIM are conserved between C . elegans and mammals .", "Among previously reported mutations , unc-13 ( s69 ) is a null allele for the entire locus , and unc-13 ( e1091 ) is a null allele for the long isoform only ( Figure 1A , Supplementary file 1A ) .", "Both unc-13 ( s69 ) and unc-13 ( e1091 ) mutants are severely paralyzed ( Kohn et al . , 2000 ) ( Figure 1B ) .", "In contrast , unc-13 ( n2609 ) animals exhibited moderate slowing of locomotion ( Figure 1B , Figure 1—figure supplement 3 ) .", "unc-13 ( n2609 ) /unc-13 ( s69 ) or unc-13 ( n2609 ) /unc-13 ( e1091 ) animals showed more movement impairment than did unc-13 ( n2609 ) homozygous mutants ( data not shown ) .", "These observations indicate that unc-13 ( n2609 ) is a recessive partial loss of function mutation .", "The mild behavioral defects of unc-13 ( n2609 ) suggest that some UNC-13L proteins are produced in this mutant .", "Indeed , immunostaining using an antibody raised against amino acids 106–528 of the UNC-13L N-terminus ( Kohn et al . , 2000 ) revealed strong staining in unc-13 ( n2609 ) ( described later ) , while no immunostaining signal was detected in unc-13 ( s69 ) and unc-13 ( e1091 ) mutants ( Kohn et al . , 2000 ) ( data not shown ) .", "We performed RT-PCR for unc-13 transcripts , and confirmed that the n2609 mutation did not alter unc-13 splicing or cause skipping of the mutation-containing exon 3 ( Figure 1—figure supplement 2 ) .", "The Q46 to stop codon mutation was present in cDNA clones generated from the mutant strain .", "Thus , the observed expression of UNC-13L proteins in unc-13 ( n2609 ) mutant must be due to translation from an ATG codon ( s ) downstream of the amino acid Q46 ( Figure 1A , Figure 1—figure supplement 1 ) .", "To rule out the possibility that loss of additional N-terminal sequences in UNC-13L might contribute to the phenotypes in unc-13 ( n2609 ) , we next generated single-copy insertion transgenes ( designated Si ) expressing the full length UNC-13L or a mutant UNC-13LC2A- lacking only the C2A domain ( deleting amino acids 1–152 , Figure 1—figure supplement", "3 ) driven by pan-neuronal promoter , using the transposon Mos-mediated insertion ( MosSCI ) technique ( Frøkjær-Jensen et al . , 2008 ) .", "While Si ( UNC-13L ) transgene fully rescued the locomotion of unc-13 ( s69 ) null mutants , Si ( UNC-13LC2A- ) rescued the movement deficits of unc-13 ( s69 ) to a level similar to unc-13 ( n2609 ) mutants ( Figure 1—figure supplement 3 ) .", "Thus , we conclude that the unc-13 ( n2609 ) mutant is specifically deficient in the UNC-13L C2A domain , and provides a genetic background to investigate the role of the C2A domain in a physiological setting .", "To define how lacking the C2A domain of UNC-13 affects synaptic physiology , we assessed synaptic transmission at the cholinergic neuromuscular junctions ( NMJs ) by electrophysiological recordings of muscle cells .", "SV release at these synapses occurs in a graded manner in response to membrane potential change ( Liu et al . , 2009 ) .", "Under depolarizing condition , evoked excitatory post-synaptic currents ( eEPSCs ) represent simultaneous release of hundreds of SVs .", "unc-13 ( s69 ) null animals exhibit no eEPSCs ( Richmond et al . , 1999 ) ( Figure 1D ) .", "In unc-13 ( n2609 ) mutants the amplitude of eEPSCs was reduced to 50% of that in wild type animals ( Figure 1C , E ) .", "We also performed electrophysiological recording in the unc-13 ( s69 ) null animals expressing full-length UNC-13L or UNC-13LC2A− from the same integrated genomic locus .", "We observed a full rescue of the amplitude of eEPSC by Si ( UNC-13L ) expression in unc-13 ( s69 ) ( Figure 1D , E ) .", "Si ( UNC-13LC2A- ) ; unc-13 ( s69 ) animals showed significantly reduced amplitude of eEPSC , similar to unc-13 ( n2609 ) .", "To address that the C2A domain is directly responsible for the observed physiological defect , not secondary due to reduced protein levels , we overexpressed full-length UNC-13L and UNC-13LC2A− in unc-13 ( s69 ) mutants .", "While both transgenes rescued the paralysis of unc-13 ( s69 ) , NMJ recordings showed that simply elevating the levels of UNC-13LC2A- did not fully rescue the eEPSC amplitude , compared to overexpression of the full-length UNC-13L ( Figure 1—figure supplement 4B ) .", "Thus , these analyses strongly support that the C2A domain is required for Ca2+ influx evoked SV release .", "The reduced presynaptic release in unc-13 ( n2609 ) could be due to defective priming of SVs or a weak response of SVs to Ca2+ influx at the presynaptic terminal .", "A classic assay to analyze SV priming is by the application of hypertonic sucrose solution to induce vesicle exocytosis in a Ca2+-independent manner , which is often used to assess readily releasable pool ( RRP ) ( Rosenmund and Stevens , 1996 ) .", "Previous reports have shown that SV priming under brief ( 1 s ) sucrose application is almost abolished in unc-13 ( s69 ) mutants and severely inhibited in unc-13 ( e1091 ) mutants ( Richmond et al . , 1999; Madison et al . , 2005 ) .", "Here we applied a prolonged sucrose stimulation protocol to release the majority of primed vesicles .", "This protocol enabled us to assess the charge transfer with better time resolution at the first second and initial 5 s of sucrose application .", "Under this protocol , the charge transfer during the first second was 23 . 7 ± 2 . 9 pC in wild type animals and was 1 . 7 ± 0 . 5 pC in unc-13 ( s69 ) mutants ( Figure 1G ) , comparable to previous reports using a brief sucrose stimulation ( Gracheva et al . , 2006; McEwen et al . , 2006 ) .", "Prolonged sucrose application did not induce further release in unc-13 ( s69 ) .", "Sucrose-induced charge transfers in the time windows of first one and 5 s were similar between wild type and unc-13 ( n2609 ) ( Figure 1F , G ) .", "Both Si ( UNC-13L ) and Si ( UNC-13LC2A- ) transgenes rescued SV priming in unc-13 ( s69 ) null mutants to the level of wild-type .", "These results indicate that SVs are fully competent for release in the absence of the C2A domain of UNC-13L .", "The extended current evoked by sucrose stimulation under our protocol may reflect continuous release of refilled SVs to RRP ( Deng et al . , 2011; Watanabe et al . , 2013 ) .", "Because the preparation for C . elegans NMJ recording cannot endure multiple stimulations , it is not feasible to record reliable responses for multiple stimulations to compare the charge transfers during eEPSC and sucrose application in the same animal .", "We therefore calculated the ratio of mean charge transfers during eEPSC and sucrose application for a given genotype .", "This ratio may not directly represent the release probability , but is positively correlated with release probability .", "In unc-13 ( n2609 ) mutants and unc-13 ( s69 ) ; Si ( UNC-13LC2A− ) transgenic animals , this ratio was severely reduced ( Figure 1—figure supplement 4A ) .", "As unc-13 ( n2609 ) mutants display reduced evoked release but unaltered SV priming , we conclude that the C2A domain of UNC-13L regulates the release probability of SVs .", "SV priming and release probability are generally correlated with the number of docked SVs ( Schikorski and Stevens , 2001; Holderith et al . , 2012 ) .", "The requirement of UNC-13 for SV docking at the active zone has been revealed by ultrastructural analyses of synapses using high pressure freezing fixation ( Weimer et al . , 2006; Hammarlund et al . , 2007 ) .", "In unc-13 ( s69 ) and unc-13 ( e1091 ) mutants , fewer docked SVs are present within 231 nm from presynaptic dense projections , while slightly more SVs are accumulated at distal regions ( >330 nm from presynaptic dense projections ) ( Hammarlund et al . , 2007 ) .", "To address if the C2A domain of UNC-13L influences docking of SVs at active zones , we examined the distribution of SVs using the high pressure freezing fixation protocol ( Weimer et al . , 2006; Hammarlund et al . , 2007 ) .", "Neuromuscular synapses in unc-13 ( n2609 ) showed normal ultrastructural organization ( Figure 2A ) .", "Consistent with the normal SV priming in the unc-13 ( n2609 ) mutant , the number of total SVs and that of total docked SVs were similar to those in wild type animals ( Figure 2C ) .", "However , fewer docked SVs were present in the central active zone ( 0–165 nm ) and more docked SVs were present distally ( >330 nm ) ( Figure 2B , D ) .", "Although this SV docking defect in unc-13 ( n2609 ) is less severe than those in unc-13 ( s69 ) and unc-13 ( e1091 ) mutants ( Hammarlund et al . , 2007 ) , the mild reduction in the centrally docked SV in unc-13 ( n2609 ) may partially account for the reduced release probability . 10 . 7554/eLife . 01180 . 008Figure 2 . The C2A domain of UNC-13L promotes the docking of synaptic vesicles at the active zone .", "( A ) Ultrastructural organization of cholinergic presynaptic terminals in wild type and unc-13 ( n2609 ) .", "The dense projections were outlined by light green .", "The 165 nm , 231 nm and 330 nm regions along the plasma membrane from the edge of dense projection were marked by ticks with different colors .", "Docked synaptic vesicles are indicated by white arrowheads .", "( B ) The histogram of docked vesicle number per profile located at different distances to the dense projection in wild type and unc-13 ( n2609 ) .", "Insert , Normalized accumulative distribution of docked vesicles in wild type and unc-13 ( n2609 ) .", "( C ) The average number of total synaptic vesicles ( left ) and docked synaptic vesicles ( right ) in single profiles of cholinergic synapse containing a dense projection are similar between wild type and unc-13 ( n2609 ) .", "( D ) .", "The average docked vesicle number per profile from each synapse in specific regions ( <165 nm , <231 nm , 232–330 nm and >330 nm ) .", "Data were collected from one wild type animal ( 21 synapses , 122 profiles and 501 docked synaptic vesicles ) and one unc-13 ( n2609 ) animal ( 25 synapses 115 profiles and 485 docked synaptic vesicles ) .", "Error bars indicate SEM in C and D . Statistics , two-tailed Student’s t test .", "*p<0 . 05 . DOI: http://dx . doi . org/10 . 7554/eLife . 01180 . 008 UNC-13/Munc13 proteins are core components of the presynaptic active zone and interact with multiple active zone proteins ( Kohn et al . , 2000; Andrews-Zwilling et al . , 2006; Südhof , 2012b ) .", "The ultrastructural appearance of the presynaptic dense projection was grossly normal in unc-13 ( n2609 ) .", "To further test whether the recruitment of active zone proteins might be affected , we examined the localization of several active zone proteins , including the C . elegans RIM protein UNC-10 ( Koushika et al . , 2001 ) , ELKS-1 ( Deken et al . , 2005 ) , and the α1 subunit of presynaptic voltage-gated Ca2+ channels ( VGCCs ) UNC-2 ( Jospin et al . , 2007 ) .", "We found that the co-localization pattern of UNC-10 with ELKS-1 ( Figure 3A1–4 ) and of UNC-10 with UNC-2::GFP ( Figure 3—figure supplement", "1 ) were indistinguishable between wild type and unc-13 ( n2609 ) animals .", "UNC-13L proteins , recognized by the antibodies against the N-terminus of UNC-13L , showed a punctate pattern in unc-13 ( n2609 ) mutants .", "However , UNC-13L puncta displayed significantly reduced co-localization with UNC-10/RIM ( Figure 3B1 , B3 ) .", "The distance from an UNC-13L punctum to the nearest UNC-10/RIM punctum was significantly increased in unc-13 ( n2609 ) , compared to wild type ( Figure 3B2 , B4 ) .", "We observed similarly altered UNC-13L and UNC-10/RIM colocalization in unc-13 ( s69 ) ; Si ( UNC-13LC2A− ) animals , comparing to unc-13 ( s69 ) ; Si ( UNC-13L ) ( Figure 3C1–4 ) .", "As UNC-10/RIM , ELKS-1 and UNC-2/VGCC are correctly recruited to the active zone in unc-13 ( n2609 ) mutants , these data indicate that lacking the C2A domain causes UNC-13L to be shifted away from the active zone where Ca2+ entry sites reside . 10 . 7554/eLife . 01180 . 009Figure 3 . The C2A domain of UNC-13L is required for the precise localization of UNC-13L at active zones .", "( A1 )", "Representative confocal Z-stack images of co-immunostaining for ELKS-1 and UNC-10/RIM from wild type and unc-13 ( n2609 ) .", "( A2 )", "Average fluorescence intensities in six-pixel wide regions along a line drawn down the dorsal nerve cord ( DNC ) shown in A1 corresponding to ELKS-1 and UNC-10/RIM signals .", "Peaks from ELKS-1 and UNC-10/RIM signals are differentially marked .", "The distances between the nearest peaks from fluorescence traces of ELKS-1 and UNC-10/RIM , which are less than 800 nm , are plotted against the positions of peaks along the line drawn down the DNC .", "The color broken lines indicate intensity thresholds to detect peaks from corresponding channels .", "The grey broken line indicates the average peak distance from that sample .", "( A3 )", "Average pixel-by-pixel fluorescence intensity correlation coefficients between paired signals or shuffled data from ELKS-1 and UNC-10/RIM in wild type and unc-13 ( n2609 ) .", "( A4 )", "Summary of the peak distances between ELKS-1 and UNC-10/RIM signals in wild type and unc-13 ( n2609 ) .", "( B1-4 )", "Representative confocal Z-stack images ( B1 ) , average pixel-by-pixel fluorescence intensity correlation coefficients ( B3 ) , peak distance calculation from images shown in B1 ( B2 ) and summary ( B4 ) of co-immunostaining for UNC-13L and UNC-10/RIM from wild type and unc-13 ( n2609 ) .", "( C1-4 )", "Representative confocal Z-stack images ( C1 ) , average pixel-by-pixel fluorescence intensity correlation coefficients ( C3 ) , peak distance calculation from images shown in C1 ( C2 ) and summary ( C4 ) of co-immunostaining for UNC-13L and UNC-10/RIM from unc-13 ( s69 ) ; Si ( UNC-13L ) and unc-13 ( s69 ) ; Si ( UNC-13LC2A− ) .", "Scale bar: 5 µm in pictures and 0 . 5 µm in inserts for A1 , B1 and C1 .", "For each intensity correlation comparison , a shuffled data set was also used to calculate the extent of random correlation between images ( see ‘Materials and methods’ ) .", "AFU , arbitrary fluorescence units .", "The number of animals analyzed is indicated for each genotype .", "Error bars indicate SEM .", "Statistics , two-tailed Student’s t test .", "***p<0 . 001 for comparison between genotypes; ###p<0 . 001 for comparison between paired data set and shuffled data set for each genotype . DOI: http://dx . doi . org/10 . 7554/eLife . 01180 . 00910 . 7554/eLife . 01180 . 010Figure 3—figure supplement 1 . Loss of C2A domain does not change the co-localization between Ca2+ channel and UNC-10/RIM .", "( A1–4 )", "Representative confocal Z-stack images ( A1 ) , average pixel-by-pixel fluorescence intensity correlation coefficients ( A3 ) , peak distance calculation from images shown in A1 ( A2 ) and summary ( A4 ) of co-immunostaining for UNC-2-GFP and UNC-10/RIM from wild type and unc-13 ( n2609 ) .", "Scale bar: 5 µm in A1 , 0 . 5 µm in inserts of A1 .", "For intensity correlation comparison , a shuffled data set was also used to calculate the extent of random correlation between images ( see ‘Materials and methods’ ) .", "AFU , arbitrary fluorescence units .", "The color broken lines indicate intensity thresholds to detect peaks from corresponding channels in A2 .", "The grey broken line indicates the average peak distance from that sample .", "The number of animals analyzed is indicated for each genotype .", "Error bars indicate SEM .", "Statistics , two-tailed Student's t test .", "###p<0 . 001 for comparison between paired data set and shuffled data set . DOI: http://dx . doi . org/10 . 7554/eLife . 01180 . 01010 . 7554/eLife . 01180 . 011Figure 3—figure supplement 2 . Presynaptic localization of UNC-13 is not solely dependent on UNC-10/RIM . Representative confocal Z-stack images of co-immunostaining for UNC-13L and UNC-10/RIM from unc-10 ( md1117 ) null mutants .", "Scale bar: 5 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 01180 . 011 The distribution pattern of UNC-13L proteins is grossly punctate in unc-10/rim mutants ( Koushika et al . , 2001 ) ( Figure 3—figure supplement 2 ) , suggesting that the presynaptic localization of UNC-13L is not solely dependent on UNC-10/RIM .", "Supporting this idea , GFP tagged C2A domain ( N1–157 ) displayed a diffuse pattern throughout the axon ( Figure 4A ) .", "It has been shown that the UNC-13S isoform , which has a different N-terminal domain , is diffusely localized throughout the cytoplasm ( Nurrish et al . , 1999 ) .", "These observations imply that additional protein sequences of the N-terminus of UNC-13L may contribute to its active zone localization .", "Indeed , we found that the entire N-terminal region ( N1–607 ) of UNC-13L tagged with GFP showed a punctate pattern similar to the full-length UNC-13L::GFP ( Figure 4A ) .", "Conversely , removing the N-terminal domain , UNC-13LN− , resulted in diffuse axonal localization .", "These data are consistent with the recent report ( Hu et al . , 2013 ) and show that both the C2A domain and additional N-terminal sequences of UNC-13L are responsible for its precise position in the presynaptic active zone . 10 . 7554/eLife . 01180 . 012Figure 4 . The N-terminal region of UNC-13L determines the presynaptic active zone localization of UNC-13L and is necessary for fast kinetics of evoked release .", "( A ) Schematics and images in dorsal nerve cords of GFP tagged full length UNC-13L , UNC-13LN− lacking the entire N-terminal region ( amino acids 632–1816 ) , N-terminal amino acids 1–157 fragment and N-terminal amino acids 1–607 fragment driven by pan-neuronal promoter Prgef-1 .", "Scale bar: 5 µm .", "( B and C )", "Average recording traces , mean peak amplitudes ( B ) and 90–10% decay time ( C ) of eEPSCs in animals of genotype indicated .", "The wild type data are the same data set in Figure 1 .", "( D ) The normalized cumulative charges of eEPSCs within 50 ms after electrical stimuli , time constants fitted with a double exponential function and relative fractions of fast component in animals of genotypes indicated .", "( E ) Average recording traces ( left ) , and transferred charges ( right ) of 0 . 5 M hypertonic sucrose solution induced vesicle release in animals of genotype indicated .", "The wild type data are the same data set in Figure 1 .", "The number of animals analyzed is indicated for each genotype .", "Error bars in B–E indicate SEM .", "Statistics , one way ANOVA .", "***p<0 . 001; **p<0 . 01; *p<0 . 05 . DOI: http://dx . doi . org/10 . 7554/eLife . 01180 . 01210 . 7554/eLife . 01180 . 013Figure 4—figure supplement 1 . Locomotion speeds of unc-13 ( s69 ) rescue strains . The locomotion speeds in animals of genotypes indicated without OP50 bacteria .", "Number of animals analyzed is indicated for each genotype .", "Error bars indicate SEM .", "Statistics , one way ANOVA .", "***p<0 . 001; **p<0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 01180 . 01310 . 7554/eLife . 01180 . 014Figure 4—figure supplement 2 . Ratios of mean charge transfers during eEPSC and during sucrose application . Summary of ratios of mean charge transfers during eEPSCs within 50 ms and during sucrose applications within 5 s after triggers in animals of genotype indicated . DOI: http://dx . doi . org/10 . 7554/eLife . 01180 . 01410 . 7554/eLife . 01180 . 015Figure 4—figure supplement 3 . Higher [Ca2+]ex partially rescue eEPSC of unc-13 ( n2609 ) .", "( A ) Shown are average recording traces of eEPSCs from wild type and unc-13 ( n2609 ) in 2 mM and 5 mM extracellular Ca2+ concentrations , as well as peak amplitudes of eEPSCs and fractions of increased eEPSC amplitude at 5 mM Ca2+ normalized to 2 mM condition .", "( B ) Shown are normalized cumulative charges of eEPSCs within 50 ms after electrical stimuli from wild type and unc-13 ( n2609 ) in 2 mM and 5 mM extracellular Ca2+ concentrations , time constants fitted with a double exponential function , relative fractions of fast component , and fractions of increased fast component at 5 mM normalized to 2 mM condition .", "Number of animals analyzed is indicated for each genotype .", "Error bars indicate SEM .", "Statistics , two-tailed Student's t test .", "***p<0 . 001; **p<0 . 01 , *p<0 . 05 . DOI: http://dx . doi . org/10 . 7554/eLife . 01180 . 015 It has been proposed that the distance of release competent SVs to sites of Ca2+ influx strongly influences the release probability and kinetics of SV exocytosis ( Wadel et al . , 2007; Hoppa et al . , 2012 ) .", "Among presynaptic active zone proteins , UNC-13/Munc13 is unique in that it directly interacts with the SV fusion apparatus ( Betz et al . , 1997; Ma et al . , 2011 ) .", "To address the significance of UNC-13L localization in function , we first compared the activity of UNC-13LN− , UNC-13LC2A− , and full-length UNC-13L driven by the same pan-neuronal promoter from the same genomic insertion locus in rescuing the paralysis of unc-13 ( s69 ) .", "Quantitative analysis of locomotion speed showed a poor rescuing activity of unc-13 ( s69 ) paralysis by Si ( UNC-13LN− ) , compared to Si ( UNC-13L ) and Si ( UNC-13LC2A− ) transgenes ( Figure 4—figure supplement 1 ) .", "We next investigated how altered UNC-13L localization affected SV release kinetics .", "We analyzed eEPSCs with 90–10% decay time , which mainly reflects the duration of slow phase of evoked release , as well as the cumulative charge transfer of eEPSCs by fitting with a double-exponential function ( see ‘Materials and methods’ ) .", "In wild type animals , eEPSCs lasted around 50 ms and decayed close to the baseline with 90–10% decay time being 18 . 56 ± 2 . 22 ms ( Figure 4B–C ) .", "The charge transfer during eEPSC manifested two kinetic components with the time constants for the fast and slow components differing by a factor of around ten: τfast = 5 . 29 ± 0 . 20 ms , and τslow = 40 . 30 ± 2 . 61 ms ( Figure 4D ) .", "The relative fraction of the fast component was 77 . 21 ± 4 . 33% , indicating that the fast component of evoked release is dominant in C . elegans cholinergic NMJs .", "To assess the specific contribution of UNC-13L localization in SV release kinetics , we analyzed unc-13 ( s69 ) null animals expressing each UNC-13L variant .", "The time constants in unc-13 ( s69 ) ; Si ( UNC-13L ) were faster than those in wild type ( Figure 4D ) , suggesting that other endogenous UNC-13 isoforms contribute to SV release with a slower kinetics .", "The time constants and the relative fraction of the fast component in unc-13 ( s69 ) ; Si ( UNC-13LC2A− ) were both significantly changed , compared to those in unc-13 ( s69 ) ; Si ( UNC-13L ) ( Figure 4D ) .", "unc-13 ( s69 ) ; Si ( UNC-13LN− ) animals showed a more prolonged SV release .", "The time constants and the decay time of eEPSC were further affected , compared to unc-13 ( s69 ) ; Si ( UNC-13LC2A− ) ( Figure 4C , D ) .", "In C . elegans NMJs , the decays of tonic excitatory postsynaptic current ( tEPSC ) , which represent spontaneous release of individual SVs , are general short ( Figure 4C , Figure 5—figure supplement 1A ) .", "Moreover , the decay times of tEPSC are not altered in UNC-13L transgenic lines that have prolonged eEPSCs ( see below , and [Hu et al . , 2013] ) .", "Therefore , the observed changes in the evoked release kinetics are unlikely due to the kinetic change of postsynaptic ACh receptor response; and instead , the slower decay time of eEPSC reflects desynchronisation of presynaptic release .", "These results show that the C2A domain of UNC-13L is required for the fast release kinetics of SVs , and additional N-terminal protein sequences of UNC-13L further contribute to accelerating the Ca2+-triggered evoked release .", "Interestingly , Si ( UNC-13LN− ) transgene rescued the amplitude of eEPSCs in unc-13 ( s69 ) to the wild type level , while Si ( UNC-13LC2A− ) showed a partial rescue ( Figure 4B ) .", "To address what might account for this difference , we performed recordings using sucrose application .", "We found that Si ( UNC-13LN− ) showed significantly increased sucrose evoked SV release , comparing to Si ( UNC-13L ) and Si ( UNC-13LC2A− ) ( Figure 4E , Figure 4—figure supplement 2 ) .", "Since UNC-13LN− shows diffused localization throughout presynaptic axons , the rescue of eEPSC amplitude by Si ( UNC-13LN− ) likely reflects recruitment of SVs located distally from active zones .", "Together , with the enhanced slow release in unc-13 ( s69 ) ; Si ( UNC-13LN− ) , these observations indicate that these SVs are competent for release , but with lower release probability and slower release kinetics .", "unc-13 ( s69 ) ; Si ( UNC-13LN− ) animals showed significantly slower locomotion than unc-13 ( s69 ) expressing either full-length UNC-13L or UNC-13LC2A− ( Figure 4—figure supplement 1 ) , suggesting that SV release probability and kinetics , rather than the total vesicle supply in RRP , are functional relevant determinants for synaptic transmission efficiency in these cholinergic neuromuscular junctions .", "Lastly , as a further test to our conclusion that the distance of UNC-13L to calcium entry site directly influences SV release property , we performed recordings of unc-13 ( n2609 ) in 5 mM extracellular Ca2+ concentration .", "While high [Ca2+]ex resulted in an increase in eEPSC amplitudes in both wild type and unc-13 ( n2609 ) mutants , 5 mM [Ca2+]ex had a stronger effect in unc-13 ( n2609 ) than in wild type animals ( Figure 4—figure supplement 3A ) .", "Furthermore , we analyzed cumulative charge transfer kinetics of unc-13 ( n2609 ) ( Figure 4—figure supplement 3B ) .", "In normal 2 mM [Ca2+]ex , unc-13 ( n2609 ) showed release kinetics defects similar to unc-13 ( s69 ) ; Si ( UNC-13LC2A− ) .", "5 mM [Ca2+]ex increased the fraction of fast component in unc-13 ( n2609 ) , compared to wild type animals , although the time constant of fast component in unc-13 ( n2609 ) remains slower than that in wild type .", "Nonetheless , this result shows that higher [Ca2+]ex can compensate for the lengthened distance to UNC-13L to calcium microdomain .", "UNC-13 is also essential for spontaneous release ( Richmond et al . , 1999 ) .", "To analyze the effects of UNC-13L active zone localization on spontaneous release , we recorded tonic excitatory post-synaptic currents ( tEPSCs ) from the cholinergic motor neurons .", "unc-13 ( n2609 ) mutants showed a strong reduction in tEPSC frequency , compared to wild type ( Figure 5A ) .", "The amplitude and kinetics of tEPSCs was not altered ( Figure 5—figure supplement 1A ) .", "Similarly , reduced tEPSC frequency was also observed in unc-13 ( s69 ) ; Si ( UNC-13LC2A− ) , comparing to unc-13 ( s69 ) ; Si ( UNC-13L ) .", "Overexpression of UNC-13LC2A− in unc-13 ( s69 ) did not fully rescue the defects of tEPSC frequency , while overexpression of UNC-13L displayed an enhanced tonic release ( Figure 5—figure supplement 1C ) .", "We also recorded tEPSCs in unc-13 ( s69 ) ; Si ( UNC-13LN− ) animals , in which UNC-13 proteins are diffuse throughout the axon , and observed reduced tEPSC frequency to a level similar to that in unc-13 ( s69 ) ; Si ( UNC-13LC2A− ) ( Figure 5A ) , indicating that the C2A domain alone accounts for the specific effect of the active zone localized UNC-13L in tonic release .", "Since loss of the C2A domain caused UNC-13L to be shifted away from the center of the active zone ( Figures", "3 ) and SVs docked in regions distal to the active zone were competent for release ( Figures 1F and 4E ) , these results suggest that a major proportion of tonic release may occur in regions proximal to the active zone .", "To test this idea further , we examined double mutants of unc-13 ( n2609 ) and cpx-1/complexin .", "CPX-1/complexin is a key regulator of SV release by acting as a clamp on SNARE ( Reim et al . , 2001; Xue et al . , 2007; Giraudo et al . , 2009; Maximov et al . , 2009 ) .", "Loss of CPX-1 significantly enhances tEPSC frequency ( Hobson et al . , 2011; Martin et al . , 2011 ) , but it is not clear where within the synapse the increased tEPSC events occur .", "We found that tEPSC frequency in cpx-1 ( ok1552 ) unc-13 ( n2609 ) double mutants was significantly reduced , compared to cpx-1 ( ok1552 ) mutants ( Figure 5B ) , indicating that the enhanced spontaneous release caused by loss of complexin requires the function of the UNC-13 C2A domain .", "We next recorded evoked release in cpx-1 ( ok1552 ) unc-13 ( n2609 ) double mutants .", "cpx-1 ( ok1552 ) single mutant showed dramatically reduced eEPSCs ( Figure 5C ) , in part due to loss of a facilitating function of complexin on SV release ( Reim et al . , 2001; Xue et al . , 2007; Maximov et al . , 2009; Hobson et al . , 2011; Martin et al . , 2011 ) .", "The amplitude of eEPSC in cpx-1 ( ok1552 ) unc-13 ( n2609 ) double mutants was significantly reduced , compared to wild type or unc-13 ( n2609 ) , but was moderately increased , compared to cpx-1 single mutants .", "Analysis of charge transfer further showed a noticeable increase primarily in the fast phase of release , within 20 ms after electrical stimulation ( Figure 5D ) .", "Based on these observations , we infer that in these cholinergic synapses SV populations involved in spontaneous release may be mainly from the region proximal to the active zone , which , in cpx-1 ( ok1552 ) unc-13 ( n2609 ) mutants , were converted to account for the fast phase of evoked release . 10 . 7554/eLife . 01180 . 016Figure 5 . The C2A domain of UNC-13L is required for tonic synaptic vesicle release .", "( A and B )", "Representative recording traces ( left ) and summary ( right ) of tEPSC frequency in animals of genotype indicated .", "( C ) Average recording traces and mean peak amplitudes of eEPSCs in animals of genotype indicated .", "( D ) Superposed average recording traces , 0–20 ms transferred charge and 25–50 ms transferred charge of eEPSCs from cpx-1 ( ok1552 ) and cpx-1 ( ok1552 ) unc-13 ( n2609 ) .", "The number of animals analyzed is indicated for each genotype .", "Error bars indicate SEM .", "Statistics , one way ANOVA for multiple groups in A–C and two-tailed Student’s t test in D . ***p<0 . 001; **p<0 . 01; *p<0 . 05 . DOI: http://dx . doi . org/10 . 7554/eLife . 01180 . 01610 . 7554/eLife . 01180 . 017Figure 5—figure supplement 1 . Tonic EPSC amplitudes and decay times of unc-13 ( s69 ) rescue strains and cpx-1 mutants , and the rescue effects of overexpression of UNC-13L and UNC-13LC2A− on tEPSC in unc-13 ( s69 ) .", "( A and B )", "Summary of the amplitudes and decay times of tEPSCs in animals of genotypes indicated .", "( C ) Overexpression UNC-13LC2A− did not fully rescue tEPSC frequency , but had no effects on tEPSC amplitudes and decay times .", "The number of animals analyzed is indicated for each genotype .", "Error bars indicate SEM .", "Statistics , one way ANOVA in A and C , and two-tailed Student's t test in B . DOI: http://dx . doi . org/10 . 7554/eLife . 01180 . 017 To further address the temporal and spatial requirement of active zone localization of UNC-13L in SV exocytosis , we next employed the InSynC ( Inhibition of Synapses with CALI , for Chromophore-assisted light inactivation ) technique ( Lin et al . , 2013 ) .", "This method takes advantage of the singlet oxygen production by the genetically encoded photosensitizer miniSOG ( mini singlet oxygen generator ) to acutely ablate tagged proteins in vivo upon blue light illumination .", "We constructed miniSOG tagged UNC-13L , which localizes to the active zone , and UNC-13LN− , which is diffuse in axons ( Figure 4A ) .", "In unc-13 ( s69 ) , both UNC-13L-miniSOG and UNC-13LN−-miniSOG rescued the paralysis to different degrees ( Figure 6—figure supplement 1A ) , indicating miniSOG tagged UNC-13L proteins are functionally incorporated into the SV release apparatus .", "Upon pulsed blue-light illumination , both miniSOG transgenic animals exhibited fast paralysis to a similar degree ( Figure 6—figure supplement 1A ) , indicating miniSOG-mediated chromophore-assisted light inactivation ( CALI ) can inactivate UNC-13L and UNC-13LN– equally effectively .", "By NMJ recordings , without blue light , both UNC-13L-miniSOG and UNC-13LN--miniSOG fully rescued the amplitude of eEPSCs ( Figure 6—figure supplement 1B ) .", "CALI by 2–3 min blue light illumination resulted in a severe inhibition of eEPSCs in both transgenic animals , while the same illumination had little effect on wild type animals expressing miniSOG tagged free YFP ( miniSOG-Citrine ) ( Figure 6A , B ) . 10 . 7554/eLife . 01180 . 018Figure 6 . MiniSOG-mediated acute abalation supports a specific role of UNC-13L in fast phase of evoked release and in tonic release .", "( A ) Average recording traces of eEPSCs in animals of genotype indicated without or with blue light treatment .", "( B and C )", "Summaries of the peak amplitude , transferred charge of fast component and slow component ( B ) and 90–10% decay time ( C ) of eEPSCs from genotypes shown in A . ( D1–2 ) Representative recording traces of tEPSC with blue light illumination ( D1 ) , enlarged recording traces in 1 s duration before and after 2 min blue light illumination ( D2 ) in animals of genotype indicated .", "( E and F )", "Average frequencies of tEPSCs during blue light illumination ( E ) and normalized tEPSC frequencies after 2 min illumination to mean tEPSC frequencies before illumination ( F ) from genotypes shown in D . The number of animals analyzed is indicated for each genotype .", "Error bars indicate SEM .", "Statistics , one way ANOVA among different genotypes and two-tailed Student’s t test for a given genotype with or without blue light .", "***p<0 . 001; **p<0 . 01; *p<0 . 05; n . s . , not significant . DOI: http://dx . doi . org/10 . 7554/eLife . 01180 . 01810 . 7554/eLife . 01180 . 019Figure 6—figure supplement 1 . Effects of acute miniSOG-mediated CALI of UNC-13L and UNC-13LN− on locomotion speeds and on SV release in unc-13 ( s69 ) .", "( A ) Summary of the effects of blue light treatment on normalized locomotion speeds in L4 stage animals of genotype indicated .", "( B and C )", "Rescue and inactivation effect of miniSOG tagged UNC-13L and UNC-13LN− on eEPSC ( B ) and tEPSC ( C ) in unc-13 ( s69 ) .", "The number of animals analyzed is indicated for each genotype .", "Error bars indicate SEM .", "Statistics , paired two-tailed Student's t test for a given genotype before or after blue light and unpaired two-tailed Student's t test between different genotypes or for a given genotype with or without blue light .", "***p<0 . 001; **p<0 . 01; *p<0 . 05 . DOI: http://dx . doi . org/10 . 7554/eLife . 01180 . 019 We next tested whether acute photo-inactivation of UNC-13L-miniSOG and UNC-13LN−-miniSOG could cause specific inhibition of synapse transmission in wild type animals .", "Our assumption is that transgenically over-expressed UNC-13L-miniSOG or UNC-13LN−-miniSOG would interact with native protein interacting partners by competing with endogenous UNC-13 .", "Wild type animals carrying UNC-13L-miniSOG transgenes showed rapid movement impairment upon CALI by blue light ( Figure 6—figure supplement 1A ) .", "Notably , animals with UNC-13L-miniSOG showed much slower movement than those with UNC-13LN−-miniSOG ( Figure 6—figure supplement 1A ) , supporting our assumption that UNC-13L-miniSOG and UNC-13LN−-miniSOG are incorporated into the endogenous SV release apparatus in different subsynaptic domains .", "We then performed NMJ recordings .", "Without blue light treatment , overexpression of UNC-13L-miniSOG in wild type animals caused increased eEPSC amplitude and charge transfer in the fast phase of release , compared to control animals expressing miniSOG-Citrine ( Figure 6A , B ) .", "CALI of UNC-13L-miniSOG dramatically reduced the amplitude of eEPSCs , and resulted in a strong decrease in the transferred charge of the fast phase , but little effect on the slow phase of eEPSCs ( Figure 6B ) .", "In contrast , overexpression of UNC-13LN−-miniSOG in wild type animals , without blue light illumination , resulted in a large slow phase of evoked release ( Figure 6A–B ) , which is consistent with the report that UNC-13LN− is able to induce release competent SVs with slow release kinetics ( Hu et al . , 2013 ) .", "CALI of UNC-13LN−-miniSOG caused a mild reduction in the amplitude and the fast phase of eEPSCs , but nearly abolished the slow phase .", "Importantly , inactivation of UNC-13L-miniSOG resulted in a much slower 90–10% decay time of eEPSCs than the control animals expressing free miniSOG-Citrine , whereas inactivation of UNC-13LN−-miniSOG had an opposite effect ( Figure 6C ) .", "Since UNC-13L and UNC-13LN− reside in different subdomains of synapses and likely interact with the release machinery for different pools of SVs , we interpret that the differential effects of acute ablation of UNC-13 protein variants reflect the consequence of inhibiting or damaging themselves and their immediately associated protein interacting partners that are necessary for their action in situ .", "These results are consistent with the conclusion that the active zone localization of UNC-13L , hence close proximity to the Ca2+ entry site , is critical for the fast phase of evoked release .", "We further tested the specificity of acute ablation of UNC-13 functional complex in spontaneous release .", "All transgenic animals showed stable levels of tEPSC frequency after 2 min illumination ( Figure 6D , E ) .", "In unc-13 ( s69 ) mutants , UNC-13L-miniSOG fully rescued tEPSC frequency , while UNC-13LN−-miniSOG partially rescued it ( Figure 6—figure supplement 1C ) .", "Blue light illumination caused a strong inhibition on both rescued lines .", "In wild type background , inactivation of UNC-13L-miniSOG dramatically reduced the frequency of tEPSCs by 70% in the presence of endogenous proteins , compared to the pre-light condition ( Figure 6D , F ) .", "Inactivation of UNC-13LN−-miniSOG had a weak effect on the frequency of tEPSCs .", "Together , these analyses suggest that UNC-13LN−-miniSOG , being diffusely localized in axons , has a less role in interacting with the release apparatus for tonic release , and provide further support for the conclusion that the precise localization of UNC-13L to the active zone is crucial for spontaneous release .", "We isolated unc-13 ( n2609 ) allele as a genetic suppressor of the behavior deficits caused by acr-2 ( n2420gf ) , which causes over-excitation in the locomotion circuit and exhibits spontaneous and frequent whole body muscle contractions ( Jospin et al . , 2009 ) ( Figure 7A ) .", "A similar amino acid change in a non α-subunit of acetylcholine receptors in the human brain has been reported to cause epilepsy ( Phillips et al . , 2001 ) .", "unc-13 ( n2609 ) strongly suppresses acr-2 ( gf ) -induced convulsions ( Figure 7A ) .", "This behavioral suppression is likely due to reduced over-excitation as unc-13 ( n2609 ) ; acr-2 ( gf ) double mutants showed reduced tEPSCs compared to acr-2 ( gf ) ( Figure 7—figure supplement 1 ) .", "Interestingly , unc-13 ( n2813 ) , which contains a missense mutation in the C-terminal MUN domain and reduces SV priming to less than half of that in wild type ( Richmond et al . , 1999 ) , showed much weaker suppression on convulsions in acr-2 ( gf ) animals ( Figure 7A ) .", "These observations are consistent with our overall conclusion that the C2A domain-containing full length of UNC-13L and the C-terminal region of UNC-13L mediate different modes of synaptic transmission , and suggest that specific modes of synaptic transmission involving the C2A domain may underlie synaptic dysfunction in acr-2 ( gf ) animals . 10 . 7554/eLife . 01180 . 020Figure 7 . The C2A domain-containing N-terminal region of UNC-13L is required for acr-2 ( gf ) -induced epileptic-like convulsions .", "( A ) Summary of the suppression of unc-13 ( n2609 ) , unc-13 ( n2813 ) on acr-2 ( gf ) -induced convulsions , and the effects of unc-13 genomic DNA cosmid C44E1 , UNC-13L and UNC-13N− transgene on convulsions in acr-2 ( gf ) mutants .", "***p<0 . 001 , **p<0 . 01 and *p<0 . 05 ( red ) , compared to acr-2 ( gf ) .", "( B ) Summary of the effects of blue light treatment on convulsions in L4 stage animals of genotype indicated .", "The number of animals analyzed is indicated for each genotype .", "Error bars indicate SEM .", "Statistics , one way ANOVA in A and paired two-tailed Student’s t test for a given genotype with or without blue light in B . ***p<0 . 001; **p<0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 01180 . 02010 . 7554/eLife . 01180 . 021Figure 7—figure supplement 1 . Tonic release in acr-2 ( gf ) mutants is reduced by unc-13 ( n2609 ) .", "Representative recording traces and mean frequencies of tEPSCs in animals of genotype indicated .", "The number of animals analyzed is indicated for each genotype .", "Error bars indicate SEM .", "Statistics , one way ANOVA .", "***p<0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 01180 . 02110 . 7554/eLife . 01180 . 022Figure 7—figure supplement 2 . Recovery of convulsions in acr-2 ( gf ) ; UNC-13L-miniSOG after blue light treatment . Recovery of convulsions in acr-2 ( gf ) ; UNC-13L-miniSOG animals after blue light treatment . DOI: http://dx . doi . org/10 . 7554/eLife . 01180 . 022 To further investigate the contributions of the N- and C-terminal regions of UNC-13L to acr-2 ( gf ) -induced convulsions , we expressed UNC-13L variants in acr-2 ( gf ) animals .", "Interestingly , overexpression of UNC-13L or genomic unc-13 in acr-2 ( gf ) animals exacerbated convulsions ( Figure 7A ) .", "In contrast , overexpression of UNC-13LN− in acr-2 ( gf ) animals had no augmentative effect on convulsions .", "Lastly , to address whether the suppression of acr-2 ( gf ) -induced convulsions by unc-13 mutant is due to an acute effect to inhibit over-excitation , we introduced UNC-13L-miniSOG and UNC-13LN−-miniSOG into acr-2 ( gf ) animals .", "Upon blue light illumination , UNC-13L-miniSOG expressing animals showed a strong suppression of convulsions , while UNC-13LN−-miniSOG expressing animals continued to convulse similarly to acr-2 ( gf ) animals ( Figure 7B ) .", "After blue light treatment , acr-2 ( gf ) animals expressing UNC-13L-miniSOG gradually restored convulsions , showing a full recovery after 16 hr ( Figure 7—figure supplement 2 ) , which may reflect the time course of presynaptic UNC-13L protein turnover .", "This latter analysis also shows that the effect of miniSOG-mediated CALI is reversible , and suggests the possibility of temporal interference of specific aspects of synaptic transmission in controlling synapse dysfunction underlying some neurological disorders ." ], [ "Differential expression and function of UNC-13/Munc13 isoforms endow synapses with distinct release properties ( Augustin et al . , 2001; Rosenmund et al . , 2002 ) .", "Several recent studies have begun to unveil the function specificity mediated by the N-terminal domains in different Munc13 isoforms ( Deng et al . , 2011; Chen et al . , 2013; Hu et al . , 2013; Lipstein et al . , 2013 ) .", "The non-calcium binding C2A domain of UNC-13/Munc13 serves as protein interacting domain to bind itself or the active zone protein RIM ( Betz et al . , 2001; Lu et al . , 2006 ) .", "In this study , taking advantage of the unc-13 ( n2609 ) mutation as well as single-copy expression of UNC-13L variants in unc-13 null mutants , we have uncovered specific roles of the C2A domain in SV release probability and spontaneous release .", "The precise active zone localization depends on the C2A-containing N-terminal region unique to UNC-13L , and directly contributes to SV release kinetics .", "Our data support a conclusion that the proximity of UNC-13/Munc13 to the Ca2+ entry site plays a critical role in SV release probability and release kinetics , and also suggest that spontaneous release and the fast phase of evoked release may share a common pool of synaptic vesicles at the active zone .", "Previous studies , largely based on overexpression of mutant Munc13/UNC-13 proteins in cultured neurons or transgenic animals , have suggested that N-terminal C2A domain is necessary for their localization at the active zone ( Andrews-Zwilling et al . , 2006; Hu et al . , 2013 ) .", "Here , we show that lack of C2A domain causes a delocalization of UNC-13L from UNC-10/RIM , resulting in a shift of UNC-13L from the center of the active zone .", "Homodimerization of the C2A domain of Munc13 is recently shown to inhibit its function in SV priming , whereas RIM binding to the C2A domain converts this priming-inhibitory state to a priming-promoting state ( Deng et al . , 2011 ) .", "A monomeric Munc13 lacking the C2A domain-containing N-terminal region can rescue the priming defects of synapses lacking majority of Munc13 , but does not fully rescue evoked release ( Deng et al . , 2011 ) .", "Consistent with this study , we find that SV priming is normal in synapses lacking specifically the C2A domain of UNC-13L .", "We further show that lack of C2A domain specifically reduces the release probability of SV release .", "Based on the immunostaining of UNC-13L and ultrastructural analysis of unc-13 ( n2609 ) , we think that this effect is likely due to both mispositioning of the remaining UNC-13L in the active zone as well as a mild effect on SV docking at the proximal region to active zones .", "The SV release kinetics has been primarily attributed to the intrinsic Ca2+ sensitivity modulated by distinct Ca2+ sensor proteins ( Südhof , 2012a ) .", "The distance between SV release sites to Ca2+ influx sites also significantly influences release kinetics ( Neher and Sakaba , 2008 ) .", "Among the core SV fusion apparatus , including SNARE and Munc18 proteins , UNC-13/Munc13 exhibits the most restricted localization at the active zone ( Südhof , 2012b ) .", "In C . elegans the precise active zone localization of UNC-13L is regulated by the C2A domain-containing N-terminal region ( this study , and [Hu et al . , 2013] ) .", "Overexpression of a chimeric protein with only the C2A domain attached to the C-terminal common region of UNC-13 shortens the latency of SV release ( Hu et al . , 2013 ) .", "We find that lacking the C2A domain of UNC-13L causes reduced amplitude of evoked release , which can be partially suppressed by increasing extracellular calcium , supporting that the primary defect in unc-13C2A− animals is the increased distance between UNC-13 and calcium entry site .", "Our results that complete loss of the N-terminus of UNC-13L dramatically alters the time constants of charge transfer are completely consistent with the recent report that the non-active zone localized UNC-13S or the N-terminus truncated UNC-13L mediate slow evoked release of SVs ( Hu et al . , 2013 ) .", "Together , our two studies demonstrate that the localization of UNC-13 at the active zone is a crucial determinant for Ca2+ influx accelerating SV release .", "The functional effect of differential localization of murine Munc13s has also been tested in the calyx of Held where Munc13-2/3 isoforms , which are much less localized in the active zone , are selectively involved in slowly releasing vesicles pool , while C2A domain-containing Munc13–1 is the dominant priming factor by electrophysiological recording ( Chen et al . , 2013 ) .", "Investigation of synaptic transmission in C . elegans has traditionally relied on the use of genetic mutations that perturb gene function at birth .", "Comparing to the previously reported synapse CALI methods ( Marek and Davis , 2002; Snellman et al . , 2011 ) , the miniSOG mediated InSynC technology has the advantage to reversibly remove protein function in vivo without addition of exogenous cofactors ( Lin et al . , 2013 ) .", "The molecular basis of InSynC in wild type animals remains to be investigated , and likely involves dominant negative effects on the SV release apparatus containing miniSOG-tagged proteins .", "Our study here provides further evidence for the specificity and utility of this methodology .", "UNC-13L-miniSOG and UNC-13LN−-miniSOG are differentially localized in presynaptic terminals .", "UNC-13L-miniSOG is likely to be associated with proximal SV release apparatus close to Ca2+ entry sites , while UNC-13LN−-miniSOG can interact with distal SV release apparatus .", "We find that inactivation of UNC-13L-miniSOG and UNC-13LN−-miniSOG preferentially inhibited the fast phase and the slow phase of evoked release in wild type background , respectively .", "Moreover , the fact that animals can recover after CALI indicates significant level of protein turnover at synapses , suggesting possible applications of miniSOG-based optogenetic tools in investigating protein homeostasis in vivo and in situ .", "Our analysis that the C2A domain of UNC-13L has a specific role in spontaneous release also sheds some light to the source of SV pools in distinct release mode .", "Since the discovery of spontaneous release by Fatt and Katz ( Fatt and Katz , 1952 ) , many studies have revealed that spontaneous release contributes to physiological processes .", "For example , spontaneous release regulates the initiation of action potential in hippocampal pyramidal neurons and firing rates in cerebellar interneurons ( Carter and Regehr , 2002; Sharma and Vijayaraghavan , 2003 ) , influences dendritic spine morphology ( McKinney et al . , 1999 ) , inhibits local dendritic protein translation ( Sutton et al . , 2006 ) and modulates homeostatic synaptic plasticity ( Aoto et al . , 2008 ) .", "Several molecules such as Doc2b ( Groffen et al . , 2010 ) and Vti1a ( Ramirez et al . , 2012 ) appear to function preferentially in spontaneous release .", "However , the question of whether spontaneous release and evoked release utilize distinct SV populations remains difficult to resolve ( Alabi and Tsien , 2012 ) .", "Studies using the similar preparations and measurements often reach different conclusions ( Sara et al . , 2005; Groemer and Klingauf , 2007 ) .", "We find that removing C2A domain , as in unc-13 ( n2609 ) animals , specifically reduces spontaneous release and the fast phase of evoked release to about 50% of wild type animals , and suppresses the increased spontaneous release in cpx-1 ( null ) mutants .", "The reduced tonic release in unc-13 ( n2609 ) cpx-1 ( null ) is accompanied with a noticeably enhanced fast phase of evoked release .", "CALI of UNC-13L shows a strong effect of the active zone localized UNC-13L in spontaneous release and the fast phase of evoked release , but not slow phase .", "In contrast , UNC-13LN− shows diffuse distribution and accounts for an enhanced slow phase of evoked release , and rescues the spontaneous release defect of unc-13 ( null ) to a similar level to that of UNC-13LC2A− .", "These results suggest that SVs associated with diffused UNC-13LN− , the majority of which may be positioned distally from the active zone , mainly undergo evoked release with slow kinetics , and may not contribute to spontaneous release .", "Our data are generally consistent with the idea that spontaneous release and evoked release use the same pool of SVs , and further suggest that at the C . elegans cholinergic NMJs SV populations involved in spontaneous release and the fast phase of evoked release may likely reside at regions proximal to the active zone ( Figure 8 ) . 10 . 7554/eLife . 01180 . 023Figure 8 . Model for the C2A domain-containing N-terminal region of UNC-13L support spontaneous release and fast kinetics of evoked release . N-terminal sequences subsequent to the C2A domain interact with unknown targets ( represented by a question mark ) to facilitate the presynaptic localization of UNC-13L .", "The C2A domain binding to the zinc finger domain ( ZF ) of UNC-10/RIM promotes UNC-13L to be concentrated at active zones , where Ca2+ channels reside .", "The UNC-13L anchored at active zones supports both spontaneous and the fast phase of evoked release .", "SVs in regions distal to the active zone are mainly involved in the slow phase of evoked release .", "The N-terminal region of UNC-13L facilitates the spontaneous and fast synchronous release by promotes UNC-13L and possibly SVs close to Ca2+ influx sites . DOI: http://dx . doi . org/10 . 7554/eLife . 01180 . 023" ], [ "C . elegans strains were maintained on Nematode Growth Medium ( NGM ) plates at room temperature ( 20–22°C ) as described ( Brenner , 1974 ) .", "Double mutants were constructed following standard procedures , and genotypes were confirmed by allele-specific sequence polymorphism .", "Supplementary file 1A and 1B show the details for each mutation and strains; and Supplementary file 1C lists the genotypes of all transgenic strains .", "n2609 was isolated as a suppressor of the convulsion behavior caused by acr-2 ( gf ) , in a previously described EMS mutagenesis screen ( Jospin et al . , 2009 ) .", "Genetic mapping placed n2609 on chromosome I . Whole genome sequencing analysis ( Sarin et al . , 2008 ) revealed that n2609 contains a single nucleotide C to T change in exon 3 of the unc-13 long isoform transcript , changing UNC-13L glutamine 46 to a stop codon .", "Molecular biology was performed according to standard methods ( Sambrook et al . , 1989 ) .", "The constructs of miniSOG tagged UNC-13 were made using the Gibson assembly method ( Gibson et al . , 2009 ) .", "All other DNA expression constructs were made using Gateway cloning technology ( Invitrogen , CA ) , following the manufacturer’s procedures .", "DNA sequences were verified using restriction enzyme digestion and sequencing .", "Supplementary file 1C lists constructs and transgenes .", "Cosmid C44E1 was made by Sanger center , and obtained from James Rand ( Oklahoma Medical Research Foundation ) .", "High-copy number transgenes were generated following standard procedures ( Mello et al . , 1991 ) .", "In general , plasmid DNAs of interest were used at 10 ng/µl and co-injection markers Pttx-3-RFP at 50–90 ng/µl .", "For each construct , multiple independent transgenic lines were analyzed .", "Mos1-mediated single copy insertions were at the insert into ttTi5605 site of chromosome II as described ( Frøkjær-Jensen et al . , 2008 ) .", "L4 larvae were placed on freshly seeded NGM plates .", "The following day , individual young adults were transferred to fresh plates and recorded by video for 90 s .", "8–10 animals were recorded for each genotype per trial and at least two trials were performed per genotype .", "A convulsion was defined as a visible sudden shortening in the animal’s body length ( Jospin et al . , 2009 ) .", "Locomotion speeds were measured using Worm Tracker 2 . 0 ( W . Schafer’s laboratory , MRC Laboratory of Molecular Biology , Cambridge , UK ) ( Ben Arous et al . , 2010 ) , and animals were prepared as described ( Qi et al . , 2012 ) .", "If locomotion speeds were measured with food , the NGM plates were seeded with OP50 bacteria on the day before experiments and were kept at room temperature overnight .", "Immediately before transferring the worms , about 300 µl of 100 mM CuCl2 was poured and swirled on the rim of the NGM plate to form a ‘copper ring’ , and excessive CuCl2 solution was removed .", "Individual young adults grown on an OP50 lawn were gently transferred to M9 solution using an eyelash .", "Any bacteria were rinsed off using an aspiration micropipette , and the worm was then transferred onto a fresh tracking plate using the same micropipette .", "The plate was placed on the tracker platform , and tracking started about 90 s after the puddle of M9 with the worm was absorbed into the agar and the worm had started crawling .", "Each tracking movie lasted 5 min with 10 frames per second .", "Movies were analyzed using the algorithms modified by Suk-Ryool Kang ( Department of ECE , University of California , San Diego ) .", "Before transferring Larva 4 stage worms to plates , 3-cm NGM plates were spread with 15 µl concentrated OP50 to form thin OP50 lawn by waiting for a short while .", "About 100 µl of 100 mM CuCl2 was poured and swirled on the rim of the plate to form a ‘copper ring’ to keep worms away from the edge of plates , and excessive CuCl2 solution was removed .", "Plates containing worms were illuminated with blue LED light source ( 460 nm , spectrum half width 27 nm , Prizmatix , Givat Shmuel , Israel ) .", "The diameter of illumination area is 5 . 8 cm to cover the entire surface of plates .", "The light intensity was measured to be 2 . 07 mW/mm2 with a power sensor D10MM connected to an Optical Power Meter PM50 ( Thorlabs , Newton , New Jersey , US ) .", "Animals were exposed to 1 Hz pulsed blue light for 12 min to avoid high heat accumulation on plates ( Qi et al . , 2012 ) .", "The frequency of pulsed light is controlled by TTL signals provided with PASCO digital function generator PI-9587C ( Roseville , California , US ) .", "Before the locomotion or convulsion measurement , worms were transferred back to normal OP50 seeded NGM plates for recovery for around 5–10 min after blue light treatment .", "Young adult worms were immobilized by high-pressure freezing at −176°C in the BAL-TEC HPM 010 .", "Then frozen worms were freeze substituted in the Leica EM AFS2 system with 2% osmium tetroxide and 0 . 1% uranyl acetate in acetone for 4 days at −90°C and 16 hr at −20°C .", "After infiltration and embedding in Durcupan ACM resin blocks were polymerized at 60°C for 48 hr .", "Serial sections of 33 nm thicknesses were collected using the ultramicrotome Leica ULTRACUT UCT and stained for 5 min in 2 . 5% uranyl acetate in 70% methanol , followed after washing by 3 min in Reynold’s lead citrate .", "All images of synapses with density from ventral nerve cord were obtained on a JEOL-1200 EX transmission electron microscope using Gatan 4 MP digital camera and DigitalMicrograph acquisition software .", "Distances from the edge of the dense projection to all docked vesicles along membrane were measured using ImageJ software .", "The distance from the dense projection to each docked vesicle was sorted into 33 nm bins .", "The number of vesicles in each bin was divided by the number of profiles to yield an average number of vesicles per profile in each bin to generate the histogram of docked vesicles .", "Only vesicles in profiles containing a dense projection were included .", "The histogram of docked synaptic vesicles was integrated and normalized to generate the accumulative fraction .", "Each contiguous set of serial profiles containing a dense projection was considered as a single data point , that is a synapse .", "The number of docked vesicles in specific regions ( <165 nm , <231 nm , 232–330 nm and >330 nm ) within each such set was divided by the number of profiles in the set , resulting in a number of docked vesicles per profile for that set .", "The mean and SEM of all data points within each genotype was determined and used to calculate p values in two-tailed Student’s t test .", "Whole-mount staining was conducted using 1% paraformaldehyde fixation as previously described ( Finney and Ruvkun , 1990 ) .", "Primary antibodies used were mouse anti-UNC-10/RIM ( RIM2-s from Developmental Studies Hybridoma Bank , Iowa City , IA ) ( Hadwiger et al . , 2010 ) at 1:3 dilution , rabbit anti-UNC-13 Rab598 at 1:35 ratio ( gift from James Rand ) ( Kohn et al . , 2000 ) , rabbit anti-ELKS-1 Rb237 at 1:200 ( gift from Michael Nonet ) ( Deken et al . , 2005 ) and rabbit anti-GFP ( A11122 from Invitrogen , CA ) at 1:500 .", "Secondary antibodies were goat anti-mouse Alexa Fluor 488 ( A11001 ) , goat anti-rabbit Alexa Fluor 594 ( A11012 ) , goat anti-mouse Alexa Fluor 594 ( A11005 ) and goat anti-rabbit Alexa Fluor 488 ( A11008 ) from Invitrogen , and used at 1:2000 dilution .", "Confocal images were taken on a Zeiss LSM 510 with 488 nm and 594 nm lasers .", "Laser output was set to 40% and transmission was optimized for detection and minimum bleed-through .", "Single 0 . 5 µm confocal planes were captured , merged using LSM software and exported as lsm file .", "To compare the correlation of signals from two channels , signals in each channel were separated with MetaMorph ( Sunnyvale , CA ) and exported as TIFF file .", "After thresholds were set , pixel-by-pixel intensity correlation analysis was performed and plotted automatically by Metamorph between two channels in the same animal ( paired correlation ) .", "Since some fluorescence overlapping could have arisen by chance , the images from green and red channels were shuffled to determine green-red correlations between animals ( shuffled correlation ) .", "In all cases , the shuffled correlation coefficient was nearly zero , confirming that the measured paired correlation is not due to chance .", "To calculate the distance from the center of a punctum in one channel to the center of the nearest punctum in another channel , average fluorescence intensities in six or eight-pixel wide segment regions along a line drawn down the dorsal nerve cord ( DNC ) were calculated by MetaMorph and plotted against the pixel position along the scan-line in IGOR Pro ( WaveMetrics , Lake Oswego , OR ) .", "The peaks of each channel signal above the threshold were automatically found in IGOR , which representing the center of puncta .", "To determine the threshold , the lowest non-zero point of a given signal trace in the region without punctum fluorescence was identified in IGOR .", "The standard deviation ( SD ) of the signal trace within 600 nm region with the lowest non-zero point as the center was calculated .", "The threshold was initially set as the value of 3 . 5-fold of SD plus the lowest non-zero value .", "If needed , the threshold was then manually adjusted to include all peaks from fluorescence puncta for the analysis .", "For each peak of RIM signal , the nearest peak from another channel with 800 nm was identified and the distance between the two peaks along scan-line was calculated .", "Neuromuscular dissection methods were adapted from previous studies ( Richmond et al . , 1999 ) .", "Adult worms were immobilized on Sylgard-coated cover slips with cyanoacrylate glue .", "A dorsolateral incision was made with a sharp glass pipette and the cuticle flap was folded back and glued down to expose the ventral medial body wall muscles .", "The preparation was then treated by collagenase type IV ( Sigma-Aldrich , St . Louis , MO ) for ∼ 30 s at a concentration of 0 . 4 mg/ml at room temperature .", "The bath solution containing ( in mM ) : 127 NaCl , 5 KCl , 26 NaHCO3 , 1 . 25 NaH2PO4 , 2 CaCl2 , 4 MgCl2 , 10 glucose , and sucrose to 340 mOsm , bubbled with 5% CO2 , 95% O2 at 20°C .", "The pipette solution containing ( in mM ) : 120 CH3O3SCs , 4 CsCl , 15 CsF , 4 MgCl2 , 5 EGTA , 0 . 25 CaCl2 , 10 HEPES and 4 Na2ATP , adjusted to pH 7 . 2 with CsOH .", "The extracellular calcium concentration is otherwise indicated if it is not 2 mM .", "Conventional whole-cell recordings from muscle cells were performed at 20°C with 2–3 MΩ pipettes .", "An EPC-10 patch-clamp amplifier was used together with the Patchmaster software package ( HEKA Electronics , Lambrecht , Germany ) .", "Tonic EPSCs were recorded at −60 mV .", "For record evoked EPSCs , a second glass pipet filled with bath solutions was put on the ventral nerve cord as stimulating electrode .", "The stimulating electrode gently touched the anterior region of ventral nerve cord to form loose patch configuration , which is around 1 muscle distance from recording pipets .", "A 0 . 5 ms , 85 μA square current pulse was generated by the isolated stimulator ( WPI A320RC , Sarasota , FL ) as stimulus to obtain the maximal responses .", "For RRP depletion , the 0 . 5 M sucrose in bath solution was applied to ventral nerve cord near the recorded muscles by Picospritzer with 8 psi for 7 s .", "Under this prolonged stimulation protocol , we could observe the current decay over the stimulation window .", "The sucrose-evoked responses have been compensated for the basal activities by subtracting basal line current level prior to sucrose application .", "For miniSOG mediated CALI , illumination ( 15 or 30 mW/mm2 ) was provided with a Sutter Instrument Lambda LS fitted with a Lamda 10–2 filter wheel for shuttering ( Novato , CA ) .", "The excitation light was filtered with an eGFP filter set with 480 nm excitation ( Chroma N41012; Chroma , Bellows Falls , VT ) and focused on the specimen with a 63 × water immersion objective ( Olympus , Center Valley , PA ) .", "Light intensity was measured with a calibrated photometer with an integrating sphere detector ( International Light Technologies , Newburyport , MA ) .", "A glass slide with a semi-spherical lens containing a water drop was placed under the objective and was used to direct the light into the integrating sphere .", "The area of illumination was measured with a stage micrometer for the calculation of illumination intensity .", "The 2–3 min continuous blue light was illuminated on the prep including the ventral nerve cord and recorded muscle for miniSOG-mediated CALI .", "All current traces were imported to IGOR Pro ( WaveMetrics , Lake Oswego , OR ) for further analysis .", "The cumulative transferred charge of eEPSC was integrated over 50 ms after the electrical stimulation .", "The charge trace was fitted with a following double-exponential function to derive the time constant ( τ ) and size ( A ) of each component:Q ( t ) =Afast .", "( 1−exp ( − ( t−t0 ) /τfast ) ) +Aslow .", "( 1−exp ( − ( t−t0 ) /τslow ) ) where t0 is the time of electrical stimulation .", "The size of slow component ( Aslow ) was not directly used .", "Instead , we subtracted the Afast from the total amount of transferred charge within 50 ms . We used Graphpad Prism 5 ( GraphPad Software , La Jolla , CA ) to test significance .", "For comparisons of two groups , we used a two-tailed Student’s t test .", "For comparisons involving multiple groups , we used one-way ANOVA and Newman-Keuls post hoc test .", "***p<0 . 001; **p<0 . 01; *p<0 . 05 ." ] ]

[ "The presynaptic active zone proteins UNC-13/Munc13s are essential for synaptic vesicle ( SV ) exocytosis by directly interacting with SV fusion apparatus .", "An open question is how their association with active zones , hence their position to Ca2+ entry sites , regulates SV release .", "The N-termini of major UNC-13/Munc13 isoforms contain a non-calcium binding C2A domain that mediates protein homo- or hetero-meric interactions .", "Here , we show that the C2A domain of Caenorhabditis elegans UNC-13 regulates release probability of evoked release and its precise active zone localization .", "Kinetics analysis of SV release supports that the proximity of UNC-13 to Ca2+ entry sites , mediated by the C2A-domain containing N-terminus , is critical for accelerating neurotransmitter release .", "Additionally , the C2A domain is specifically required for spontaneous release .", "These data reveal multiple roles of UNC-13 C2A domain , and suggest that spontaneous release and the fast phase of evoked release may involve a common pool of SVs at the active zone ." ]

[ "Neurons are connected to each other by junctions called synapses .", "When an electrical signal travelling along a neuron arrives at a synapse , it causes the release of bubble-like structures called synaptic vesicles that contain chemicals called neurotransmitters .", "When released by the vesicles these neurotransmitters bind to receptors on a second neuron and allow the signal to continue on its way through the nervous system .", "The release of synaptic vesicles from the neuron depends largely on the number of calcium ions that enter this neuron via structures called ion channels , and also on the rate at which they enter .", "Vesicles are released in one of three ways: they can be released quickly ( within a few milliseconds ) in response to the influx of calcium ions; they can be released slowly ( over a period of tens or hundreds of milliseconds ) in response to the influx; or they can be released at random times that are not related to the influx .", "It is known that the sensitivity of certain calcium sensors near the synapse influences the release of the vesicles .", "It had been thought that the distance between the “active zone” where the calcium ions enter the neuron and the region where the vesicles reside might also influence rate of release , but the molecular mechanism underlying this hypothesis is poorly understood .", "Zhou et al . have now shed new light on this question by performing a series of experiments that involved manipulating a protein called UNC-13 – which is known to be involved in the release of vesicles – in neurons from C . elegans , a nematode worm .", "First it was shown that the precise position of UNC-13 in the active zone depended on a domain within the protein called the C2A domain .", "Next it was shown that the distance between the UNC-13 protein and the calcium ion channels strongly influences the quick mode of vesicle release .", "Finally , Zhou et al . showed that the C2A domain also had a significant influence on the spontaneous release of vesicles , which suggests that a common fleet of vesicles might be used for both the quick and the spontaneous modes of vesicle release .", "Zhou et al . also generated mutant worms that mimicked a neurological disease , epileptic seizure , and showed that eliminating the C2A domain can relieve some of the symptoms associated with the disease .", "Many neurological diseases are caused by signals not being transmitted properly at synapses , so in addition to providing insights into the basic mechanism underlying synaptic action , these results could also assist with the development of new strategies for managing neurological diseases ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "developmental biology", "computational and systems biology" ]

[ [ "Cells within complex organisms exist in different states that confer specific functionalities to each cell .", "Cellular states can be organized into cyclic cascades such as the G1-S-G2-M cell cycle , or into linear cascades as observed in cell differentiation .", "A common feature of cells that undergo state transitions is the apparent irreversibility of the transitions even when such transitions are triggered by transient stimuli .", "Modeling of these transitions often assumes system bistability , in which cells can be resting in one of two stable states .", "Animal cells frequently utilize transcription factors to enforce a given state , and transitions to another state are driven by increasing or decreasing the levels of these transcription factors ( Yao et al . , 2008; Kueh et al . , 2013; Laslo et al . , 2006; Park et al . , 2012 ) .", "The Rb-E2F pathway generates bistability in E2F expression , which dictates the transition from G1 to S phase ( Yao et al . , 2008 ) .", "Expression of the transcription factor PU . 1 determines lymphoid versus myeloid hematopoietic cell lineages ( Kueh et al . , 2013; Laslo et al . , 2006 ) .", "Adipocyte differentiation is controlled by differential expression of C/EBP and PPARγ proteins ( Park et al . , 2012 ) .", "Regulation by positive feedback is a hallmark of bistable systems , and in all of the above cases , the transcription factors act in one or more positive feedback circuits .", "In some systems , positive feedback is generated by two transcription factors that mutually repress each other’s expression .", "In these scenarios , cells in one state continually express a transcription factor that represses a second transcription factor , and when these cells transit to another state , they continually express the second transcription factor , which represses its antagonist .", "Fate restriction in hematopoietic , neural , pancreatic , and muscle cell lineages is regulated by such double-negative feedback circuits ( Briscoe et al . , 2000; Laslo et al . , 2006; Olguin et al . , 2007; Schaffer et al . , 2010 ) .", "Most examples of this type of bistable mechanism have transcription factors that act specifically within a handful of cell states limited to a single tissue or organ system .", "One remarkable exception to this rule is found in Drosophila .", "There , two ETS-domain transcription factors act in a wide assortment of cell types across the body and across the life cycle .", "Yan and Pointed ( Pnt ) act downstream of signals mediated by receptor tyrosine kinases ( RTKs ) that regulate cell differentiation , migration , and division in tissues ranging from ovarian follicular cells , dorsal and ventral neuroectoderm , embryonic mesoderm , the embryonic trachea , and the post-embryonic compound eye ( Dumstrei et al . , 1998; Gabay et al . , 1996; Halfon et al . , 2000; Jurgens et al . , 1984; Morimoto et al . , 1996; O'Neill et al . , 1994; Ohshiro et al . , 2002; Yao et al . , 2008 ) It is thought that Yan and Pnt control such diverse cell states by acting in concert with tissue-specific transcription factors to regulate transcription of appropriate target genes .", "For instance , transcription of even-skipped occurs in mesoderm only if Yan/Pnt act in combination with Tinman and Twist proteins ( Halfon et al . , 2000 ) , whereas transcription of prospero ( pros ) in the eye only occurs if Yan/Pnt act in combination with Lozenge , Sine Oculis , and Glass proteins ( Hayashi et al . , 2008; Xu et al . , 2000 ) .", "Several tissues show mutually exclusive expression of Yan and Pnt , suggestive of cross-repression ( Boisclair Lachance et al . , 2014 ) .", "The best characterized is the embryonic ventral ectoderm , where ventral-most cells express Pnt and more lateral cells express Yan ( Gabay et al . , 1996 ) .", "This pattern is established by secretion of a ligand for the EGF Receptor ( EGFR ) from the ventral midline ( Golembo et al . , 1996 ) .", "EGFR activation in nearby ventral cells induces the Ras-MAPK pathway to express Pnt and degrade Yan ( Gabay et al . , 1996; Melen et al . , 2005 ) .", "Cells with insufficient EGFR activation express Yan , which represses Pnt .", "Mathematical modeling has described this as a bistable system in which cells are either in a High Yan/Low Pnt state or a Low Yan/High Pnt state ( Graham et al . , 2010; Melen et al . , 2005 ) .", "Transition from one state to the other is ultrasensitive to the strength of EGFR activation ( Melen et al . , 2005 ) .", "Paradoxically , other Drosophila tissues show co-expression of Yan and Pnt ( Boisclair Lachance et al . , 2014 ) .", "The larval eye is one such tissue .", "Retinal progenitor cells initiate expression of both proteins , and when they transit to differentiated photoreceptor fates , these cells reduce expression of both proteins .", "In contrast , when retinal progenitor cells transit to differentiated cone cell fates , they maintain their expression of both proteins .", "These observations are at odds with long-standing genetic studies that support a standard bistable mechanism acting in the eye ( Lai and Rubin , 1992; O'Neill et al . , 1994; Rebay and Rubin , 1995 ) .", "Thus , new approaches to studying these transitions in the eye are needed .", "Here , we have adopted a systems-level approach to study Yan dynamics in the larval eye .", "A yellow fluorescent protein ( YFP ) — based isoform of Yan was developed as a reporter for Yan protein levels .", "Fluorescence-based microscopic imaging of cells was coupled with automated high-throughput image analysis to score fluorescence in each cell and annotate the data in a quantitative and unbiased fashion .", "Yan exhibits monostability , both in progenitor and differentiating cells , with Yan levels varying in cells in either state .", "Cell state transitions occur independent of absolute Yan concentrations , suggesting that some other mechanism allows Yan to regulate transitions .", "One such mechanism might be the noise in Yan levels , which undergoes a transient spike as cells begin to transition to differentiated states .", "Loss of EGFR signaling , which prevents cells from differentiating , causes these cells to have prolonged noisy Yan expression , and suggests that Yan noise is key for cell state transitions in the eye ." ], [ "To quantitatively test the bistable model , we used BAC recombineering to insert fast-fold yellow fluorescent protein ( YFP ) in-frame at the carboxy-terminus of the yan coding sequence ( Webber et al . , 2013 ) .", "The Yan-YFP transgene fully complemented null mutations in the endogenous yan gene and completely restored normal eye development ( Figure 1—figure supplement 1 ) , demonstrating that the YFP tag does not compromise Yan function and that all essential genomic regulatory sequences are included .", "The pattern of Yan-YFP protein expression was qualitatively similar to that of endogenous Yan ( Figure 1—figure supplement 1 ) .", "We used histone His2Av-mRFP fluorescence in fixed specimens to mark all eye cell nuclei for automated segmentation ( Figure 1D—figure supplement 2 ) .", "Nuclei were manually classified into the different cell types of the eye , which is possible because every cell undergoing differentiation can be unambiguously identified by its position and nuclear morphology without the need of cell-specific markers ( Ready et al . , 1976; Tomlinson , 1985; Tomlinson and Ready , 1987; Wolff and Ready , 1993 ) .", "To validate our identification of all cell types using this method , we cross-checked with specific cell-specific markers , and found that our classification was accurate over 98% of the time ( Figure 1—figure supplement 3 ) .", "Cells were scored for nuclear Yan-YFP concentration and their exact position within 3D coordinate space of each fixed eye sample ( Figure 1—figure supplement 2 ) .", "Yan-YFP protein levels were normalized to His2Av-mRFP , which provided some control over measurement variation ( Figure 1—figure supplement 4 ) .", "We then mapped each cell’s spatial position in the x-y plane of the eye disc onto time .", "Two reasons made this possible .", "First , the furrow moves at an approximately constant velocity forming one column of R8 cells every two hours ( Basler and Hafen , 1989; Campos-Ortega and Hofbauer , 1977 ) .", "Second , each column of R8 cells induces the other cell fates at a constant rate ( Lebovitz and Ready , 1986 ) .", "Thus even in a fixed specimen , the temporal dynamics of cell state transitions are visible in the repetitive physical organization of ommatidia relative to the furrow ( Figure 1C ) .", "Together these features allow the estimation of time based on a cell’s position relative to the furrow ( Figure 2—figure supplement 1 ) .", "This can be applied repeatedly to hundreds of cells in a single sample , creating a composite picture of the dynamics ( Figure 2B–F ) .", "Although the developmental progression of an individual cell cannot be measured by this approach , it nevertheless provides a dynamic view of hundreds of cells across a developing epithelium as they respond to signaling processes .", "From this information , individual cell behaviors can be reconstructed and modeled . 10 . 7554/eLife . 08924 . 008Figure 2 . Dynamics of Yan-YFP in eye cells .", "( A ) Average time at which initiation of differentiation is first detected by apical migration of committing cell nuclei .", "Time zero is set to when R8 differentiation initiates .", "Differentiation proceeds over a time course after commitment is initiated ( horizontal arrows ) ( B ) Yan-YFP fluorescence in multipotent progenitor cells .", "We fit a Hill function ( blue curve ) to the inductive phase and an exponential decay ( black curve ) to the decay phase .", "( C-F )", "Scatter plots of Yan-YFP levels in individual cells for R2/R5 ( C ) , R3/R4 ( D ) , R7 ( E ) , and C3/C4 ( F ) cells .", "These are overlaid with scatter plots of Yan-YFP in multipotent cells at times preceding and coincident with the appearance of the relevant differentiated cells .", "Note the similar Yan-YFP levels between multipotent cells and differentiating cells at their first appearance .", "( G ) Moving averages of Yan-YFP levels for multipotent and photoreceptor cells .", "Gaps between the multipotent and photoreceptor curves are due to the window size for line averaging .", "( H ) Moving averages of Yan-YFP in multipotent and cone cells . DOI: http://dx . doi . org/10 . 7554/eLife . 08924 . 00810 . 7554/eLife . 08924 . 009Figure 2—figure supplement 1 . Mapping identified nuclei within XY space of eye discs . Anterior is to left for each panel .", "( A–H )", "Maps of identified nuclei in an eye disc .", "Progenitor cell nuclei ( A ) ; R8 cell nuclei ( B–H ) ; R2 and R5 cell nuclei ( C ) ; R3 and R4 cell nuclei ( D ) ; R1 and R6 cell nuclei ( E ) ; R7 cell nuclei ( F ) ; C1 and C2 cone cell nuclei ( G ) C3 and C4 cone cell nuclei ( H ) .", "( I , J )", "Delauney triangulation of R8 nuclei centroids within a representative eye disc from panel B . The complete triangulation network of R8 nodes ( I ) ; the network in which links are retained for nodes that are closest to one another along the x-axis ( J ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 08924 . 009 Yan-YFP expression in multipotent progentior cells was biphasic ( Figure 2B ) .", "Cells anterior to the furrow expressed a basal level of Yan-YFP , but this level dramatically increased in cells immediately anterior to the furrow , starting eight hours before the first R8 cells were identifiable .", "Approximately eight hours after R8 definition , Yan-YFP levels peaked , and thereafter gradually decayed until Yan-YFP approached its basal level again .", "The results are surprising in two ways .", "First , Yan-YFP is not maintained at a stable steady state within progenitor cells , which would have been predicted by the bistable model .", "Rather , its dynamics are reminiscent of monostable responses to transient stimuli , with a single basal steady state .", "Second , at the peak of Yan-YFP expression , there is remarkably large heterogeneity in Yan-YFP levels across cells .", "We also followed Yan-YFP dynamics in cells as they transited into a differentiated state and thereafter .", "Again , the results did not follow the expectations predicted by the bistable model .", "First , progenitors transited to a differentiated state at levels of Yan-YFP that varied , depending upon the type of differentiated state being adopted ( Figure 2C–H ) .", "Cells entering the R3/R4 and R1/R6 states began with Yan-YFP levels that were approximately two-fold greater than cells entering the R2/R5 states .", "Cells entering the R7 state were intermediate between these two extremes .", "Despite these differences at the transition point , Yan-YFP levels decayed to a similar basal steady state irrespective of the photoreceptor type , and this basal level was at least three-fold lower than that which the progenitor cells achieved ( Figure 2G ) .", "Thus , rather than the single high Yan progenitor state previously modeled , our results suggest a dynamic range of high Yan states from which different cell fates are specified according to the spatio-temporal sequence of differentiation .", "We noted that for most photoreceptors , it took approximately 20 hr for Yan-YFP to decay to the basal steady state ( Figure 2G ) , significantly longer than had been previously modeled ( Graham et al . , 2010 ) .", "Since expression of several neural-specific genes is detected 2–8 hr after the transition ( Tomlinson and Ready , 1987; Van Vactor et al . , 1988 ) , the slower than anticipated Yan decay indicates that early differentiation does not require cells to have assumed a basal steady-state level of Yan-YFP .", "The last group of progenitors to differentiate form the non-neuronal cone cells .", "We also measured Yan-YFP in those cells .", "Yan-YFP dynamics in cone cells were more similar to progenitor cells over the same time period ( Figure 2F , H ) .", "This behavior was in contrast to photoreceptors , which exhibited different decay dynamics from progenitor cells .", "Thus , accelerated degradation of Yan-YFP is not essential for cells to transition to all retinal cell states .", "The bistable model posits that the switch from one state to another is triggered by a signal that progenitor cells receive though the EGFR protein .", "Given the unanticipated Yan-YFP dynamics , we wanted to ask whether and how they were influenced by EGFR signaling .", "EGFR null mutants are inviable; however , a temperature sensitive ( ts ) allele of EGFR enables controlled inactivation of the RTK’s activity ( Kumar et al . , 1998 ) .", "We grew EGFRts mutant larvae at a restrictive temperature for 18 hr before analyzing effects on Yan-YFP .", "Surprisingly , progenitor cells exhibited biphasic expression of Yan-YFP over time , but the amplitude of the pulse in expression was significantly reduced ( Figure 3A ) .", "This suggests that EGFR signaling contributes to the stimuli that induce the Yan-YFP peak .", "To further test this hypothesis , we misexpressed a constitutively active form of Ras protein in eye cells .", "The peak of Yan-YFP in progenitors was now prolonged ( Figure 3B ) .", "Together , these results suggest that EGFR-Ras signaling stimulates the transient appearance of Yan-YFP in progenitor cells , and that the decline in Yan-YFP within older progenitor cells is linked to a loss of signal reception by these cells over time . 10 . 7554/eLife . 08924 . 010Figure 3 . EGFR/Ras and Pnt regulate Yan-YFP levels .", "( A–D )", "Moving averages of Yan-YFP in different cell types .", "Shown with shading is the standard error of the mean for each moving average .", "( A , C )", "Wildtype and EGFRts mutants incubated at the non-permissive temperature and analyzed for progenitors ( A ) and R2/R5 cells ( C ) .", "( B , D )", "Wildtype and sev>Rasv12 mutants were analyzed for progenitors ( B ) and R2/R5 cells ( D ) .", "( E ) Optical slice through progenitor cell nuclei in a disc that contains clones of pnt2 mutant cells .", "Left , fluorescence of RFP , which positively marks wildtype cells and not pnt2 mutant cells .", "Middle , Yan-YFP fluorescence , showing reduced levels in pnt2mutant clones .", "Arrows highlight apoptotic nuclei .", "Right , merged image with Yan-YFP in green and RFP in purple .", "Clone borders are outlined .", "( F , G )", "Moving averages of Yan-YFP in R3/R4 cells that ectopically express PntP1 ( F ) or PntP2-VP16 ( G ) due to LongGMR-Gal4 driving the UAS transgenes .", "Since PntP2 requires MAPK phosphorylation to become transcriptionally active , we misexpressed a VP16 fusion of PntP2 .", "PntP1 is constitutively active ( Brunner et al . , 1994; Gabay et al . , 1996 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 08924 . 01010 . 7554/eLife . 08924 . 011Figure 3—figure supplement 1 . Yan-YFP levels in R2/R5 correlate with distance from R8 cells when EGFR is active . Correlation between log-transformed detrended Yan-YFP levels in R2 and R5 nuclei and their distances from the nearest R8 nucleus as a function of developmental time .", "( A , B )", "The Pearson correlation coefficient obtained in wildtype and EGFRts mutant .", "( C , D ) p value of correlation in wildtype and EGFRts mutant .", "( E , F )", "Proportion of variance in R2/R5 levels explained by distance to nearest R8 in wildtype and EGFRts mutant .", "Values calculated from pooled R2/R5 cells ( four wildtype and four EGFRts replicates ) within a 12 . 5 hr window centered on the corresponding developmental time . DOI: http://dx . doi . org/10 . 7554/eLife . 08924 . 011 We next examined the effects of EGFR and Ras on Yan-YFP dynamics in cells as they differentiate .", "The bistable model predicts that EGFR is required for the loss of Yan-YFP in photoreceptors .", "Indeed , EGFRts mutant R2/R5 cells delayed their initial decline in Yan-YFP levels ( Figure 3C ) .", "Conversely , misexpression of constitutively active Ras caused a premature decline in Yan-YFP ( Figure 3D ) .", "These results are consistent with EGFR-Ras stimulating the degradation of Yan-YFP as cells switch their states .", "However , Yan-YFP dropped to below-normal levels in EGFRts mutant R2/R5 cells ( Figure 3C ) .", "These complex effects suggest a dual role for EGFR signaling in photoreceptors .", "In the first few hours as cells transit to a photoreceptor state , EGFR stimulates the accelerated decay of Yan-YFP .", "Thereafter , EGFR inhibits the decay of Yan-YFP in a manner that might be related to that role that EGFR plays in boosting Yan-YFP levels in progenitor cells .", "The source of EGFR ligand originates from the R8 cell ( Freeman , 1996 ) .", "If this diffusive ligand is responsible for controlling Yan-YFP levels in other photoreceptors as they are recruited to an ommatidium , we would predict a correlation between Yan-YFP levels in differentiating cells and their distances from adjacent R8 cells .", "Indeed , at the times when R2/R5 cells differentiate ( ~0–15 hr ) , their Yan-YFP levels are positively correlated ( p<0 . 01 ) with their physical distance to the nearest R8 cells ( Figure 3—figure supplement 1 ) .", "These correlations are absent in EGFRts mutants , providing evidence that R8 cells act through EGFR to control Yan-YFP dynamics in differentiating cells .", "Pnt proteins have been hypothesized to cross-repress Yan expression , and this double negative feedback loop is thought to be necessary for bistability ( Graham et al . , 2010; Shilo , 2014 ) .", "At odds with this view , Pnt and Yan proteins are co-expressed in progenitor and differentiating cells of the eye ( Boisclair Lachance et al . , 2014 ) .", "Since Pnt proteins act downstream of many RTKs including EGFR , we wondered if Pnt mediated the positive effects of EGFR-Ras signaling on Yan-YFP in progenitor cells .", "Null mutants of the pnt gene are embryonic inviable; therefore we generated clones of eye cells that were null mutant for pnt in an otherwise wildtype animal .", "Mutant progenitor cells showed a reduction in Yan-YFP levels as they progressed through their biphasic expression pattern ( Figure 3E ) .", "Thus , Pnt possibly mediates the positive effect of EGFR signaling on Yan-YFP expression in progenitors .", "We also wished to know if Pnt mediates the complex effects of EGFR in differentiating photoreceptors .", "Pnt proteins are rapidly cleared in photoreceptors ( Boisclair Lachance et al . , 2014 ) and so loss-of-function mutant analysis would be uninformative .", "Therefore , we overexpressed PntP1 or constitutively-active PntP2 in cells as they transited into a photoreceptor state and beyond .", "The early phase of Yan-YFP decay was accelerated while the later phase of Yan-YFP decay was inhibited ( Figure 3F , G ) .", "These complex effects are precisely the opposite to those caused by loss of EGFR signaling , as would be expected if Pnt mediated EGFR’s complex effects on Yan-YFP dynamics in photoreceptor cells .", "The bistable model predicts that different cell states depend upon discrete absolute concentration of Yan present inside cells .", "To test this idea , we varied the number of Yan-YFP gene copies .", "In general , protein output is proportional to gene copy number in Drosophila ( Lucchesi and Rawls , 1973 ) .", "We increased Yan-YFP copy number from its normal diploid number to tetraploid , and monitored Yan-YFP in progenitors and differentiating cells .", "As expected , four copies caused a higher steady-state level of Yan-YFP in progenitor cells , though this increase was less than two-fold ( Figure 4A ) .", "The amplitude of the Yan-YFP pulse was also increased as progenitor cells aged .", "Strikingly , as four-copy progenitor cells transited to a differentiated state , the onset of Yan-YFP decay occurred at the same time as it occurred for two-copy cells ( Figure 4A–C ) .", "Yan-YFP levels were much greater in four-copy cells compared to two-copy cells making their transit into the identical cell states .", "To confirm that absolute Yan-YFP concentration had little effect on cell state transitions , we examined expression of a direct target of Yan in R7 cells: the pros gene ( Kauffmann et al . , 1996; Xu et al . , 2000 ) .", "Expression was monitored with an antibody specific for Pros protein .", "We observed at most a one hour delay in the onset of Pros expression in R7 cells containing four copies of Yan-YFP ( Figure 4D ) , far less than the ten-hour delay predicted if absolute concentration of Yan-YFP dictated when Pros expression begins ( Figure 4D ) . 10 . 7554/eLife . 08924 . 012Figure 4 . Cell state transitions are unaffected by Yan-YFP gene copy number .", "( A–C )", "Moving averages of Yan-YFP in eye discs containing two versus four copies of the Yan-YFP transgene .", "( A ) R2/R5 and progenitor cells .", "( B ) R3/R4 and progenitor cells .", "( C ) R7 and progenitor cells .", "( D ) Moving averages of Yan-YFP and Pros proteins in R7 cells containing either two vs . four copies of the Yan-YFP transgene . DOI: http://dx . doi . org/10 . 7554/eLife . 08924 . 01210 . 7554/eLife . 08924 . 013Figure 4—figure supplement 1 . Model fitting Yan-YFP decay in eye disc cells .", "( A ) Progenitors , ( B ) R8 cells , ( C ) R2 and R5 cells , ( D ) R3 and R4 cells , ( E ) R1 and R6 cells , ( F ) R7 cells .", "For each dataset , four models are shown: linear , exponential , second-order polynomial , and third-order polynomial .", "R squared values are indicated for each model . DOI: http://dx . doi . org/10 . 7554/eLife . 08924 . 01310 . 7554/eLife . 08924 . 014Figure 4—figure supplement 2 . Exponential decay models . Yan-YFP levels in cells within a representative eye disc .", "Each cell type is color-coded as indicated .", "( A ) Progenitors with fitted Hill ( blue line ) and exponential decay functions ( black line ) overlaid .", "The green and orange lines label Yan-YFP levels at the start of the decay and at the end ( decay asymptote ) respectively .", "The red line outlines the transition from induction to decay .", "( B-F )", "Identified photoreceptors are overlaid with the progenitors that precede and coincide with them at the point when the photoreceptors first appear .", "Best-fit exponential functions for Yan-YFP in photoreceptors are shown . DOI: http://dx . doi . org/10 . 7554/eLife . 08924 . 014 Possibly , absolute concentration of Yan is unimportant when a cell transits to a different state , but the switch to a constant basal Yan level is robustly maintained regardless of starting concentration .", "An examination of Yan-YFP decay in photoreceptor cells makes that possibility less likely; Yan-YFP decays to different basal levels in two- versus four-copy differentiated cells ( Figure 4A–C ) .", "To further test this notion , we fit the data to several plausible functional forms .", "We found that exponentially decaying functions systematically best-fit to the data ( Figure 4—figure supplement 1 ) .", "Thus , for each cell state we fit an exponential decay function to its Yan-YFP temporal profile ( Figure 4—figure supplement 2 ) .", "From these fits , we derived the average decay rate constants and half-lives of Yan-YFP for cells carrying two , four , or six copies of yan .", "As expected , we found that Yan-YFP half-life was different between progenitors and differentiating photoreceptors ( Figure 5—figure supplement 1 ) .", "The half-life in photoreceptors was two-fold lower than in progenitors , accounting for the more rapid loss of Yan-YFP in the former cells .", "Strikingly , Yan-YFP half-life was not significantly affected by yan copy number within either progenitor or photoreceptor cells ( Figure 5 ) .", "Thus , Yan-YFP concentration only affected its decay rate as a first-order function , implying that there is no higher order mechanism to accelerate Yan-YFP decay when cells contain greater concentrations of Yan-YFP . 10 . 7554/eLife . 08924 . 015Figure 5 . Yan protein half-life is largely unaffected by yan gene copy number . Exponential decay of Yan-YFP levels .", "Note the robustness of Yan-YFP half-lives across replicates and yan gene copy number .", "Note also how half-lives are nearly twice as long for progenitor cells versus photoreceptor neurons . DOI: http://dx . doi . org/10 . 7554/eLife . 08924 . 01510 . 7554/eLife . 08924 . 016Figure 5—figure supplement 1 . Mean half-lives for Yan-YFP decay versus yan gene copy number ( 2 , 4 , 6x ) .", "Error bars represent 95% confidence intervals for estimation of the mean .", "Each estimation of the mean is based on bootstrapping data from one disc sample .", "Four discs were analyzed for each copy number condition . DOI: http://dx . doi . org/10 . 7554/eLife . 08924 . 01610 . 7554/eLife . 08924 . 017Figure 5—figure supplement 2 . Compound eyes of adults carrying two , four , or six copies of the yan gene .", "Anterior is to left . DOI: http://dx . doi . org/10 . 7554/eLife . 08924 . 017 As a final test of the effects of Yan-YFP levels on cell states , we allowed 4X and 6X yan animals to complete eye development and then examined the external patterning of the fully differentiated compound eye .", "The compound eyes were completely normal in appearance ( Figure 5—figure supplement 2 ) , implying that differentiation was unaffected by the absolute concentrations of Yan inside eye cells .", "Our results indicate that Yan’s effects on retinal cell states are not dictated by uniform and stable concentrations of Yan protein .", "One potential explanation is that Yan’s effect on cell states actually depends on variability in Yan protein levels .", "A growing body of evidence is pointing to the importance of transient fluctuations in expression of factors to control cell states ( Cahan and Daley , 2013; Torres-Padilla and Chambers , 2014 ) .", "Key regulators of stem cells fluctuate in abundance over time , and this is correlated with stem cells existing in multiple connected microstates , with the overall structure of the population remaining in a stable pluripotent macrostate ( MacArthur and Lemischka , 2013 ) .", "Heterogeneity among cells is not simply due to independent noise in expression of individual genes but is due to fluctuating networks of regulatory genes ( Chang et al . , 2008; Kumar et al . , 2014 ) .", "Such fluctuations appear to be stochastic in nature , priming cells to transit into differentiated states with a certain probability that is guided by extrinsic signals .", "Our data revealed considerable heterogeneity in the level of Yan-YFP among cells at similar developmental times ( Figure 2B ) .", "To quantify the noise , we used developmental time to bin cells of similar age , and measured detrended fluctuations of Yan-YFP by calculating residuals to the fitted function and normalizing binned residuals to the function ( Goldberger et al . , 2002 ) .", "Progenitor cells showed a spike in Yan-YFP noise as they began to induce Yan-YFP expression ( Figure 6A ) .", "The noise spike was short-lived ( approximately 17 hr ) , and noise thereafter returned to a basal level with secondary minor spikes .", "The major peak in noise magnitude coincided with the time at which R8 cells are formed . 10 . 7554/eLife . 08924 . 018Figure 6 . Noise in Yan-YFP expression is highly dynamic . Moving averages of Yan-YFP levels and noise ( detrended fluctuations ) for ( A ) progenitors , ( B ) R2/R5 , ( C ) R3/R4 , ( D ) R1/R6 , and ( E ) R7 cells .", "( F ) Comparative noise dynamics for all cells analyzed in ( A–E ) .", "( G ) Moving averages of Yan-YFP noise ( coefficient of variation ) in R2/R5 cells sampled from wildtype and EGFRts mutant eyes at the non-permissive temperature .", "Shown with shading is the standard error of the mean for each moving average .", "( H ) Moving averages of Yan-YFP noise ( coefficient of variation ) in R2/R5 cells sampled from wildtype and sev>Rasv12 mutant eyes .", "Shown with shading is the standard error of the mean for each moving average . DOI: http://dx . doi . org/10 . 7554/eLife . 08924 . 01810 . 7554/eLife . 08924 . 019Figure 6—figure supplement 1 . Comparison of methods to measure Yan-YFP noise based on sensitivity to window size .", "( A–H )", "Yan-YFP levels are analyzed in sliding windows of size 20 , 150 , 250 , or 500 progenitor cells .", "Noise is estimated by detrended fluctuation ( A , C , E , G ) or coefficient of variation ( B , D , F , H ) within a sliding window that bins cells .", "( I–P )", "Yan-YFP levels are analyzed in sliding windows of size 20 , 40 , 70 , or 150 R2/R5 cells .", "Noise is estimated by detrended fluctuation ( I , K , M , O ) or coefficient of variation ( J , L , N , P ) within a sliding window that bins cells . DOI: http://dx . doi . org/10 . 7554/eLife . 08924 . 01910 . 7554/eLife . 08924 . 020Figure 6—figure supplement 2 . Measurement of Yan-YFP noise in all cell types .", "( A , C , E , G , I , K )", "Yan-YFP noise is estimated by detrended fluctuations for progenitors ( A ) , R8 ( C ) , R2/R5 ( E ) , R3/R4 ( G ) , R1/R6 ( I ) , and R7 cells ( K ) .", "( B , D , F , H , J , L )", "Yan-YFP noise is estimated by the coefficient of variation for progenitors ( B ) , R8 ( D ) , R2/R5 ( F ) , R3/R4 ( H ) , R1/R6 ( J ) , and R7 cells ( L ) .", "Window sizes used for each analysis are indicated . DOI: http://dx . doi . org/10 . 7554/eLife . 08924 . 02010 . 7554/eLife . 08924 . 021Figure 6—figure supplement 3 . Comparison of Yan-YFP and endogenous Yan protein dynamics .", "( A , C , E )", "His2Av-mRFP discs stained with anti-Yan antibody to measure endogenous Yan .", "( B , D , F )", "Yan-YFP measurements of His2Av-mRFP Yan-YFP discs in which endogenous Yan has been deleted .", "( A , B )", "Moving averages of anti-Yan ( A ) and Yan-YFP ( B ) fluorescence levels in multipotent , R8 , and cone cells .", "Gaps between the multipotent and R8 curves are due to the window size for line averaging .", "( C , D )", "Moving averages of anti-Yan ( C ) and Yan-YFP ( D ) levels and noise ( coefficient of variation ) for multipotent cells .", "( E , F )", "Moving averages of anti-Yan ( E ) and Yan-YFP ( F ) levels and noise ( coefficient of variation ) for R8 cells . DOI: http://dx . doi . org/10 . 7554/eLife . 08924 . 02110 . 7554/eLife . 08924 . 022Figure 6—figure supplement 4 . Moving averages of anti-Pros fluorescence levels and noise ( coefficient of variation ) in R7 cells . DOI: http://dx . doi . org/10 . 7554/eLife . 08924 . 022 Photoreceptor cells showed a large spike in Yan-YFP noise as they began to differentiate ( Figure 6B–E ) .", "The magnitude of each noise peak varied with the photoreceptor cell state; R3 and R4 cells exhibited the greatest amplitude in noise ( Figure 6F ) .", "These noise spikes showed a distinct temporal relationship , with spikes coinciding with the times at which individual cell states were switched ( Figure 6F ) .", "Thus , the noise spikes are not a simple consequence of a global stimulus synchronously affecting noise in all cells .", "Thereafter , all cells reduced Yan-YFP noise to a basal level that was comparable to basal noise in the progenitor cells .", "However , each cell type exhibited a secondary minor spike at 30–35 hr , which might reflect a synchronous stimulus .", "Detrended fluctuation is one method to measure expression variability , but it can suffer from distortion if the model fitting is not adequately weighted .", "Therefore , we also calculated the coefficient of variation , that is , the standard deviation of Yan-YFP intensity within a sliding window divided by its mean .", "This method yielded noise profiles with transient spikes for each cell type that was consistent with calculations using detrended fluctuation ( Figure 6—figure supplements 1 and 2 ) .", "However , while the coefficient of variation yielded results that varied strongly with bin width , the detrended fluctuations yielded profiles that were generally robust against changes in bin width ( Figure 6—figure supplement 1 ) .", "To rule out the possibility that these unexpectedly dynamic features of Yan-YFP were caused by its transgenic origins or fusion with YFP , we compared Yan-YFP dynamics to those of endogenous Yan protein that was bound with an anti-Yan antibody .", "The profiles of Yan-YFP protein levels and noise were highly similar to endogenous Yan protein levels and noise , in both multipotent and differentiating cells ( Figure 6—figure supplement 3 ) .", "Thus , transient spikes of expression heterogeneity are a fundamental feature of Yan protein .", "Since Yan regulates Pros expression in R7 cells , it was possible that Pros showed a transient noise spike as a consequence .", "Therefore , we measured Pros protein heterogeneity and found that its dynamics did not resemble that of Yan ( Figure 6—figure supplement 4 ) .", "Instead , Pros noise was high starting at the onset of expression , and thereafter gradually declined as Pros protein levels increased in R7 cells .", "We conclude that noise spikes are not a general feature of gene expression in the developing eye but might reflect unique roles of Yan in mediating cell state transitions .", "We wondered what might cause these spikes in Yan-YFP noise during cell state transitions .", "Because EGFR signaling is important for regulating Yan-YFP concentration during these transitions ( Figure 3 ) , we analyzed Yan-YFP noise when EGFR signaling was inhibited in EGFRts mutant animals raised at a non-permissive temperature .", "The noise spike in progenitor cells was not significantly affected by loss of EGFR signaling ( data not shown ) .", "We also examined the effects of EGFRts on noise in differentiating photoreceptor cells .", "Interestingly , noise increased at the normal time of transition but the elevated noise did not quickly drop to basal levels ( Figure 6G ) .", "Rather , high noise was extended for an additional 10 to 15 hr .", "Conversely , misexpressing constitutively active Ras within differentiating cells caused a premature dropdown in Yan-YFP noise ( Figure 6H ) .", "These results indicate that EGFR/Ras signaling is required for the rapid drop in Yan-YFP noise after it has peaked , creating a transient spike ." ], [ "This study relied upon a set of methods that enable systems-level analysis of Yan expression dynamics in a developing animal tissue .", "Transgene recombineering was used to insert YFP into a genomic rescue fragment of the yan gene , which fully replaced endogenous yan .", "Yan-YFP protein was quantified in thousands of individual cells by fluorescence confocal microscopy and automated cell segmentation analysis .", "Based on the unique features of Drosophila eye development , every analyzed cell was assigned an age , and composites of cells across a time-spectrum of ages were derived .", "This allowed us to reconstruct the temporal dynamics of Yan protein expression in cells as they transited from one state to another or were maintained in a given state .", "The fact that both Yan concentration and noise were easily measured using our approach indicates that it provides a powerful method for studying how other molecular determinants regulate cell states .", "Contrary to what is currently believed , the expression of Yan in progenitor cells has many hallmarks of monostability .", "A stable basal state exists in cells anterior to the furrow , and when the furrow passes , Yan rises and falls to form a biphasic profile ( Figure 7A ) .", "If cells transit towards differentiation , then the fall in Yan is accelerated but the fundamental biphasic profile is preserved .", "This monostable-like behavior is not like a behavior where progenitor cells exist in a high Yan stable-state and switch to a low Yan stable-state when they transit towards differentiation ( Figure 7A ) .", "Other lines of evidence also point away from a bistable switch mechanism .", "Yan reaches its basal steady state many hours after cells have adopted their differentiated photoreceptor state and are executing specialized gene expression programs .", "Thus , Yan levels are variable at the time when cells actually become differentiated . 10 . 7554/eLife . 08924 . 023Figure 7 . Summary of analysis .", "( A ) Top: a hypothetical bistable behavior would be where Yan is in a stable high state within progenitor cells and in a stable low state within differentiated cells .", "Bottom: the observed behavior of Yan appears monostable , with both progenitors and differentiated cells in unstable Yan states .", "( B ) Heterogeneity in Yan expression is maximal when progenitor cells enter a transition state that resolves to a more homogeneous differentiated state , dependent upon EGFR signaling .", "This heterogeneous transition state may be a primary mechanism for Yan’s effect on cells , independent of the absolute Yan concentration within progenitors . DOI: http://dx . doi . org/10 . 7554/eLife . 08924 . 023 Since cell states are indifferent to Yan stability , it would suggest that absolute Yan levels do not dictate Yan’s effects on cell behavior .", "This conclusion is bolstered by experiments in which yan gene dosage affects absolute Yan levels but not cell behavior .", "Several mechanisms could explain how Yan regulates cells in a concentration-independent manner .", "First , total Yan might vary but a specific modified form of Yan might remain constant .", "For example , MAPK phosphorylation of Yan could operate under Michaelis-Menten saturation to generate constant levels of phospho-Yan that depend upon MAPK activity .", "Second , Yan’s transcriptional activities might be independent of Yan concentration due to limiting levels of other transcription factors such as Lozenge , Glass , and Sine Oculis , which are known to act combinatorially with Yan to regulate gene transcription ( Flores et al . , 2000; Hayashi et al . , 2008; Xu et al . , 2000 ) .", "Third , cells might sense relative changes in Yan , and respond to a constant fold-change in Yan levels using integral negative feedback or incoherent feedforward loops ( Cohen-Saidon et al . , 2009; Goentoro and Kirschner , 2009; Goentoro et al . , 2009; Wartlick et al . , 2011 ) .", "Indeed , Yan is predicted to function in an incoherent feedforward loop with Pnt .", "Pnt directly activates transcription of target genes such as pros and mir-7 , but it also activates expression of Yan ( Figure 3G ) , which in turn , directly represses transcription of pros and mir-7 ( Li et al . , 2009; Xu et al . , 2000 ) .", "The most intriguing hypothesis , however , is that variation in Yan concentration might actually be exploited by cells to guide their state transition .", "Indeed , it has recently been noted that undifferentiated stem cells express highly variable levels of transcription factors such as Nanog , Myc , Otx2 , and Pax6 ( Cahan and Daley , 2013; Kumar et al . , 2014; Torres-Padilla and Chambers , 2014 ) .", "Conditions that repress variability keep these cells trapped in an undifferentiated state ( Kumar et al . , 2014 ) .", "Noisy expression is thought to place cells into a transition state where a sub-population of cells at any given time express a particular combination of factors that spontaneously trigger differentiation ( Martinez Arias and Brickman , 2011 ) .", "Such a fluctuating transition state renders the probability of cell differentiation to be greater than zero , and thus provides cells with the ability to respond to extrinsic signals .", "Consistent with this hypothesis , Yan levels not only vary over long time-scales but also show heterogeneity within a small time interval .", "This expression noise is reminiscent of the heterogeneity in Nanog expression observed in pluripotent stem cells .", "As progenitor cells transit to differentiated states , there is a sharp spike in Yan expression noise that coincides with the early stages of the transition .", "By analogy to the situation in embryonic stem cells , the spike in Yan noise could reflect the existence of a metastable intermediate state between the undifferentiated and differentiated states ( Figure 7B ) .", "A limitation to our interpretation is that because our data do not track individual cells over time , we cannot distinguish whether the expression heterogeneity reflects asynchronous fluctuations within individual cells or a wide range of stable microstates .", "Real-time single cell measurements will be required to distinguish between these possibilities .", "The purpose of the elevated noise could be to prime cells to change states upon reception of a differentiation signal .", "Without elevated noise , differentiation or de-differentiation would be blocked .", "A prediction of this hypothesis is that differentiation signals would act downstream of noise elevation and might even be important for noise reduction once cells enter an irreversible differentiated state .", "Indeed , EGFR/Ras signaling is not required for elevation of Yan noise but rather is important in promoting the rapid reduction of noise back to its baseline level after differentiation .", "In the absence of signaling , the cell may remain trapped in the intermediate ( undifferentiated ) state .", "The model raises several questions .", "What triggers the increase in the noise level of Yan as cells enter this transition state ?", "Do other determinants such as Pnt similarly fluctuate ?", "Do individual cells fluctuate over time or does this represent a population of cells that adopt stable microstates ?", "And finally , there is the question of the noise spike in progenitor cells at the time of R8 formation .", "Does this represent another transition state that early progenitor cells enter before moving into a different progenitor state ?", "One intriguing possibility is that it represents the transition from G1-arrest to synchronized cell division that progenitor cells undergo in the second mitotic wave immediately posterior to the furrow ( Wolff and Ready , 1993 ) .", "Loss-of-function studies have suggested that Yan prevents progenitors from undergoing multiple rounds of cell division rather than again arresting at G1 phase after one round of division ( Rogge et al . , 1995 ) .", "Further studies should help elucidate the causes and functions of this and the other noise peaks ." ], [ "Growth conditions were 21°C unless stated otherwise .", "The recombineered Yan-YFP BAC transgene was previously described ( Webber et al . , 2013 ) .", "It was inserted into the attP2 ( 3L ) and attP16 ( 2R ) sites in the genome .", "2 x Yan-YFP animals used for most experiments were null for endogenous yan and carried a single copy of the His2Av-mRFP transgene ( yanER443/yanE884; Yan-YFP/Yan-YFP , His2Av-mRFP ) .", "EGFR activity was conditionally reduced by placing Egfrf24 ( Clifford and Schupbach , 1989 ) in trans to the ts mutant Egfrtsla ( Kumar et al . , 1998 ) .", "Flies were raised at the permissive temperature ( 18°C ) and shifted to a semi-restrictive temperature ( 26 . 5°C ) as third instar larvae for 18-20h .", "At this temperature , developing eye cells had compromised EGFR activity since animals that were transferred back to the permissive temperature and allowed to eclose had rough eyes .", "In EGFRts eye discs , there were signs of some cells undergoing apoptosis - a significant reduction of nuclear diameter , a strong Yan-YFP brightness , and anomalous nuclear position along the apical-basal axis .", "Apoptosis was more prevalent in discs from animals treated at temperatures greater than 26 . 5°C .", "Therefore , we chose this temperature for EGFR activity reduction so as to minimize apoptosis but still achieve effects on cell differentiation .", "We only included in our analysis cells corresponding to classical anatomical positions and apical basal migration patterns .", "Ras activation was achieved using a transgene expressing a Ras1V12 mutant .", "Expression was under the control of the 3xsev enhancer and promoter ( Fortini et al . , 1992 ) .", "Clones of the pnt2 pan-isoform null allele were generated with eyFLP122 and FRT82B crossover points .", "Pnt+ tissue was labeled using Ubi>mRFPnls .", "UAS-PntP1 and UAS-PntP2::VP16 expression was driven with LongGMR-Gal4 ( Wernet et al . , 2003 ) .", "Dynamics of Yan using different yan gene copy numbers were measured in 2 x yan flies ( yanER443/yanE884; Yan-YFP , His2Av-mRFP/Yan-YFP ) , 4 x yan flies ( Yan-YFP , His2Av-mRFP/Yan-YFP ) , and 6 x yan flies ( Yan-YFP/Yan-YFP; Yan-YFP , His2Av-mRFP/Yan-YFP ) .", "Eye-antennal discs from white prepupae were fixed in 4% PFA/PBS for ~45 min .", "Discs were incubated in 1:1 ( v/v ) PBS:VectaShield ( Vector Laboratories ) for 45 min , followed by a 45 min incubation in 100% VectaShield , and then were mounted .", "For certain experiments , Yan and Prospero protein immunofluorochemistry was performed using mouse anti-Yan ( DSHB , 1:200 dilution ) and anti-Prospero ( DSHB , 1:100 dilution ) antibodies , and secondary goat anti-mouse Pacific Blue or goat anti-mouse Alexa 633 ( Life Technologies , both at 1:200 dilution ) .", "To validate cell-type identification , discs were treated with mouse anti-Rough ( DSHB ) , mouse anti-Cut ( DSHB ) , rabbit anti-Senseless ( Hugo Bellen ) , and rat anti-Elav ( DSHB ) , and secondary Pacific Blue labeled antibodies ( Life Technologies , 1:200 dilution ) .", "Samples were kept in the dark at -20°C and imaged no later than 18 hr after fixation .", "All discs for a given condition were fixed , mounted , and imaged in parallel to reduce measurement error .", "Mounted samples were imaged with an SP5 Leica confocal microscope using a 40X objective , and the 405 nm , 514 nm , 541 nm , and 630 nm lasers were used to excite Pacific Blue , Yan-YFP , His2Av-mRFP and Alexa 633 , respectively .", "Offset was set to zero .", "The gain was adjusted using the simple scanning mode without line averaging such that the brightest nuclei in the sample did not overexpose in the detector .", "Stacks of ~60 optical sections ( 1 μm ) were acquired after orienting the eye disc equatorial region parallel to the x-axis of the image .", "Sections of 2048 x 2048 pixel resolution were collected using the 8-line average bidirectional scanning mode .", "A typical nucleus was usually represented by 150–300 pixels at its widest plane .", "Zoom and all other imaging parameters were kept constant for all samples within the same experiment .", "Bleaching controls were made at the beginning and end of imaging to ensure that fluorophore bleaching was not significant .", "Images were acquired such that the eye’s equator was parallel to the x-axis of the image , and the eye’s MF was parallel to the y-axis of the image .", "We cropped images so that each region of interest ( ROI ) was a rectangle with its long axis centered at the equator ( Figure 1A ) .", "This region extended eight ommatidial rows on either side of the equator .", "It was bounded on the anterior side 30–60 μm ahead of the MF and on the posterior side two ommatidial columns from the posterior margin .", "It is known that the margins of the eye disc exhibit different MF dynamics ( Dominguez and Hafen , 1997; Legent and Treisman , 2008 ) , and so to minimize heterogeneity due to margin effects , we limited analysis to this centered region .", "We estimated the potential illumination biases of the SP5 confocal microscope by exciting and imaging VectaShield media alone .", "We divided the images into squares of 32 × 32 pixels .", "For each square , we calculated and plotted the average fluorescence in 1 and 2 dimensions .", "Based on this analysis , we found that the microscope had no significant illumination biases across the field of view .", "Previous studies of Drosophila embryos found that autofluorescence , which contributes to the measured total fluorescence , has to be subtracted to properly estimate the fluorescent protein signal of interest ( Surkova et al . , 2008 ) .", "To measure autofluorescence of eye disc samples , we fixed and imaged wildtype discs using the same conditions as for Yan-YFP discs .", "The wildtype discs had negligible fluorescence .", "Therefore , we did not subtract autofluorescence background from total fluorescence in Yan-YFP discs .", "We tested whether His2Av-mRFP fluorescence affected the measurement of Yan-YFP signal in the yellow channel .", "To measure the contribution of mRFP to the yellow channel , discs expressing only His2Av-mRFP were imaged with the same parameters as discs described above .", "We calculated the average fluorescence in 2 dimensions using a heat map to represent intensity ( Figure 1—figure supplement 4A–C ) .", "To determine if the low RFP signal detected in the yellow channel was uniform , we segmented nuclei and measured the ratio of yellow:red signals ( Figure 1—figure supplement 4D ) .", "We found that no more than 5% of total RFP fluorescence was detected in the yellow channel .", "Therefore , a complete absence of YFP fluorescence would generate 0 . 05 units of normalized fluorescence ( yellow/red ) in Yan-YFP discs .", "A pipeline was custom-built to automatically segment nuclei and manually assign their identities with GUI software .", "The pipeline is accessible for download at https://dl . dropboxusercontent . com/u/2649235/Pipeline_eye_eLife . zip .", "Segmentation of nuclei in each optical section image was performed with a custom-built Matlab program ( Qi et al . , 2013 ) .", "A detailed description of the algorithm , coding , and analysis will be published separately .", "We briefly describe it as follows .", "First , graph-cut estimates the mean overall signal and partitions the image into background and foreground , resulting in nuclear pixels clustering in the foreground .", "Such clusters are then analyzed with a mean-shift algorithm , which further partitions the objects into smaller pixel clusters in a locally optimized manner .", "Due to the tight packing of eye disc nuclei , many such segmented objects contain multiple nuclei whose fluorescence signals partially overlap .", "Thus , objects are then subjected to concavity-convexity analysis .", "According to this analysis a cluster of cells is split into multiple cells , based on a threshold that quantifies the degree of concavity of the cluster ( Figure 1—figure supplement 2 ) .", "The Matlab implementation of this algorithm contains 5 parameters .", "The developed algorithm was evaluated extensively utilizing publicly available hand-segmented benchmark datasets of microscope images .", "Towards this task a number of objective performance metrics were utilized , such as the Rand Index and the Hausdorff distance .", "In facilitating the evaluation of the various indices we utilized the following error classes:", "1 ) two reference nuclei ( “ground truth” ) are assigned to a single machine-segmented nucleus;", "2 ) one reference nucleus is segmented as two by the machine;", "3 ) machine picks up a non-existent nucleus from the background;", "4 ) a nucleus belonging to the reference image is lost in the machine-segmented image .", "In all experiments the developed algorithm outperformed existing segmentation algorithms that are widely used and referenced , such as the watershed algorithm .", "For example , with respect to the Rand index , the performance of the developed algorithm was 91% as compared to 78% achieved by the watershed algorithm ( data not shown ) .", "The developed algorithm was also compared against manual segmentations of the same eye disk images .", "In all cases it resulted in segmentations very similar to the manual ones .", "While performing manual segmentation , we observed that the mean and standard deviation of pixel intensity for a nucleus ( size >80 pixels ) was distorted by pixels close to the segmented boundary .", "This is due to light scattering .", "The phenomenon has also been reported for Drosophila embryonic nuclei ( Surkova et al . , 2008 ) .", "Thus , once the automated segmentation routine was completed , the contour of each object was shrunk using two shrink parameters - shrink and shrink level .", "The first parameter determines whether boundary shrinking occurs , and the second parameter determines the number of pixels from the boundary to be eroded .", "We find that this operation results in smaller measurement errors .", "A single set of parameters ( P1=6 , P2=30 , P3=3 , P4=5 , P5=30 , Shrink=1 , Shrinklevel=4 ) was used for all layers .", "This parameter combination produced accurate segmentations , despite the heterogeneity in size and shape of different cell types .", "We assigned cell-type identities to segmented nuclei by using nuclear position and morphology , two key features that enable one to unambiguously identify eye cell types without the need for cell-specific markers ( Wolff and Ready , 1993 ) .", "To perform this task , we displayed the confocal microscopy data in a custom-made Graphic User Interface ( GUI ) , which allowed users to visualize the contours of segmented nuclei mapped on section images , and for users to label each segmented nucleus with an ID value .", "Each ID value was then automatically assigned to the record of fluorescence intensities and positional information for the segmented object within the database ( Figure 1—figure supplement 2 ) .", "Our facilitated method of manual identification was over 98% accurate in identifying cell types ( Figure 1—figure supplement 3 ) .", "We plotted the centroid positions of identified cell types on a 2D Cartesian plane .", "These produced coordinate maps that are in complete accordance with anatomical descriptions of the eye disc ( Figure 2—figure supplement 1 ) .", "For each cell that we identified , we only used the data obtained from the optical section with the greatest nuclear contour .", "Consecutive sections in a stack could easily be scrolled through to ascertain the section having the greatest 2D nuclear contour for any given cell .", "This approach prevented oversampling of cells and minimized measurement error due to insufficient pixel number in a nuclear section .", "We tested the veracity of this 2D sampling method by comparing Yan-YFP fluorescence intensity in different optical sections of the same nucleus .", "If fluorescence is uniformly distributed throughout the nucleus volume , then fluorescence intensities will be similar across different optical sections .", "This was indeed found to be the case when we performed such sampling on 20 distinct nuclei belonging to various cell types .", "We found that His2Av-mRFP exhibits non-uniform fluorescence intensity along the anterior-posterior axis of the eye disc ( Figure 1—figure supplement 4E ) .", "Intensity particularly fluctuates in regions immediately posterior to the MF .", "We also observed fluctuations in nuclear size along the same axis ( Figure 1—figure supplement 4F ) .", "These fluctuations are somewhat anti-correlated with His2Av-mRFP intensity fluctuations .", "It suggested that perhaps nuclear volume dynamics are responsible for changes in His2Av-mRFP concentration and therefore fluorescence intensity .", "Hence , we multiplied fluorescence intensity by nuclear size to provide a measure of His2Av-mRPF content ( mass ) , which we predicted should be constant in G1-arrested cells .", "Indeed , His2Av-mRPF content was more uniform in cells along the anterior-posterior axis ( Figure 1—figure supplement 4G ) .", "However , there were two regions in which His2Av-mRFP content was greater: one region anterior to the MF and another region immediately posterior to the MF .", "These correspond to regions in which many cells are proliferative and therefore can be in S and G2 phases .", "The anterior region is where asynchronous division occurs and the posterior region is where the second mitotic wave occurs ( Wolff and Ready , 1993 ) .", "We reasoned that some of the variation in Yan-YFP intensity that we observe could be attributed to the same factors that cause His2Av-mRFP variation —DNA ploidy and nuclear volume .", "Since we wanted to measure Yan-YFP output as a function of regulatory network activity and not DNA content or volume , we normalized the fluorescence intensity of Yan-YFP to the fluorescence intensity of His2Av-mRFP .", "Normalization was done by taking the ratio of the mean pixel intensity of YFP over mean pixel intensity of RFP for each segmented nucleus .", "For each disc , we calculated a conversion factor that makes equivalent the distance travelled by the MF to the time required by the MF to travel that distance .", "This conversion is possible for several reasons .", "First , the MF moves at constant velocity , making a new column of R8 neurons every 2 hr at 21°C ( Campos-Ortega and Hofbauer , 1977 ) .", "Second , no cell migration occurs .", "There is a region where cell division occurs posterior to the MF —the second mitotic wave .", "Although this could potentially distort the physical distance between ommatidial columns , we find that it does not create a significant displacement .", "As a result , distances between R8 cells in adjacent columns do not undergo major rearrangements .", "The conversion factor was derived by finding the average distance between adjacent columns and relating this distance to the two hour time interval required to form a new column .", "Using a Delaunay triangulation , we determined the network of R8 cells within each sample ( Figure 2—figure supplement 1I ) .", "In this network , nodes are R8 nuclei centroids and links are the first R8 neighbors of each R8 cell nucleus found in the triangulation .", "The Delauney triangulation for a set P of points in a plane is a triangulation DT ( P ) in which no point in P is inside the circumcircle of any triangle in DT ( P ) , and in which the minimum angle of all the angles of the triangles in the triangulation is maximized .", "As a result , Delauney triangulations tend to avoid skinny triangles , thus providing a proper estimation of the grid of nearest neighbors for each R8 neuron in the set .", "We enumerated all the R8 neighbors for each R8 cell found in the triangulation , and for each R8 cell , we selected neighbors whose links to the R8 cell were oriented between 30° to 60° away from the anterior-posterior axis ( Figure 2—figure supplement 1J ) .", "We reasoned that these neighbors would correspond to R8 cells found within adjacent columns .", "The pairing process was repeated for every R8 cell in the network .", "The anterior-most column of R8 cells did not pair with more anterior cells since there were none .", "For each R8 pair , we decomposed the diagonal distance separating the two R8 cells using Pythagoras .", "We used the x-component of the distance separating the two R8 cells as the distance traveled by the furrow between those two adjacent columns .", "We then computed the average distance ( μ ) in pixels that the MF travels between adjacent columns as μ=1n∑i=1nxia−xib where xia and xib are the x coordinates in pixel values for the ith pair of R8 cell centroids , and n is the total number of R8 pairs .", "The distance μ and the two hours required for the MF to travel it , then allowed us to convert the distance that any disc cell was from the first column into a developmental time point for that cell: t=2μ ( xC-xC1 ) where t is the developmental time of cell c ( in hours ) , xc is the x coordinate of the cell c centroid ( in pixels ) , and xC1 is the x coordinate for the first column ( in pixels ) .", "We analyzed a minimum of four replicate eye discs for each treatment .", "A moving line average was generated for Yan-YFP intensity in progenitor cells for each disc sample of a given treatment .", "Samples from the same treatment were then aligned along the time axis such that the line average inflection points of Yan-YFP in the MF were minimized between all samples .", "We found that alignment using only the Yan-YFP inflection point caused the first columns of all samples to align with one another .", "It also caused the first appearance of photoreceptors in all samples to align with one another .", "Although we present the results of pooled samples and their analyses , these findings do not depend on sample alignment , and are reproducibly observable in individual samples when analyzed separately .", "A typical sample contained >1500 measured and cell-type assigned nuclei .", "Thus , each treatment represents close to 6000 datapoints .", "After samples were pooled according to treatment , we calculated a moving line average for each cell type class using a moving window of fixed size .", "We used different window sizes for progenitors ( n=130 cells ) and differentiating cells ( n=40 ) , and their output was smoothened with a second-order sliding window ( n=20 ) .", "We partitioned the trajectory of Yan-YFP in multipotent progenitors into two independent phases: induction and decay .", "We used a Hill function to fit the induction phase: Y ( t ) =a+btntn+kn where Y ( t ) is Yan-YFP intensity at time t , a is the intensity at t=0 , b is a constant , n is the Hill coefficient , and k is the time when Y is half-maximal .", "We implemented the minimize routine in scy . py to minimize the least squares between the data and a model estimating the values of a , b , k and n to find the best fit to the data ( Figure 4—figure supplement 2A ) .", "To fit the decay phase of Yan-YFP , we tested linear , exponential , and polynomial functions , and we found that the simplest function that best fit all of the data was an exponential function ( Figure 4—figure supplement 1 ) .", "The exponential decay function used is: Y ( t ) =A+Be- ( t/τ ) where Y ( t ) is Yan-YFP intensity at time t , A is the intensity at t=∞ , B is an amplification constant , and τ is the mean lifetime .", "These parameters were estimated using the minimize routine ( Figure 4—figure supplement 2A–F ) .", "To obtain interval estimates for these parameters , we implemented a bootstrapping routine in which 1000 fits were iteratively performed with 70% of the data randomly selected for each fit , performing data resampling without replacement .", "No significant difference was found in the results when resampling was done with replacement .", "For each fit , we calculated the half-life as T12=τln2 The mean half-life and uncertainty estimates were calculated for bootstrapped samples ( Figure 5—figure supplement 1 ) .", "Yan-YFP noise was calculated one of two independent ways .", "First , we computed the coefficient of variation ( CV = sigma/mean ) inside a sliding window .", "The window size or bin width was varied for different cell types to determine if the CV profile significantly changed ( Figure 6—figure supplement 1 ) .", "A window size of n=280 nuclei for progenitors and n=70 nuclei for differentiating cells was chosen for subsequent analysis ( Figure 6—figure supplement 2 ) .", "We calculated Pros noise in the same way .", "The second way we calculated Yan-YFP noise was to compare each datapoint to the fitted Hill and exponential functions , and estimate their residuals .", "The standard deviation of the residuals within a sliding window was divided by the mean value of the fitted function within the window , which gave the detrended fluctuation ( Goldberger et al . , 2002 ) .", "The window size was varied from n=20 to n=500 nuclei , and plotted as shown ( Figure 6—figure supplement 1 ) .", "A window size of n=280 nuclei for progenitors and n=70 nuclei for differentiating cells was chosen for subsequent analysis ( Figure 6—figure supplement 2 ) .", "Initially we carried out analysis of each cell type separately , differentiating between cell type pairs that commit almost concurrently ( R2 and R5 , R3 and R4 , R1 and R6 , C1 and C2 , C3 and C4 ) .", "We did not find significant differences between individuals with each pair in terms of their Yan-YFP dynamics .", "Therefore , we pooled the pairs to gain power in the estimations of the mean trajectory and moving averages .", "An exception was observed with the misexpression of PntP1 , where a difference between the R3/R4 cells was observed .", "But since both cells behaved by changing Yan-YFP levels in the same direction , we pooled them for the analysis shown in Figure 3 .", "To quantify the local influence of R8 cells on adjacent differentiating neurons , we calculated sliding window correlations along the anterior-posterior axis ( X-axis ) between the Yan-YFP levels in R2 and R5 nuclei and the their physical distance to the nearest R8 nuclei .", "We first detrended R2/R5 Yan-YFP levels according to their positions in X to avoid spurious spatial correlations with developmental time .", "Specifically , we performed standard LOESS detrending in X on log-transformed R2/R5 Yan-YFP levels using local second-degree polynomials and tri-cubic weighted neighborhoods with a smoothing parameter of 0 . 5 .", "The resulting residual log-transformed R2/R5 Yan-YFP levels were distributed with approximately constant variance .", "We separately calculated the physical distance from the center of each R2 and R5 nucleus to the center of its nearest R8 nucleus .", "Detrending and distance calculations were performed independently for each measured eye disc ( 4 WT and 4 EGFRts replicates ) and then pooled for correlation calculations .", "For a series of windows along the X-axis that each span 12 . 5 hr of developmental time , we selected R2 or R5 cells within those windows and calculated the Pearson product-moment correlation coefficient from their residual log-transformed Yan-YFP levels and distances to the nearest R8 .", "The p-value associated with each window is calculated from the corresponding t-test assuming an uncorrelated bivariate normal distribution for sample points .", "The explained variance reported for each window is derived from the R2 value of a standard linear fit .", "Correlation analysis was performed using the R statistical software package ( v3 . 1 . 2 ) ." ] ]

[ "Yan is an ETS-domain transcription factor responsible for maintaining Drosophila eye cells in a multipotent state .", "Yan is at the core of a regulatory network that determines the time and place in which cells transit from multipotency to one of several differentiated lineages .", "Using a fluorescent reporter for Yan expression , we observed a biphasic distribution of Yan in multipotent cells , with a rapid inductive phase and slow decay phase .", "Transitions to various differentiated states occurred over the course of this dynamic process , suggesting that Yan expression level does not strongly determine cell potential .", "Consistent with this conclusion , perturbing Yan expression by varying gene dosage had no effect on cell fate transitions .", "However , we observed that as cells transited to differentiation , Yan expression became highly heterogeneous and this heterogeneity was transient .", "Signals received via the EGF Receptor were necessary for the transience in Yan noise since genetic loss caused sustained noise .", "Since these signals are essential for eye cells to differentiate , we suggest that dynamic heterogeneity of Yan is a necessary element of the transition process , and cell states are stabilized through noise reduction ." ]

[ "As animal cells develop , they pass through different states to mature into specific cell types .", "For some cells , this development depends on the cell’s ability to switch between two stable states , a property called bistability .", "Many bistable systems operate during development and often feature proteins called transcription factors that regulate a few cell states in specific tissues .", "These proteins activate or repress a range of target genes , and cells can adjust the levels of the transcription factors to bring about transitions between states .", "In fruit flies , two transcription factors , called Yan and Pnt , regulate numerous processes throughout development .", "In the developing embryo of a fruit fly , Yan and Pnt are regulated by signals induced by a receptor called EGFR .", "When EGFR is activated , Pnt is produced and Yan is degraded .", "When this receptor is not activated , Yan is produced and represses Pnt .", "Mathematical modelling of these interactions has concluded that this is a bistable system: that is , cells should either have high levels of Yan and low levels Pnt , or low Yan levels and high Pnt levels .", "However , larval eye cells first activate , and then turn off , both proteins together .", "This argues against bistability and raises questions about how these proteins regulate cell fate transitions in the eye , and perhaps in other organs .", "To investigate this question , Peláez et al . tagged Yan with a fluorescent marker to track its activity in the eyes of fruit fly larvae as they develop .", "A combination of fluorescence-based microscopy and an automated imaging analysis system were then used to score fluorescence in thousands of individual eye cells and assess the changes in the levels of Yan over time .", "This approach revealed some unexpected results .", "Yan levels were seen to vary in both immature and maturing cells .", "Thus eye cells transition between states as Yan levels increase rapidly , suggesting the need for a mechanism that is distinct from bistability .", "Peláez et al . suggest that larger changes in the seemingly random fluctuations in the levels of the transcription factor ( also known as “expression noise” ) might play a role in this mechanism .", "In particular , Yan expression noise briefly peaks as cells transition to a more mature state , and the transient nature of this ‘noisy’ response requires the activation of EGFR .", "One possible explanation for these observations is that Yan’s effect on cell states depends on this variability in its levels , which might prime cells to change states when they receive another signal .", "These findings also raise many questions for future studies to explore , including how this increase in the noise level of Yan is triggered as cells begin their transitions towards specific cell types ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "developmental biology" ]

[ [ "DNA repair is crucial to maintain genomic integrity in the face of exogenous and endogenous challenges .", "Cells express an arsenal of DNA repair enzymes that maintain genomic integrity , and mouse mutants have revealed a critical role of DNA repair in both organismic ageing and disease ( Jacobs and Schär , 2012; Lombard et al . , 2005 ) .", "In addition , there is now compelling evidence that DNA repair enzymes function not only in lesion control but have been co-opted in epigenetic gene regulation via active DNA demethylation ( Bellacosa and Drohat , 2015; Schuermann et al . , 2016; Wu and Zhang , 2017 ) .", "The best understood active demethylation mechanism involves TET dioxygenases , which iteratively oxidize the methyl group at C5 to yield 5-hydroxymethylcytosine ( 5hmC ) ( Kriaucionis and Heintz , 2009; Tahiliani et al . , 2009 ) , 5-formylcytosine ( 5fC ) ( Ito et al . , 2011; Pfaffeneder et al . , 2011 ) , and 5-carboxylcytosine ( 5caC ) ( He et al . , 2011; Ito et al . , 2011 ) .", "Thymine DNA glycosylase ( TDG ) excises 5fC and 5caC and the ensuing abasic site intermediate is processed by BER to restore unmethylated C ( Cortázar et al . , 2011; Cortellino et al . , 2011; He et al . , 2011; Maiti and Drohat , 2011; Shen et al . , 2013; Song et al . , 2013 ) .", "The need for abasic site processing during active DNA demethylation therefore places BER enzymes center stage in epigenetic gene regulation .", "Deficiency of the BER enzymes ( e . g . TDG , APEX1 , POLB , LIG3 , XRCC1 ) can lead to abnormalities or lethality during embryogenesis ( Cortázar et al . , 2011; Cortellino et al . , 2011; Puebla-Osorio et al . , 2006; Sugo et al . , 2000; Tebbs et al . , 1999; Xanthoudakis et al . , 1996 ) , but the etiology of the physiological defects is often poorly understood .", "Notably , it is unclear if the phenotypic abnormalities are due to accumulating DNA damage or impaired DNA demethylation .", "An example for BER enzymes acting both in lesion control and in epigenetic gene regulation are the endonuclease VIII-like glycosylases 1 and 2 ( NEIL1 and NEIL2 ) .", "These enzymes process oxidative DNA base lesions ( Bandaru et al . , 2002; Hazra et al . , 2002a; Hazra et al . , 2002b; Takao et al . , 2002 ) , but recently they have also been implicated in the machinery that removes 5-methylcytosine ( 5mC ) from DNA during epigenetic DNA demethylation ( Müller et al . , 2014; Schomacher et al . , 2016; Slyvka et al . , 2017; Spruijt et al . , 2013 ) .", "NEIL1 and NEIL2 are bifunctional enzymes , which not only excise the damaged base but introduce a DNA single strand break via their AP lyase activity ( Hazra et al . , 2002a; Hazra et al . , 2002b ) , while NEIL3 is mainly a monofunctional DNA glycosylase ( Krokeide et al . , 2013 ) .", "NEIL1 is involved in prereplicative repair during S-phase ( Hegde et al . , 2013 ) , and NEIL2 preferentially processes oxidized bases from transcribing genes via transcription-coupled BER ( Banerjee et al . , 2011 ) .", "During epigenetic DNA demethylation NEIL1 and NEIL2 cooperate with TDG to excise oxidized 5mC intermediates generated by TET enzymes ( Schomacher et al . , 2016; Slyvka et al . , 2017 ) .", "Mice deficient of NEIL1 are viable but display metabolic syndrome and brain dysfunction ( Canugovi et al . , 2012; Rolseth et al . , 2017; Vartanian et al . , 2006 ) .", "Neil2 null mice are also viable but are susceptible to inflammation ( Chakraborty et al . , 2015 ) , while Neil2-deficient frog embryos display neural crest defects ( Schomacher et al . , 2016 ) .", "This raises the question why and how does a defect in NEIL DNA glycosylases lead to these diverse and tissue-specific phenotypes ?", "Both epigenetic regulation and DNA damage can , in principle , impact neural crest development ( Hu et al . , 2014; Sakai and Trainor , 2016; Simões-Costa and Bronner , 2015 ) , thus what is the relative contribution of oxidative lesion control and epigenetic DNA demethylation to the NEIL phenotypes ?", "To address these questions we have investigated Neil-deficient Xenopus embryos and created and characterized seven mouse embryonic stem cell ( mESC ) lines deficient for Neil1 , 2 , 3 ( triple and single knockouts ) , AP-endonuclease 1 ( Apex1 ) , Thymine DNA glycosylase ( Tdg ) and Neil1/Tdg .", "We describe a mechanism where NEIL-deficiency elicits an oxidative stress-induced , TP53-dependent DNA damage response ( DDR ) , which induces apoptosis and impairs early cNCC specification .", "We show that Neil1- and Neil2-deficiency leads to accumulation of oxidative DNA damage in mitochondria .", "Our work demonstrates how impaired removal of oxidative lesions can lead to a selective lineage defect during embryonic development .", "Our study contributes to the understanding of aberrant cNCC development , the root cause of congenital craniofacial malformations ( Wilkie and Morriss-Kay , 2001 ) ." ], [ "We showed previously that in Xenopus embryos knockdown of Neil2 with an antisense morpholino oligonucleotide ( neil2 MO ) induces head and tail abnormalities at tailbud stage , which are caused by impaired cranial neural crest cell ( cNCC ) specification at neurula stage ( Schomacher et al . , 2016 ) .", "Of note , MOs are the loss-of-function approach of choice in model systems with large maternal stores of mRNA such as Xenopus , which can be targeted by MOs but not by for example TALEN or CRISPR/Cas9 approaches ( Blum et al . , 2015; El-Brolosy et al . , 2019; El-Brolosy and Stainier , 2017; Rossi et al . , 2015 ) .", "The specificity of the neil2 MO had been documented ( Schomacher et al . , 2016 )", "i ) by phenocopy with a second non-overlapping morpholino , and", "ii ) by rescue of the head and tail abnormalities with orthologous human NEIL2 mRNA , which was not targeted by neil2 MO ( Figure 1A ) .", "Proper differentiation of cNCCs is crucial for the development of craniofacial cartilage and bone structures ( Sakai and Trainor , 2016 ) .", "Indeed , Alcian blue staining of head cartilage in neil2 MO-injected embryos ( single blastomere injection at 2-cell stage ) revealed head cartilage , notably branchial cartilage defects at tadpole stage ( Figure 1B ) , which were rescued by simultaneous injection of human NEIL2 mRNA ( Figure 1B ) .", "To gain insight into the underlying mechanism leading to the cNCC phenotype , we microinjected Xenopus embryos with neil2 MO and carried out RNA-seq gene expression profiling at early tailbud stage ( Supplementary file 1 ) .", "Differential expression analysis yielded a similar number of a few hundred up- and downregulated genes ( Figure 1C ) .", "Interestingly , pathway enrichment analysis revealed significant results only for the upregulated genes with the top hits ‘Tp53 pathway’ and ‘Tp53 pathway feedback loops’ suggestive of a DNA damage response ( DDR ) ( Figure 1D ) .", "Indeed , protein levels of both Tp53 and its upstream regulator phospho-Chk1 ( pChk1 ) were induced in neural plates of Neil2 morphants , indicative for a DDR ( Figure 1E ) .", "RT-qPCR confirmed upregulation of tp53 and its target genes , including ccng1 , eda2r , aen , and riok3 ( Figure 1F ) .", "Furthermore , co-injection of human NEIL2 mRNA rescued induced pChk1 in Neil2 morphants , ruling out an unspecific Tp53-response to MO injection ( Figure 1G ) .", "Notably , human NEIL2 mRNA also reduced basal pChk1 levels .", "The DDR in Neil2 morphants induced direct Tp53 targets characteristic for apoptosis ( e . g . eda2r , aen ) .", "Apoptosis is linked to cNCC developmental defects since ablation of Tp53 and concomitant block of apoptosis suppress cranial facial abnormalities in Tcof1 mouse mutants ( Jones et al . , 2008 ) .", "Indeed , while cell proliferation seemed unaffected in neil2 MO-injected embryos as judged by phospho-histone H3 levels ( Figure 1H ) , we observed elevated Caspase-3 cleavage in dissected neural plates but less in non-neural tissue ( Figure 1I ) , indicative of cNCC-specific apoptosis in Neil2 morphants .", "Consistently , human NEIL2 mRNA injection in Neil2 morphants rescued elevated Caspase-3 cleavage to endogenous levels , corroborating the specificity of the neil2 MO-induced apoptosis in neural plates .", "Human NEIL2 mRNA injection also decreased basal levels of cleaved Caspase-3 , both in neural plates and non-neural tissue ( Figure 1I ) , suggesting that elevated Neil2 expression protects against apoptosis in whole embryos .", "Whole mount TUNEL assay of unilaterally neil2 MO-injected embryos confirmed elevated apoptosis by Neil2-deficiency in stage 16 embryos ( Figure 1J ) .", "We conclude that Neil2 protects against Tp53-mediated cell apoptosis in Xenopus embryos , notably in neural plate tissue .", "To test if the phenotypic malformations in Neil2-morphants are related to elevated apoptosis , we blocked the apoptosis pathway by co-injection with bcl2l1 mRNA .", "Bcl2l1 is a Xenopus homologue of mammalian BCL2 , an anti-apoptotic factor acting downstream of TP53 ( Hemann and Lowe , 2006; Tsujimoto , 1998 ) .", "Importantly , bcl2l1 overexpression substantially reduced phenotypic abnormalities of Neil2 morphants ( Figure 2A ) , and rescued elevated cleaved Caspase-3 levels in a dose-dependent manner ( Figure 2B ) .", "As expected , bcl2l1 expression had no effect on endogenous nor induced Tp53 protein levels ( Figure 2B ) .", "BCL2 regulates the intrinsic apoptosis pathway that is associated with mitochondria and results in mitochondrial dysfunction .", "Upregulation of mitochondrial ( mt ) gene expression is a characteristic response to mitochondrial dysfunction ( Heddi et al . , 1999; Reinecke et al . , 2009 ) .", "Indeed , we observed increased expression of mt-Nd1 , 4 and 5 ( NADH dehydrogenase 1 , 4 and 5 ) and mt-co3 ( cytochrome c oxidase III ) in Neil2-morphant whole embryos , supporting ongoing intrinsic apoptosis and mitochondrial dysfunction ( Figure 2C ) .", "What leads to the induction of a Tp53 DDR in Neil2 morphants ?", "NEIL2 processes oxidative base lesions induced by reactive oxygen species ( ROS ) , such as 8-oxoguanine ( 8oxoG ) , 5-hydroxyuracil ( 5hU ) , thymine glycol , and the formamidopyrimidines FapyG and FapyA ( Hailer et al . , 2005; Jacobs and Schär , 2012 ) , suggesting that accumulation of ROS DNA damage may account for embryonic abnormalities in Neil2-deficient Xenopus embryos .", "We therefore analyzed if Xenopus embryos are competent to mount a DDR following oxidative damage and if a DDR results in developmental abnormalities .", "Embryo treatment with the ROS producer pyocyanin ( Zhao et al . , 2014 ) upregulated Tp53 and pChk1 protein ( Figure 3A ) , induced expression of tp53 and its target genes ( Figure 3B ) , and elevated Caspase-3 cleavage ( Figure 3C ) .", "Moreover , pyocyanin-treated embryos phenocopied Neil2 morphants , displaying similar head and tail abnormalities ( Figure 3D ) .", "Neurula stage embryos showed cNCC specification defects , where the cNCC markers sox10 , twist , and snail2 were downregulated , while the markers sox3 ( pan-neural ) , en2 ( midbrain ) , and rx1 ( eye ) were unaffected ( Figure 3E ) .", "Elevated ROS levels sensitized Neil2 morphants since pyocyanin treatment of embryos injected with a sub-critical dose of neil2 MO , which alone did not yield abnormalities , elicited exacerbated abnormalities ( Figure 3F ) .", "We conclude that Neil2-deficiency and ROS damage induce a Tp53 DDR in Xenopus embryos , leading to cNCC defects .", "Importantly , treatment of embryos with Vitamin C , a prominent antioxidant ( Arrigoni and De Tullio , 2002 ) , attenuated the severe malformations of Neil2 morphants ( Figure 3G ) , supporting ROS as the basis of cNCC defects in the absence of Neil2 .", "The Tp53 response to DNA damage is a widespread cellular phenomenon ( Ciccia and Elledge , 2010 ) .", "Hence , what restricts the DDR mostly to neuroectoderm during Xenopus development ?", "Among the most upregulated genes in Neil2 morphants was the direct Tp53 target gene cyclin-G1 ( ccng1 ) ( Supplementary file", "1 ) ( Okamoto and Beach , 1994 ) .", "Ccng1 interacts with Mdm2 , another Tp53 target gene , and is involved in a negative feedback loop regulating Tp53 protein levels induced by DNA damage ( Kimura and Nojima , 2002; Okamoto et al . , 2002 ) .", "Unilateral injection of neil2 MO in Xenopus embryos with the lineage tracer β-galactosidase confirmed upregulation of ccng1 on the injected side ( Figure 4A ) .", "Ccng1 induction was restricted to the neural plate even in embryos where the lineage tracer extended to mesoderm and endoderm ( Figure 4A ) .", "To test if the spatial restriction of ccng1 expression reflects tissue-specificity of the DDR , we provoked a systemic DDR using pyocyanin , which induced strong ccng1 expression ( Figure 4B ) .", "As observed in Neil2 morphants , ccng1 expression was spatially restricted to the neural plate .", "Spatial restriction of the DDR may be related to patterned expression of tp53 itself , as in situ hybridization showed preferential tp53 expression in the neural plate , notably in the anterior wherefrom cNCCs arise ( Figure 4C ) .", "High-level expression of tp53 in the embryonic CNS is observed in diverse vertebrates , including zebrafish , Xenopus , chick , and mouse ( Hoever et al . , 1997; Lee et al . , 2008; Rinon et al . , 2011 ) .", "The results suggest that the cNCC defects in Neil2 morphants reflect a neural plate-restricted Tp53-response to oxidative DNA damage .", "Consistently , tp53 mRNA injection downregulated the cNCC marker snail2 at mid neurula stage ( Figure 4D ) and induced head and tail abnormalities ( Figure 4E ) .", "Importantly , injection of a tp53 antisense MO ( Takebayashi-Suzuki , 2003 ) not only blocked induction of Tp53 target genes in Neil2 morphants but also rescued phenotypic abnormalities substantially ( Figure 4F–G ) .", "In sum , we propose that Neil2 protects against an oxidative DNA damage-induced Tp53 response and intrinsic apoptosis in the neural plate , thereby safeguarding cNCC development ( Figure 4H ) .", "We asked if other BER factors have roles similar to Neil2 in Xenopus embryogenesis .", "APEX1 ( Apurinic/Apyrimidinic Endodeoxyribonuclease", "1 ) functions downstream of DNA glycosylases , processing the abasic ( apurinic/apyrimidinic ( AP ) ) sites produced during BER ( Abbotts and Madhusudan , 2010 ) , and Apex1-deficiency in mice leads to early embryonic lethality ( Xanthoudakis et al . , 1996 ) .", "Injection of apex1 MO at similar dosage as neil2 MO induced severe abnormalities and early lethality confirming essentiality of Apex1 also for Xenopus embryonic development ( data not shown ) .", "Interestingly , injection of reduced amounts of apex1 MO phenocopied Neil2 morphants , with embryos displaying microcephaly , and reduced or absent dorsal and tail fins ( Figure 5A ) .", "Human APEX1 mRNA partially rescued the phenotype confirming specificity of the apex1 MO ( Figure 5A ) .", "While Tp53 and phospho-Chk1 levels were unaltered ( Figure 5B ) , Tp53 target genes were induced in Apex1 morphants ( Figure 5C ) .", "As in Neil2 morphants , phospho-histone H3 levels were unchanged and Caspase-3 cleavage was induced ( Figure 5D–E ) .", "Hence , the BER enzymes Neil2 and Apex1 exhibit similar functions in Xenopus neural crest development .", "We next investigated if the role of NEIL DNA glycosylases to protect against ROS damage and safeguard neural crest development is conserved in mammals , and used mouse embryonic stem cells ( mESCs ) as a model system .", "We generated Neil1 , 2 , 3 triple-knockout ( TKO ) mESCs by CRISPR/Cas9 genome editing ( Cong et al . , 2013 ) .", "We included NEIL3 in addition to NEIL1 and NEIL2 to account for any possible functional redundancy among the NEIL family .", "The biochemical and biological properties of NEIL3 , however , are quite distinct from those of NEIL1 and NEIL2 ( Krokeide et al . , 2013; Liu et al . , 2013 ) .", "We flanked and deleted the coding region of the catalytic domains with two gRNAs for each Neil gene ( Figure 6—figure supplement 1A ) , as validated by genotyping PCR ( Figure 6—figure supplement 1B ) .", "Gene inactivation was further confirmed by western blot analysis for NEIL1 and RT-qPCR for Neil2 and Neil3 ( Figure 6—figure supplement 1C-D ) .", "Expression of pluripotency markers was unaltered for Pou5f1 and Klf4 , whereas Nanog expression was slightly reduced in Neil1 , 2 , 3 TKO mESCs compared to control cells that originated from mock transfections lacking specific guide RNAs ( Figure 6—figure supplement 1E ) .", "We subjected control and Neil-TKO mESCs to teratoma assays ( Ralston and Rossant , 2010 ) using three independent clones of each , to average-out clonal variation .", "When transplanted subcutaneously into immunodeficient mice , teratomas grew from all six mESC lines .", "Histological analysis of control and Neil-TKO teratomas revealed derivatives of ectoderm , endoderm and mesoderm in all samples confirming pluripotency of Neil-deficient mESCs ( Figure 6A ) .", "However , transcriptome analysis by RNA-seq uncovered thousands of genes differentially expressed between control and Neil-TKO teratomas ( Figure 6B ) .", "Intriguingly , pathway enrichment analysis of >2 fold differentially expressed genes yielded one significant hit for the up- and downregulated genes each , ‘PluriNetWork’ and ‘neural crest differentiation’ , respectively ( Figure 6C ) .", "The term ‘PluriNetWork’ refers to the genes regulating pluripotency in mouse stem cells ( Som et al . , 2010 ) .", "We confirmed upregulation of pluripotency markers ( Pou5f1 , Nanog and Klf4 ) , suggesting incomplete silencing of the pluripotent state in Neil-TKO teratomas ( Figure 6D ) .", "Downregulated genes included neural crest effectors such as Pax3 ( LaBonne and Bronner-Fraser , 1998 ) , Tfap2b , Phox2b , Dbh , Crabp1 , Neurog1 and Wnt3a , besides a suite of downregulated Hox genes ( Hoxa2 , Hoxa3 , Hoxa4 , Hoxa5 , Hoxa9 , Hoxb1 , Hoxb2 , Hoxb3 , Hoxb4 , Hoxb5 , Hoxc4 , Hoxc5 , Hoxd3 ) , which are prominently expressed during neural crest/pharyngeal arch patterning , where they control head skeletal development ( Trainor and Krumlauf , 2001 ) ( Supplementary file 2 ) .", "In fact , marker gene analysis for endoderm ( Gata6 ) , mesoderm ( Eomes ) , neuroectoderm ( Pax6 , Nestin , Sox1 and Pax2 ) , and neural crest ( Pax3 , Hoxa2 , Tfap2b and Neurog1 ) indicated mild neural and severe neural crest differentiation defects ( Figure 6E and Figure 6—figure supplement 2 ) .", "Moreover , the TP53 target genes Ccng1 , Mdm2 , Sesn2 and Eda2r were significantly upregulated in Neil TKO teratomas ( Figure 6F ) indicative of a TP53 DDR .", "These results indicate that the requirement for NEIL function in cNCC development is evolutionarily conserved between amphibians and mammals .", "To analyze the individual requirement of NEILs for neural and cNCC specification , we generated single Neil1 , −2 , and −3 mutant mESCs ( Figure 7—figure supplement 1A–D ) .", "Neil-mutant mESCs were subjected to in vitro differentiation for eight days in embryoid bodies ( EB ) in absence or presence of retinoic acid ( RA ) , the latter favoring neural differentiation ( Bibel et al . , 2007 ) .", "Neil3 mutants were largely unaffected for all markers tested ( Figure 7A ) .", "In contrast , single Neil1 and Neil2 mutants showed significant reduction in neural crest marker ( Pax3 , Hoxa2 , Tfapb2 , Neurog1 ) and also pan-neuroectodermal marker ( Pax6 , Nestin , Sox1 , Pax2 ) expression , while endoderm- and mesoderm markers were unaffected ( Figure 7A and Figure 7—figure supplement 2 ) .", "Importantly , Pax3 and Pax6 induction during differentiation was partially restored in Neil1- and Neil2-deficient cells by stable transfection with catalytically active- but not inactive human NEIL1- and NEIL2-encoding constructs ( Figure 7—figure supplement 3A ) .", "Note that the degree of rescue was likely limited by the low expression of the transfected NEIL constructs ( Figure 7—figure supplement 3B ) .", "This rescue not only confirms specificity of the Neil knockout approach but also demonstrates that the neural and cNCC differentiation defects are not due to mutant mESCs having undergone irreversible changes/DNA damage .", "Instead , the rescue indicates an acute requirement for NEIL1 and NEIL2 during cNCC differentiation .", "To corroborate neural and cNCC developmental defects , we carried out RNA-seq analysis of undifferentiated- and embryoid-body differentiated Neil1 and Neil2 single-mutant mESCs .", "While we observed hundreds of differentially expressed ( DE ) genes in mutant Neil1 and Neil2 mESCs ( Figure 7B and Supplementary file 3–4 ) , they did not cluster when subjected to pathway enrichment analysis .", "Upon differentiation , the number of DE genes increased in Neil1- and Neil2-deficient cells substantially to several thousand , notably in EBs treated with RA , with up- and downregulated genes equally distributed ( Figure 7B and Supplementary file 3–4 ) .", "The majority of DE genes overlapped between Neil1 and −2 mutant EBs and EBs + RA ( Figure 7C ) , supporting functional commonality between NEIL1 and NEIL2 .", "Importantly , pathway enrichment analysis of the downregulated genes revealed ‘neural crest differentiation’ as the top hit in both genotypes and in both differentiation regimes ( Figure 7D and Figure 7—figure supplement 4A ) .", "Downregulated genes included the neural crest effectors Pax3 , Tfap2b , Phox2b , Crabp1 , Neurog1 and a series of Hox genes ( Supplementary file 3–4 ) similarly as for Neil-TKO teratomas .", "Downregulated genes from Neil-TKO teratomas significantly overlapped with downregulated genes from either Neil1- or Neil2-mutant EBs and EBs + RA ( Figure 7—figure supplement 4B and Figure 7E ) .", "We conclude that in vitro differentiation of Neil1- and Neil2 single-mutant mESCs recapitulates neural and cNCC differentiation defects observed in Neil-TKO teratomas .", "Besides , the upregulated genes of both Neil1- and Neil2-deficient EBs + RA were significantly enriched for the pathway term ‘TYROBP causal network’ ( Figure 7—figure supplement 4C ) , associated with late-onset Alzheimer’s disease ( Zhang et al . , 2013 ) .", "We asked if Apex1-deficiency in mESC differentiation phenocopies Neil-deficiency as observed in Xenopus embryos .", "Hence , we generated and validated an Apex1 mESC knockout-line ( Apex1 #46 , Figure 8—figure supplement 1A–D ) and subjected it to in vitro differentiation .", "Expression of germ layer marker genes was reduced for all tested tissues in the Apex1-mutant line ( Figure 8A ) , indicating a more severe differentiation defect than in Neil1 and Neil2 mutants .", "Yet , RNA-seq analysis revealed a substantial overlap of commonly deregulated genes between Neil1- , Neil2- and Apex1-deficient EBs treated with RA ( Figure 8B–C and Supplementary file 5 ) .", "Moreover , pathway enrichment analysis of the downregulated genes in Apex1 EBs + RA once again resulted in ‘neural crest development’ as the top hit ( Figure 8D ) .", "Thus , Apex1-deficient mESCs substantially phenocopy cNCC differentiation defects of Neil mutants similar to Xenopus embryos .", "We tested if active removal of the oxidative demethylation intermediates 5fC and 5caC is required for neural and cNCC differentiation in EBs .", "To this end , we generated a Tdg mESC knockout-line using CRISPR/Cas9 ( Tdg #25 , Figure 9—figure supplement 1A–C ) .", "Tdg knockout mice are embryonic lethal and Tdg-deficient mESCs fail to undergo terminal neuronal differentiation ( Cortázar et al . , 2011 ) .", "As expected , Tdg-deficient mESCs had 3–4-fold increased genomic 5fC and 5caC levels ( Shen et al . , 2013; Steinacher et al . , 2019 ) , but they showed no change in pluripotency marker expression ( Figure 9—figure supplement 1D–E ) .", "Moreover , Tdg-deficient cells subjected to EB differentiation induced marker gene expression of all germ layers with no significant difference from control cells ( Figure 9A ) .", "We conclude that oxidative Tdg-dependent DNA demethylation is not required for early cNCC differentiation in embryoid bodies .", "NEIL1 and NEIL2 are involved in handover and processing of abasic sites during oxidative DNA demethylation after 5fC/5caC excision by TDG ( Schomacher et al . , 2016 ) .", "Since abasic sites are genotoxic , Neil-deficiency may trigger a DDR because of accumulation of unprocessed TET/TDG-demethylation intermediates .", "If so , preventing 5fC/5caC excision in the first place should rescue the differentiation defects in Neil-deficient cells .", "To block 5fC/5caC excision , we generated a Tdg-knockout mESC line in a Neil1-deficient background ( Neil1 #7/Tdg #11 , Figure 9—figure supplement 2A–E ) .", "However , in the Neil1/Tdg double-knockout line there was no rescue of cNCC differentiation , while the Tdg-single mutant line expectedly showed normal neural and cNCC marker gene expression ( Figure 9B ) .", "We conclude that the differentiation defects induced by deficiency of Neils are independent of a role in oxidative DNA demethylation .", "To test if defective neural and cNCC differentiation is related to elevated oxidative DNA damage as in Xenopus , we differentiated mESCs in the presence of pyocyanin .", "Consistently , pyocyanin inhibited neural and cNCC gene expression upon RA-induced EB differentiation , without affecting endoderm or mesoderm differentiation ( Figure 10A ) .", "In addition , upon differentiation , TP53 target gene expression significantly increased in presence of pyocyanin compared to mock treatment ( Figure 10B ) .", "The results align with the observation that oxidative stress impairs cNCC differentiation ( Chen and Sulik , 1996; Sakai and Trainor , 2016; Yan et al . , 2010 ) .", "Hence , the Xenopus and mESC data converge on the conclusion that NEIL1 and NEIL2 are required to repair oxidative base lesions during early neural development .", "Since NEIL1 and NEIL2 localize to- and maintain genomic stability in the nucleus as well as in mitochondria ( Hu et al . , 2005; Mandal et al . , 2012; Prakash and Doublié , 2015; Vartanian et al . , 2006 ) , this raised the question , in which of these two compartments NEILs may be required during early embryogenesis .", "To quantify NEIL-processed lesions , we developed a novel protocol .", "We isolated gDNA and mtDNA ( >14 fold enriched for mtDNA , Figure 11—figure supplement 1A ) , and digested it with E . coli EndoIII , a bifunctional DNA glycosylase/AP lyase that excises a similar spectrum of oxidatively damaged DNA bases as NEIL1 and NEIL2 ( Dizdaroglu et al . , 2000 ) .", "EndoIII base excision at oxidative lesions generates abasic sites ( Hatahet et al . , 1994 ) , which are then quantified by LC-MS/MS mass spectrometry ( Rahimoff et al . , 2017 ) .", "Since steady state levels of endogenous abasic sites in DNA are more abundant than oxidative base damages ( Swenberg et al . , 2011 ) we pretreated the DNA with recombinant APEX1 prior to the EndoIII reaction in order to reduce the background from preexisting abasic sites ( Figure 11A and Figure 11—figure supplement 1B ) .", "Quantification of abasic sites on synthetic oligonucleotides mixed with known amounts of AP sites accurately matched the expected result ( Figure 11—figure supplement 1C ) , validating the method .", "We noted , though , that APEX1-treatment did not completely erase abasic sites on oligonucleotides under our reaction conditions .", "Similarly , EndoIII processed the oxidative damage 5hU on oligonucleotides to abasic sites , but also not completely ( Figure 11—figure supplement 1C ) .", "APEX1-treatment of purified gDNA from mESCs reduced endogenous abasic sites by two-fold , from ~229 . 000 sites per genome to ~120 . 000 sites per genome .", "Subsequent EndoIII incubation led to a statistically significant increase of ~29 . 000 abasic sites per genome ( Figure 11—figure supplement 1D ) .", "Thus , steady state levels of EndoIII-reactive DNA damage sites were ~8 fold lower than the level of abasic sites , in agreement with previous reports ( Swenberg et al . , 2011 ) .", "Levels of endogenous abasic sites ( without APEX1/EndoIII treatment ) were unaffected by Neil1- and Neil2-deficiency in gDNA and mtDNA , regardless of whether mESCs were differentiated or not ( Figure 11—figure supplement 1E ) .", "Likewise , there was no significant increase in EndoIII-processed sites in gDNA from Neil1- and Neil2-knockout cells ( Figure 11B , top ) .", "In contrast , in mtDNA from Neil1- and Neil2-deficient mESCs , EndoIII-created abasic sites were elevated only upon neural differentiation ( EB + RA ) , but not in EBs or undifferentiated mESCs ( Figure 11B , bottom ) .", "We confirmed elevated mtDNA damage in Neil1- and Neil2-deficient EBs + RA by an independent method ( Gureev et al . , 2017 ) .", "Using long-range PCR , levels of DNA damage are monitored based on the fact that DNA lesions inhibit DNA polymerase and slow down accumulation of the PCR product .", "Therefore , the rate of product amplification is inversely proportional to the number of damaged DNA molecules .", "Comparing Neil-deficient to control cells , this approach revealed increased mtDNA damage in EBs treated with RA , and to lesser extend in EBs without RA ( Figure 11C ) .", "Moreover , as in Xenopus embryos we detected significant upregulation of mitochondrial and nuclear genes encoding components of oxidative phosphorylation as sign of mitochondrial dysfunction in Neil1- and Neil2-knockout cells , specifically under EB + RA treatment ( Figure 11D ) ( Babenko et al . , 2018; Heddi et al . , 1999; Reinecke et al . , 2009 ) .", "Together , the results suggest that NEIL1 and NEIL2 are specifically required for processing of oxidative lesions occurring in mitochondrial DNA during neural differentiation .", "We asked if Neil-deficiency in mESCs elicits a DNA damage response as in Xenopus embryos .", "Indeed , we found a ~ 40% overlap between the 116 top TP53 target genes ( Fischer , 2017 ) and the upregulated genes in Neil1- and Neil2-mutant EBs + RA ( Figure 12A ) , but no significant overlap with Neil1- and Neil2-deficient EBs and mESCs ( Figure 12—figure supplement 1A–B ) .", "Furthermore , differentiation of control mESCs in presence of the TP53 stabilizer NSC 146109 ( Berkson et al . , 2005 ) resulted in specific neural and cNCC differentiation defects , thus mimicking Neil-deficiency ( Figure 12B ) .", "Moreover , we tested if TP53 inhibition could rescue impaired differentiation of Neil1- and Neil2-mutant mESCs .", "We differentiated Neil1 and Neil2 single mutant mESCs in the presence of Pifithrin-α , an inhibitor of TP53 ( Komarov et al . , 1999 ) .", "Strikingly , upon Pifithrin-α treatment the neuronal marker Pax6 and neural crest marker Pax3 in Neil1- and Neil2-mutant EBs + RA regained expression levels of control cells , while endoderm and mesoderm differentiation was marginally inhibited in all tested genotypes ( Figure 12C ) .", "In control EBs + RA Pifithrin-α treatment did not affect Pax6 and Pax3 expression .", "We conclude that an upregulated TP53 DDR impairs neural and cNCC differentiation in Neil1- and Neil2-mutant cells .", "Since we observed mtDNA damage accumulation and mitochondrial dysfunction in Neil1- and Neil2-deficient cells , we tested specifically for a mitochondrial TP53 DDR ( Vaseva and Moll , 2009 ) as in Xenopus .", "Expression of the anti-apoptotic factor Bcl2 was strongly downregulated in Neil-mutant EBs and EBs + RA , while expression of Bak1 , a pro-apoptotic factor of the Bcl2 family ( Graupner et al . , 2011; Tsujimoto , 1998 ) , was significantly induced in Neil-deficient EBs + RA ( Figure 12D ) , consistent with an intrinsic/mitochondrial TP53 response .", "Pifithrin-α treatment reversed repression of Bcl2 and induction of Bak1 in Neil-deficient EBs + RA , confirming a TP53-dependent regulation of both genes ( Figure 12—figure supplement 1C ) .", "The apoptosis effector CASPASE-3 is induced upon- and required for mESC neural differentiation ( Fujita et al . , 2008 ) .", "Concordantly , levels of CASPASE-3 ( cleaved and uncleaved ) were systematically decreased in Neil-deficient EBs + RA ( Figure 12—figure supplement 2A ) , thus different from Caspase-3-effected apoptosis in Xenopus ( Figure 1I ) .", "Levels of CASPASE-7 , the alternative effector caspase of the intrinsic apoptosis pathway ( Lakhani et al . , 2006 ) , were unchanged ( Figure 12—figure supplement 2B ) .", "Among the upregulated TP53-target genes in Neil-deficient EBs + RA were effectors of cell cycle arrest ( e . g . Cdkn1a ) .", "We therefore tested for cell cycle differences in Neil1- and Neil2-deficient cells by flow cytometry analysis .", "However , while cell cycle profiles of EBs and EBs + RA were clearly distinguishable from mESCs ( more G1-phase and fewer S- and G2/M-phase cells upon mESC differentiation; White and Dalton , 2005 ) , there were no significant differences in cell cycle profiles between the control and Neil1- or Neil2-deficient cells , arguing against a TP53-induced cell cycle arrest ( Figure 12—figure supplement 2C ) .", "In line , cell cycle effects are also absent after forced TP53 induction in neural crest cells in mice ( Bowen et al . , 2019 ) .", "Collectively , the results support a model in which NEIL1 and NEIL2 function as mitochondrial DNA repair glycosylases to counteract an increased oxidative stress during neurogenesis .", "Thereby , NEIL1 and NEIL2 protect against a mitochondrial-induced TP53-DDR and an intrinsic apoptosis pathway , and safeguard neural differentiation ( Figure 12E ) ." ], [ "One-third of all congenital birth defects are craniofacial malformations that arise by perturbations in cNCC development ( Sakai and Trainor , 2016 ) .", "Hence , understanding the environmental and genetic causes leading to perturbations of cNCC development is important for the development of potential therapeutic avenues for their prevention .", "The main finding of our study is the elucidation of a mechanism whereby disruption of the ubiquitous DNA glycosylases NEIL1 and NEIL2 leads to neural and cNCC differentiation defects during embryonic development .", "Our study indicates that cNCC defects caused by NEIL1- and NEIL2-deficiency are attributable primarily to their role in protecting against oxidative DNA lesions , in particular of the mitochondrial genome , rather than in promoting epigenetic DNA demethylation .", "Our study , therefore , links mitochondrial BER to neural cell differentiation .", "The physiological role of NEIL1 and NEIL2 DNA glycosylases has previously been analyzed in mouse mutants .", "Neil1 mutants present metabolic syndrome and show impaired brain function and neuronal stress resistance in adults ( Canugovi et al . , 2012; Canugovi et al . , 2015; Vartanian et al . , 2006 ) .", "Neil2-null mice are susceptible to innate inflammation ( Chakraborty et al . , 2015 ) .", "These abnormalities were accompanied by BER defects in both types of mutants .", "Neil1 , 2 , 3 triple-mutant mice were recently reported to be viable , but mice were only analyzed- and reported negative for cancer predisposition ( Rolseth et al . , 2017 ) .", "In contrast , we found that cNCC development showed a surprisingly specific vulnerability towards NEIL-deficiency both in Xenopus embryos ( Neil2 ) as well as in differentiating mESCs ( NEIL1 and NEIL2 ) .", "Deficiency of NEIL3 , which processes a different spectrum of lesions compared to NEIL1 and NEIL2 and is not found in mitochondria ( Prakash and Doublié , 2015 ) , had no effect on neural and cNCC development .", "The cNCC abnormalities in Xenopus were mirrored in the transcriptome of differentiating Neil1- and Neil2-deficient mESCs .", "In Xenopus embryos and mESCs , we elucidated the mechanism as a TP53-mediated DNA damage response , which induced apoptosis in Xenopus ( Caspase-3-dependent ) and mouse EBs ( downregulation of Bcl2 and induction of Bak1 ) without major effects on the cell cycle in both model systems .", "Moreover , in Xenopus and mESCs we found that elevated ROS reduced cNCC specification , supporting a conserved mechanism whereby an oxidative DNA damage response impairs cNCC differentiation .", "Similarly , Apex1 mutant mESCs and Apex1 Xenopus morphants displayed cNCC differentiation defects , although gene misregulation ( mESCs ) and malformations ( Xenopus ) were more severe than in Neil-mutant mESCs and Neil2 morphants , consistent with early embryonic lethality of Apex1 mutant mice ( Ludwig et al . , 1998; Xanthoudakis et al . , 1996 ) .", "In contrast , neither did Tdg-deficiency affect cNCC differentiation in mESCs , nor did combined Tdg/Neil1-deficiency rescue cNCC differentiation defects , arguing against oxidative DNA demethylation as the primary cause of the differentiation defect .", "The question arises , why are neural and neural crest cells particularly sensitive to oxidative DNA damage ?", "Similarly , why is the systemic oxidative stress response to pyocyanin in Xenopus embryos limited to neuroectoderm ( Figure 4B ) ?", "Neurogenesis is accompanied by a metabolic switch from glycolysis to oxidative phosphorylation and , hence , escalated oxidative stress ( Khacho and Slack , 2018 ) .", "High intrinsic ROS levels may render neural crest cells particularly vulnerable to oxidative DNA damage and thus dependent on efficient damage repair .", "Consistently , neuroectoderm expresses high levels of DNA repair factors ( Albino et al . , 2011 ) and of Tp53 itself ( Cheng et al . , 1997; Hoever et al . , 1994; Rinon et al . , 2011 ) ; this study ) , suggesting a specific adaptation to a lesion-prone environment .", "Intriguingly , we identified mitochondrial DNA as the primary target for oxidative DNA damage in the absence of NEIL1 and NEIL2 and specifically upon mESC neural differentiation .", "This suggests a model whereby NEIL1 and NEIL2 function to repair and protect the mitochondrial genome against oxidative damages that accompany the metabolic switch upon neural differentiation .", "Thereby , NEIL1 and NEIL2 shelter neural specification from an intrinsic , mitochondrial-induced apoptosis pathway ( Figure 12E ) .", "NEIL1 and NEIL2 both localize in mitochondria besides the nucleus ( Hu et al . , 2005; Mandal et al . , 2012 ) .", "Moreover , Neil1-mutant mice harbor increased mtDNA damage in liver tissue , which might be related to the metabolic syndrome observed in these mice ( Vartanian et al . , 2006 ) .", "Our study therefore corroborates the physiological relevance of NEIL1 and NEIL2 in mtDNA repair and ties mitochondrial BER to cell differentiation .", "Similarly , the observed neural and cNCC differentiation defects of Apex1-mutant cells ( Figure 8 ) and Xenopus Apex1 morphants ( Figure 5 ) might partly be due to inefficient abasic site repair of mtDNA , as a truncated APEX1 is present in mitochondria ( Chattopadhyay , 2006 ) .", "Our findings align with studies , which documented a role of TP53 in vertebrate neural crest formation .", "In chick embryos , TP53 stabilization decreases cNCC differentiation , while dominant-negative TP53 increases the number of cNCC progenitors ( Rinon et al . , 2011 ) .", "TP53 activation in mouse embryos specifically causes severe neural crest defects ( Bowen et al . , 2019; Van Nostrand et al . , 2014 ) .", "Moreover , Treacher Collins syndrome ( TCS ) , a congenital disorder characterized by severe cNCC and craniofacial anomalies , is caused by impaired ribosomal biogenesis due to deficiency in the Pol I transcription machinery , nucleolar dysfunction and/or rDNA damage , resulting in TP53 activation ( Calo et al . , 2018 ) .", "Consequently , partial TP53 deficiency or pharmacologic TP53 inhibition ameliorates the craniofacial defects in TCS-mutants ( Jones et al . , 2008; Sakai et al . , 2016 ) , emphasizing the role of TP53 in cNCC development .", "Our results support the observation that neural tissue is particularly vulnerable to Neil1-deficiency in adults and is linked to neurodegenerative disease ( Canugovi et al . , 2012; Canugovi et al . , 2015; Vartanian et al . , 2006 ) .", "Why then do Neil1- and Neil2-mutant mice develop without obvious neural malformations ?", "First , genetic compensation of Neil-mutants may occur via Nthl1 , which has an overlapping substrate spectrum and also localizes both in the nucleus and mitochondria ( Ikeda et al . , 2002; Jacobs and Schär , 2012 ) .", "Single-knockout mice deficient for Neil1 and Nthl1 are phenotypically inconspicuous , the combined knockout , however , is highly cancer prone , indicative of mutual compensation of both factors for oxidative DNA damage repair ( Chan et al . , 2009 ) .", "mESC differentiation in vitro may not reproduce the complexity of in utero development where transcriptomic fine-tuning can buffer genetic ablations in the developing embryo .", "Second , differentiating mESCs in vitro experience gas phase oxygen partial pressure ( pO2 ) of 142 mmHg , whereas embryonic cells in vivo are exposed to pO2 values of 0–30 mmHg ( Powers et al . , 2008 ) .", "Similarly , one millimeter-sized Xenopus embryos developing close to the air-aquatic interface likely experience pO2 levels closer to ambient partial pressure .", "Hence , cNCCs in differentiating mESCs as well as frog embryos have to cope with higher pO2 and hence ROS levels than is the case for mouse embryos in utero .", "Increased basal ROS levels might sensitize cultured cells and frog embryos when challenged by Neil-deficiency during neural and cNCC differentiation .", "Third , apparently none of the Neil-mutant studies has specifically investigated whether Neil-deficient mice exhibit cNCC development-related cranial malformations , which may require special bone- and cartilage staining procedures for detection .", "Hence , our results call for a ( re- ) analysis of Neil-mutant mice for cranial abnormalities possibly under high fat diet , which favors basal ROS production ( Vial et al . , 2011 ) .", "Our findings also support the proposition that antioxidant supplementation may be beneficial for the prevention of craniofacial defects ( Sakai et al . , 2016 ) , since environmental factors such as alcohol and nicotine promote ROS formation ( Wright et al . , 1999; Zhao and Reece , 2005 ) .", "We also note that Neil1 is one of 11 genes affected in a chromosomal micro-deletion of a patient presenting craniofacial defects ( Li and Bodamer , 2014 ) .", "Finally , while our study demonstrates the importance of NEIL1 and NEIL2 to protect cNCCs against oxidative DNA damage , it does not exclude that NEIL1 and NEIL2 play a physiological role in TET/TDG-mediated gene regulation in other embryonic processes or adult tissues .", "Elucidating such direct gene-regulatory roles may require genome-wide monitoring of 5mC and its oxidation products in Neil-mutants .", "The here-established Neil- , Apex1- and Tdg-mutant mESCs will be useful for this and other investigations into the biology and mechanisms of BER enzymes ." ], [ "Human NEIL1 , NEIL2 and APEX1 cDNAs ( BC010876 . 1 , BC013964 . 2 and BC008145 . 1 , respectively ) were from the ORFeome clone collection .", "For in vitro transcription NEIL2 and APEX1 cDNA was inserted into pCS2FLAG ( Addgene plasmid 16331 ) .", "Additionally , NEIL1 and NEIL2 cDNAs were inserted into pcDNA3 . 1 ( + ) ( Invitrogen ) as C-terminal ( catalytically active ) and N-terminal ( catalytically inactive ) 2xFLAG-tag expression constructs .", "pCMV-Sport6-xt . bcl2l1 encoding wild type Xenopus bcl2l1 was purchased from Dharmacon ( MXT1765-202788918 ) .", "pCS105-xp53 encoding wildtype Xenopus tp53 was a kind gift from S . Piccolo ( University of Padua , Italy ) .", "Western blot analysis of X . laevis samples was essentially as described ( Kirsch et al . , 2017 ) .", "Mouse embryoid bodies were incubated in lysate buffer ( 20 mM Tris pH 7 . 5 , 150 mM NaCl , 5 mM EDTA , 2% NP-40 and Complete Mini Protease Inhibitor Cocktail ( Roche ) ) .", "Lysates were cleared by centrifugation and protein concentrations were estimated by bicinchoninic acid ( BCA ) assay using BSA as standard followed by SDS-PAGE and western blotting .", "Antibodies are depicted in key resources table .", "Total RNA was prepared by RNeasy Mini Kit ( Qiagen ) including an on-column DNase digestion according to the manufacturer’s instructions .", "Complementary DNA ( cDNA ) was synthesized using SuperScript II reverse transcriptase ( Life Technologies ) .", "Quantitative real time PCR was performed on a LightCycler 480 ( Roche ) in technical duplicates using the Universal ProbeLibrary technology ( Roche ) including the supplier’s LightCycler 480 Probes Master .", "Quantitative analysis was performed with LightCycler 480 software ( Roche ) .", "Primer sequences and hydrolysis probe numbers are listed in Supplementary file 6 .", "Animal experiments with X . laevis were approved by state authorities ( Landesuntersuchungsamt Rheinland-Pfalz , reference number 23177–07/A12-5-001 ) .", "No blinding or randomization was performed .", "Embryos were obtained by in vitro fertilization as described ( Gawantka et al . , 1995 ) and cultivated in 0 . 1x Barth’s solution ( Wang et al . , 2010 ) .", "Human and Xenopus expression constructs as depicted above were used as templates to generate mRNAs with the MEGAscript SP6 Transcription Kit ( Invitrogen ) according to the manufacturer’s instructions .", "Morpholino antisense oligonucleotides ( MOs , see Supplementary file 6 ) were designed to block translation of the respective gene .", "MOs and mRNAs were injected two times into animal blastomeres at one-cell stage with a total volume of 10 nl per embryo .", "For overexpression , each embryo was injected with: human NEIL2 mRNA , 2 ng; Xenopus tp53 mRNA , 200 pg; Xenopus bcl2l1 mRNA , 2 ng .", "Total amounts of single MOs injected per embryo were as follows: neil2 MO , 40 ng and tp53 MO , 20 ng .", "For double MO injections , each embryo was injected with a mixture of 40 ng neil2 MO and 20 ng p53 MO .", "For cartilage staining , one blastomere of two-cell stage embryos was injected with 5 nl ( total volume ) of neil2 MO ( 7 . 5 ng ) and human NEIL2 mRNA ( 375 pg ) .", "Control mRNA used for injections was preprolactin .", "For ROS induction embryos were grown in 0 . 1x Barth’s solution supplemented with pyocyanin ( Sigma , P0046; final concentration 10–25 µM ) .", "For Vitamin C treatment embryos were grown in 0 . 1x Barth’s solution supplemented with 100 µM L-Ascorbic acid 2-phosphate ( Sigma , A8960 ) .", "Embryos were fixed at the indicated developmental stage in freshly prepared MEMFA ( 100 mM MOPS pH 7 . 4 , 2 mM EGTA , 1 mM MgSO4 , 4% formaldehyde ) for 1 hr at RT .", "After fixation , embryos were washed twice in 100% ethanol at RT for 5 min and stored in 100% ethanol at −20°C .", "Whole mount in situ hybridization was performed as described ( Bradley et al . , 1996 ) .", "In situ hybridization probes were generated from cDNAs for X . laevis neuronal marker genes , ccng and tp53 using the Dig RNA labeling Kit ( Roche ) .", "For lineage tracing , lacZ mRNA was co-injected ( 250 pg/blastomere ) and β-gal staining was performed as described ( Sive et al . , 2000 ) using X-gal as substrate .", "Neural plates were dissected at stage 14 with Dumont No .", "five forceps .", "Cartilage staining was performed as described ( Nie and Bronner , 2015 ) .", "TUNEL assays were carried out as previously described ( Hensey and Gautier , 1998 ) .", "Images were taken on a Zeiss SteREO Discovery . V20 microscope .", "Mouse E14TG2a embryonic stem cells ( mESCs ) were obtained from ATCC , number CRL-1821 .", "Identity has been authenticated by ATCC .", "E14TG2a were initially tested Mycoplasma-positive , decontaminated using MycoZap Elimination Reagent ( Lonza ) and subsequently used for the study .", "E14TG2a cells were cultured on plates coated with 0 . 1% Gelatin ( Millipore ) in DMEM supplemented with 15% PANSera ES FBS ( PAN Biotech ) , 2 mM L-Glutamine , 100 µM non-essential amino acids ( NEAA , Gibco ) , 1 mM sodium pyruvate ( Gibco ) , 100 µM 2-mercaptoethanol ( Sigma ) , 1000 U/ml Leukemia inhibitory factor ( LIF , Millipore ) , 100 U/ml PEN-STREP at 37°C in 5% CO2 and 20% O2 .", "To generate mESCs deficient for Neil1 , 2 , 3 , Neil1 , Neil2 , Neil3 , Apex1 , Tdg , and Neil1/Tdg , 1 × 106 mESCs were seeded and transfected the next day with either empty or gRNA encoding pX330-U6-Chimeric_BB-CBh-hSpCas9 ( Addgene #42230 ) mixed with the selection plasmid pPGKPuro ( Addgene #11349 ) using Lipofectamine 2000 ( Invitrogen ) according to manufacturer’s instruction .", "Cells were selected with 2 µg/ml puromycin for 6 days , colonies picked , passaged and subjected to genotyping PCR using primers flanking the anticipated deletion region ( see Supplementary file 6 ) .", "Positive clones were expanded for further analyses .", "3 . 5 × 106 mESCs were plated on non-adherent 10 cm bacterial dishes ( Greiner ) in 15 ml CA medium ( Bibel et al . , 2007 ) .", "CA medium was changed every second day .", "For retinoic acid induced neural EB differentiation , CA medium was supplemented with all-trans-Retinoic acid ( R2625 , Sigma; final concentration 5 µM ) at days 4 and 6 of differentiation .", "EBs were harvested after 8 days of differentiation .", "For EB differentiation in presence of ROS inducer pyocyanin ( Sigma , P0046; final concentration 2 µM ) , TP53 stabilizer NSC 146109 ( Santa Cruz Biotechnology , sc-203652; final concentration 40 nM ) and TP53 inhibitor Pifithrin-α ( Sigma , P4359; final concentration 50 µM ) drug treatment of cells was started 24 hr prior to plating mESCs on non-adherent dishes and was continued throughout differentiation .", "Stable transfection mESCs were transfected with empty vector or human NEIL1 and NEIL2 expressing pcDNA3 . 1 constructs ( see ‘Expression constructs’ ) using Lipofectamine 2000 ( Invitrogen ) according to manufacturer’s instructions .", "Following selection for 6 days with 500 µg/ml G-418 ( Gibco ) single colonies were picked , expanded in selection medium and analyzed by RT qPCR for expression of the transgene .", "Transplantation of mESCs into immunodeficient ( NSG ) mice , animal husbandry and tumor preparation was performed by EPO Berlin GmbH .", "Resulting tumors were split and either shock frozen , or formalin fixed , paraffin embedded , sectioned and stained with hematoxylin and eosin for histological analysis .", "Tumors from each three independent control and Neil1 , 2 , 3−/− mESC lines were grown in technical triplicates .", "Genomic DNA for analysis of 5mC , 5hmC , 5fC and 5caC was prepared using the DNeasy Kit ( Qiagen ) according to manufacturer’s instructions .", "Genomic DNA for abasic site analysis was prepared with the Qiagen Blood and Cell Culture DNA Midi Kit essentially as described ( Rahimoff et al . , 2017 ) but using buffers G2 , QC , QF supplemented with each 400 µM of the antioxidants 2 , 6-Di-tert-butyl-4-methylphenol ( BHT , Sigma B1378 ) and deferoxamine mesylate ( DFOM , Sigma D9533 ) .", "DNA was stored at −20°C in H2O supplemented with 40 µM BHT and DFOM .", "Preparation of mtDNA was performed using the QIAprep Spin Miniprep Kit ( Qiagen ) following manufacturer’s instructions .", "Buffers P1 , P2 , N3 , PB and PE were supplemented with 400 µM BHT and DFOM .", "DNA was eluted with H2O containing 40 µM BHT and DFOM .", "Quantitative real time PCR as described above was used to calculate the enrichment of mtDNA over gDNA by the ΔΔCp method ( Quispe-Tintaya et al . , 2013 ) .", "PCR mixture contained 1 ng of DNA and gDNA- and mtDNA-specific primers ( mmActB and mmCytB , see Supplementary file 6 for sequences ) .", "Endogenous abasic sites on genomic and mtDNA ( 3 µg each ) were processed by incubation with 20 units APE 1 ( NEB , M0282 ) in a 50 µl reaction volume for 2 hr at 37°C .", "After phenol/chloroform extraction , DNA was further incubated with 20 units Endonuclease III ( Nth , NEB , M0268 ) in a 50 µl reaction volume for 2 hr at 37°C , phenol/chloroform extracted , ethanol precipitated and resuspended in H2O supplemented with 40 µM BHT and DFOM .", "Synthetic oligonucleotides were resuspended in H2O supplemented with 40 µM BHT and DFOM .", "The unmodified ( 40mer ) and 5hU-containing oligo ( 40mer_5hU ) were hybridized in 1x SSC ( 150 mM NaCl , 15 mM trisodium citrate , pH 7 . 0 ) to the complementary strand ( 40mer_complementary ) in a 1:1 molar ratio .", "For abasic site production 500 pmoles of the single-stranded uracil-containing oligo ( 40mer_U ) were incubated with 25 units UDG ( NEB , M0280 ) in a 50 µl reaction volume for 30 min at 37°C .", "After phenol/chloroform extraction and ethanol precipitation the abasic site oligo was hybridized to the complementary strand as described above .", "APE 1- and Endonuclease III-treatment was performed as depicted above with 3 µg of double-stranded oligonucleotides .", "DNA was purified by phenol/chloroform , ethanol precipitated and resuspended in H2O supplemented with 40 µM BHT and DFOM for abasic site derivatization as outlined below .", "Oligonucleotide sequences are listed in Supplementary file 6 .", "Quantification of 5mC and oxidative derivatives was carried out as described before ( Schomacher et al . , 2016 ) .", "Quantification of abasic sites by LC-MS/MS was performed according to the published protocol ( Rahimoff et al . , 2017 ) with specific changes: Derivatization was performed with 1–2 µg of DNA and 30 nmoles of reagent 1a for 60 min at 37°C .", "After an additional incubation with 30 nmoles of reagent 1a for 60 min at 37°C reaction was stopped , the DNA ethanol precipitated , dissolved in 15 µl H2O and digested as described ( Schomacher et al . , 2016 ) .", "After digest , DNA was mixed with an equal volume of isotopic standards , and 5 μL were injected for LC-MS/MS analysis .", "The chromatographic separation was performed on a ZORBAX SB-C18 column ( Agilent , 5 μm , 2 . 1 ×50 mm ) .", "Elution was performed with 5 mM Ammonium acetate pH 6 . 9 and Acetonitrile ( ACN ) , the flow rates were 0 . 4 ml/min for 0–7 min , 0 . 5 ml/min for 7–9 min and 0 . 4 ml/min for 9–10 min at 30°C with the following gradient: 0–2 min , 0% ACN; 2–5 min , 0–5% ACN; 5–9 min , 5–50% ACN; 9–10 min , 0% ACN .", "Transitions corresponding to dG , 9a_1 , 10a_1 and their respective isotopic standards 15N5-dG , 9b_1 , 10b_1 were monitored ( for compounds 9a_1 , 9_b1 , 10_a1 , 10_b1 see Rahimoff et al . , 2017 ) .", "The source‐dependent parameters were as follow: gas temperature 110°C , gas flow 19 l/min ( N2 ) , Nebulizer 25 psi , sheath gas heater 375°C , sheath gas flow 11 l/min ( N2 ) , capillary voltage 2000 V ( positive mode ) , nozzle voltage 0 V , fragmentor voltage 300 V . Compound dependent parameters were as previously described ( Rahimoff et al . , 2017 ) except that MS1 resolution for dG and 15N5-dG were enhanced and MS2 was unit .", "For the rest of ions all MS1 and MS2 resolution were set to unit .", "Abasic sites were initially quantified over total dG and subsequently calculated over total N using a GC content of 42% of the mouse genome .", "Preparation of mtDNA was performed as described above .", "Quantitative PCR was essentially as described ( Gureev et al . , 2017 ) using 500 pg of mtDNA on a LightCycler 480 ( Roche ) in technical duplicates .", "Short fragments were amplified each with 20 s , long fragments with 2 min elongation steps in 35 cycles .", "SYBR Green was from Sigma ( S9430 ) .", "Calculation of lesions per 10 kb was as described ( Gureev et al . , 2017 ) .", "For primer sequences and amplicon lengths see Supplementary file 6 .", "Cells were detached using 0 . 25% trypsin and washed with PBS containing 1% ESC grade FBS .", "Cells were fixed by adding dropwise ice-cold ethanol and subsequent incubation at −20°C for 30 min or storage at this point .", "For propidium iodide staining , cells were washed twice with PBS containing 0 . 1% ESC grade FBS and 100 µg/ml RNAse A ( Qiagen ) .", "Cells were then resuspended in PBS containing 50 µg/ml propidium iodide ( Sigma ) according to the cell number and incubated at room temperature for 10 min in the dark .", "Stained cells were then analysed by the BD LSRFortessaSORP flow cytometry system using FACSDiva software .", "Data analysis was performed with FlowJo software v . 10 . 5 . 3 ( BD ) .", "Data presented are displayed as arithmetic mean , error bars represent standard deviations ( s . d . ) of the indicated replicates .", "Statistical significance as shown in bar diagrams was determined by two-tailed unpaired Student’s t-test .", "Significance of overlapping groups of genes presented in Venn Diagrams was calculated by hypergeometric distribution ( http://nemates . org/MA/progs/overlap_stats . html ) using a total number of 17 000 expressed genes per calculation .", "In triple overlaps significance was calculated on the basis of commonly deregulated genes in Neil1 and Neil2-deficient cells .", "Significances are displayed as *p<0 . 05 , **p<0 . 01 , ***p<0 . 005 .", "NS , not significant ." ] ]

[ "Base excision repair ( BER ) functions not only in the maintenance of genomic integrity but also in active DNA demethylation and epigenetic gene regulation .", "This dual role raises the question if phenotypic abnormalities resulting from deficiency of BER factors are due to DNA damage or impaired DNA demethylation .", "Here we investigate the bifunctional DNA glycosylases/lyases NEIL1 and NEIL2 , which act in repair of oxidative lesions and in epigenetic demethylation .", "Neil-deficiency in Xenopus embryos and differentiating mouse embryonic stem cells ( mESCs ) leads to a surprisingly restricted defect in cranial neural crest cell ( cNCC ) development .", "Neil-deficiency elicits an oxidative stress-induced TP53-dependent DNA damage response , which impairs early cNCC specification .", "Epistasis experiments with Tdg-deficient mESCs show no involvement of epigenetic DNA demethylation .", "Instead , Neil-deficiency results in oxidative damage specific to mitochondrial DNA , which triggers a TP53-mediated intrinsic apoptosis .", "Thus , NEIL1 and NEIL2 DNA glycosylases protect mitochondrial DNA against oxidative damage during neural crest differentiation ." ]

[ "The face of animals with a backbone is formed in great part by a group of cells called cranial neural crest cells .", "When too few of these cells are made , the skull and the face can become deformed .", "For example , the jaw- or cheekbones can be underdeveloped or there may be defects in the eyes or ears .", "These types of abnormalities are among the most common birth defects known in humans .", "NEIL1 and NEIL2 are mouse proteins with two roles .", "On the one hand , they help protect DNA from damage by acting as so-called ‘base excision repair enzymes’ , meaning they remove damaged building blocks of DNA .", "On the other hand , they help remove a chemical group known as a methyl from DNA building blocks in a process called demethylation , which is involved both in development and disease .", "Previous research by Schomacher et al . in 2016 showed that , in frogs , the absence of a similar protein called Neil2 , leads to deformities of the face and skull .", "Han et al . – who include some of the researchers involved in the 2016 study – have now used frog embryos and mouse embryonic stem cells to examine the role of the NEIL proteins in cranial neural crest cells .", "Stem cells can become any type of cell in the body , but when NEIL1 and NEIL2 are missing , these cells lose the ability to become cranial neural crest cells .", "To determine whether the effects of removing NEIL1 and NEIL2 were due to their role in DNA damage repair or demethylation , Han et al . removed two proteins , each involved in one of the two processes .", "Removing APEX1 , which is involved in DNA damage repair , had similar effects to the removal of NEIL1 and NEIL2 , while removing TDG , which only works in demethylation , did not .", "This indicates that NEIL1 and NEIL2’s role in DNA damage repair is likely necessary for stem cells to become cranial neural crest cells .", "Although NEIL1 and NEIL2 are part of the DNA repair machinery , Han et al . showed that when stem cells turn into cranial neural crest cells , these proteins are not protecting the cell’s genomic DNA .", "Instead , they are active in the mitochondria , the compartments of the cell responsible for producing energy , which have their own DNA .", "Mitochondria use oxygen to produce energy , but by-products of these reactions damage mitochondrial DNA , explaining why mitochondria need NEIL1 and NEIL2 .", "These results suggest that antioxidants , which are molecules that protect the cells from the damaging oxygen derivatives , may help prevent deformities in the face and skull .", "This theory could be tested using mice that do not produce proteins involved in base excision repair , which could be derived from the cells lacking NEIL1 and NEIL2 ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "epidemiology and global health", "medicine" ]

[ [ "On March 23 , 2014 , the Ministry of Health Guinea notified the World Health Organization ( WHO ) of a rapidly evolving outbreak of Ebola virus disease ( EVD ) , now believed to have begun in December 2013 .", "The epidemic spread through West Africa and reached Europe and the United States .", "As of November 4 , 2015 , WHO reported more than 28 , 000 cumulative cases and 11 , 000 deaths in Guinea , Liberia , and Sierra Leone , where transmission had been most intense ( World Health Organization , 2016 ) .", "As the emergency progressed , researchers developed mathematical models of the epidemiological dynamics .", "Modelers have assessed ongoing epidemics previously , but the prominence of recent EVD work , enabled by existing research programs for infectious disease modeling ( National Institutes of Health , 2016a; National Institutes of Health , 2016b ) and online availability of EVD data via WHO ( World Health Organization , 2016 ) , Ministries of Health of affected countries , or modelers who transcribed and organized public WHO or Ministry of Health data ( Rivers C ) may be unprecedented .", "The efforts for this outbreak have been numerous and diverse , with major media incorporating modeling results in many pieces throughout the outbreak .", "U . S . Government decision making has benefited from modeling results at key moments during the response ( Robinson R ) .", "We draw on this vigorous response of the epidemiological modeling community to the EVD epidemic to review ( Moher et al . , 2009 ) the application of modeling to public health emergencies , and identify lessons to guide the modeling response to future emergencies ." ], [ "We identified 66 publications meeting inclusion criteria ( Figure 1 ) . 10 . 7554/eLife . 09186 . 003Figure 1 . Literature search flow . DOI: http://dx . doi . org/10 . 7554/eLife . 09186 . 003 Models addressed 6 key uncertainties about the EVD epidemic: transmissibility , typically represented by the reproduction number ( R , the average number of people each infected person infects; assessed in 41 publications ) ; effectiveness of various interventions that had been or might be implemented ( in 29 publications ) ; epidemic forecast ( in 29 publications ) ; regional or international spreading patterns or risk ( in 15 publications ) ; phylogenetics of EVD viruses ( in 9 publications ) ; and feasibility of conducting vaccine trials in West Africa ( in 2 publications ) ( Table 1 , Supplementary file 1 ) . 10 . 7554/eLife . 09186 . 004Table 1 . Overview of modeling publications on the 2013-present EVD epidemic . DOI: http://dx . doi . org/10 . 7554/eLife . 09186 . 004Ref . Date of latest EVD dataDate publishedEVD data was pre-existing and publicUncertainties addressedRInterventionsForecastSpreadPhylogeneticsClinical trialsBaize et al . , 2014 3/20/144/16/14No*Dudas and Rambaut , 2014 3/20/145/2/14Yes*Alizon et al . , 2014 6/18/1412/13/14Yes**Gire et al . , 2014 6/18/148/28/14No**Stadler et al . , 2014 6/18/1410/6/14Yes*Volz and Pond , 2014 6/18/1410/24/14Yes*Pandey et al . , 2014 8/7/1410/30/14Yes***Gomes et al . , 2014 8/9/149/2/14Yes***Valdez et al . , 2015 8/15/147/20/15Yes****Merler et al . , 2015 8/16/141/7/15Yes****Rainisch et al . , 2015 8/16/142/18/15Yes*Althaus , 2014 8/20/149/2/14Yes*Fisman et al . , 2014 8/22/149/8/14Yes**Nishiura and Chowell , 2014 8/26/149/11/14Yes**Poletto et al . , 2014 8/27/1410/23/14Yes**Meltzer et al . , 2014 8/28/149/26/14Yes***Agusto et al . , 2015 8/29/144/23/15Yes**Althaus , 2015 8/31/144/19/15Yes*Scarpino et al . , 2014 8/31/1412/15/14Yes*Weitz and Dushoff , 2015 8/31/143/4/15Yes**Drake et al . , 2015 9/2/1410/30/14Yes***Towers et al . , 2014 9/8/149/18/14Yes**Bellan et al . , 2014 9/14/1410/14/14Yes*Chowell et al . , 2015 9/14/141/19/15Yes*Cooper et al . , 2015 9/14/144/14/15Yes*Read et al . , 2015 9/14/1411/12/14Yes**WHO Ebola Response Team , 2014 9/14/149/23/14No***Faye et al . , 2015 9/16/141/23/15No**Bogoch et al . , 2015 9/21/1410/21/14Yes**Yamin et al . , 2014 9/22/1410/28/14No**Lewnard et al . , 2014 9/23/1410/24/14Yes***Webb et al . , 2015 9/23/141/30/15Yes***Shaman et al . , 2014 9/28/1410/27/14Yes**Chowell et al . , 2014 10/1/1411/20/14Yes**Fasina et al . , 2014 10/1/1410/9/14Yes*Khan et al . , 2015 10/1/142/24/15Yes*Rivers et al . , 2014 10/5/1410/16/14Yes***Xia et al . , 2015 10/7/149/8/15Yes**Majumder et al . , 2014 10/11/144/28/15Yes**Kiskowski , 2014 10/15/1411/13/14Yes**Fisman and Tuite , 2014 10/18/1411/21/14Yes***Althaus et al . , 2015 10/20/141/15/15Yes***Simon-Loriere et al . , 2015 10/25/146/24/15No*Rainisch et al . , 2015 10/31/146/16/15Yes*Fast et al . , 2015 11/1/145/15/15Yes*Kucharski et al . , 2015 11/1/142/18/15Yes***Tong et al . , 2015 11/11/145/13/15No**Hoenen et al . , 2015 11/21/143/26/15No*Cope et al . , 2014 12/3/1412/10/14Yes**White et al . , 2014 12/3/141/30/15Yes***WHO Ebola Response Team , 2015 12/14/1412/24/14No**Chowell et al . , 2014 12/17/141/21/15Yes*Siettos et al . , 2014 12/21/143/9/15Yes**Park et al . , 2015 12/26/146/18/15No*Nadhem and Nejib , 2015 12/30/146/14/15Yes*Camacho et al . , 2015 1/18/152/10/15Yes**Carroll et al . , 2015 1/31/156/17/15No**Bellan et al . , 2015 2/9/154/15/15Yes**Barbarossa et al . , 2015 2/13/157/21/15Yes***Kugelman et al . , 2015 2/14/156/12/15No*Cleaton et al . , 2015 2/28/159/3/15Yes*Wang and Zhong , 2015 3/18/153/24/15Yes*Toth et al . , 2015 3/31/157/14/15Yes**Dong et al . , 2015 4/3/159/5/15Yes**Browne et al . , 2015 4/12/155/14/15Yes**Zinszer et al . , 2015 5/13/159/1/15Yes* The number of publications with models to estimate R increased rapidly early in the epidemic , along with those including intervention , forecasting , and regional and international spread models; the growth rate of publications with phylogenetic modeling applications and clinical trial models increased later in the epidemic ( Figure 2 ) . 10 . 7554/eLife . 09186 . 005Figure 2 . Cumulative number of modeling applications by date of most recent EVD data used . The figure includes 125 modeling applications across the 66 publications . DOI: http://dx . doi . org/10 . 7554/eLife . 09186 . 005 Of the 125 models reported across the studies , 74% included mechanistic assumptions about disease transmission ( e . g . , compartmental , agent-based , or phylogenetic models ) , while 26% were purely phenomenological ( Supplementary file 2 ) .", "For 54 ( 82% ) of the 66 publications , the only EVD data used was pre-existing and publicly-available ( Table 1 ) .", "Typically , these were aggregate case data posted online by the WHO or affected countries , or Ebola virus genetic data released previously during the epidemic .", "Twelve studies used original EVD epidemiological data ( Baize et al . , 2014; WHO Ebola Response Team , 2014; 2015; Faye et al . , 2015; Yamin et al . , 2014 ) or genomic data ( Baize et al . , 2014; Gire et al . , 2014; Simon-Loriere et al . , 2015; Tong et al . , 2015; Hoenen et al . , 2015; Park et al . , 2015; Carroll et al . , 2015; Kugelman et al . , 2015 ) .", "Examples of additional data used for some modeling applications include official reports of social mobilization efforts ( Fast et al . , 2015 ) , media reports of case clusters ( Cleaton et al . , 2015 ) , media reports of events that may curtail or aggravate transmission ( Majumder et al . , 2014 ) , and international air travel data ( Gomes et al . , 2014; Poletto et al . , 2014; Read et al . , 2015; Bogoch et al . , 2015; Rainisch et al . , 2015; Cope et al . , 2014 ) .", "Several studies incorporated spatial data on EVD cases into models of regional EVD spread ( Gire et al . , 2014; Merler et al . , 2015; Rainisch et al . , 2015; Tong et al . , 2015; Carroll et al . , 2015; Zinszer et al . , 2015 ) .", "Of the 12 studies that collected original EVD data , 9 released those data before or at the time of publication ( 8 with Ebola virus genetic data deposited in GenBank ( Baize et al . , 2014; Gire et al . , 2014; Simon-Loriere et al . , 2015; Tong et al . , 2015; Hoenen et al . , 2015; Park et al . , 2015; Carroll et al . , 2015; Kugelman et al . , 2015 ) and 1 with detailed epidemiological data in the online publication ( Yamin et al . , 2014 ) .", "Many publications used results from the WHO Ebola Response Team investigations ( WHO Ebola Response Team , 2014; 2015 ) ( for example , estimates of the generation time , case fatality rate , or other epidemiological parameters as model inputs ) , but the detailed epidemiological data from these studies , to date , are not publicly available .", "Accumulation of shared EVD data over successive studies was evident especially in the phylogenetic analyses .", "For example , all phylogenetic studies published after release of the initial Ebola virus sequences by ( Baize et al . , 2014 ) ( Guinea ) and ( Gire et al . , 2014 ) ( Sierra Leone ) incorporated those sequence data .", "Across all studies , the publication lag ( defined as date of most recent EVD data used to date of online publication ) was almost 3 months ( median [interquartile range] = 85 [30–157] days ) .", "The lag varied across modeling applications , and was considerably shorter in studies that included models to estimate R ( median = 58 days for publications with R estimation versus 118 days for others ) or to forecast ( median = 50 versus 125 days ) ( Figure 3 ) . 10 . 7554/eLife . 09186 . 006Figure 3 . Publication lag by type of modeling application . The vertical red and turquoise lines indicate the median lag for publications including and not including , respectively , the type of modeling application . DOI: http://dx . doi . org/10 . 7554/eLife . 09186 . 006 Lags were longest for studies with phylogenetic and clinical trials applications ( median = 125 and 108 days , respectively ) , although there were fewer publications with these models .", "Forty-one publications characterized epidemic dynamics using epidemiological ( N=36 ) , genomic ( N=4 ) , or news report data ( N=1 ) .", "Twenty-four of these provided estimates of the basic reproduction number ( R0 ) for Guinea , Liberia , Sierra Leone , or West Africa , using epidemiological or genomic data ( Figure 4 , Supplementary file 3 ) . 10 . 7554/eLife . 09186 . 007Figure 4 . R0 estimates by type of model input data . Aggregate , case counts released by the WHO or Ministries of Health; Line-level , individual-level data from epidemiological investigations; Genomic , Ebola virus sequence data .", "The Figure excludes an outlier estimate of 8 . 33 for Sierra Leone ( Fisman et al . , 2014 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 09186 . 007 There were 16 country-specific estimates of R0 for Guinea , Liberia , or Sierra Leone that used EVD epidemiological data ( aggregate or line-level ) and provided 95% confidence or credible intervals ( CIs ) .", "Median CI width was about 85% smaller for models that used cumulative EVD counts ( N=11 models in 5 publications ) than for models that used disaggregated EVD case data , such as weekly counts ( N=5 models in 3 publications ) ( Figure 5 ) . 10 . 7554/eLife . 09186 . 008Figure 5 . R0 estimates and CIs by type of epidemiological input data . Disaggregated data typically were weekly counts .", "Top row: Vertical lines indicate 95% CIs .", "Bottom row: Horizontal bars indicate median CI width . DOI: http://dx . doi . org/10 . 7554/eLife . 09186 . 008 Although CIs were also narrower for models when deterministic rather than stochastic methods were used to estimate parameter uncertainty , all of the deterministic results came from a single study ( Figure 6 ) . 10 . 7554/eLife . 09186 . 009Figure 6 . R0 estimates and CIs by model fitting method . Top row: Vertical lines indicate 95% CIs .", "Bottom row: Horizontal bars indicate median CI width . DOI: http://dx . doi . org/10 . 7554/eLife . 09186 . 009 Fifteen publications provided numerical forecasts of cumulative EVD incidence for West African countries .", "Of 22 models that assumed no additional response measures beyond those implemented at the time ( i . e . , 'status quo' assumptions ) , 18 overestimated the future number of cases ( Figure 7 , Supplementary file 4 ) . 10 . 7554/eLife . 09186 . 010Figure 7 . Accuracy of cumulative incidence forecasts . Accuracy is shown as the ratio of predicted incidence to incidence subsequently reported by the WHO .", "'Dampening' refers to various approaches to restrict the growth of forecasted incidence over time .", "Top row: Accuracy by date of forecast .", "Bottom row: Accuracy by forecast lead time ( 'Horizon' ) .", "The Figure excludes one forecast with horizon > 1 year ( Fisman and Tuite , 2014 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 09186 . 010 In multivariate analysis , forecast error was lower for forecasts made later in the outbreak ( 14% reduction in mean absolute percentage error [MAPE] per week , P<0 . 001 ) , higher for forecasts with longer time horizons ( 29% increase in MAPE per week , P<0 . 01 ) , and lower for forecasts that used decay terms , spatially heterogeneous contact patterns , or other methods that served to constrain projected incidence growth ( 90% reduction in MAPE , P<0 . 01 ) .", "Country and number of parameters in the model were not statistically significant predictors of forecast accuracy ." ], [ "We identified 66 modeling publications during approximately 18 months of the EVD response that assessed trends in the intensity of transmission , effectiveness of control measures , future case counts , regional and international spreading risk , Ebola virus phylogenetic relationships and recent evolutionary dynamics , and feasibility of clinical trials in West Africa .", "We found a heavy dependence on public data for EVD modeling , and identified factors that might have influenced model performance .", "To our knowledge , this review is one of the most comprehensive assessments of mathematical modeling applied to a single real-world public health emergency .", "An important caveat of our review is that it only captures published results .", "We are aware of additional EVD epidemiological investigations and modeling not yet published .", "Some modelers providing direct support to operational response efforts have not published results , possibly because of operational demands .", "Also , we could not account comprehensively for the sources of variation across studies .", "For example , studies that estimated R0 using the same data sources at about the same time reported varied results .", "Such variation may , in part , reflect the problem of identifiability , with different R0 estimates possible for models that perform equally well depending on other parameter values ( Weitz and Dushoff , 2015 ) .", "Ideally , an investigation into this heterogeneity would include implementation of models in a common testing environment .", "Our review suggests several possible steps for improving the application of epidemiological modeling during public health emergencies .", "First , agreement on community best practices could improve the quality of modeling support to decision-makers .", "For example , our analysis is consistent with simulation studies showing underestimation of uncertainty in estimating R0 with cumulative ( as opposed to disaggregated ) incidence data , and supports the recommendation to use disaggregated data and stochastic models ( King et al . , 2015 ) .", "Additionally , incidence forecasts provided reasonable prospective estimates several weeks forward in time during the initial phase; however , given available data and methodologies these forecasts became progressively more inaccurate as they projected dynamics beyond several weeks .", "Validation of incidence forecasts against other relevant data , such as hospital admissions and contacts identified , also could provide evidence that the assumptions are sound .", "The 2014 onwards ebola outbreak in West Africa clearly highlights the need for a better understanding of how increasing awareness of severe infections within a community decreases their transmissibility even in the absence of specific interventions .", "Advancing methodological approaches to capture this effect , such as dampening approaches , might help account for behavioral changes , interventions , contact heterogeneity , or other factors that can be expected in a public health emergency which likely will improve forecasting accuracy .", "Establishing best practices within the community will allow decision-makers the ability to more quickly accept methodologies and results that have been generated via these best practices .", "Hence , decisions based on these results can happen more quickly .", "Second , modeling coordination could facilitate direct comparison of modeling results , identifying issues on which diverse approaches agree and areas of greater uncertainty .", "Epidemiological modelers might learn from comparison initiatives in modeling of influenza ( Centers for Disease Control and Prevention , 2013 ) , dengue ( US Department of Commerce ) , and HIV ( HIV modeling consortium ) ; and in other fields such as climate forecasting Intergovernmental Panel on Climate Change , 2010 ) .", "For epidemiological application , an ensemble approach should preserve methodological diversity to exploit the full range of state-of-the-art modeling methods , but include enough standardization to enable cross-model comparison .", "Establishing an initial architecture for a coordinated , ensemble effort now could assist the response to EVD , and future public health emergencies .", "Perhaps most importantly , outbreak modeling efforts would be much more fruitful if data and analytical results could be made available more quickly to all interested parties ( Yozwiak et al . , 2015 ) .", "The publication timelines for academic journals typically will not be consistent with decision-making needs during public health emergencies like the EVD epidemic , where the epidemiological situation was highly dynamic and the usefulness of data and forecasts time-constrained .", "Establishing mechanisms for modelers without special access to the official epidemiological teams to share interim results would expand the evidence base for response decision-making .", "Ideally , data should be made available online in machine-readable form to facilitate use in analyses .", "Modelers and other analysts expended enormous effort during the EVD epidemic transcribing data posted online in pdf documents .", "New norms for data-sharing during public health emergencies ( World Health Organization , 2015 ) would remove the most obvious hurdle for model comparison .", "The current situation where groups either negotiate bilaterally with individual countries or work exclusively with global health and development agencies is understandable , but highly ineffective .", "The EVD outbreak highlights again – after the 2003 Severe Acute Respiratory Syndrome epidemic and 2009 influenza A ( H1N1 ) pandemic – that an independent , well-resourced global data observatory could greatly facilitate the public health response in many ways , not least of which would be the enablement of rapid , high quality , and easily comparable disease-dynamic studies ." ], [ "For this review , we adapted the PRISMA methodology ( Moher et al . , 2009 ) to identify quantitative modeling studies of the 2013-present West Africa EVD epidemic .", "We searched PubMed on September 24 , 2015 , for publications in English since December 1 , 2013 , using the term ‘Ebola’ in any field .", "We reviewed all returned abstracts and selected ones for confirmatory , full-text review that mentioned use of quantitative models to characterize or predict epidemic dynamics or evaluate interventions .", "We included studies that met this criterion in full-text review .", "We excluded studies of clinical prediction models , viral or physiological function models , ecological niche models , animal reservoir models , and publications that did not use data from the 2013-present West Africa EVD epidemic .", "For included publications , we recorded the geographic settings , date of most recent EVD data used and date of publication , type of EVD data used , questions the models addressed , modeling approaches , and key results , including estimates of the basic reproduction number ( R0 ) and forecasts of future EVD incidence provided in the main text of the publications .", "To assess forecast accuracy , we compared predictions of models made under ‘status quo’ assumptions ( i . e . , without explicit inclusion of additional interventions or behavioral changes ) to EVD incidence data subsequently released by the WHO ( World Health Organization , 2016 ) , using the WHO figures dated soonest after the forecast target date ." ] ]

[ "As of November 2015 , the Ebola virus disease ( EVD ) epidemic that began in West Africa in late 2013 is waning .", "The human toll includes more than 28 , 000 EVD cases and 11 , 000 deaths in Guinea , Liberia , and Sierra Leone , the most heavily-affected countries .", "We reviewed 66 mathematical modeling studies of the EVD epidemic published in the peer-reviewed literature to assess the key uncertainties models addressed , data used for modeling , public sharing of data and results , and model performance .", "Based on the review , we suggest steps to improve the use of modeling in future public health emergencies ." ]

[ "The outbreak of Ebola that started in West Africa in late 2013 has caused at least 28 , 000 illnesses and 11 , 000 deaths .", "As the outbreak progressed , global and local public health authorities scrambled to contain the spread of the disease by isolating those who were ill , putting in place infection control processes in health care settings , and encouraging the public to take steps to prevent the spread of the illness in the community .", "It took a massive investment of resources and personnel from many countries to eventually bring the outbreak under control .", "To determine where to allocate people and resources during the outbreak , public health authorities often turned to mathematical models created by scientists to predict the course of the outbreak and identify interventions that could be effective .", "Many groups of scientists created models of the epidemic using publically available data or data they obtained from government officials or field studies .", "In some instances , the models yielded valuable insights .", "But with various groups using different methods and data , the models didn’t always agree on what would happen next or how best to contain the epidemic .", "Now , Chretien et al . provide an overview of Ebola mathematical modeling during the epidemic and suggest how future efforts may be improved .", "The overview included 66 published studies about Ebola outbreak models .", "Although most forecasts predicted many more cases than actually occurred , some modeling approaches produced more accurate predictions , and several models yielded valuable insights .", "For example , one study found that focusing efforts on isolating patients with the most severe cases of Ebola would help end the epidemic by substantially reducing the number of new infections .", "Another study used real-time airline data to predict which traveler screening strategies would be most efficient at preventing international spread of Ebola .", "Furthermore , studies that obtained genomic data showed how specific virus strains were transmitted across geographic areas .", "Chretien et al . argue that mathematical modeling efforts could be more useful in future pubic health emergencies if modelers cooperated more , and suggest the collaborative approach of weather forecasters as a good example to follow .", "Greater data sharing and the creation of standards for epidemic modeling would aid better collaboration ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "developmental biology", "cell biology" ]

[ [ "Mutations within the P53 pathway occur in all human cancers ( Hanahan and Weinberg , 2011 ) .", "While the mutation of TP53 itself is a common event , how this contributes to the initiation of cancer is incompletely understood .", "The most prevalent mutations are point mutations that result in proteins with altered function ( Olivier et al . , 2010 ) .", "Extensive analysis of these mutations using mouse models has revealed the pervasive cellular consequences of mutant P53 ( Bieging and Attardi , 2012; Bieging et al . , 2014; Goh et al . , 2011 ) .", "In osteosarcoma ( OS ) , the most common primary tumour of bone , unique genomic rearrangements and other mutation types most often result in null alleles of P53 ( Ribi et al . , 2015; Chen et al . , 2014 ) .", "The reason for this distinct TP53 mutational preference in osteoblastic cells , the lineage of origin of OS , is not understood , nor are the signaling cascades that are altered in p53-deficient osteoblastic cells that facilitate the initiation of OS .", "Understanding how the loss of P53 modifies osteoblast precursor cells to enable OS initiation will provide new avenues to improve clinical outcomes .", "OS occurs predominantly in children and teenagers and 5 year survival rates have plateaued at ~70% for patients with localised primary disease and ~20% for patients with metastatic or recurrent disease ( Janeway et al . , 2012; Mirabello et al . , 2009 ) .", "The advances in the understanding of OS biology and genetics have brought limited patient benefit to date or changes in clinical management .", "Sequencing of OS using both whole genome and exome approaches identified the universal mutation of TP53 accompanied by recurrent mutation of RB1 , ATRX and DLG2 in 29%-53% of cases ( Ribi et al . , 2015; Chen et al . , 2014; Perry et al . , 2014 ) .", "The OS predisposition of Li-Fraumeni patients and mouse models support the key role of p53 mutation in OS: Trp53+/- and Trp53-/- mice develop OS in addition to other tumors while conditional deletion of Trp53 in the osteoblastic lineage results in full penetrance OS , largely in the absence of other tumor types ( Mutsaers and Walkley , 2014; Donehower et al . , 1992; Quist et al . , 2015; Wang et al . , 2006; Lengner et al . , 2006; Zhao et al . , 2015 ) .", "The consequence of p53 loss in osteoblastic cells is only understood to a limited extent .", "A more complete understanding of the pathways impacted by loss of p53 will be important to understanding the rewiring of osteoblastic cells that underlies OS initiation .", "Genetic association studies ( GWAS ) in OS have identified changes in cyclic AMP ( cAMP ) related processes as predisposing to OS .", "A GWAS defined two OS susceptibility loci in human: the metabotropic glutamate receptor GRM4 and a region on chromosome 2p25 . 2 lacking annotated transcripts ( Savage et al . , 2013 ) .", "GRM4 has a role in cAMP generation .", "A GWAS in dogs with OS identified variants of GRIK4 and RANK ( TNFRSF11A ) , both involved in cAMP pathways ( Karlsson et al . , 2013 ) .", "Using a murine OS model induced by an osteocalcin promoter-driven SV40T/t , OS were identified that deleted a regulatory subunit of the cAMP-dependent protein kinase ( PKA ) complex , Prkar1a , or with amplification of Prkaca , the PKA catalytic component ( Molyneux et al . , 2010 ) .", "A recent transposon mediated mutagenesis OS model recovered both activating and inactivating mutations within cAMP related pathways , but no functional analysis was performed ( Moriarity et al . , 2015 ) .", "Evidence from multiple species implicates enhanced cAMP-PKA activity in OS .", "Osteoblastic cells are highly sensitive to cAMP levels , and major regulators of the osteoblast lineage such as PTHrP/PTH increase cAMP and activate cAMP-dependent signaling ( Juppner et al . , 1991 ) .", "The requirement for these pathways in OS initiation and maintenance has not been tested .", "Parathyroid hormone ( PTH ) and PTH-related protein ( PTHrP ) are key regulators of osteoblast and skeletal homeostasis ( McCauley and Martin , 2012; Martin , 2016 ) .", "PTHrP and PTH activate their common receptor , PTHR1 ( Suva et al . , 1987; Juppner et al . , 1988 ) .", "Binding to PTHR1 on osteoblasts or OS cells activates adenylyl cyclase , stimulates cAMP production , followed by PKA activation , leading to many of the transcriptional changes associated with PTH/PTHrP treatment ( Gardella and Jüppner , 2001; Pioszak and Xu , 2008; Swarthout et al . , 2002; Partridge et al . , 1981 ) .", "In normal osteoblasts PTHrP is produced by osteoblastic lineage cells and acts in a paracrine manner upon other osteoblastic cells at different stages of differentiation ( Martin , 2005; Miao , 2005 ) .", "The long-term administration of PTH ( 1–34 ) resulted in a high incidence of OS in rats ( Vahle et al . , 2002 ) .", "Elevated expression of PTHR1 is a feature of human and rodent OS ( Martin et al . , 1976; Yang et al . , 2007 ) .", "PTHrP was expressed in murine OS subtypes ( Ho et al . , 2015 ) , placing it as a plausible ligand for activating PTHR1 signaling .", "It is presently unknown how PTHrP might act in OS .", "The consequence of PTH/PTHrP signaling via cAMP-PKA is phosphorylation of the cAMP response element binding ( CREB1 ) protein ( Datta and Abou-Samra , 2009 ) .", "CREB1 regulates gene expression through the activation of cAMP-dependent or -independent signal transduction pathways ( Mayr and Montminy , 2001 ) .", "We sought to understand how loss of p53 leads to the initiation of OS .", "We identified that an early consequence of p53 deletion in osteoblastic cells was increased cAMP levels and the autocrine activation of cAMP signalling via PTHrP .", "This same signaling node is active in OS and was important in both the initiation and maintenance of OS .", "The activation of the PTHrP-cAMP-CREB1 axis was required for the hyperproliferative phenotype of Trp53 deficient osteoblasts and the maintenance of established OS , identifying this as a tractable pathway for therapeutic inhibition in OS ." ], [ "As inactivating mutations of TP53 are universal in conventional OS , we used this to model an OS initiating lesion ( Chen et al . , 2014 ) .", "Primary osteoblasts were isolated from R26-CreERT2ki/+Trp53+/+ ( WT ) and R26-CreERT2ki/+Trp53fl/fl ( KO ) animals and in vitro tamoxifen treatment was used to induce deletion of p53 .", "Over 20 days culture , a loss of expression of p53 target genes in the KO cultures + tamoxifen occurred , compared to both WT and non-tamoxifen treated isogenic R26-CreERT2ki/+Trp53fl/fl cultures ( Figure 1A ) .", "Given the strong association between osteoblastic differentiation , OS and cAMP signaling , we assessed if pathways were impacted by loss of p53 .", "CREB1 transcriptional target genes were identified from ChIP and ChIP-Chip studies of CREB genomic occupancy ( Kenzelmann Broz et al . , 2013; Ravnskjaer et al . , 2007 ) .", "Only those targets that associated with CREB1 in response to cAMP activation were considered .", "Analogously , p53 target genes were defined from a ChIP-seq dataset from human HCT116 cells ( Sánchez et al . , 2014 ) and further refined against a second independent dataset of p53 ChIP-seq from murine embryonic fibroblasts ( Kenzelmann Broz et al . , 2013 ) .", "Strikingly , the expression of CREB1 target genes was increased , inversely paralleling the reduction in p53 target genes ( Figure 1A , Figure 1—figure supplement 1A–B ) .", "Similar gene expression results were obtained using shRNA against Trp53 in primary WT osteoblasts , demonstrating that the observed changes did not result from proliferation differences ( Figure 1—figure supplement 1C–E ) .", "The altered transcript levels were reflected at the protein level , where loss of p53 was associated with an increase in total CREB1 and phosphorylated CREB1 ( pCREB1 ) in the KO cells ( Figure 1B ) .", "Interestingly , the KO cultures had increased cAMP levels compared to WT or isogenic controls ( Figure 1C ) .", "Collectively , these results demonstrate that derepression of cAMP/CREB1 pathways is an early event following Trp53 mutation in osteoblasts . 10 . 7554/eLife . 13446 . 003Figure 1 . Intact PTHrP and CREB1 are necessary for hyperproliferation of p53-deficient primary osteoblasts .", "( A ) Heat map of qPCR data .", "Expression of the PTHrP/CREB1 and p53 target genes between indicated cell types .", "Data from >3 independent cell lines for each , expressed as fold change relative to non-tamoxifen treated isogenic culture .", "( B ) Western blot of p53 , pCREB1 and CREB1 , β-ACTIN used as a loading control .", "Data representative of 2–3 independent cell lines from each .", "( C ) Quantification of cAMP levels ( +IBMX ) in the R26-CreERT2p53+/+ ( vehicle and tamoxifen treated ) and R26-CreERT2p53fl/fl vehicle and tamoxifen treated ( p53△/△ ) primary osteoblasts , and day 5 , 10 , 15 and 20 days post tamoxifen .", "Data from 2 R26-CreERT2p53+/+ and 4 R26-CreERT2p53fl/fl independent cultures; mean ± SEM .", "( D ) Experimental outline for proliferation assay; Western blot of p53 , pCREB1 and CREB1 in indicated cell types , β-ACTIN used as a loading control at Day 0 of culture .", "Proliferation assays performed in the indicated genotype post CREB1 ( E ) and PTHrP ( F ) knockdown with tamoxifen treatment commencing at day 0; shLuc = control shRNA; Data from 4 independent R26-CreERT2p53fl/fl and 2 R26-CreERT2p53+/+ cultures; mean ± SEM and statistics = area under the curve across the time course .", "( G ) AnnexinV/7-AAD profiles of R26-CreERT2p53fl/fl +/- tamoxifen treatment infected with control ( shLuc ) , shCreb1_A or shPthlh_A .", "( H ) Percent apoptotic cells in each culture +/- tamoxifen .", "( I ) Heat map of qPCR data .", "Expression of the p53 and PTHrP/CREB1 target genes between cell types; 3 independent cell cultures for each condition .", "Data expressed as mean ± SEM ( n=3 ) .", "For all panels: *p<0 . 05 , **p<0 . 01 , ***p<0 . 001 .", "See Figure 1—figure supplement 1 and Figure 1—figure supplement 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 13446 . 00310 . 7554/eLife . 13446 . 004Figure 1—figure supplement 1 . Expression of p53 and PTHrP/CREB1 target genes in R26-CreER p53+/+ and R26-CreER p53fl/fl cultures +/- tamoxifen .", "( A ) Expression of p53 ( Mdm2 , Cdkn1a1 , Atp9a1 , Atf3 , Bax ) and PTHrP/CREB1 ( Nr4a1 , Nr4a2 , Nr4a3 , Rgs2 , Areg ) target genes at the indicated time points +/- tamoxifen in WT ( R26-CreER p53+/+ ) primary osteoblast cultures ( n=2 independent cultures ) .", "( B ) Expression of p53 ( Mdm2 , Cdkn1a1 , Atp9a1 , Atf3 , Bax ) and PTHrP/CREB1 ( Nr4a1 , Nr4a2 , Nr4a3 , Rgs2 , Areg ) target genes at the indicated time points +/- tamoxifen in p53 deficient ( R26-CreER p53fl/fl ) primary osteoblast cultures ( n=4 independent cultures ) .", "QPCR data normalized to β2m , for all the experiments above Students t-test was used , *p<0 . 05 .", "( C ) Expression of p53 , CREB1 , ATF1 and ACTIN after infection with a control ( sh-Luc ) or p53 targeting shRNA in primary long bone osteoblasts .", "( D ) Expression of p53 ( Mdm2 , Cdkn1a1 , Atp9a1 , Atf3 , Bax ) and PTHrP/CREB1 ( Nr4a1 , Nr4a2 , Nr4a3 , Rgs2 , Areg ) target genes at the indicated time points 48 hr after selection of cells infected with control ( sh-Luc ) or p53 targeting shRNA ( n=3 independent cultures ) .", "qPCR data normalized to β2m .", "( E ) Raw data normalized to β2m , for data in panel D; Students t-test was used , *p<0 . 05 . .", "DOI: http://dx . doi . org/10 . 7554/eLife . 13446 . 00410 . 7554/eLife . 13446 . 005Figure 1—figure supplement 2 . Effects of shRNA against Pthrp and Creb1 in R26-CreER p53+/+ and R26-CreER p53fl/fl cultures +/- tamoxifen .", "( A ) Expression of Pthlh , Creb1 and p53 in WT ( R26-CreER p53+/+ ) primary osteoblast cultures ( n=2 independent cultures ) at Day 0 .", "( B ) Expression of Pthlh , Creb1 and p53 in p53 deficient ( R26-CreER p53fl/fl ) primary osteoblast cultures ( n=4 independent cultures ) at Day 0 of tamoxifen addition .", "( C ) Expression of CREB1 and p53 at Day 15 and 21 in WT ( R26-CreER p53+/+ ) primary osteoblast cultures ( n=2 independent cultures ) .", "( D ) Genomic PCR for p53 locus at Day 15 and 21 in WT ( R26-CreER p53+/+ ) primary osteoblast cultures ( n=2 independent cultures ) .", "( E ) Expression of CREB1 and pCREB1 at Day 15 and 21 in p53 deficient ( R26-CreER p53fl/fl ) primary osteoblast cultures ( n=4 independent cultures ) .", "( F ) Genomic PCR for p53 locus at Day 15 and 21 in p53 deficient ( R26-CreER p53fl/fl ) primary osteoblast cultures ( n=4 independent cultures ) .", "( G ) Expression of p53 target genes and ( H ) PTHrP/CREB1 target genes 72 hrs after infection of isogenic p53 deficient ( R26-CreER p53fl/fl ) +/- tamoxifen primary osteoblast cultures at day 21 post tamoxifen/vehicle addition ( n=3 independent cultures ) .", "Data expressed as fold change relative to shLuc vehicle control cultures .", "qPCR data normalized to β2m , for all the experiments above Students t-test was used , *p<0 . 05 , **p<0 . 001 , ***p<0 . 0001 . DOI: http://dx . doi . org/10 . 7554/eLife . 13446 . 005 The tamoxifen treated Trp53-KO osteoblasts hyperproliferate after ~15 days ( Ng et al . , 2015 ) , coinciding with the loss of p53 .", "Coinciding with the increased proliferation of the p53-deficient cultures was an increase in cAMP per cell and an activation of CREB1 target genes , potentially explained by the increased Pthlh expression ( also known as Pthrp , Figure 1A–C ) .", "As elevated PTHrP would increase cAMP levels , the involvement of both CREB1 and PTHrP in the p53-deficient response was assessed .", "Primary osteoblasts from the respective genotypes were infected with two independent shRNAs against either Creb1 or Pthlh then cultured ± tamoxifen .", "Efficient and stable knockdown of Creb1 and Pthlh mRNA respectively was confirmed in both WT and KO cultures before tamoxifen treatment ( Figure 1D , Figure 1—figure supplement 2A–B ) .", "The p53-WT cells were largely unaffected by the shRNA’s independent of tamoxifen treatment , except for an initial delayed proliferation in shCreb1 cultures ( Figure 1E–F ) .", "The control ( shLuc ) infected KO osteoblasts hyperproliferated following tamoxifen treatment from day 15 onward ( Figure 1E–F ) .", "In contrast , knockdown of either Creb1 or Pthlh completely prevented the hyperproliferation of the KO + tamoxifen cells ( Figure 1E–F , Figure 1—figure supplement 2C–F ) .", "Therefore , intact PTHrP and CREB1 signaling are required for the hyperproliferation of p53 deficient osteoblasts .", "Finally , to assess the requirements for PTHrP and CREB1 in p53-deficient osteoblasts , we infected cells with the respective shRNAs after they had been cultured for 21 days with tamoxifen , such that the cells had already undergone the hyperproliferative transformation prior to knockdown .", "The p53-KO osteoblasts underwent apoptosis within 48 hr of knockdown with either shCreb1 or shPthlh whilst the isogenic control ( -tam ) cultures were minimally affected ( Figure 1G–H ) .", "The knockdown of PTHrP/CREB1 led to an expected downregulation of PTHrP-CREB1 targets ( Figure 1I , Figure 1—figure supplement 2G–H ) .", "The hyperproliferative effect of loss of Trp53 in osteoblastic cells required PTHrP and CREB1 .", "Having established the necessity of PTHrP and CREB1 for the hyperproliferation and survival of p53-deficient osteoblasts , we sought to understand the contribution of this pathway in OS .", "We systematically profiled the contribution of PTHrP , cAMP and CREB1 in primary cell cultures derived from murine OS models compared to primary osteoblasts .", "We made use of the Sp7 ( Osx ) -Cre Trp53fl/flRb1fl/fl model ( Cre:lox deletion of Trp53 and Rb1; referred to as fibroblastic OS ) which yields a OS characterised by predominant areas of fibroblastic or poorly differentiated/undifferentiated ( Berman et al . , 2008 ) histology and a cell surface phenotype consistent with immature osteoblasts ( Walkley et al . , 2008; Mutsaers et al . , 2013 ) .", "The second model was the Sp7 ( Osx ) -Cre TRE_shp53 . 1224Rb1fl/fl model ( shRNA knockdown of Trp53; referred to as osteoblastic OS ) which histologically resemble osteoblastic OS with large mineralized areas , appreciated by von Kossa staining or microCT , and a cell surface phenotype of mature osteoblasts ( Mutsaers et al . , 2013 ) .", "The early passage cells from both models have comparable genetic and pharmacological sensitivities to those of primary human patient derived OS cultures where tested ( Gupte et al . , 2015; Baker et al . , 2015 ) .", "As a control population ( referred to herein as “primary osteoblasts” ) , we isolated osteoblastic cells from the collagenase digested long bones of wild-type C57BL/6 mice .", "These cells are negative for haematopoietic markers ( lineage markers , CD45 , CD11b , F4/80 ) , negative for the endothelial cell surface marker CD31 and co-express CD51 and Sca-1 .", "The majority of the cells have a cell surface phenotype consistent with pre-osteoblasts ( lin-CD45-CD31-CD51+Sca1+ ) when the cultures are initiated , and when induced to differentiate acquire a mature osteoblast/osteocyte gene expression profile .", "We first assessed PTHrP given that PTHrP stimulates cAMP generation following activation of PTHR1 , ultimately leading to CREB1 phosphorylation and transcriptional activation ( Figure 2A ) .", "Osteoblastic OS cells expressed high levels of Pthlh transcript ( Figure 2B ) , consistent with our previous data identifying substantial levels of intracellular PTHrP in OS cells ( Ho et al . , 2015 ) .", "As other GPCRs are expressed on OS cells , such as β-adrenergic receptors ( Figure 2—figure supplement 1A ) , we sought to determine if PTHrP was an OS autocrine ligand .", "Cells were treated with the phosphodiesterase inhibitor , IBMX , without adding exogenous PTHrP , thus assaying the cAMP induced by autocrine activation of receptor-linked adenylyl cyclase by ligand ( s ) provided by the OS cells .", "Treatment with a neutralising anti-PTHrP antibody significantly and substantially reduced cAMP levels ( Onuma et al . , 2004 ) ( Figure 2C , Figure 2—figure supplement 1B ) .", "Using the shRNAs against Pthlh , a >50% reduction in the cAMP accumulation was observed ( Figure 2D–E , Figure 2—figure supplement 1C ) .", "The reduction of cAMP levels by Pthlh knockdown or by antibody mediated PTHrP neutralization are consistent with OS–derived PTHrP as an endogenous ligand promoting cAMP accumulation . 10 . 7554/eLife . 13446 . 006Figure 2 . Cell autonomous stimulation of cAMP by PTHrP in OS .", "( A ) Cartoon of PTHrP-PTHR1-cAMP-CREB1 axis .", "( B ) qPCR expression of Pthlh normalized to β2m; mean ± SEM ( n=3 ) .", "( C ) cAMP levels after anti-PTHrP antibody treatment for fibroblastic OS ( light grey ) and osteoblastic OS ( dark grey ) .", "Expressed as normalized mean cAMP ± SEM ( ( n=3/subtype ) .", "( D ) Knockdown of Pthlh transcript using 2 independent shRNA ( A and B ) in indicated OS subtypes .", "Data normalized to β2m , expressed as mean ± SEM ( n=3/subtype ) .", "( E ) Fold reduction of cAMP levels in sh-Pthlh infected OS subtype cells .", "IBMX in all treatments , data displayed as normalized mean cAMP ± SEM ( n=3/subtype ) .", "The data is the mean of 3 independent cell cultures for each subtype .", "( F ) pCREB1/CREB1 protein levels following knockdown of PTHrP .", "Pan-ACTIN/ATF-1 used as a loading control .", "Data are representative of 2 independent cell cultures from each OS subtype .", "( G ) Expression of indicated CREB1 target gene transcripts following Pthlh knockdown .", "Means ± SEM ( n=3/subtype ) .", "( H ) AnnexinV/7-AAD staining of indicated cells following infection with two independent sh-Pthlh ( A and B ) or sh-Luc control .", "( I ) Quantitation of dead cells in indicated cell type .", "The data represents 3 independent cell cultures for each type , mean ± SEM ( n=3 ) .", "*p<0 . 05 , **p<0 . 001 , ***p<0 . 0001 .", "( J ) In vivo bilateral grafts of independent fibrobastic OS lines OS80 and 494H with control ( sh-Luc ) on one flank and sh-Pthlh_A on the other flank .", "Data expressed as mean weight ± SEM ( n=3 tumours per shRNA per cell line; performed once ) ; P value as indicated .", "See Figure 2—figure supplement 1 and Figure 2—figure supplement 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 13446 . 00610 . 7554/eLife . 13446 . 007Figure 2—figure supplement 1 . PTHrP , an endogenous ligand for cAMP signaling in OS .", "( A ) Average number of reads ( TMM normalised , RNA-seq ) representing the GPCRs expressed in fibroblastic and osteoblastic OS .", "( B ) cAMP assays in 3 independent cell cultures within 2 OS subtypes depicting the effect of PTHrP blockade using anti-PTHrP antibody .", "( C ) cAMP assays in 3 independent cell cultures within 2 OS subtypes post Pthlh knockdown .", "( D ) Western blot showing the loss of PTHrP protein upon knockdown as compared to control cells ( E ) Cell proliferation post knockdown of Pthlh in two fibroblastic OS cultures .", "( F , G )", "Expression of genes in fibroblastic OS cultures ( OS80 and 494H ) by qPCR and normalized to Hprt .", "Data expressed as relative expression . DOI: http://dx . doi . org/10 . 7554/eLife . 13446 . 00710 . 7554/eLife . 13446 . 008Figure 2—figure supplement 2 . PTHrP overexpression alone does not initiate OS .", "( A ) Quantification of PTHrP levels in the media of primary osteoblasts infected with control retrovirus ( MSCV_Control; empty vector ) or PTHrP expressing retrovirus ( MSCV_PTHrP ) .", "Media from infected cells was applied to UMR106 . 01 cells and cAMP levels measured by radioimmunoassay .", "( B ) Representative AnnexinV/7-AAD staining profiles of primary osteoblasts following infection with control or PTHrP overexpressing retrovirus .", "( C ) Quantitation of cell death from AnnexinV/7-AAD staining following infection with control or PTHrP overexpressing retrovirus .", "Data expressed as mean ± SEM , Students t-test was used , *p<0 . 05 , **p<0 . 001 , ***p<0 . 0001 . DOI: http://dx . doi . org/10 . 7554/eLife . 13446 . 008 Knockdown of Pthlh ( Figure 2—figure supplement 1D ) reduced the proliferation ( Figure 2—figure supplement 1E ) , transcription of known target genes ( Figure 2—figure supplement 1F–G ) and the levels of pCREB1 , and surprisingly , total CREB1 in OS cells at early time points post infection ( Figure 2F ) .", "Correspondingly there was a significant reduction in the basal expression of CREB1 target genes ( Figure 2G ) .", "Next , cell survival 48–72 hr after shRNA infection was assessed .", "Primary osteoblasts were largely unaffected by Pthrp knockdown .", "In contrast there was a rapid induction of apoptosis following Pthlh knockdown in OS cells ( Figure 2H–I ) .", "To determine the contribution of elevated PTHrP expression on OS initiation , we retrovirally overexpressed PTHrP in wild-type primary osteoblasts .", "Surprisingly , the cells overexpressing high levels of PTHrP failed to thrive and a significant proportion underwent cell death indicating that PTHrP overexpression alone is not sufficient to support OS initiation ( Figure 2—figure supplement 2A–C ) .", "Two different fibroblastic OS lines infected with control ( sh-Luc ) or sh-Pthlh_A were grafted subcutaneously in vivo and both had significantly reduced proliferation as measured by tumor weight ( Figure 2J ) , comparable to the effects of shPthr1 knockdown in the same OS lines ( Ho et al . , 2015 ) .", "These results demonstrate that PTHrP is a critical , OS cell-derived stimulus of the elevated cAMP in OS .", "We next assessed basal cAMP levels in normal osteoblasts and the two OS subtypes in unstimulated proliferating cultures ( with and without phosphodiesterase inhibition but no exogenous ligand treatment ) .", "OS cells produced significantly greater amounts of intracellular cAMP compared to primary murine osteoblasts ( Figure 3A , Figure 3—figure supplement 1A ) .", "Furthermore , treatment with the direct cAMP agonist forskolin in the presence of IBMX resulted in an increased and sustained accumulation of cAMP in OS compared to primary osteoblasts ( Figure 3B ) .", "Without IBMX the relative responses to forskolin remained the same , albeit with lower cAMP levels ( Figure 3—figure supplement 1B ) . 10 . 7554/eLife . 13446 . 009Figure 3 . Persistent , elevated cAMP production in OS compared to primary osteoblasts .", "( A ) cAMP levels in indicated cells ( 1000 cells per well ) with and without IBMX treatment .", "Data from 3 independent cultures per type , mean ± SEM .", "( B ) Intracellular cAMP levels in indicated cell type following treatment with forskolin; mean ± SEM ( n=3 per cell type; 1000 cells per well ) ; statistical significance for OS vs normal Ob; all points of fibroblastic vs osteoblastic OS not significantly different .", "( C ) Western blot and ( D ) quantification of CREB1/pCREB1 during a time course of cAMP activation by forskolin .", "Data representative of 2 independent cultures each .", "( E ) Heat map of qPCR data .", "CREB1 target gene expression in indicated cells; data expressed as relative expression .", "( F ) ChIP analysis of CREB1/pCREB1 on the promoters of indicated genes over a 2 hr time course following stimulation with forskolin .", "Data is represented as percentage of input .", "The data from 2 independent cell lines for each subtypes mean occupancy ± SEM ( n=2–3 assays per line ) .", "( G ) Western blot of CREB1/pCREB1 expression in proliferating non stimulated cultures , β-ACTIN used as a loading control .", "Data representative of 3–4 independent cell lines from each type .", "( H ) CREB1 transcript expression in human osteoblasts and osteosarcoma ( data taken from PMID: 25961939 ) .", "( I ) Western CREB1/pCREB1 expression in indicated cell types under differentiative conditions , ATF-1 used as a loading control .", "( J ) Relative expression of negative regulators of cAMP in OS subtypes compared to primary osteoblasts by qPCR and normalized to β2m represented as a heat map ( n=3/cell type ) .", "*p<0 . 05 , **p<0 . 001 , ***p<0 . 0001 .", "See Figure 3—figure supplement 1 and Figure 3—figure supplement 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 13446 . 00910 . 7554/eLife . 13446 . 010Figure 3—figure supplement 1 . cAMP is constitutive in mouse OS leading to continuous phosphorylation of CREB1 . ( A ) Quantification of cAMP in primary osteoblasts , and OS with and without IBMX treatment ( 500 cells/well ) .", "Data represents 3 independent cell cultures for each type , mean ± SEM .", "( B ) Kinetics of cAMP accumulation in OS subtypes as compared to primary osteoblasts in the absence of IBMX treated with 10 μM forskolin ( using 500 cells/well ) .", "( C-D ) qPCR validation of CREB1 target gene expression following 10 μM forskolin treatment over a time course of 2 hr .", "The data represents 3 independent cell cultures for each subtype ± SEM ( n=3 ) .", "( E ) Expression of CREB1 target genes post knockdown of Creb1 using siRNA by qPCR and normalized to β2m .", "Means ± SEM ( n=3 ) .", "The data represents 3 independent cell lines for each subtype ± SEM ( n=3 ) .", "Effect of Creb1 knockdown on Crem1 expression in fibroblastic OS ( F ) and osteoblastic OS ( G ) , respectively .", "Expression of Crem1 by qPCR and normalized to β2m .", "Means ± SEM ( n=3 ) .", "( H ) Expression of Creb1 in primary osteoblasts compared to each OS subtype by qPCR and normalized to β2m .", "Means± SEM ( n=3 ) .", "For all the experiments above Students t-test was used to assess statistical significance , *p<0 . 05 , **p<0 . 001 , ***p<0 . 0001DOI: http://dx . doi . org/10 . 7554/eLife . 13446 . 01010 . 7554/eLife . 13446 . 011Figure 3—figure supplement 2 . Altered Creb1 dynamics in osteoblasts and OS .", "( A ) Expression of Creb1 during in vitro differentiation of primary osteoblasts , data expressed as mean ± SEM ( n=3 ) .", "( B ) Expression of Creb1 during in vitro differentiation of fibroblastic OS , data expressed as mean ± SEM ( n=3 ) .", "( C ) Expression of CREB1 protein during in vitro differentiation of primary human osteoblasts isolated from normal healthy donors ( 17–35 year old ) .", "( D ) Expression of CREB1 transcript during in vitro differentiation of primary human osteoblasts and markers of osteoblast differentiation state as indicated , data expressed as normalized gene expression compared to β2microglobulin expression; graphed as mean ± SEM ( n=5 independent donor samples ) .", "( E ) Expression of negative regulators of cAMP in OS subtypes compared to primary osteoblasts by qPCR and normalized to β2m ( n=3/cell type ) .", "*p<0 . 05 , **p<0 . 001 , ***p<0 . 0001 . DOI: http://dx . doi . org/10 . 7554/eLife . 13446 . 011 Based on the elevated cAMP in OS , we tested the dynamics of CREB1 phosphorylation in serum starved cells to acute elevation of cAMP induced by forskolin .", "Induction of pCREB1 was rapid in normal osteoblasts , peaking at 30 min , then reducing throughout the 120 min time course as expected ( Figure 3C–D ) .", "In contrast , OS cells displayed continuous and persistent activation of pCREB1 , consistent with the cAMP levels ( Figure 3C–D ) .", "The OS-specific altered dynamics of cAMP and pCREB1 resulted in aberrantly extended transcriptional activation of known CREB1 target genes based on both transcript expression and chromatin occupancy of CREB1/pCREB1 ( Figure 3E–F , Figure 3—figure supplement 1C–D ) .", "The requirement for CREB1 in the transcription of these targets in fibroblastic OS was confirmed using siRNA ( Figure 3—figure supplement 1E ) .", "Importantly , there was no evidence of compensation for loss of Creb1 by the related Crem1 in either OS subtype ( Figure 3—figure supplement 1F–G ) ( Mantamadiotis et al . , 2002 ) .", "We assessed the levels of pCREB1 , the downstream transcriptional effector of cAMP signaling , in proliferating OS cells compared to primary osteoblasts .", "CREB1 was more prominently phosphorylated in the osteoblastic OS than in either the fibroblastic OS or primary osteoblasts ( Figure 3G ) .", "qRT-PCR using independent OS cultures and primary osteoblasts demonstrated that the mean Creb1 expression was 2–3 fold higher in osteoblastic OS compared to fibroblastic OS and primary osteoblasts ( Figure 3—figure supplement 1H ) .", "Analysis of RNA-seq from human OS revealed a significant increased in the expression level of Creb1 in OS compared to normal osteoblasts ( Figure 3H ) ( Moriarity et al . , 2015 ) .", "During culture in differentiation inductive conditions , CREB1 levels reduced over the first 7 days in osteoblasts and stayed low for the remainder of the culture ( Figure 3I , Figure 3—figure supplement 2A ) .", "In contrast , OS cells maintained CREB1 expression under the same conditions ( Figure 3I , Figure 3—figure supplement 2B ) .", "The decrease in CREB1 expression ( both transcript and protein levels ) upon differentiation was confirmed in primary human osteoblasts ( Figure 3—figure supplement 2C–D ) .", "In human OS , somatic SNV mutations in negative regulators of cAMP levels were described , including members of the phosphodiesterases ( PDE ) , A kinase anchoring proteins ( AKAP ) and protein phosphatases ( PP ) ( Chen et al . , 2014 ) .", "There was a 2–3 fold decreased expression of several members of these gene families in the murine OS cells compared to primary osteoblasts ( Figure 3J , Figure 3—figure supplement 2E ) .", "The reduced expression of PDE , AKAPs and PPs would be expected to favour the accumulation and action of cAMP following GPCR activation .", "As intracellular cAMP increased following p53 deletion in primary osteoblasts , we sought to determine the effect of elevated cAMP levels on normal osteoblast differentiation .", "Primary osteoblasts were treated with the forskolin and their response compared to that of OS cells ( Walkley et al . , 2008; Mutsaers et al . , 2013 ) .", "Forskolin stimulates cAMP independently from cell surface GPCRs so was used instead of PTHrP , allowing a meaningful comparison of the isolated consequences of elevated cAMP as undifferentiated primary osteoblasts express less PTHR1 compared to the OS cells ( Mutsaers et al . , 2013 ) .", "After 72 hr treatment , primary osteoblasts had altered cell surface phenotypes and reduced expression of Runx2 and Osx ( Figure 4A–C ) ( Mutsaers et al . , 2013 ) .", "Brief exposure to forskolin ( 24 hr ) yielded the same result ( Figure 4—figure supplement 1A–B ) .", "During differentiation , cAMP activation led to decreased expression of differentiation markers and failure to normally mineralise ( Figure 4D–E , Figure 4—figure supplement 1C–D ) .", "Therefore continuously elevated cAMP increased features associated with immature osteoblasts .", "In OS cells , the cell surface phenotypes and expression of Runx2 and Sp7 were the inverse of primary osteoblasts after 72 hr forskolin treatment ( Figure 4F–H , Figure 4—figure supplement 1E ) .", "Under differentiation conditions , forskolin induced less profound changes in the expression of markers of osteoblast maturation , with the exception of Osteocalcin ( Figure 4I ) , and resulted in increased mineralization ( Figure 4J , Figure 4—figure supplement 1D–G ) .", "To determine the consequences of CREB1 retention in OS cells , CREB1 was knocked down using both shRNAs ( 3’UTR , CDS ) in fibroblastic OS cells and differentiation evaluated ( Figure 4—figure supplement 1J–L ) .", "Both early and late markers of maturation were reduced with sh-Creb1 ( Figure 4—figure supplement 1J ) .", "Mineralization was significantly reduced in sh-Creb1 expressing cells compared to controls ( Figure 4—figure supplement 1K–L ) .", "The level of intracellular cAMP achieved with forskolin treatment is significantly higher that that achieved by cell derived autocrine/paracrine stimuli , such as secreted PTHrP ( Figure 4—figure supplement 2A–C ) .", "These levels likely reflect maximal stimulation through the cAMP pathway in these cells which yields a distinct biological effect on primary osteoblastic cells compared to OS derived primary cultures .", "Therefore continuously elevated cAMP has distinct effects on the behaviour of normal osteoblasts and OS . 10 . 7554/eLife . 13446 . 012Figure 4 . Constitutively elevated cAMP differentially affects primary osteoblasts and osteosarcoma cells .", "( A ) Primary osteoblasts treated with DMSO or forskolin for 72 hr and assessed for expression of Sca-1 , CD51 , PDGFRα , representative results shown , n=3 independent experiments .", "( B ) Quantitation of cell surface markers from each treatment ( n=3 independent cultures ) ( C ) Expression of Sp7 ( Osterix ) and Runx2 by qPCR after 72 hr of forskolin treatment .", "Expression levels normalized to β2m; mean ± SEM ( n=3 ) .", "( D ) Expression level of indicated genes over 21 days of treatment with DMSO or forskolin .", "Expression normalized to β2m; mean ± SEM ( n=3 ) .", "( E ) Mineralisation analysis of primary osteoblasts at day 21 after treatment .", "Images are representative of 3 independent experiments .", "( F ) Fibroblastic OS cells were treated with DMSO or forskolin for 72 hr and assessed for expression of Sca-1 , CD51 , PDGFRα , representative results shown .", "( G ) Quantitation of cell surface markers from ( n=3 independent cultures of fibroblastic OS ) from each treatment .", "( H ) Expression of Sp7 ( Osterix ) and Runx2 in fibroblastic OS by qPCR following 72 hr treatment .", "Expression levels normalized to β2m; mean ± SEM ( n=3 ) .", "( I ) Expression of indicated genes in fibroblastic OS over 21 days from each treatment .", "Expression normalized to β2m; mean ± SEM ( n=3 ) .", "( J ) Representative images of alizarin red stained fibroblastic OS cells treated with DMSO or forskolin for 21 day; n=3 independent OS cultures; *p<0 . 05 , **p<0 . 001 , ***p<0 . 0001 .", "See Figure 4—figure supplement 1 and Figure 4—figure supplement 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 13446 . 01210 . 7554/eLife . 13446 . 013Figure 4—figure supplement 1 . cAMP has different effects in OS and primary osteoblasts .", "( A , B )", "Expression of Runx2 and Osterix by qPCR and normalized to β2m; means ± SEM ( n=3 ) .", "( C , D )", "Quantitation of elution of Alizarin red stain from primary osteoblasts and fibroblastic OS cells treated with DMSO or 10 μM forskolin for 21 days .", "The data is representative of 3 independent experiments with each .", "( E ) Cell surface profiling for Sca-1 and CD51 ( αV Integrin ) , PDGFRα on osteoblastic OS cells ( F ) Quantitation of Sca-1/CD51 , Sca-1/PDGFRα and CD51/PDGFRα populations in osteoblastic OS ( n>3 ) with ( blue bars ) and without ( black bars ) forskolin treatment .", "( G ) Mineralisation assay showing alizarin red staining of osteoblastic OS cells .", "( H ) Quantitation of elution of Alizarin red staining of osteoblastic OS cells treated with DMSO or 10 μM forskolin for 21 days .", "The data is representation of 3 independent experiments .", "( I ) Representative western blot for CREB1 following infection with 2 independent shRNA or control shRNA ( shLuc ) .", "( J ) Gene expression analysis of fibroblastic OS infected with the indicated shRNA and placed in differentiating conditions for the indicated periods of time .", "qPCR data normalized to β2m ( n=3/cell type ) .", "( K-L )", "Representative alizarin stained wells and quantitation of alizarin elution from day 21 cultures in differentiating conditions with fibroblastic OS .", "For all the experiments above Students t-test was used , *p<0 . 05 , **p<0 . 001 , ***p<0 . 0001 . DOI: http://dx . doi . org/10 . 7554/eLife . 13446 . 01310 . 7554/eLife . 13446 . 014Figure 4—figure supplement 2 . Level of cAMP in primary osteoblasts and osteosarcoma cells +/- forskolin .", "( A ) Intracellular cAMP levels in primary osteoblasts and fibroblastic OS cells at day 0 and day 21 of culture in differentiation inductive conditions with no exogenous agonist added ( IBMX treated only ) ; data expressed as cAMP level ( pmol ) per μg protein; graphed as mean ± SEM ( n=3 ) .", "( B ) Intracellular cAMP levels in primary osteoblasts and fibroblastic OS cells at day 21 of culture in differentiation inductive conditions with forskolin ( continuously present in the culture ) ; data expressed as cAMP level ( pmol ) per μg protein; graphed as mean ± SEM ( n=3 ) .", "( C ) Intracellular cAMP levels in primary osteoblasts and fibroblastic OS cells at day 21 of culture in differentiation inductive conditions with acute forskolin treatment at day 21 ( 20 min exposure to forskolin only ) ; data expressed as cAMP level ( pmol ) per μg protein; graphed as mean ± SEM ( n=3 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 13446 . 014 Reducing PTHrP levels caused apoptosis of OS cells and also reduced levels of CREB1/pCREB1 ( Figure 2F , Figure 2H–J ) .", "To determine if loss of CREB1 impacted OS cell survival similarly we assessed the effects of Creb1 knockdown .", "In all cohorts there was loss of CREB1 protein ( Figure 5A–C ) .", "CREB1 knockdown in primary osteoblasts caused reduced proliferation in the first week then the cells recovered and proliferated similarly to control infected cells thereafter , with no apparent effect on survival ( Figure 5A ) .", "Loss of CREB1 in fibroblastic OS cells resulted in sustained proliferation impairment , yet cell survival was not appreciably impacted ( Figure 5B ) .", "Knockdown of CREB1 in osteoblastic OS , the most common clinical subtype , caused profound proliferation arrest and apoptosis ( Figure 5C ) .", "The effect was so complete that we have not been able to establish stable sh-Creb1 expressing cultures from the osteoblastic OS .", "The phenotype was observed with both sh-Creb1 constructs and in ≥3 independent cultures .", "Therefore , CREB1 is dispensable for normal osteoblast function yet required for proliferation of fibroblastic OS and survival of osteoblastic OS . 10 . 7554/eLife . 13446 . 015Figure 5 . CREB1 is differentially required for proliferation and survival by OS subtypes .", "( A ) CREB1 in primary osteoblasts 72 hr after infection with indicated shRNA construct .", "ATF1 was used as a loading control; representative blot from 3 independent cultures; proliferation plotted as mean ± SEM ( n=3 ) .", "( B ) Western blot of CREB1 and proliferation kinetics of shCreb1 knockdown and sh-Luc fibroblastic OS; representative blot from 3 independent OS lines; proliferation as mean ± SEM ( n=3 ) .", "( C ) Western of CREB1 in osteoblastic OS cells 72 hr after infection; ATF1 = loading control .", "Viability ( annexinV/7AAD ) of indicated OS subtype following infection with each shRNA .", "Data are representative of 3 independent cell lines/type; quantitation of dead cells .", "Data from 3 independent cell lines/subtypes; mean ± SEM .", "*p<0 . 05 , **p<0 . 001 , ***p<0 . 0001DOI: http://dx . doi . org/10 . 7554/eLife . 13446 . 015 Given the subtype specific effects of CREB1 knockdown , we sought to determine if CREB1 gene signatures could be used to appreciate differences between the subtypes .", "We modelled our analysis on the evidence that PTHrP was the endogenous ligand leading to cAMP accumulation and CREB1 activation .", "We defined a PTHrP-specific gene signature bioinformatically from previous microarrays comparing PTHrP ( 1–141 ) to PTH ( 1–34 ) in differentiating osteoblasts ( Allan et al . , 2008 ) .", "The top 45 candidates from the signature were validated .", "Using proliferating primary osteoblasts , fibroblastic OS and osteoblastic OS cells , 32 of the 45 genes were most highly expressed in the osteoblastic OS ( Figure 6A ) .", "Archetypal CREB1 target genes were all more highly expressed in osteoblastic OS ( Figure 6B , Figure 6—figure supplement 1A–C ) .", "Chromatin immunoprecipitation-PCR ( ChIP-qPCR ) demonstrated enrichment of active pCREB1 and serine 2 phosphorylated RNA polymerase II ( pPolII ) , a mark of active transcription , on CREB1 target genes in osteoblastic OS ( Figure 6C , Figure 6—figure supplement 1D ) ( Ho and Shuman , 1999 ) .", "Promoter binding was generally lower in the fibroblastic OS , consistent with the expression patterns of the target genes .", "The enhanced binding of CREB1 in osteoblastic OS corresponded to the elevated levels of cAMP and osteoblastic OS is characterised by increased CREB1 activity . 10 . 7554/eLife . 13446 . 016Figure 6 . CREB1 signatures discriminate OS subtypes .", "( A ) Heat map of qPCR data .", "Expression of the PTHrP/CREB1 gene set between indicated cell types .", "Data from 3 independent cultures for each , expressed as fold change relative to primary osteoblasts .", "( B ) Examples of CREB1 target gene expression between the indicated cell types .", "Expression levels normalized to β2m and depicted as relative expression ± SEM ( n=3 ) .", "( C ) ChIP-qPCR for the indicated target genes from proliferating cells ( no exogenous ligand/stimulus of cAMP applied ) with CREB1 , pCREB1 and pPolII .", "Data represented as fold occupancy relative to Dpp10 promoter , expressed as mean ± SEM .", "*p<0 . 05 , **p<0 . 001 , ***p<0 . 0001 .", "See also Figure 6—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 13446 . 01610 . 7554/eLife . 13446 . 017Figure 6—figure supplement 1 . CREB1 defines OS subtypes by driving specific gene signatures .", "( A , B , C )", "Expression of Creb1 signatures ( upregulated , downregulated and unchanged ) were assessed in mouse OS subtypes compared to primary osteoblast cells by qPCR .", "Expression normalized to β2m respectively .", "Data represents average relative expression ± SEM ( n≥3 ) .", "( D ) ChIP experiments depicting the binding of CREB1 , pCREB1 and Pol II on CREB1 target genes .", "Enriched DNA by ChIP was amplified using primers against the promoters of mentioned targets .", "Data is represented as fold occupancy calculated with respect to binding over a cold promoter DPP10 . DOI: http://dx . doi . org/10 . 7554/eLife . 13446 . 017 Mutations and oncogenic effects of the cAMP pathway have been described in the context of other tumor types , including breast ( Kok et al . , 2011; Miller , 2002; Beristain et al . , 2015; Pattabiraman et al . , 2016 ) and haematological malignancies ( Pigazzi et al . , 2013; Sandoval et al . , 2012; Shankar et al . , 2005; Smith et al . , 2005; Mullighan et al . , 2011 ) amongst other tumors .", "The mutational landscape of OS has been recently defined , identifying 1704 somatic single nucleotide variations ( SNV mutations ) across 20 cases of sporadic conventional OS ( Chen et al . , 2014 ) .", "To determine if these somatic SNV mutations were functionally related , we assessed pathways enriched within the somatic SNV mutations ( Figure 7A ) .", "In the top 20 pathways were signatures associated with ion channel complexes , transmembrane transporter complexes , PI3K signaling , calcium channel signaling , and protein kinase A ( PKA ) activity , all of which are related to cAMP ( Figure 7A ) .", "Based on this result , we compared the somatic SNV mutations of human OS to the 169 genes comprising the KEGG cAMP interactome .", "To determine if this was OS specific or a more generalised feature of tumors , we further compared the cAMP interactome with whole genome sequencing that identified SNV mutations of other human cancers ( Ellis et al . , 2012; Morin et al . , 2013; Berger et al . , 2012; Cazier et al . , 2014; Tirode et al . , 2014 ) .", "Genes within the cAMP interactome were most highly enriched within the somatic SNV mutations of human OS ( Figure 7B ) .", "These data suggest that , despite the diverse mutational pattern of somatic SNV mutations in human OS , recurrent and enriched changes in the cAMP and CREB1 pathways occur in OS . 10 . 7554/eLife . 13446 . 018Figure 7 . A high proportion of the cAMP interactome are somatic SNV mutations in human osteosarcoma .", "( A ) Analysis of functional pathways within the somatic SNV mutations of human OS using Cytoscape .", "Brown color indicates a cAMP related pathway , blue color indicates non cAMP related pathways .", "( B ) Analysis of the enrichment for somatic SNV mutations within the cAMP interactome in each of the indicated tumor types .", "Based on somatic SNV mutations identified by whole genome sequencing .", "P value defined using hypergeometric distribution test .", "( C ) Graphical summary of the differences between primary osteoblasts and OS subtypes regarding cAMP and CREB1 function .", "See also Figure 7—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 13446 . 01810 . 7554/eLife . 13446 . 019Figure 7—figure supplement 1 . Analysis of cAMP and cGMP pathway enrichment of somatic SNV tumor mutations . Table comparing the enrichment of somatic SNV tumor mutations within OS , Ewings and Breast cancer in the cAMP pathway , cGMP pathway and those unique to the cAMP and cGMP pathway respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 13446 . 019" ], [ "There remains an incomplete understanding of the cellular rewiring that accompanies the loss or null mutation of TRP53 .", "Although the p53 pathway can be targeted , most interventions aim to activate or restore function of the mutant P53 protein , an approach not feasible in null settings ( Khoo et al . , 2014 ) , the most common case in OS ( Chen et al . , 2014 ) .", "The identification of critical cellular pathways that are activated/altered in response to TRP53 deficiency may yield novel avenues to test therapeutically .", "Using the fact that loss/mutation of TRP53 is essentially universal in OS , we modelled an initiating lesion in primary osteoblastic cells and used this to understand the consequences in these cells .", "Several lines of evidence have implicated the cAMP pathway in OS , yet the functional requirement for this pathway has not been evaluated .", "We recently demonstrated a role for PTHR1 in OS proliferation and maintenance of the undifferentiated state ( Ho et al . , 2015 ) .", "We therefore sought to determine if these pathways intersected in the p53 deletion dependent initiation and maintenance of OS .", "There was a co-ordinated increase in Pthlh , cAMP per cell and CREB1 levels and transcriptional activity when osteoblasts became functionally p53-deficient .", "This was unexpected , as it suggested that a very early event in the initiation of OS following the loss of p53 is the activation of the PTHrP→cAMP→CREB1 signaling axis .", "Whilst the detailed processes through which p53 loss activates this pathway is to be resolved , these results demonstrate that this is an essential pathway in the manifestations of the p53-deficient phenotype in osteoblastic cells .", "Furthermore , we demonstrated that this pathway was also required for the survival of osteoblasts rendered p53-deficient prior to loss of PTHrP or CREB1 .", "Therefore , activation of the PTHrP→cAMP→CREB1 signaling axis appears to be a core component of the rewiring of osteoblastic lineage cells in response to loss of Trp53 ( Figure 7C ) .", "Our results , together with prior studies implicating cAMP pathways in OS , indicate that elevated cAMP signaling could be considered oncogenic in OS .", "Forcibly increasing intracellular cAMP in normal osteoblasts retained them in an immature state , consistent with the recent report identifying forskolin as an inducer of pluripotency ( Hou et al . , 2013 ) .", "Analysis of human OS identified somatic SNV mutations in a number of negative regulators of cAMP levels ( Chen et al . , 2014 ) .", "Inactivating mutations in these would be predicted to elevate PKA and CREB1 activity .", "Khokha and colleagues identified mutations in Prkar1a using a murine model of OS , with a corresponding PRKAR1A low human OS subset defined ( Molyneux et al . , 2010 ) .", "In the same study , mutually exclusive amplification of the α-subunit of PKA ( Prkarca ) was also reported .", "Our data reconcile these observations and indicate that the increased PKA-CREB1 activity is ultimately important for this tumor , in that it has evolved mechanisms ensuring elevated and persistent cAMP levels mediated primarily by autocrine production of PTHrP and modulated by reduced expression of negative regulators of cAMP activity .", "In normal physiology , PTHrP acts in a paracrine manner whilst in OS , as reported here , there is capacity for PTHrP to act in an autocrine and paracrine manner , as well as intracrine activities .", "Reducing PTHrP levels was tolerated by normal osteoblasts but not OS cultures .", "While mutations promoting accumulation of cAMP are important , the stimulus of cAMP had not been defined .", "We demonstrate that PTHrP is a key OS cell-intrinsic inducer of cAMP .", "PTHrP is required for normal bone homeostasis via its actions upon osteoblastic cells ( Miao , 2005 ) .", "The functions of PTHrP in malignant osteoblast biology , however , have not been resolved .", "Reducing PTHR1 expression on OS cells enhanced differentiation and mineralization in vivo ( Ho et al . , 2015 ) .", "There was a trend to greater levels of PTHrP , notably intracellular , in osteoblastic OS compared to fibroblastic OS ( Ho et al . , 2015 ) .", "These data are consistent with the present measurements of pCREB1 levels in the subtypes and the CREB1 dependence of the osteoblastic OS .", "We did not previously impact OS in vivo using a neutralizing anti-PTHrP antibody ( Ho et al . , 2015 ) .", "We reconcile the failure to achieve a therapeutic dose of antibody in vivo with high concentrations of PTHrP likely within the immediate cell environment in OS ( Ho et al . , 2015 ) .", "The reduction in CREB1 levels when PTHrP was reduced was unexpected , raising the possibility to be explored , that PTHrP may contribute to maintenance of CREB1 levels in an analogous manner to the role of JAK2 in preventing proteasomal degradation of CREB1 ( Lefrancois-Martinez et al . , 2011 ) .", "Coupled with the present data , targeting PTHrP is a candidate therapeutic strategy in OS , although any intracrine contribution of PTHrP needs to be evaluated .", "The therapeutic targeting of components downstream of PTHrP/PTHR1 signalling may also be feasible , with several recent reports of inhibitors of CREB signalling activity ( Mitton et al . , 2016; Xie et al . , 2015 ) , although these are yet to progress to preclinical evaluation .", "The management of OS has not substantively changed for the last three decades .", "The identification of the pathways that are utilised during the evolution from osteoblastic lineage cells to OS cells may reveal new means to target this tumour .", "Recent sequencing and modeling has revealed the genetic complexity and diverse mutational patterns of OS , yet underlying these data is the recurrent and universal inactivation of the P53 pathway ( Chen et al . , 2014; Moriarity et al . , 2015 ) .", "The Notch pathway has been implicated in OS and is potentially druggable , however the evidence from human OS is equivocal as mutations in this pathway are not common in sporadic OS ( Tao et al . , 2014 ) .", "Recent work from two groups using whole genome screening identified the PI3K/mTOR pathway as a conserved therapeutic vulnerability in OS , demonstrating the power of understanding the biological networks underpinning OS ( Perry et al . , 2014; Gupte et al . , 2015 ) .", "The identification of pathways synthetically lethal with p53-deficiency , or that are required for the maintenance of p53 deficient phenotypes , will yield new means to target these cells .", "Using this approach we have defined a requirement within the osteoblast lineage for continuous PTHrP and CREB1 activity for the initiation and maintenance of OS , yet normal osteoblasts could tolerate the depletion of both of these factors .", "A striking feature of the requirement for CREB1 was the differences between OS subtypes .", "The OS subtypes could be discriminated from normal osteoblasts and each other based on the progressive enrichment of CREB1 gene signatures that reflected their dependence on this pathway for proliferation and survival .", "These observations raise the largely unexplored possibility that OS subtypes are at some level genetically distinct and have different biological dependences .", "Collectively , the constitutive activity of the PTHrP-cAMP-CREB1 axis , tightly coupled to loss of p53 , represents an essential node in OS that is amenable to therapeutic inhibition at multiple levels ." ], [ "All experiments involving animals were approved by the Animal Ethics Committee of St . Vincent’s Hospital , Melbourne .", "Primary human osteoblasts were isolated from bone marrow aspirates from the posterior iliac crest of de-identified healthy human adult donors with informed consent and consent to publish ( IMVS/SA Pathology normal bone marrow donor program RAH#940911a , Adelaide , South Australia ) .", "RNA-Sequencing ( RNA-Seq ) was conducted at the Ramaciotti Centre for Genomics ( University of New South Wales , Australia ) on the Illumina HiSeq 2000 with 100 bp paired-end reads .", "Reads were aligned to the mouse genome build mm9/NCBI37 using Casava 1 . 7 and Bowtie v0 . 12 . 2 mapping software , normalized using Voom linear modeling ( Law et al . , 2014 ) and transcript abundance measured as reads per kilobase of exon per million mapped reads ( RPKM ) ( Chepelev et al . , 2009 ) .", "The datasets are deposited in GEO ( accession number GSE58916 ) ." ] ]

[ "Mutations in the P53 pathway are a hallmark of human cancer .", "The identification of pathways upon which p53-deficient cells depend could reveal therapeutic targets that may spare normal cells with intact p53 .", "In contrast to P53 point mutations in other cancer , complete loss of P53 is a frequent event in osteosarcoma ( OS ) , the most common cancer of bone .", "The consequences of p53 loss for osteoblastic cells and OS development are poorly understood .", "Here we use murine OS models to demonstrate that elevated Pthlh ( Pthrp ) , cAMP levels and signalling via CREB1 are characteristic of both p53-deficient osteoblasts and OS .", "Normal osteoblasts survive depletion of both PTHrP and CREB1 .", "In contrast , p53-deficient osteoblasts and OS depend upon continuous activation of this pathway and undergo proliferation arrest and apoptosis in the absence of PTHrP or CREB1 .", "Our results identify the PTHrP-cAMP-CREB1 axis as an attractive pathway for therapeutic inhibition in OS ." ]

[ "Bone cancer ( osteosarcoma ) is caused by mutations in certain genes , which results in cells growing and dividing uncontrollably .", "In particular , a gene that produces a protein called P53 in humans is lost in all bone cancers .", "However , we don’t understand what happens to the bone cells when they lose P53 .", "Although a number of studies have identified several molecular pathways that are changed in bone cancers – such as the cyclic AMP ( cAMP ) pathway – how these interact to cause a cancer is not well understood .", "Walia et al . compared bone-forming cells from normal mice with cells from mutant mice from which the gene that produces the mouse p53 protein could be removed .", "This revealed that the loss of p53 causes these cells to grow faster .", "The activity of the cAMP pathway also increases in p53-deficient cells .", "Further investigation revealed that the cells grow faster only if they are able to activate the cAMP pathway , and that this pathway needs to stay active for bone cancer cells to grow and survive .", "This suggests that inhibiting this pathway could present a new way to treat bone cancer .", "Walia et al . confirmed several of their findings in human cells .", "Future studies will now investigate how the loss of the P53 protein in humans activates the cAMP pathway , which will be important for understanding how this cancer forms .", "It will also be worthwhile to begin testing ways to block this pathway to determine whether it is a useful target for therapies ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "neuroscience" ]

[ [ "Multisensory information offers substantial benefits for behavior .", "For example , acoustic and visual cues can be combined to derive a more reliable estimate of where an object is located ( Alais and Burr , 2004; Ernst and Banks , 2002; Körding et al . , 2007; Wozny and Shams , 2011b ) .", "Yet , the process of multisensory perception does not end once an object is removed .", "In fact , multisensory information can be exploited to calibrate subsequent perception in the absence of external feedback ( Frissen et al . , 2012; Wozny and Shams , 2011a ) .", "In a ventriloquist paradigm , for example , the sight of the puppet and the actor’s voice are combined when localizing the speech source , and both cues influence the localization of subsequent unisensory acoustic cues , if probed experimentally ( Bosen et al . , 2017; Bosen et al . , 2018; Bruns and Röder , 2015; Bruns and Röder , 2017; Callan et al . , 2015; Radeau and Bertelson , 1974; Recanzone , 1998 ) .", "This trial-by-trial recalibration of perception by previous multisensory information has been demonstrated for spatial cues , temporal cues , and speech signals ( Kilian-Hütten et al . , 2011a; Lüttke et al . , 2016; Lüttke et al . , 2018; Van der Burg et al . , 2013 ) , and has been shown to be modulated by attention ( Eramudugolla et al . , 2011 ) .", "Despite the importance of both facets of multisensory perception for adaptive behavior - the combination of information within a trial and the trial-by-trial adjustment of perception - it remains unclear whether they originate from shared neural mechanisms .", "In fact , the neural underpinnings of trial-by-trial recalibration remain largely unclear .", "Those studies that have investigated neural correlates of multisensory recalibration mostly focused on the adaptation following long-term ( that is , often minutes of ) exposure to consistent multisensory discrepancies ( Bruns et al . , 2011; Zierul et al . , 2017 ) .", "However , we interact with our environment using sequences of actions dealing with different stimuli , and thus systematic sensory discrepancies as required for long-term effects are possibly seldom encountered .", "Hence , while the behavioral patterns of multisensory trial-by-trial recalibration are frequently studied ( Bosen et al . , 2017; Bruns and Röder , 2015; Delong et al . , 2018; Van der Burg et al . , 2018; Wozny and Shams , 2011a ) it remains unclear when and where during sensory processing their neural underpinnings emerge .", "In contrast to this , the neural underpinnings of multisensory integration of simultaneously received information have been investigated in many paradigms and model systems ( Angelaki et al . , 2009; Bizley et al . , 2016; Fetsch et al . , 2013 ) .", "Studies on spatial ventriloquist-like paradigms , for example , demonstrate contributions from auditory and parietal cortex ( Bonath et al . , 2014; Bruns and Röder , 2010; Bruns and Röder , 2015; Callan et al . , 2015; Harvey et al . , 2014; Bonath et al . , 2007; Starke et al . , 2017 ) .", "A series of recent studies demonstrates that posterior parietal regions automatically fuse multisensory information , while anterior parietal regions give way to a more flexible spatial representation that follows predictions from Bayesian causal inference ( Cao et al . , 2019; Rohe et al . , 2019; Rohe and Noppeney , 2015b; Rohe and Noppeney , 2016 ) .", "Given that parietal regions also contribute to the maintenance of sensory information within or between trials ( Harvey et al . , 2012; Morcos and Harvey , 2016; Raposo et al . , 2014; Schott et al . , 2018; Uncapher and Wagner , 2009; Vilberg and Rugg , 2008 ) this raises the possibility that parietal regions are in fact mediating both the combination of sensory information within a trial , and the influence of such an integrated representation on guiding subsequent adaptive behavior .", "To link the neural mechanisms underlying multisensory integration and trial-by-trial recalibration , we measured whole-brain activity using magnetoencephalography ( MEG ) while human participants performed a spatial localization task ( Figure 1A ) .", "The paradigm was designed to reveal the behavioral correlates of audio-visual integration ( i . e . the ventriloquist effect , VE ) and the influence of this on the localization of a subsequent unisensory sound ( the ventriloquist aftereffect , VAE ) ( Wozny and Shams , 2011a ) .", "Using single-trial classification we determined the relevant neural representations of auditory and visual spatial information and quantified when and where these are influenced by previous sensory evidence .", "We then modeled the influence of these candidate neural representations on the participant-specific trial-by-trial response biases .", "As expected based on previous work , our results reveal neural correlates of sensory integration in superior temporal and parietal regions .", "Importantly , of these , only activity within the superior parietal cortex encodes current multisensory information and retains information from preceding trials , and uses both to guide adaptive behavior within and across trials ." ], [ "Behavioral responses in AV trials revealed a clear ventriloquist effect ( VE ) as a function of the presented audio-visual discrepancy ( ΔVA = VAV - AAV ) , whereby the visual stimulus biased the perceived sound location ( Figure 1B ) .", "The VE was computed as the difference between participant’s response ( R ) and the actual sound location for that trial ( i . e . , RAV – AAV , where subscript denotes the trial type ) .", "Model comparison revealed that both stimuli had a significant influence on the participants’ responses ( relative BIC values of three candidate models ,", "c . f .", "Materials and methods Section: mi1: 938 , mi2: 3816 , mi3: 0; relative AIC values; mi1: 945 , mi2: 3823 , mi3: 0; protected exceedance probability ( Rigoux et al . , 2014 ) ; mi1: 0 , mi2: 0 , mi3: 1; winning model: mi3: VE ~ 1 + β⋅AAV + β⋅VAV ) , with significant contributions from both the auditory ( AAV ) , and visual stimuli ( VAV ) ( βA_AV = -0 . 48 , βV_AV = 0 . 22 , tA_AV = -70 . 0 , tV_AV = 31 . 7 , pA_AV , pV_AV <0 . 01 ,", "d . f .", "= 8064 ) .", "Across participants , the VE bias was significant for each non-zero audio-visual discrepancy ( all p<10−4; Wilcoxon signed rank tests , corrected for multiple tests with the Holm procedure ) .", "Participants localized the sound in the A trials reliably ( Figure 1C , black graph ) , with the data exhibiting a well-known central bias ( Rohe and Noppeney , 2015a ) .", "This confirms that the convolution with HRTFs indeed led to sounds that were perceived as spatially dispersed .", "Behavioral responses in A trials revealed a significant ventriloquist aftereffect ( VAE; Figure 1D ) as a function of the audio-visual discrepancy ( ΔVA ) in the previous AV trial , demonstrating that the preceding multisensory stimuli had a lasting influence on the localization of subsequent sounds .", "The VAE for each sound location was computed as RA – mean ( RA ) ; whereby mean ( RA ) reflects the mean over all localization responses for this position and participant .", "This approach ensures that any bias in pure auditory localization does not confound the VAE effect ( Rohe and Noppeney , 2015a; Wozny and Shams , 2011a ) .", "Model comparison revealed that both previous stimuli had a significant influence on the VAE ( relative BIC values of three candidate models ,", "c . f .", "Materials and methods section; mr1: 27 , mr2: 357 , mr3: 0; relative AIC values: mr1: 34 , mr2: 364 , mr3: 0; protected exceedance probability; mr1: 0 , mr2: 0 , mr3: 1; winning model: mr3: VAE ~ 1 + β⋅AAV + β⋅VAV ) , with significant contributions from the previous sound ( AAV ) , and the previous visual stimulus ( VAV ) ( βA_AV = -0 . 09 , βV_AV = 0 . 03 , tA_AV = -19 . 4 , tV_AV = 6 . 0 , pA_AV , pV_AV <0 . 01 ,", "d . f .", "= 8064 ) .", "Note that because the VAE was defined relative to the average perceived location for each sound position , the actual sound position ( AA ) does not contribute to the VAE .", "Across participants , the VAE bias was significant for each non-zero audio-visual discrepancy ( all p<10−2; two-sided Wilcoxon signed rank tests , corrected for multiple tests with the Holm procedure ) .", "We performed two control analyses to further elucidate the nature of the VAE .", "First , we asked whether the shift in the perceived sound location was the result of a bias towards the previous visual stimulus location , or a bias induced specifically by the previous audio-visual discrepancy ( Wozny and Shams , 2011a ) .", "To dissect these hypotheses , we selected trials for which the expected biases arise from the direction of the VE , and not from a visual bias towards VAV ( Figure 1E ) .", "The data were clearly in favor of a genuine multisensory bias , as the VAE remained significant for these trials ( all p<0 . 05; except for +25 . 5 condition; two-sided Wilcoxon signed rank tests , corrected for multiple tests; Figure 1F ) ( Wozny and Shams , 2011a ) .", "Second , we asked whether the response bias in the A trial was better accounted for by the sensory information in the previous trial ( i . e . the previous multisensory discrepancy: ΔVA ) or the participant’s response in that trial ( RAV ) .", "Formal model comparison revealed that the model RA ~1 + AA + ΔVA provided a better account of the data than a response-based model RA ~1 + AA + RAV ( relative BIC: 0 , 393; BIC weights: 1 , 0 ) , supporting the notion that recalibration is linked more to the physical stimuli than the participants response ( Van der Burg et al . , 2018 ) .", "The analysis of the MEG data was designed to elucidate the neural underpinnings of the VAE and to contrast these to the neural correlates of the VE .", "Specifically , we first determined neural representations of the task-relevant sensory information , or of the upcoming participant’s response .", "We then used these representations in a neuro-behavioral analysis to probe which neural representations of acoustic or visual spatial information are directly predictive of the participant-specific VE and VAE single trial biases .", "We applied linear discriminant analysis to the time-resolved MEG source data to determine neural representations of the spatial lateralization of the auditory and visual stimuli ( Figure 2 ) .", "From the MEG activity during the A trials , we obtained significant classification ( cluster-based permutation test , correcting for multiple comparisons , for details refer to Materials and methods - Statistical Analysis ) performance for the current sound ( AA; peaking at 80 ms in the left inferior parietal and at 160 ms in the middle temporal gyrus ) and for the location of the sound in the previous trial ( AAV; peaking around 120 ms in the left middle occipital lobe and the bilateral precuneus; at p≤0 . 01 FWE corrected for multiple comparisons in source space ) .", "This characterizes neural representations of acoustic spatial information currently received and persisting from the previous trial in a wider network of temporal and parietal brain regions .", "Classification of the lateralization of previous visual stimuli ( VAV ) was not significant at the whole brain level in the activity of the A trial , suggesting that persistent visual information was weaker than that of the acoustic information .", "However , the whole brain classification maps revealed meaningful clusters in early left inferior temporal areas and the right inferior/superior parietal areas .", "Classification of the upcoming response ( RA ) was significant with a similar pattern as observed for the current sound ( AA ) in the A trial .", "To reveal the neural correlates of the VAE we investigated three regression models capturing different aspects of how current and previous sensory information shape", "i ) the neural encoding of current sensory information in the A trial ( i . e . AA ) ,", "ii ) the encoding of the upcoming response ( RA ) , and", "iii ) how neural representations of previous sensory information contribute to the single trial VAE bias .", "The first model tested how the previous stimuli affect the encoding of the current sound , that is , how the encoding of sound AA in the MEG activity of the A trial was affected by the previous stimulus positions AAV and VAV ( Figure 3A; Table 1 ) .", "There was a significant ( cluster-based permutation test FWE corrected at p≤0 . 05 ) influence of AAV , starting around 80 ms in the cingulum , precuneus , shifting towards inferior/superior parietal areas around 220 ms . There was also significant influence of VAV in the left occipital/parietal areas around 160 ms . Importantly , the significant effects from the previous acoustic and visual stimuli overlapped in the left parietal areas ( Figure 3A; red inset ) .", "The second model revealed that the previous stimuli also influenced neural activity discriminative of the participants’ response ( RA; Figure 3B; Table 1 ) .", "In particular , both previous sound and visual stimulus influenced the activity predictive of the current response around 80 ms in the right parietal cortex ( precuneus in particular ) , with the effect of AAV including also frontal and temporal regions .", "The significant effects of AAV and VAV overlapped in the cingulum and precuneus ( Figure 3B; red inset ) .", "These results demonstrate that parietal regions represent information about previous multisensory stimuli , and this information affects the neural encoding of the currently perceived sound .", "Using the third model , we directly tested whether these neural signatures of the previous stimuli in the MEG activity during the A trial are significantly related to the participants’ single trial response bias ( Figure 3C; Table 1 ) .", "The significant influences of the neural representations of previous acoustic and visual stimuli overlapped again in parietal cortex ( angular gyrus , precuneus; Figure 3C; red inset ) .", "The converging evidence from these three analyses demonstrates that the same parietal regions retain information about both previously received acoustic and visual spatial information , and that single trial variations in these neural representations directly influence the participants’ bias of subsequent sound localization .", "To be able to directly compare the neural correlates of the ventriloquist aftereffect to multisensory integration ( i . e . the VE effect ) , we repeated the same analysis focusing on the MEG activity in the AV trial .", "As expected from the above , classification for both auditory ( AAV ) and visual ( VAV ) locations was significant in a network of temporal and occipital regions ( Figure 4—figure supplement 1 ) .", "To directly link the encoding of multisensory information to behavior , we again modeled the single trial VE response bias based on the representations of current acoustic and visual information ( Figure 4 ) .", "This revealed overlapping representations of both stimuli that directly correlated with the response bias within superior parietal regions ( precuneus and superior parietal lobule ) , and , in a separate cluster , within inferior temporal areas ( Figure 4; Table 2 ) .", "The above reveals neural representations of audio-visual information in parietal regions that either contribute to integration within a trial ( VE bias ) or that shape the localization of auditory information based on previous sensory information ( VAE bias ) .", "Given that each effect was localized independently ( as overlapping clusters in Figure 3C and Figure 4 , respectively ) , we asked whether the same neural sources significantly contribute to both effects .", "To this end we subjected the above identified clusters to both neuro-behavioral models ( VAE and VE; Equations 4/5 ) to assess the significance of each regressor and to compare the strength of the VAE and VE effects between clusters ( Table 3 ) .", "This revealed that the spatially selective activity contributing to the VE effect ( CPAR , from Figure", "4 ) also significantly contributes to the VAE effect .", "That is , the single trial variations in the encoding of auditory and visual information in this cluster also contributed significantly ( at p≤0 . 05 ) to the recalibration effect .", "Further , the effect strength in this cluster for recalibration did not differ from that observed in the cluster directly identified as significantly contributing to the VAE bias ( CVAE , at p<0 . 05; FDR adjusted; Table 3 ) .", "Vice versa , we found that the parietal sources mediating recalibration ( cluster CVAE; from Figure 3C ) also significantly contributed to sensory integration within the AV trial ( Table 3 ) .", "These results confirm that spatially selective activity within superior parietal regions ( identified by both clusters , CPAR , and CVAE , comprising precuneus and superior parietal regions ) is significantly contributing to both sensory integration and trial-by-trial recalibration .", "While the cluster predictive of the recalibration effect comprised significant grid points in both hemispheres , the model predicting the VE bias in the AV trial based on brain activity was significant only within the left hemisphere ( clusters CTEMP and CPAR ) .", "We performed an additional analysis to directly test whether this effect is indeed lateralized in a statistical sense , that is whether the underlying effect is significantly greater in the left vs . the right hemisphere .", "First , we compared the ability to discriminate stimulus locations ( AUC values ) between the actual cluster and the corresponding grid points in the opposite hemisphere: there was no significant difference for either cluster for discriminating the auditory ( AAV ) ( CTEMP; p=0 . 10 , CPAR; p=0 . 65 , FDR corrected ) or visual stimulus locations ( VAV ) ( CTEMP; p=0 . 24 , CPAR; p=0 . 35 , FDR corrected ) .", "Second , we compared the contributions of each cluster to the VE bias .", "The auditory contribution ( regression beta for AAV ) differed significantly between hemispheres for CTEMP ( p=0 . 03 , FDR corrected ) but not for CPAR ( p=0 . 56 , FDR corrected ) .", "The visual contribution differed for neither cluster ( regression beta for VAV , p=0 . 68 , FDR corrected ) .", "Hence , the overall evidence for the neural correlates of the VE bias to be lateralized was weak , and absent for the parietal contribution .", "The results so far demonstrate that medial superior parietal activity reflects both , integration and recalibration .", "Given that the behavioral recalibration in the A trial was driven by the combined audio-visual information in the preceding AV trial ( c . f . Figure 1F ) this raises the question as to whether the neurally encoded information ( in the MEG activity in the A trial ) about the previous stimuli ( from the AV trial ) reflects previous unisensory information , or the behaviorally combined information .", "To test this , we compared the classification performance of the MEG activity in each cluster of interest ( CVAE , CTEMP , CPAR ) for the location of the previous sound ( AAV ) and the combined sensory information , as predicted by each participant’s behavioral weighting model ( i . e . , the VE bias predicted by mi3: βs*AAV + βs*VAV , s: participant", "c . f .", "Figure 1B ) .", "For parietal activity in the AV trial ( clusters CVAE and CPAR ) discriminant performance was significantly higher for the weighted multisensory than for unisensory AAV information ( two-sided paired t-test , p≤3*10−6 , for both comparisons , FDR corrected; Figure 5 ) , confirming that these regions indeed encode the integrated multisensory information .", "This difference was no longer significant when tested using the brain activity in the A trial , possibly because the overall classification performance was lower for previous than for current stimuli .", "In contrast , temporal activity ( CTEMP ) was equally sensitive to unisensory and combined multisensory information in both trials , in line with temporal regions participating both in sensory integration and unisensory processing ( Beauchamp et al . , 2004 ) .", "Given potential influences of eye position on behavioral sound localization ( Kopco et al . , 2009; Razavi et al . , 2007 ) it is important to rule out that eye movements consistently influenced the above results .", "Because eye tracking was technically impossible due to the close participant-to-screen distance , which was essential in order to promote audio-visual co-localization , we used the MEG data to confirm that participants indeed maintained fixation .", "We extracted ICA components known to reflect eye movement related artifacts based on their topographies and time courses ( Hipp and Siegel , 2013; Keren et al . , 2010 ) and determined the presence of potential EOG signals during the fixation- , stimulus- , and post-stimulus periods .", "This revealed that only 4 . 9 ± 1 . 7% ( mean ± s . e . m ) of trials exhibited evidence of potential eye-movements across all participants .", "In particular , eye-movements during the stimulus presentation period were rare ( 0 . 35 ± 0 . 12% , max 2 . 2% ) .", "Given that participants fixated the central fixation dot in both AV and A trials , this suggests that sound localization performance , or the VAE bias , are not affected by systematic differences in eye position across trials ." ], [ "Despite many behavioral studies demonstrating the robustness of multisensory trial-by-trial recalibration ( Bosen et al . , 2017; Bruns and Röder , 2015; Wozny and Shams , 2011a ) , little is known about the underlying neural substrate .", "Reasoning that recalibration relies on the persistence of information about previous stimuli , our study was guided by the quantification of where in the brain sensory information experienced during one trial can be recovered during the subsequent trial .", "This revealed persistent representations of previous acoustic information in a temporal-parietal network , suggesting that the regions known to reflect auditory spatial information within a trial also retain previous sensory evidence to mediate behavior ( At et al . , 2011; Bizley and Cohen , 2013; Lewald et al . , 2008; Zimmer and Macaluso , 2005 ) .", "Neural representations of previous visual information were also strongest in temporal and parietal regions , although classification performance was not significant at the whole brain level .", "Importantly , the behavioral data clearly revealed a lasting effect of visual information on behavior .", "Furthermore , the neuro-behavioral analysis revealed a significant contribution to behavior of neural representations of previous visual information in the superior parietal cortex .", "One reason for the weaker classification performance for previous visual stimuli could be a bias towards acoustic information in the participant’s task , which was to localize the sound rather than the visual stimulus in both trials .", "Alternatively , it could be that the visual information is largely carried by neural activity reflecting the combined audio-visual information , and hence persists directly in form of a genuine multisensory representation .", "This integrated multisensory representation comprises a stronger component of acoustic over visual spatial information , as reflected by the ventriloquist bias in the present paradigm .", "Our data indeed support this conclusion , as parietal activity within the audio-visual trial was encoding the behaviorally combined information more than the acoustic information .", "Previous work has shown that parietal activity combines multisensory information flexibly depending on task-relevance and crossmodal disparity ( Eramudugolla et al . , 2011; Rohe and Noppeney , 2016; Rohe and Noppeney , 2018 ) and the focus on reporting the sound location in our task may have led to the attenuation of the combined visual information in the subsequent trial .", "An interesting approach to test this could be to reverse the VE and thus task-relevance with very blurred visual stimulus ( Alais and Burr , 2004 ) and investigate if a symmetry in the VAE and its neural correlates hold .", "Furthermore , it is also possible , that eye movements during the response period may have contributed to reducing a persistent representation of visual information , in part as additional visual information was seen and processed during the response period ( e . g . the response cursor ) .", "Future work is required to better understand the influence of task-relevance on uni- and multisensory representations and how these are maintained over time .", "Several brain regions have been implied in the merging of simultaneous audio-visual spatial information ( Atilgan et al . , 2018; Bizley et al . , 2016; Sereno and Huang , 2014 ) .", "Our results suggest that the perceptual bias induced by vision on sound localization ( i . e . the ventriloquist effect ) is mediated by the posterior middle temporal gyrus and the superior parietal cortex , with parietal regions encoding the perceptually combined multisensory information .", "These regions are in line with previous studies , which have pinpointed superior temporal regions , the insula and parietal-occipital areas as hubs for multisensory integration ( Bischoff et al . , 2007; Bonath et al . , 2014; Callan et al . , 2015; Bonath et al . , 2007 ) .", "In particular , a series of studies revealed that both temporal and parietal regions combine audio-visual information in a reliability- and task-dependent manner ( Aller and Noppeney , 2019; Rohe et al . , 2019; Rohe and Noppeney , 2015b; Rohe and Noppeney , 2016 ) .", "However , while posterior parietal regions reflect the automatic fusion of multisensory information , more anterior parietal regions reflect an adaptive multisensory representation that follows predictions of Bayesian inference models .", "These anterior regions ( sub-divisions IPS3 and 4 in Rohe and Noppeney , 2015b ) combine multisensory information when two cues seem to arise from a common origin and only partially integrate when there is a chance that the two cues arise from distinct sources , in accordance with the flexible use of discrepant multisensory information for behavior ( Cao et al . , 2019; Körding et al . , 2007 ) .", "Noteworthy , the peak effect for the ventriloquist aftereffect found here was located at the anterior-posterior location corresponding to the border of IPS2 and IPS3 ( Wang et al . , 2015 ) , albeit more medial .", "While the significant clusters were more pronounced on the left hemisphere , a direct assessment did not provide evidence for these effects to be lateralized in a strict sense ( Liégeois et al . , 2002 ) .", "Our results hence corroborate the behavioral relevance of superior-anterior parietal representations and fit with an interpretation that these regions mediate the flexible use of multisensory information , depending on task and sensory congruency , to mediate adaptive behavior .", "In contrast , very little is known about the brain regions implementing the trial-by-trial recalibration of unisensory perception by previous multisensory information .", "In fact , most studies have relied on prolonged adaptation to multisensory discrepancies .", "Hence these studies investigated long-term recalibration , which seems to be mechanistically distinct from the trial-by-trial recalibration investigated here ( Bosen et al . , 2017; Bruns et al . , 2011; Bruns and Röder , 2010; Bonath et al . , 2007; Zierul et al . , 2017 ) .", "The study most closely resembling the present one suggested that the ventriloquist after-effect is mediated by an interaction of auditory and parietal regions ( Zierul et al . , 2017 ) , a network centered view also supported by work on the McGurk after-effect ( Kilian-Hütten et al . , 2011a; Kilian-Hütten et al . , 2011b ) .", "Yet , no study to date has investigated the direct underpinnings of multisensory recalibration at the trial-by-trial level , or attempted to directly link the neural signature of the encoded sensory information about previous stimuli to the participant-specific perceptual bias .", "Our results close this gap by demonstrating the behavioral relevance of anterior medial parietal representations of previous multisensory information , which seem to reflect the flexible combination of spatial information following multisensory causal inference , and have a direct influence on participants’ perceptual bias in localizing a subsequent unisensory stimulus .", "The retention of information about previous stimuli in parietal cortex directly links to animal work , which has revealed a mixed pattern of neural selectivity in parietal cortex , with individual neurons encoding both unisensory and multisensory information , reflecting the accumulation of this over time , and the transformation into perceptual choice ( Raposo et al . , 2014 ) .", "For example , parietal neurons are involved in maintaining the history of prior stimulus information , a role that is directly in line with our results ( Akrami et al . , 2018 ) .", "Also , the observation that the previously experienced stimuli shape both the neural encoding of the subsequent sound and neural correlates of the upcoming response ( Figure 3A , B ) suggests that these representations of prior evidence emerge in a neural system intermediate between pure sensory and pure motor ( or choice ) representations .", "Again , this fits with the observation of mixed neural representation in parietal neurons .", "Our results set the stage to directly probe the correlates of multisensory recalibration at the single neuron level , for example , to address whether integration and recalibration are mediated by the very same neural populations .", "In humans , the medial superior parietal cortex pinpointed here as mediator of recalibration has been implied in maintaining spatial and episodic memory ( Müller et al . , 2018; Pollmann et al . , 2003; Schott et al . , 2018; Uncapher and Wagner , 2009; Vilberg and Rugg , 2008 ) used for example during navigation , spatial updating or spatial search ( Brodt et al . , 2016; Pollmann et al . , 2003 ) .", "Our results broaden the functional scope of these parietal regions in multisensory perception , by showing that these regions are also involved in the integration of multiple simultaneous cues to guide subsequent spatial behavior .", "This places the medial parietal cortex at the interface of momentary sensory inference and memory , and exposes multisensory recalibration as a form of implicit episodic memory , mediating the integration of past and current information into a more holistic percept .", "Similar to other forms of memory , the history of multisensory spatial information influences perception across a range of time scales ( Bosen et al . , 2017; Bosen et al . , 2018; Bruns and Röder , 2015 ) .", "In particular , recalibration emerges on a trial-by-trial basis , as investigated here , and after several minutes of exposure to consistent discrepancies ( Frissen et al . , 2012; Kopco et al . , 2009; Mendonça et al . , 2015; Radeau and Bertelson , 1974; Razavi et al . , 2007; Recanzone , 1998 ) .", "Behavioral studies have suggested that the mechanisms underlying the trial-by-trial and long-term effects may be distinct ( Bruns and Röder , 2015; Bruns and Röder , 2017 ) .", "Yet , it remains possible that both are mediated by the same neural mechanisms , such as the same source of spatial memory ( Müller et al . , 2018; Schott et al . , 2018 ) .", "Indeed , an EEG study on multisensory long-term recalibration has reported neural correlates compatible with an origin in parietal cortex ( Bruns et al . , 2011 ) .", "The finding that medial parietal regions are involved in spatial and episodic memory and mediate trial-by-trial perceptual recalibration clearly lends itself to hypothesize that the very same regions should also contribute to long term recalibration as well .", "Eye movement patterns can affect sound localization .", "For example , changes in fixation affect early auditory cortex ( Fu et al . , 2004; Werner-Reiss et al . , 2003 ) , visual prism-adaptation results in changes in sound localization behavior ( Zwiers et al . , 2003 ) , and both prolonged peripheral fixation and the continuous use of fixation while searching for sounds can bias sound localization ( Razavi et al . , 2007 ) .", "This raises the question as to whether audio-visual recalibration emerges in head- or eye-centered coordinate systems ( Kopco et al . , 2009 ) .", "Spatial representations in the brain emerge in a number of coordinate systems ( Chen et al . , 2018; Schechtman et al . , 2012; Town et al . , 2017 ) raising the possibility that visual and auditory information is combined in any , or possibly multiple , coordinate systems .", "Indeed , one study suggested that the long-term ventriloquist after-effect arises in mixed eye-head coordinates ( Kopco et al . , 2009 ) .", "However , this study also noted that a transition from head- to mixed-spatial representations emerges slowly and only after prolonged exposure , while recalibration after a few exposure trials is best explained in head coordinates .", "With respect to the trial-by-trial recalibration investigated here , this suggests an origin in head-centered coordinates .", "The present study was not designed to disentangle different types of spatial representations , as the participants maintained fixation before , during and after the stimulus , and hence eye and head coordinates were aligned .", "Any , if present , systematic biases in fixations were eliminated on a trial-by-trial level by randomizing stimulus locations across trials and allowing participants to move their eyes while responding ( Razavi et al . , 2007 ) .", "This makes it unlikely that systematic patterns of eye movements would have affected our results , and calls for further work to elucidate the precise coordinate systems encoding the different forms or recalibration .", "The use of virtual sound locations , rather than for example an array of speakers , may have affected the participants’ tendency to bind auditory and visual cues ( Fujisaki et al . , 2004 ) .", "While the use of HRTFs is routine in neuroimaging studies on spatial localization ( Rohe and Noppeney , 2015b ) , individual participants may perceive sounds more ‘within’ the head in contrast to these being properly externalized .", "While this can be a concern when determining whether audio-visual integration follows a specific ( e . g . Bayes optimal ) model ( Meijer et al . , 2019 ) , it would not affect our results , as these are concerned with relating the trial specific bias expressed in participants behavior with the underlying neural representations .", "Even if visual and acoustic stimuli were not perceived as fully co-localized , this may have reduced the overall ventriloquist bias , but would not affect the neuro-behavioral correlation .", "Indeed , the presence of both the ventriloquist bias and the trial-by-trial recalibration effect suggests that participants were able to perceive the spatially disparate sound sources , and co-localize the sound and visual stimulus when the disparity was small .", "Navigating an ever-changing world , the flexible use of past and current sensory information lies at the heart of adaptive behavior .", "Our results show that multiple regions are involved in the momentary integration of spatial information , and specifically expose the medial superior parietal cortex as a hub that maintains multiple sensory representations to flexibly interface the past with the environment to guide adaptive behavior ." ], [ "The paradigm was based on an audio-visual localization task ( Wozny and Shams , 2011a ) .", "Trials and conditions were designed to probe both the ventriloquist effect and the ventriloquist-aftereffect .", "A typical sequence of trials is depicted in Figure 1A .", "The participants’ task was to localize a sound during either Audio-Visual ( AV ) or Auditory ( A ) , trials , or , on a subset of trials ( ~8% of total trials ) , to localize a visual stimulus ( V trials ) .", "The locations of auditory and visual stimuli were each drawn semi-independently from five locations ( −17° , –8 . 5° , 0° , 8 . 5° , 17° of visual angle from the midline ) , to yield nine different audio-visual discrepancies ( −34° , –25 . 5° , −17° , –8 . 5° , 0° , 8 . 5° , 17° , 25 . 5° , 34° ) .", "Importantly , AV and A trials were always presented sequentially ( AV trial preceding A trial ) to probe the influence of audio-visual integration on subsequent unisensory perception .", "Acoustic stimuli were spatially dispersed white noise bursts , created by applying head related transfer functions ( HRTF ) ( PKU and IOA HRTF database , Qu et al . , 2009 ) to white noise ( duration = 50 ms ) defined at specific azimuths , elevations and distances .", "Here we used a distance of 50 cm and 0 elevation .", "The behavioral data obtained during A trials confirm that participants perceived these sounds as lateralized , Sounds were sampled at 48 kHz , delivered binaurally by an Etymotic ER-30 tube-phone at ~84 . 3 dB ( root-mean-square value , measured with a Brüel and Kjær Type 2205 sound-level meter , A-weighted ) .", "An inverse filtering procedure was applied to compensate for the acoustic distortion introduced by plastic tubes required for the use in the MEG shield-room ( Giordano et al . , 2018 ) .", "The visual stimulus was a white Gaussian disk of 50 ms duration covering 1 . 5° of visual angle ( at full-width half-maximum ) .", "This was back-projected onto a semi-transparent screen located 50 cm in front of the participant , via a DLP projector ( Panasonic D7700 ) .", "Stimulus presentation was controlled with Psychophysics toolbox ( Brainard , 1997 ) for MATLAB ( The MathWorks , Inc , Natick , MA ) , with ensured temporal synchronization of auditory and visual stimuli .", "In total we repeated each discrepancy ( within or between trials ) 40 times , resulting in a total of 360 AV-A trial pairs .", "In addition , 70 visual trials were interleaved to maintain attention ( V trials always came after A trials , thus not interrupting the AV-A pairs ) , resulting in a total of 790 trials ( 2 × 360 + 70 ) for each participant .", "Trials were pseudo-randomized , and divided into 10 blocks of ~8 mins each .", "Each trial started with a fixation period ( uniform range 800 ms–1200 ms ) , followed by the stimulus ( 50 ms ) .", "After a random post-stimulus period ( uniform range 600 ms–800 ms ) the response cue emerged , and was shown as a horizontal bar along which participants could move a cursor .", "Participants responded by moving a trackball mouse ( Current Designs Inc , Philadelphia , PA 19104 USA ) with their right hand by moving the cursor to the location of the perceived stimulus and clicking the button .", "A letter ‘S’ was displayed on the cursor for ‘sound’ , and ‘V’ for the visual trials .", "There was no constraint on response times .", "Inter-trial intervals varied randomly ( uniform 1100 ms–1500 ms ) and the experiment lasted about 3 . 5 hr including preparation and breaks .", "Importantly , participants were asked to maintain fixation during the entire pre-stimulus fixation period , the stimulus , and the post-stimulus period until the response cue appeared .", "During the response itself , they could freely move their eyes .", "Participants were seated in a magnetically shielded room , 50 cm in front of a screen ( 30 . 5 cm x 40 . 5 cm , 1024 × 768 resolution ) .", "The MEG data was recorded with a 248-magnetometer , whole-head MEG system ( MAGNES 3600 WH , 4-D Neuroimaging , San Diego , CA ) at a sampling rate of 1017 . 25 Hz .", "Head positions were measured at the beginning and end of each block , using five coils marking fiducial landmarks on the head of the participants , to monitor head movements .", "Coil positions were co-digitized with the head shape ( FASTRAK , Polhemus Inc , Colchester , VT ) .", "Mean head movement across all participants for all blocks was 2 . 7 mm ± 0 . 2 mm ( mean ± s . e . m . ) .", "MEG data were preprocessed with MATLAB ( The MathWorks , Inc , Natick , MA ) using the Fieldtrip toolbox ( version 20171001 , Oostenveld et al . , 2011 ) .", "Each block was preprocessed individually ( ft_preprocessing ) .", "Epochs of −0 . 6 s ~ 0 . 6 s ( 0 = stimulus onset ) were extracted from the continuous data , and denoised using the MEG reference ( ft_denoise_pca ) .", "Resulting data was filtered between 1 ~ 48 Hz ( 4-order Butterworth filter , forward and reverse ) , and down-sampled to 100 Hz .", "Known faulty channels ( N = 3 ) were removed .", "Then , variance , maximum , minimum , and range of data across trials were calculated for each channel , and channels with extreme data were excluded .", "Outliers were defined based on interquartiles ( IQR ) ( Tukey , 1977 ) ; Q1 – 4 . 5 × IQR or above Q3 +4 . 5 × IQR .", "Weight 4 . 5 instead of the standard 1 . 5 was used since 1 . 5 was eliminating too many channels .", "Overall about 6% of all channels were excluded ( 15 . 1 ± 5 . 8 channels per participant; mean ± SD ) .", "Heart and eye-movement artifacts were removed using independent component analysis ( ICA ) with Fieldtrip ( ft_componentanalysis , ft_rejectcomponent ) , which was calculated based on 30 principal components .", "Trials with SQUID ( superconducting quantum interference device ) jumps were detected and removed ( ft_artifact_jump ) with a cutoff z-value of 20 .", "Finally , the data was manually inspected using the interquartile method ( across channels weight 2 . 5 ) to exclude outlier trials .", "On average about 2% of trials had to be discarded ( 18 . 3 ± 7 . 7 trials per participant; mean ± SD ) , including very few trials on which the mouse button did not react properly ) .", "Source reconstruction was performed using Fieldtrip ( Oostenveld et al . , 2011 ) , SPM8 ( Wellcome Trust , London , United Kingdom ) , and the Freesurfer toolbox ( Fischl , 2012 ) .", "For each participant whole-brain T1-weighted structural magnetic resonance images ( MRIs , 192 sagittal slices , 256 × 256 matrix size , 1 mm3 voxel size ) were acquired using a Siemens 3T Trio scanner ( 32-channel head coil ) .", "These were co-registered to the MEG coordinate system using a semi-automatic procedure .", "Individual MRIs were segmented and linearly normalized to a template brain ( MNI space ) .", "Next , a volume conduction model was constructed using a single-shell model based on an 8 mm isotropic grid .", "We projected the sensor-level waveforms into source space using a linear constraint minimum variance ( LCMV ) beamformer with a regularization parameter of 7% .", "Then the data was collapsed onto the strongest dipole orientation based on singular value decomposition .", "Source reconstruction was performed on each block separately , and then concatenated for further analyses .", "To extract neural signatures of the encoding of different variables of interest we applied a cross-validated regularized linear discriminant analysis ( LDA ) ( Blankertz et al . , 2011; Parra and Sajda , 2003 ) to the single trial MEG source data .", "LDA was applied to the data aligned to stimulus onset in 60 ms sliding windows , with 10 ms time-steps , using a spatial searchlight around each voxel consisting of the 27 neighboring voxels .", "For each source point υ , the LDA identifies a projection of the multidimensional source data , xv ( t ) , that maximally discriminates between the two conditions of interest , defined by a weight vector , wv , which describes a one dimensional combination of the source data , Yvt: ( 1 ) Yvt=∑i27wv∙xv ( t ) +cwith i summing over grid points within a spatial searchlight , and c being a constant .", "Classification performance was quantified using the area under the receiver operator characteristic ( AUC ) based on 6-fold cross validation .", "We identified clusters with significant classification performance at the group level by applying a cluster-based permutation procedure ( see below ) .", "Having established a set of discriminant weights , wv , one can derive single trial predictions of the neurally encoded information , Yvt , using equation ( Equation 1 ) .", "Importantly , using cross-validation one can determine the classification weights on one set of trials , and then predict the discrimination performance for a separate trials .", "The value , Yvt , of such an LDA projection can serve as a proxy to the neurally encoded signal trial information about a specific stimulus variable , and can be related for example to behavioral performance ( Grootswagers et al . , 2018; Grootswagers et al . , 2017; Kayser et al . , 2016; Philiastides et al . , 2014 ) .", "We computed separate linear discriminant classifiers based on MEG activity in either the AV trial or the A trial , and for different to-be-classified conditions of interest .", "These were the location of the auditory and visual stimuli in the AV trial ( AAV , VAV ) , the auditory stimulus in the A trial ( AA ) , and the responses in either trial ( RAV , RA ) .", "For each stimulus location or response variable , we classified whether this variable was left- or right-lateralized .", "That is , we reduced the five potential stimulus locations , or the continuous response , into two conditions to derive the classifier: we grouped trials on which the respective stimulus was to the left ( −17° , –8 . 5° ) or right ( 17° , 8 . 5° ) of the fixation point , or grouped trials on which the response was to the left ( <0° ) or the right ( >0° ) .", "The center stimuli were not used to derive the LDA classifiers .", "We used these classifiers in different neuro-behavioral models to elucidate neural mechanisms of the VE and VAE .", "We investigated models capturing potential influences of each stimulus on the neural representation of sensory information , or on the neural representation of the upcoming participant’s response .", "In addition , we investigated models directly capturing the neuro-behavioral relation between the encoded sensory information and the response bias .", "First , we determined when and where neural signatures of the encoding of single trial information , as reflected by their LDA discriminant values ( c . f . Equation 1 ) , were influenced by the sensory stimuli .", "For each searchlight and time point within a trial we determined the following models: ( 2 ) LDAAAV∼ 1+βAAV⋅AAV+βVAV⋅VAV , which captures sensory integration within the AV trials , and , ( 3 ) LDAAA∼ 1+βAA⋅AA+βAAV⋅AAV+βVAV⋅VAV , which captures how the encoding of the current sound in the A trial is influenced by previous audio-visual stimuli .", "Second , and analogously , we investigated models for the encoding of the participant’s response ( i . e . LDAR_A and LDAR_AV ) .", "It is important to note that here , the MEG activity from the stimulus- and post-stimulus period in the AV trial was used for Equation 2 , while the MEG activity from the A trial was used for Equation 3 .", "Third , we determined the contribution of the single trial representations of acoustic and visual information to the single trial behavioral response bias with the following models: ( 4 ) VE~ 1+βLDAA_AV∙LDAAAV+βLDAV_AV∙LDAVAV ( 5 ) VAE~ 1+βLDAA_AV∙LDAAAV+βLDAV_AV∙LDAVAV In these models , the response biases VE and VAE were the continuous localization data ( in visual degrees ) obtained from the participant response , while the LDA components ( from Equation 1 ) reflect the continuous-valued prediction of the degree of lateralization of the respective stimulus extracted from the MEG activity .", "Importantly , each model is computed based on the MEG activity and the behavioral data in the respective trials ( VE in the AV trial , VAE in the A trial .", "That is , the VE model ( Equation 4 ) uses the behavioral response ( VE ) from the AV trial and the representation ( LDA component ) of the two stimuli in the MEG activity from the same trial .", "In contrast , the VAE model ( Equation 5 ) uses the behavioral response ( VAE ) from the A trial , and the representation ( LDA component ) of the two stimuli presented in the previous AV trial , reflected in the MEG activity from the A trial .", "Hence , each model uses the neural representation of stimuli from a different trial .", "A predictor of the current sound ( AA ) was not included in Equation 5 , as the VAE bias quantifies the deviation of the behavioral response from the current sound ( c . f . Figure 1 ) .", "To avoid overfitting , we computed these models based on 6-fold cross-validation , using distinct sets of trials to determine the weights of the LDA and to compute the regression models .", "We then computed group-level t-values for the coefficients for each regressor at each grid and time point , and assessed their significance using cluster-based permutation statistics ( below ) .", "Note that for AV trials , we excluded the AV pairs with the most extreme discrepancies ( ±34° ) , as these were inducing strong correlations between the regressors .", "To test the significance of the behavioral biases we used two-sided Wilcoxon signed rank tests , correcting for multiple tests using the Holm procedure with a family-wise error rate of p=0 . 05 .", "Group-level inference on the 3-dimensional MEG source data was obtained using randomization procedures and cluster-based statistical enhancement controlling for multiple comparisons in source space ( Maris and Oostenveld , 2007; Nichols and Holmes , 2002 ) .", "First , we shuffled the sign of the true single-subject effects ( the signs of the chance-level corrected AUC values; the signs of single-subject regression beta’s ) and obtained distributions of group-level effects ( means or t-values ) based on 2000 randomizations .", "We then applied spatial clustering based on a minimal cluster size of 6 and using the sum as cluster-statistics .", "For testing the LDA performance , we thresholded effects based on the 99th percentile of the full-brain distribution of randomized AUC values .", "For testing the betas in regression models , we used parametric thresholds corresponding to a two-sided p=0 . 01 ( tcrit = 2 . 81 , d . f . = 23; except for the analysis for the visual location in the AV trial ( tcrit = 6 . 60;", "c . f .", "Figure 4 ) .", "The threshold for determining significant clusters for classification performance was p≤0 . 01 ( two-sided ) , that for significant neuro-behavioral effects ( Equation 2-5 ) p≤0 . 05 ( two-sided ) .", "To simplify the statistical problem , we tested for significant spatial clusters at selected time points only .", "These time points were defined based on local peaks of the time courses of respective LDA AUC performance ( for models in Equations 2-3 ) , and the peaks for the beta time-course for the behavioral models ( Equations 4-5 ) .", "Furthermore , where possible , to test for the significance of individual regressors we applied a spatial a priori mask derived from the significance of the respective LDA AUC values to further ensure that neuro-behavioral effects originate from sources with significant encoding effects ( Giordano et al . , 2017 ) .", "Note that this was not possible for LDAV_AV for which we used the full brain to test for significant model effects .", "The behavioral data presented in Figure 1 and LDA performance data and source regression data used to calculate the t-values in Figures 2–5 , as well as data for Figure 4—figure supplement 1 , have been deposited to Dryad ( https://doi . org/10 . 5061/dryad . t0p9c93 ) ." ] ]

[ "Perception adapts to mismatching multisensory information , both when different cues appear simultaneously and when they appear sequentially .", "While both multisensory integration and adaptive trial-by-trial recalibration are central for behavior , it remains unknown whether they are mechanistically linked and arise from a common neural substrate .", "To relate the neural underpinnings of sensory integration and recalibration , we measured whole-brain magnetoencephalography while human participants performed an audio-visual ventriloquist task .", "Using single-trial multivariate analysis , we localized the perceptually-relevant encoding of multisensory information within and between trials .", "While we found neural signatures of multisensory integration within temporal and parietal regions , only medial superior parietal activity encoded past and current sensory information and mediated the perceptual recalibration within and between trials .", "These results highlight a common neural substrate of sensory integration and perceptual recalibration , and reveal a role of medial parietal regions in linking present and previous multisensory evidence to guide adaptive behavior ." ]

[ "A good ventriloquist will make their audience experience an illusion .", "The speech the spectators hear appears to come from the mouth of the puppet and not from the puppeteer .", "Moviegoers experience the same illusion: they perceive dialogue as coming from the mouths of the actors on screen , rather than from the loudspeakers mounted on the walls .", "Known as the ventriloquist effect , this ‘trick’ exists because the brain assumes that sights and sounds which occur at the same time have the same origin , and it therefore combines the two sets of sensory stimuli .", "A version of the ventriloquist effect can be induced in the laboratory .", "Participants hear a sound while watching a simple visual stimulus ( for instance , a circle ) appear on a screen .", "When asked to pinpoint the origin of the noise , volunteers choose a location shifted towards the circle , even if this was not where the sound came from .", "In addition , this error persists when the visual stimulus is no longer present: if a standard trial is followed by a trial that features a sound but no circle , participants perceive the sound in the second test as ‘drawn’ towards the direction of the former shift .", "This is known as the ventriloquist aftereffect .", "By scanning the brains of healthy volunteers performing this task , Park and Kayser show that a number of brain areas contribute to the ventriloquist effect .", "All of these regions help to combine what we see with what we hear , but only one maintains representations of the combined sensory inputs over time .", "Called the medial superior parietal cortex , this area is unique in contributing to both the ventriloquist effect and its aftereffect .", "We must constantly use past and current sensory information to adapt our behavior to the environment .", "The results by Park and Kayser shed light on the brain structures that underpin our capacity to combine information from several senses , as well as our ability to encode memories .", "Such knowledge should be useful to explore how we can make flexible decisions ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "developmental biology", "structural biology and molecular biophysics" ]

[ [ "Wnt morphogens , secreted cysteine-rich palmitoleoylated glycoproteins , play critical roles in cell-fate determination , tissue homeostasis and embryonic development ( Clevers and Nusse , 2012; Malinauskas and Jones , 2014 ) .", "Aberrant Wnt signalling leads to cancer , osteoporosis and degenerative illnesses ( Anastas and Moon , 2013 ) .", "Norrie Disease Protein ( NDP ) gene encodes Norrin ( Berger et al . , 1992; Chen et al . , 1992 ) , a secreted cystine-knot like growth factor , distinct from the lipid-modified Wnt ( Willert et al . , 2003 ) .", "Norrin activates the canonical Wnt/β-catenin pathway by interaction with Wnt receptor Frizzled4 cysteine-rich domain ( Fz4CRD ) , and co-receptor low density lipoprotein receptor related protein 5/6 ectodomain ( Lrp5/6ECD ) , plus the auxiliary four-pass transmembrane protein Tetraspanin-12 ( Tspan-12 ) and glycosaminoglycans ( GAGs ) of heparan sulfate proteoglycans ( HSPGs ) ( Xu et al . , 2004; Junge et al . , 2009; Ke et al . , 2013 ) .", "The Norrin mediated pathway maintains the blood-retina and blood-brain barriers ( Wang et al . , 2012 ) and regulates angiogenesis in the cochlea and uterus ( Rehm et al . , 2002; Ye et al . , 2011 ) as well as neuroprotective effects on retinal neurons ( Ohlmann et al . , 2010; Seitz et al . , 2010 ) .", "Mutations in the NDP gene and the receptor genes , FZ4 , LRP5 , and TSPAN-12 , have been identified for vitreoretinal diseases including Norrie Disease , Familial Exudative Vitreoretinopathy , and Coats' Disease ( Nikopoulos et al . , 2010; Ye et al . , 2010; Ohlmann and Tamm , 2012 ) .", "NDP , FZ4 , LRP5 , and TSPAN-12 knock-out mice experiments further support the notion that dysfunctional Norrin signalling results in impaired retinal angiogenesis ( Richter et al . , 1998; Kato et al . , 2002; Robitaille et al . , 2002; Xu et al . , 2004; Junge et al . , 2009 ) .", "Unlike Wnts which have promiscuous interactions with Fz receptors , Norrin specifically binds to Fz4CRD , but not to the 14 other CRDs of Fz and secreted Frizzled-related protein ( sFRP ) family members ( Hsieh et al . , 1999; Smallwood et al . , 2007 ) .", "Similar to Wnt , Norrin ( 1 ) binds to Lrp5/6ECD ( Ke et al . , 2013 ) ; ( 2 ) interacts with HSPGs and shows limited spatial diffusion ( Perez-Vilar and Hill , 1997; Xu et al . , 2004; Smallwood et al . , 2007; Ohlmann et al . , 2010 ) .", "As well as being a potential target for therapeutic interventions , an understanding of Norrin mediated signalling will also provide insights into the fundamental features required to trigger canonical Wnt/β-catenin signalling .", "Structural analyses of the extracellular components and interactions mediating Norrin signalling were considered to be challenging because of the difficulties of generating recombinant Norrin ( Perez-Vilar and Hill , 1997; Shastry and Trese , 2003; Ohlmann et al . , 2010 ) .", "Ke et al . ( 2013 ) reported a refolding method ( from Escherichia coli inclusion bodies ) to produce active recombinant Norrin fused with a N-terminal maltose binding protein ( MBP-Norrin ) , an advance that enabled them to determine the crystal structure of MBP-Norrin .", "Here , we develop an efficient mammalian cell expression method to produce active untagged recombinant Norrin and detail the structural and functional properties of this potential therapeutic agent .", "Our crystallographic and solution studies further reveal that dimeric Norrin forms a complex with two copies of monomeric Fz4CRD .", "Our molecular level analysis of the Norrin–Fz4CRD complex bound with GAG analogue , in combination with structure-guided biophysical and cell-based studies , defines the basis for ligand recognition .", "Structural comparison with the Xenopus Wnt8 in complex with mouse Fz8CRD ( Janda et al . , 2012 ) shows that Norrin uses its β-strands to mimic a finger-like loop in Wnt for binding to the Fz receptor CRD .", "Finally , we note that engineered Norrin mutants resulting from our analyses may be of use as agents for blocking Wnt receptor activation ." ], [ "To address the challenge of producing Norrin in large quantities , we screened conditions and constructs for Norrin expression ( Figure 1A ) .", "We found that fusion of Norrin to the C-terminus of small ubiquitin-like modifier ( SUMO ) ( Peroutka et al . , 2008 ) , in combination with addition of valproic acid ( Backliwal et al . , 2008 ) , a putative histone deacetylase inhibitor , substantially boosted expression of the secreted protein in human embryonic kidney ( HEK ) 293T cells ( Figure 1B , C ) .", "After removal of the SUMO fusion tag , the recombinant Norrin shows a monodispersed state in size-exclusion chromatography ( SEC; Figure 1D ) and is biologically active in a cell-based luciferase reporter assay ( Figure 1E ) . 10 . 7554/eLife . 06554 . 003Figure 1 . Expression and purification of biologically active recombinant Norrin .", "( A ) Schematic diagrams of the expression constructs including Norrin ( a signal peptide , SP , followed by Norrin and Rho-1D4 tag at C-terminus ) and SUMO-Norrin ( a SP followed by a Strep-tag II , an octahistidine , SUMO , HRV 3C protease cleavage site , Norrin , and Rho-1D4 tag at C-terminus ) .", "( B and C )", "Conditioned media from transfected HEK293T cells were immunoblotted ( IB ) with the anti-Rho-1D4 antibody .", "( B ) SUMO fusion improves Norrin secreted expression .", "( C ) The expression level of SUMO tagged Norrin was further boosted for HEK-293T cells treated with valproic acid .", "( D ) SEC elution profile and SDS-PAGE under reducing conditions with fractions analysed marked by red lines .", "( E ) Purified recombinant untagged Norrin actives the canonical Wnt/β-catenin pathway in the luciferase reporter assay .", "RLU: relative light unit .", "Error bars indicate standard deviations ( n = 3 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06554 . 003 We determined three crystal structures of Norrin ( Figure 2A and Table 1 ) , using selenomethionine-labeled protein for phasing ( Figure 2—figure supplement 1 ) .", "The Norrin protein fold is identical to that of the previously reported MBP-Norrin crystal structure ( Ke et al . , 2013 ) .", "Each Norrin monomer comprises three β-hairpins ( β1-β2 , β3-β4 and β5-β6 ) , a β7 strand at the C-terminus , and four intramolecular disulphide bonds ( Figure 2—figure supplement 2 ) .", "The two monomers assemble as an elongated , head-to-tail , dimer ( Figure 2A ) stabilized by three intermolecular disulphide bridges ( Cys93-Cys95 , Cys95-Cys93 , and Cys131-Cys131 ) , in agreement with small-angle X-ray scattering ( SAXS ) measurements which showed Norrin dimer in solution ( Figure 2—figure supplement 3A and Table 2 ) .", "The dimer interface is further stabilized by extensive hydrogen bonds and hydrophobic interactions ( Figure 2—figure supplement 3B , C ) .", "Superposition of all molecules in the asymmetric units from our three crystal forms with the MBP-Norrin structure ( Ke et al . , 2013 ) showed an average root-mean-square ( r . m . s . ) deviation of 1 . 5 Å over 190 equivalent Cα atoms ( Figure 2—figure supplement 3D ) .", "On inspection the superpositions revealed a high degree of conformational plasticity in the β1-β2 , β3-β4 and β5-β6 loops ( Figure 2B ) .", "The flexibility inherent in these regions is consistent with the relatively high crystallographic B factor values ( Figure 2C ) .", "Conversely , the structural comparisons underscore the conserved nature of the interface at the dimer core .", "It has previously been noted that disruption of the dimer by either Cysteine-to-Alanine mutations of intermolecular disulphide bonds or mutations of hydrophobic residues at the dimer interface results in a loss of Norrin-mediated signalling ( Smallwood et al . , 2007; Ke et al . , 2013 ) . 10 . 7554/eLife . 06554 . 004Figure 2 . Crystal structure and structural analysis of apo Norrin .", "( A ) Schematic diagram of Norrin is rainbow coloured and disulphide bonds are drawn as lines .", "Cartoon representation of dimeric Norrin .", "Four intramolecular disulphide bonds are shown as magenta sticks .", "Cys93 , Cys95 , and Cys131 ( forming intermolecular disulphide bridges ) are shown as cyan , blue , and green sticks , respectively .", "Two cystine-knot motifs are marked with dotted boxes and the filled circles denote the N- and C-termini .", "( B ) Ribbon diagram of superpositions of Norrin molecules from the asymmetric unit of crystal form I ( green , chain A and B; cyan , chain C and D; magenta , chain E and F ) , crystal form II ( yellow , chain A and B; blue , chain C and D ) , crystal form III ( grey , chain A and B; purple , chain C and D ) , and MBP-Norrin ( cyan; PDB ID: 4MY2 ) .", "The flexible regions are highlighted as red lines .", "Loop regions ( β1-β2 loop , β3-β4 loop , and β5-β6 loop ) show structural plasticity .", "The well ordered regions include the cystine-knot motifs plus intermolecular disulphide linked areas ( black circle ) and two Fz4 binding sites ( cyan dotted circle ) .", "( C ) Representative Norrin dimer displayed with the diameter of the Cα tube defined by Cα atom B factor ( small tube means structural rigidity; large tube indicates structural flexibility ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06554 . 00410 . 7554/eLife . 06554 . 005Figure 2—figure supplement 1 . Electron density map of Norrin structure . The initial density modified map from PHENIX AUTOSOL ( Terwilliger , 2000 , Terwilliger et al . , 2009 ) calculated with experimental Se-Met SAD phases is contoured at 1 . 5 σ and shown as blue meshes .", "The initial model from BUCCANEER ( Cowtan , 2006 ) is shown as magenta ribbon diagram .", "The anomalous difference map for Se-Met is contoured at 4 σ as green meshes with Se-Met residues labelled . DOI: http://dx . doi . org/10 . 7554/eLife . 06554 . 00510 . 7554/eLife . 06554 . 006Figure 2—figure supplement 2 . Multiple sequence alignment of Norrin . Secondary structure element colouring corresponds to Figure 2A .", "The magenta boxes represent conserved cysteine residues in the cystine-knot growth factor superfamily , whereas the yellow boxes denote Norrin specific conserved cysteine residues .", "Disulphide bridges are numbered and are drawn in magenta lines for the intramolecular disulphide bonds .", "Three cysteine residues ( highlighted in cyan ) that form intermolecular disulphide bridges are drawn in cyan , blue and green lines .", "Triangles indicate the mutation sites for SPR binding and luciferase reporter assays .", "Residues involved in binding of Fz4 , GAG , and Lrp5/6 are marked with blue , green , and yellow filled boxes , respectively .", "Residues involved in dimerization are highlighted with orange filled boxes .", "Black boxes denote the region of the Wnt8 index finger involved in Fz8CRD binding , which overlaps with Norrin for Fz4CRD binding ( Figure 9 ) .", "Disease-associated residues are marked by coloured dots according to the types of mutations ( purple , missense; cyan , frame-shift; black , nonsense ) .", "Cysteine residues associated with diseases are marked below the sequence with red filled boxes .", "NCBI accession numbers: Human Norrin , NP_000257; Monkey Norrin , XP_528948; Panda Norrin , XP_002928194; Pig Norrin , NP_001106528; Dog Norrin , XP_855261; Bovine Norrin , AAI12739; Horse Norrin , XP_001490401; Rabbit Norrin , XP_002719919; Mouse Norrin , NP_035013; Chicken Norrin , XP_416765; Frog Norrin , NP_001154869; Fish Norrin , XP_001338820 . DOI: http://dx . doi . org/10 . 7554/eLife . 06554 . 00610 . 7554/eLife . 06554 . 007Figure 2—figure supplement 3 . Norrin solution structure and structural analyses .", "( A ) SAXS analysis of Norrin .", "The experimental scattering data ( black circles ) and calculated scattering pattern ( green line ) are shown and the Norrin solution structure model is shown in cartoon representation .", "The upper right inset shows the experimental ( black circles ) and calculated ( green line ) Guinier region .", "The dashed lines delimit the range of fitting for Radius of gyration ( Rg ) analysis ( Rg·S ≤ 1 . 3 ) .", "The bottom right inset shows the experimental ( black line ) and calculated ( green line ) pair distance distribution P ( r ) curve .", "( B ) Hydrophobic interactions for Norrin dimerization beyond the cystine-knot motif .", "Resides on hairpin A and C ( yellow sticks ) form hydrophobic contacts with the residues on hairpin B ( cyan sticks ) from another monomer .", "Residues associated with diseases are boxed .", "( C ) Table of hydrogen bond and salt-bridge interactions in the Norrin dimer interface .", "Missense and nonsense mutations are highlighted as filled grey and purple backgrounds , respectively .", "( D ) Detailed information of structural comparison shown as root mean square ( r . m . s ) deviation ( Å ) values and the number of aligned Cα atoms ( in brackets ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06554 . 00710 . 7554/eLife . 06554 . 008Table 1 . Data collection , phasing and refinement statisticsDOI: http://dx . doi . org/10 . 7554/eLife . 06554 . 008Norrin–Fz4CRD–SOSMethylated Norrin–Fz4CRDNorrinNorrin Se-MetMethylated NorrinCrystal formIIIIData collectionSpace groupP6122P4322P212121P212121P212121Cell dimensions a , b , c ( Å ) 119 . 1 , 119 . 1 , 119 . 298 . 9 , 98 . 9 , 120 . 446 . 4 , 79 . 1 , 243 . 345 . 8 , 78 . 8 , 232 . 8102 . 7 , 53 . 1 , 96 . 1 α , β , γ ( ° ) 90 , 90 , 9090 , 90 , 12090 , 90 , 9090 , 90 , 9090 , 90 , 90PeakWavelength0 . 92000 . 97950 . 96860 . 97950 . 9795Resolution ( Å ) 47 . 34–3 . 00 ( 3 . 18– 3 . 00 ) 49 . 46–2 . 30 ( 2 . 38–2 . 30 ) 65 . 56–2 . 40 ( 2 . 49–2 . 40 ) 116 . 39–3 . 18 ( 3 . 26–3 . 18 ) 33 . 65–2 . 00 ( 2 . 05–2 . 00 ) Rpim ( % ) 3 . 1 ( 54 . 8 ) 4 . 5 ( 56 . 1 ) 6 . 1 ( 42 . 3 ) 2 . 8 ( 23 . 4 ) 4 . 1 ( 58 . 3 ) I/σI14 . 6 ( 1 . 6 ) 10 . 7 ( 1 . 4 ) 7 . 8 ( 1 . 9 ) 20 . 2 ( 3 . 0 ) 9 . 1 ( 1 . 7 ) Completeness ( % ) 100 ( 100 ) 98 . 9 ( 97 . 2 ) 99 . 9 ( 100 ) 99 . 9 ( 99 . 9 ) 100 ( 100 ) Redundancy19 . 6 ( 20 . 6 ) 6 . 0 ( 5 . 6 ) 5 . 6 ( 5 . 7 ) 33 . 3 ( 9 . 9 ) 5 . 6 ( 5 . 8 ) RefinementResolution ( Å ) 47 . 34–3 . 00 ( 3 . 18–3 . 00 ) 49 . 46–2 . 30 ( 2 . 38–2 . 30 ) 65 . 56–2 . 40 ( 2 . 49–2 . 40 ) 33 . 65–2 . 00 ( 2 . 05–2 . 00 ) No .", "reflections10 , 503 ( 1648 ) 26 , 816 ( 2514 ) 34 , 722 ( 3384 ) 36 , 272 ( 2635 ) Rwork/Rfree21 . 5/26 . 719 . 7/22 . 121 . 6/26 . 223 . 3/24 . 8No .", "atoms Protein1759255749303187 Ligand/ion833910110 Water0115164122B-factors Protein113637057 Ligand/ion133719273 Water0575551R . m .", "s deviations Bond lengths ( Å ) 0 . 0050 . 0040 . 0090 . 005 Bond angles ( ° ) 1 . 180 . 931 . 081 . 07Ramachandran plot Favored ( % ) 95 . 597 . 096 . 797 . 2 Allowed ( % ) 4 . 53 . 03 . 32 . 8PDB code5BQC5BQE5BPU5BQ8NorrinFz4CRDFz4CRDCrystal formIIIIIIData collectionSpace groupC121P212121P61Cell dimensions a , b , c ( Å ) 86 . 8 , 38 . 1 , 177 . 272 . 6 , 102 . 1 , 116 . 576 . 1 , 76 . 1 , 204 . 5 α , β , γ ( ° ) 90 , 94 , 9090 , 90 , 9090 , 90 , 90Wavelength0 . 97950 . 96860 . 9686Resolution ( Å ) 44 . 19–2 . 30 ( 2 . 38–2 . 30 ) 41 . 77–2 . 20 ( 2 . 27–2 . 20 ) 47 . 37–2 . 40 ( 2 . 49–2 . 40 ) Rpim ( % ) 2 . 8 ( 36 ) 4 . 1 ( 49 . 5 ) 2 . 6 ( 33 . 9 ) I/σI16 . 7 ( 2 . 0 ) 12 . 8 ( 2 . 0 ) 14 . 5 ( 2 . 2 ) Completeness ( % ) 99 . 2 ( 97 . 7 ) 99 . 2 ( 99 . 7 ) 99 . 5 ( 99 . 4 ) Redundancy5 . 8 ( 6 . 0 ) 4 . 3 ( 4 . 4 ) 4 . 0 ( 4 . 1 ) RefinementResolution ( Å ) 44 . 19–2 . 30 ( 2 . 38–2 . 30 ) 41 . 77–2 . 20 ( 2 . 27–2 . 20 ) 47 . 37–2 . 40 ( 2 . 49–2 . 40 ) No .", "reflections26 , 073 ( 2538 ) 44 , 268 ( 3802 ) 25 , 975 ( 2724 ) Rwork/Rfree22 . 1/25 . 017 . 7/22 . 320 . 3/24 . 3No .", "atoms Protein310438663877 Ligand/ion727099 Water5414869B-factors Protein914776 Ligand/ion726772 Water1424368R . m .", "s deviations Bond lengths ( Å ) 0 . 0060 . 010 . 005 Bond angles ( ° ) 1 . 031 . 350 . 94Ramachandran plot Favored ( % ) 96 . 099 . 097 . 0 Allowed ( % ) 4 . 01 . 03 . 0PDB code5BQB5BPB5BPQAll structures were determined from one crystal . Values in parentheses are for highest-resolution shell . 10 . 7554/eLife . 06554 . 009Table 2 . Molecular properties of the proteins determined by SAXSDOI: http://dx . doi . org/10 . 7554/eLife . 06554 . 009ProteinsN-Glyc stateRg ( nm ) *Dmax ( nm ) †Volume porod ( Vp [nm3] ) MWTheoretical ( kDa ) ‡MWMeasured ( KDa ) §MWMeasured ( KDa ) #Fz4CRDdeglyc¶1 . 986 . 9333 . 017 . 1 ( monomer ) 15 . 919 . 9Fz4CRDglyc**2 . 247 . 8441 . 121 . 4 ( monomer ) 23 . 724 . 7Norrin2 . 749 . 1837 . 427 . 2 ( dimer ) 33 . 522 . 5Norrin–Fz4CRDdeglyc¶3 . 4111 . 9293 . 861 . 3 ( 2:2 complex ) 57 . 956 . 5*Rg is Radius of gyration , calculated from Guinier plot using AutoRg ( Petoukhov et al . , 2012 ) .", "†Dmax is the maximum dimension of the particle , calculated by GNOM ( Svergun , 1992 ) .", "‡The theoretical molecular weight ( MWTheoretical ) is predicated from amino acid sequence plus the molecular weight of N-linked glycans ( see ‘Materials and methods’ , SEC-MELS analysis for detailed information of calculation ) .", "§The measured molecular weight ( MWMeasured ) is calculated from forward scattering of sample ( I ( 0 ) ) by comparison with reference bovine serum albumin ( BSA ) .", "#The measured molecular weight ( MWMeasured ) is obtained by dividing the Volume Porod ( Vp [nm3] ) by 1 . 66 ( Rambo and Tainer , 2011 ) .", "¶The proteins were produced from HEK293T cells in the presence of kifunensine with limited glycosylation and treated with endoglycosidase-F1 . **The proteins were produced from HEK293T cells with full glycosylation .", "We determined two crystal structures of Fz4CRD ( Figure 3 and Table 1 ) .", "Similar to mouse Fz8CRD ( Dann et al . , 2001 ) the Fz4CRD fold comprises four α helices ( Figure 3A and Figure 3—figure supplement 1A ) stabilized by five disulphide bridges ( Cys45–Cys106 , Cys53–Cys99 , Cys90–Cys128 , Cys117–Cys158 , Cys121–Cys145 ) .", "The N-acetylglucosamines on two N-linked glycosylation sites at Asn59 and Asn144 are visible in the electron density map ( Figure 3A ) .", "Superposition of all Fz4CRD molecules in the asymmetric units from two crystal forms revealed a well-ordered protein fold ( Figure 3—figure supplement 1B ) .", "The conserved disulphide bonds in FzCRD superfamily members are essential for functional activity .", "Familial Exudative Vitreoretinopathy disease mutant C45Y results in misfolded protein retained in the endoplasmic reticulum , similar to the effects of Cysteine-to-Alanine mutations in the related CRD of Drosophila Smoothened ( Smo ) ( Zhang et al . , 2011; Rana et al . , 2013 ) .", "Structural comparison showed Fz4CRD closely resembles the CRDs of mouse Fz8 and secreted Frizzled-related protein 3 ( sFRP3 ) with an average r . m . s . deviation of 1 . 2 Å over 115 equivalent Cα atoms ( Figure 3—figure supplement 1C–E ) and approximate sequence identity of 35% .", "Comparisons with the CRDs of muscle-specific kinase ( MuSk ) and Smo showed more substantial structural differences with an average r . m . s . deviation of 2 . 3 Å over 86 equivalent Cα atoms ( Figure 3—figure supplement 1F–H ) . 10 . 7554/eLife . 06554 . 010Figure 3 . Crystal and solution structures of unliganded Fz4CRD .", "( A ) Schematic domain organization ( SP , signal peptide; TM . transmembrane domain; CD , cytoplasmic domain ) .", "Crystallization constructs are rainbow coloured .", "Disulphide bonds are drawn and blue hexagons denote N-linked glycosylation sites .", "Cartoon representation of Fz4CRD in rainbow colouring .", "N-linked N-acetyl-glucosamines ( GlcNAc ) and disulphide bonds are shown as blue sticks .", "( B ) SEC-MALS experiments .", "The red line represents the molecular weight ( left ordinate axis ) and black lines show the differential refractive index ( right ordinate axis ) as well as SDS-PAGE ( Inset ) .", "The numbers denote the corresponding molecular weights of each peak .", "( C and D )", "SAXS analyses of deglycosylated and glycosylated Fz4CRD solution structures .", "The experimental scattering data ( black circles ) and calculated scattering patterns ( coloured lines ) are shown and the Fz4CRD solution structure model is presented .", "The upper right inset shows the experimental ( black circles ) and calculated ( coloured lines ) Guinier region .", "The dashed lines delimit the range of fitting for Rg analysis ( Rg·S ≤ 1 . 3 ) .", "The bottom right inset shows the experimental ( black circles ) and calculated ( coloured lines ) pair distance distribution P ( r ) curve . DOI: http://dx . doi . org/10 . 7554/eLife . 06554 . 01010 . 7554/eLife . 06554 . 011Figure 3—figure supplement 1 . Multiple sequence alignment and structural analysis of cysteine-rich like domains .", "( A ) Secondary structure assignment colouring corresponds to Figure 3A .", "The conserved cysteine residues ( highlighted in cyan ) form five disulphide bridges , drawn in black lines and labelled as I–V .", "Notably , Smo has a different cysteine pair arrangement in disulphide bridge IV .", "Cysteine residues of Fz4 are numbered with blue filled boxes below the sequence alignment .", "The red boxes denote the residues of Fz4 that contact with Norrin , whereas residues of Fz8 are boxed in blue to indicate binding to Wnt8 index finger ( site", "2 ) and in yellow for interaction with Wnt8 PAM ( site 1 ) .", "Coloured lines indicate Fz4CRD loops that interact with Norrin .", "The N-glycosylation sites are highlighted in green and those of Fz4 are marked by blue hexagons .", "The purple dots mark residues associated with human retinal diseases ( missense mutations; http://www . uniprot . org/uniprot/Q9ULV1 ) .", "Red asterisks denote the residues of Fz4 involving in GAG binding .", "The black arrows indicate residues potentially determining ligand-binding specificity ( Figure 9D-F ) .", "Sequences are from the following UniProt entries: hFz1 , Q9UP38; hFz2 , Q14332; hFz3 , Q9NPG1; hfz4 , Q9ULV1; hfz5 , Q13467; hFz6 , O60353; hFz7 , O75084; hfz8 , Q9H461; hfz9 , O00144; hfz10 , Q9ULW2; sfrp1 Q8N474; sfrp2 , Q96HF1; sfrp3 Q92765; sfrp4 , Q6FHJ7; sfrp5 , Q5T4F7; hSmo , Q99835; rMuSk , Q62838; hROR1 , Q01973; hROR2 , Q01974 .", "( B ) Superimposition of all molecules of unliganded Fz4CRD ( crystal form I and II ) reveals no major conformational changes .", "The main difference between Fz4CRD structures is the point at which the C-terminal region becomes disordered ( black , green , and cyan arrows indicate residue 162 , 165 , and 172 , respectively ) .", "The N-linked glycans are shown as stick models .", "The encircled N and C indicate the N- and C- termini .", "( C–H )", "Structural comparison of Fz4CRD with CRDs of Fz-like proteins .", "Ribbon diagram of superposition of Fz4CRD crystal form I ( magenta ) with ( C ) complexed mouse Fz8CRD ( cyan; PDB ID: 4F0A ) , ( D ) unliganded mouse Fz8CRD ( cyan; PDB ID: 1IJY ) , ( E ) mouse sFRP3CRD ( green; PDB ID: 1IJX ) , ( F ) rabbit MuSkCRD ( yellow; PDB ID: 3HKL ) , ( G ) zebrafish SmoCRD ( red; PDB ID: 4C79 ) , and ( H ) drosophila SmoCRD ( red; PDB ID: 2MAH ) .", "( sfrp = secreted frizzled related protein; Smo = Smoothened; MuSk = muscle-specific kinase; ROR = tyrosine-protein kinase transmembrane receptor ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06554 . 01110 . 7554/eLife . 06554 . 012Figure 3—figure supplement 2 . Distinct dimeric assembly of Fz4CRD and mouse Fz8CRD observed from crystal structures .", "( A ) The superimposition of Fz4CRD structures from two crystal forms , coloured as pink and magenta for crystal form I and green and gray for crystal form II .", "( B ) The structure of dimeric mouse Fz8CRD ( PDB: 1IJA ) coloured as cyan and blue .", "The encircled N and C denote the N- and C-termini . DOI: http://dx . doi . org/10 . 7554/eLife . 06554 . 012 Fz receptors are members of the GPCR family ( Nichols et al . , 2013 ) , known for formation of receptor dimers , although it is unclear whether dimerization is mediated by the CRD , transmembrane helices or intracellular domain .", "In the case of Fz4 , β-galactosidase complementation in combination with bioluminescence resonance energy transfer and split-yellow fluorescence protein assays suggest that Fz4 exists as dimer on the cell membrane in the absence of Norrin or Wnts ( Kaykas et al . , 2004; Ke et al . , 2013 ) .", "However , ligand-independent receptor dimerization of Fz4 is not sufficient to activate signalling ( Xu et al . , 2004; Ke et al . , 2013 ) .", "Interestingly , we found that our Fz4CRD structures form the same dimeric assembly in two crystal lattices ( r . m . s . deviation of 0 . 7 Å over 238 equivalent Cα atoms from two crystal forms; Figure 3—figure supplement 2A ) .", "The dimer interface has an average 1330 Å2 buried surface area , in agreement with the characteristics of known protein–protein interfaces ( Lawrence and Colman , 1993 ) .", "However , this Fz4CRD dimer ( front-to-front ) is distinct from the previously reported crystal structure of mouse Fz8CRD dimer ( back-to-back; Figure 3—figure supplement 2B ) ( Dann et al . , 2001 ) .", "We were therefore curious to assess the dimerization characteristics of the CRDs of Fz receptors .", "Size-exclusion chromatography coupled to multi-angle light scattering ( SEC-MALS ) results ( Figure 3B and Table", "3 ) showed Fz4CRD , Fz5CRD and Fz8CRD exist as monomers in solution at 50 μM concentration , in agreement with previously reported SEC studies of Fz8CRD and SEC-MALS analyses of MuSKCRD and SmoCRD ( Stiegler et al . , 2009; Nachtergaele et al . , 2013 ) .", "SAXS measurements further support the conclusion that Fz4CRD is monomeric in solution at 290 μM concentration ( Figure 3C , D ) .", "Taken together , our results suggest that the CRDs of Fz receptors exist as monomers and may not be involved in receptor dimerization; multiple GPCRs dimerize through their hepta-helical transmembrane domains ( Rios et al . , 2001 ) .", "However , we cannot exclude the possibility that in the environment of the cellular membrane the weak interaction propensities of the CRDs , in combination with the transmembrane domains , are important for the dimerization of Fz receptors . 10 . 7554/eLife . 06554 . 013Table 3 . Molecular properties of the proteins determined by SEC-MALSDOI: http://dx . doi . org/10 . 7554/eLife . 06554 . 013ProteinNumber of N-glyc sitesN-Glyc stateMWTheoretical ( kDa ) ‡MWMeasured ( KDa ) Fz4CRD2deglyc*17 . 1 ( monomer ) 15 . 7 ± 0 . 4Fz4CRD2glyc†21 . 4 ( monomer ) 23 . 6 ± 0 . 3mFz5CRD2glyc†22 . 2 ( monomer ) 23 . 9 ± 0 . 9mFz8CRD2glyc†22 . 1 ( monomer ) 23 . 7 ± 0 . 2Norrin–Fz4CRD4 ( 2:2 complex ) deglyc*61 . 3 ( 2:2 complex ) 60 . 1 ± 0 . 4Norrin–Fz4CRD4 ( 2:2 complex ) glyc†69 . 9 ( 2:2 complex ) 61 . 3 ± 0 . 5*The proteins were produced from HEK293T cells in the presence of the N-glycosylation processing inhibitors , kifunensine resulting in limited glycosylation and were treated with endoglycosidase-F1 . †The proteins were produced from HEK293T cells with full glycosylation .", "‡The measured molecular weight ( MWMeasured ) is in general agreement with theoretical molecular weight ( MWTheoretical ) predicated based on the primary sequence plus the molecular weight of N-linked glycans ( see ‘Materials and methods’ , SEC-MELS analysis for detailed information of calculation ) .", "We purified Norrin–Fz4CRD complex ( Figure 4—figure supplement 1A ) and determined the crystal structures of methylated Norrin–Fz4CRD ( dimethylated surface-exposed lysine residues; Figure 4—figure supplement 1B , C ) and Norrin–Fz4CRD–SOS ( complex bound with heparin mimic sucrose octasulfate , SOS; Figure 4A and Figure 4—figure supplement 1D ) at 2 . 3 Å and 3 . 0 Å resolution , respectively ( Table 1 ) .", "These two complex structures show different stoichiometries: a 2:1 complex for the methylated Norrin–Fz4CRD and a 2:2:2 stoichiometry for the Norrin–Fz4CRD–SOS complex , the architecture of which resembles a butterfly ( Figure 4A ) .", "The Norrin–Fz4CRD binding interface is conserved between the complex structures ( Figure 4—figure supplement 1E ) .", "Each Fz4CRD interacts one-to-one with a separate Norrin chain , burying on average 1680 Å2 of surface area .", "To investigate the preference for complex formation in a 2:1 or 2:2 stoichiometry , we performed SEC-MALS ( Figure 4B ) and SAXS ( Figure 4C ) measurements in the absence of SOS .", "Both methods show Norrin interacts with Fz4CRD in a 2:2 stoichiometry .", "Lysine methylation of the Norrin–Fz4CRD complex was used to facilitate crystal lattice formation ( Walter et al . , 2006; Malinauskas et al . , 2011 ) , and on close inspection of the structure we found Lys102 and Lys104 , two residues which contribute to the Norrin–Fz4CRD interface , ( see next section ) are dimethylated in the uncomplexed subunit of the Norrin dimer ( Figure 4—figure supplement 1C ) , and contribute instead to a lattice contact .", "This observation suggests that the 2:1 stoichiometry merely reflects the favourable crystallization characteristics of a sub population of asymmetrically methylated Norrin–Fz4CRD complexes .", "Thus although the methylated Norrin–Fz4CRD structure usefully provides high-resolution information for the ligand–receptor interface ( Figure 4—figure supplement 1F ) , the Norrin–Fz4CRD–SOS structure defines the overall architecture of the native complex ( Figure 4A ) .", "The two Fz4CRD diverge from the Norrin dimer without contacting each other ( Figure 4A ) , and with their C-termini suitably oriented for attachment to the same cell surface . 10 . 7554/eLife . 06554 . 014Figure 4 . Crystal structure and solution behaviour of Norrin–Fz4CRD complex .", "( A ) Ribbon representation of Norrin ( magenta and pink ) in a 2:2:2 complex with Fz4CRD ( cyan and pale cyan ) and SOS ( green ) .", "( B ) SEC-MALS analyses .", "The profile of molecular weight ( left ordinate axis ) and differential refractive index ( right ordinate axis ) are shown as thick and thin lines , respectively .", "SDS-PAGE ( Inset ) shows Norrin in complex with Fz4CRD ( triplet band for glycosylated Fz4CRD , marked as green circles , represents glycosylation heterogeneity ) .", "( C ) SAXS experiments .", "Experimental scattering data ( black circles ) and calculated scattering patterns ( coloured lines ) are shown to a maximal momentum transfer of q = 0 . 35 Å−1 .", "Individual data: fit pairs are displaced along an arbitrary y axis to allow for better visualization .", "Bottom curve: Norrin–Fz4CRD 1:2 complex crystal structure ( red line ) .", "Middle curve: Norrin–Fz4CRD 2:2 complex crystal structure ( blue line ) .", "Top curve: modelled Norrin–Fz4CRD 2:2 complex crystal structure ( missing regions for Norrin and Fz4CRD N- and C-termini are modeled into the crystal complex structure; green line ) .", "Structural models are shown in cartoon representation .", "The bottom left inset shows the experimental ( black circles ) and calculated ( coloured lines ) Guinier region .", "The bottom right inset shows the experimental ( black circles ) and calculated ( coloured lines ) pair distance distribution P ( r ) curves . DOI: http://dx . doi . org/10 . 7554/eLife . 06554 . 01410 . 7554/eLife . 06554 . 015Figure 4—figure supplement 1 . Protein complex production and structural properties of Norrin–Fz4CRD complex .", "( A ) Norrin forms a stable complex with Fz4CRD in solution .", "SEC elution profiles and SDS-PAGE under reducing condition are presented .", "SEC fractions analysed by SDS-PAGE are marked as red lines for Norrin–Fz4CRD complex and cyan lines for uncomplexed Fz4CRD .", "( B ) The crystal structure of methylated Norrin ( pink and magenta ) –Fz4CRD ( cyan ) forms a 2:1 complex , which loses one molecule of Fz4CRD during crystal lattice formation .", "The N-linked glycans are coloured in green .", "( C ) Dimethylated lysine ( MLY ) residues on the protein surface of methylated Norrin–Fz4CRD are shown as sphere models .", "Close-up view of sigmaA-weighted 2|FO| − |FC| electron density maps for MLY102 and MLY104 are contoured at 1 . 0 σ as blue meshes .", "( D ) The sigmaA-weighted |FO| − |FC| electron density maps were calculated with omission of the SOS molecules from the refined model , followed by several cycles of refinement and contoured at 4 σ level as green meshes .", "( E ) Methylated Norrin ( yellow ) –Fz4CRD ( cyan ) was superposed onto the Norrin ( magenta ) –Fz4CRD ( blue ) –SOS ( wheat ) complex using Norrin as a reference .", "( F ) The overall sigmaA-weighted 2|FO| − |FC| electron density map after refinement is contoured at 1 . 0 σ level as blue meshes for the complex of methylated Norrin–Fz4CRD ( stick model ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06554 . 01510 . 7554/eLife . 06554 . 016Figure 4—figure supplement 2 . Structural comparison of cystine-knot growth factor monomers and their ternary complexes . Norrin has a unique three intermolecular disulphide bonds arrangement and a specific intramolecular disulphide bond ( black dotted circle ) .", "( A ) Cartoon representation of a single chain of cystine-knot growth factors , coloured as rainbow from blue N-terminus to red C-terminus .", "Each structure was superposed with the Norrin monomer and presented in the same view .", "( B ) Comparison of ternary complex formation is shown as ribbon diagrams , including Norrin ( dimer , magenta and pink ) and Fz4CRD ( cyan and blue ) , TGF-β3 ( dimer , magenta and pink ) in complex with Type I ( green and lemon ) and Type II ( blue and cyan ) receptors , BMP-2 ( dimer , magenta and pink ) bound with Type I ( green and lemon ) and Type II ( blue and cyan ) receptors , PDGF ( dimer , magenta and pink ) in complex with its receptors ( PDGFR; blue and cyan ) , VEGF-C ( dimer , magenta and pink ) in complex with its receptors ( VEGFR-3; blue and cyan ) , myostatin ( dimer , magenta and pink ) and its antagonists ( follistatin 288; blue and cyan ) , and BMP-7 ( dimer , magenta and pink ) bound with its antagonists ( Noggin; orange and yellow ) .", "PDB identifiers are shown below the structural representations . DOI: http://dx . doi . org/10 . 7554/eLife . 06554 . 01610 . 7554/eLife . 06554 . 017Figure 4—figure supplement 3 . No large conformational changes upon complex formation .", "( A ) Structural comparisons Fz4CRD ( gray ) from Norrin–Fz4CRD–SOS complex with Fz4CRD ( cyan ) from methylated Norrin–Fz4CRD and unliganded Fz4CRD ( magenta ) crystal form II ( chain D ) are presented as a ribbon diagram .", "N-linked glycans are shown as stick models .", "The encircled N and C denote the N- and C-termini .", "( B ) Representative Fz4CRD displayed with diameter of the Cα tube defined by the B factor of the Cα atoms ( small tube means structural rigidity; large tube indicates structural flexibility ) .", "Fz4CRD loops for Norrin binding are highlighted as blue ( loop I ) , green ( loop II ) , and yellow ( loop III ) .", "The flexibilities of loop I and III are reduced upon complex formation and SOS binding , respectively .", "( C ) Structural comparisons of Norrin ( magenta ) from Norrin–Fz4CRD–SOS complex , Norrin ( yellow ) from methylated Norrin–Fz4CRD and uncomplexed Norrin ( blue ) crystal form I ( chain A and B ) are shown as ribbon diagram .", "The cyan dotted circles denote the Fz4 binding site on Norrin .", "( D ) Representative Norrin displayed according to the B factor of the Cα atoms .", "The SOS binding site ( mainly on β5-β6 loop ) becomes rigid upon SOS binding ( ligand induced fit ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06554 . 01710 . 7554/eLife . 06554 . 018Figure 4—figure supplement 4 . Structural comparison of Norrin–Fz4CRD complex with MBP-Norrin .", "( A ) Superposition of MBP-Norrin ( green; PDB ID: 4MY2 ) onto Norrin in the Norrin ( magenta ) –Fz4CRD ( blue ) –SOS ( wheat ) and methylated Norrin ( yellow ) –Fz4CRD ( cyan ) complex structures .", "( B ) Close-up view of steric clashes ( indicated by a red arrow ) between Fz4CRD and MBP . DOI: http://dx . doi . org/10 . 7554/eLife . 06554 . 018 The Norrin–Fz4CRD complex has a novel architecture; the mode of interaction of Norrin is distinct from that of other cystine-knot secreted growth factors ( transforming growth factor-β , bone morphogenetic protein , platelet-derived growth factor , and vascular endothelial growth factor ) with either their receptors or antagonists ( Figure 4—figure supplement 2 ) .", "Neither Norrin nor Fz4CRD undergoes large conformational changes upon complex formation , although the flexibility of residues involved in the binding interface is reduced ( Figure 4—figure supplement 3 ) .", "Interestingly , superposition of the Norrin–Fz4CRD complex and the previously reported MBP-Norrin structure resulted in steric clashes between the Fz4CRD and the MBP ( Figure 4—figure supplement 4 ) .", "This suggests that MBP hinders Norrin interaction with Fz4CRD consistent with MBP-Norrin only having half of the signalling activity of untagged Norrin ( Ke et al . , 2013 ) .", "At the ligand–receptor interface ( Figure 5A ) two β-hairpins in Norrin ( β1-β2 and β5-β6 ) contact three loops in Fz4CRD ( I , II , and III ) .", "Fz4CRD loop I hydrogen bonds to Norrin ( Figure 5B ) .", "Fz4CRD loop II makes extensive hydrophobic contacts plus one salt-bridge ( Fz4CRD Lys109 with Norrin Asp46; Figure 5C ) .", "Fz4CRD loop III interacts with Norrin via an extensive hydrogen bond network as well as hydrophobic contacts ( Figure 5D ) .", "Interactions with SOS involve the positively charged residues of Lys58 , Arg107 , Arg109 , and Arg115 on Norrin , plus His154 and Asn155 on Fz4CRD loop III ( Figure 5E ) .", "These residues define a likely binding site for GAGs , in agreement with previous reports of Norrin interactions with extracellular matrix and heparin ( Xu et al . , 2004; Ohlmann et al . , 2010 ) . 10 . 7554/eLife . 06554 . 019Figure 5 . Structural details of binding sites in the Norrin–Fz4CRD–SOS complex .", "( A ) Side-view of complex .", "Fz4CRD loops involved in Norrin binding are coloured blue ( loop I ) , green ( loop II ) , and yellow ( loop III ) .", "( B–E )", "Views detailing the interfaces .", "Selected residues involved in binding are shown as sticks and coloured magenta ( Norrin ) , blue ( loop I ) , green ( loop II ) , yellow ( loop III ) , and cyan ( Phe96 of Fz4CRD ) and those associated with disease mutations are highlighted in boxes .", "Dotted lines denote hydrogen bonds .", "( B ) Interactions between Fz4CRD loop I and Norrin .", "( C ) Hydrophobic interactions of Norrin with Fz4CRD loop II and part of loop III .", "( D ) Interactions of Fz4CRD loop III with Norrin .", "( E ) SOS binding to Norrin and Fz4CRD loop III . DOI: http://dx . doi . org/10 . 7554/eLife . 06554 . 019 The Norrin–Fz4 interface revealed in our crystal structures ( Figure 6A , B ) is in excellent agreement with reported disease-associated mutations ( Figure 6C ) and surface residue conservation ( Figure 6D ) .", "We performed mutagenesis and functional assays to verify this Fz4 binding site .", "Surface plasmon resonance ( SPR ) experiments ( Figure 6E and Figure 6—figure supplement 1A ) show a micromolar equilibrium dissociation constant between Norrin and Fz4CRD .", "Mutations of either H43N/V45T or L61N/A63S , which resulted in the introduction of an N-linked glycosylation site in the Fz4 binding site on Norrin , completely abolish the interaction ( Figure 6—figure supplement 1B ) .", "Norrin disease-associated mutants V45E and L61P/A63D lose binding affinity for Fz4CRD ( Figure 6—figure supplement 1C ) , as do mutants R41E/H43E and R38E/R41S/H43E/K102E/K104E ( Figure 6—figure supplement 1D ) .", "In contrast , Norrin mutants L52N/K54S and M114N/L116S ( to introduce an N-linked glycosylation site in the β1-β2 loop or β5-β6 loop , respectively ) , predicted to lie outside the Fz4 binding site ( Figure 6A ) , show the same binding affinity as wild-type ( Figure 6—figure supplement 1E ) .", "Cell-based Wnt/β-catenin responsive luciferase assays ( Figure 6F ) further support the significance of the Fz4 binding site .", "Norrin mutants that lose binding to Fz4CRD also fail to induce the luciferase reporter activity , in agreement with the SPR results ( Figure 6—figure supplement", "1 ) and prior genetic data ( Xu et al . , 2004; Smallwood et al . , 2007 ) .", "Taken together , our structural and functional results suggest that Norrin uses β strands ( β1-β2 and β5-β6 ) for Fz4CRD binding rather than , as proposed by Bazan et al . ( 2012 ) using the loop between β1 and β2 ( Bazan et al . , 2012 ) . 10 . 7554/eLife . 06554 . 020Figure 6 . Biophysical and functional characterisation of Fz4 binding site . Surface representation of Norrin–Fz4CRD complex in open book view .", "( A ) Interface residues are coloured orange ( Norrin ) and blue ( loop I ) , green ( loop II ) , yellow ( loop III ) , and cyan ( Phe96 ) on Fz4CRD .", "Norrin mutation sites used in functional assays are labelled ( red , residues involved in Fz4CRD binding; grey filled box , residues associated with diseases; black , residues located outside the Fz4 binding site ) .", "( B ) Norrin and Fz4CRD coloured by electrostatic potential from red ( acidic; −7 kbT/ec ) to blue ( basic; 7 kbT/ec ) .", "( C ) Disease-associated mutations mapped onto the surface of Norrin and Fz4CRD ( purple , missense mutations; red , missense mutations of cysteine residues ) .", "( D ) Surfaces colour-coded according to sequence conservation from white ( not conserved ) to black ( conserved ) .", "( E ) SPR results for Fz4CRD binding to Norrin wild-type ( WT ) and Norrin V45E mutant .", "Inset SPR sensorgrams are of equilibrium-based binding assays with reference subtraction .", "( F ) Luciferase reporter assays histograms with Kd values from SPR measurements ( Figure 6—figure supplement", "1 ) shown above .", "Residues involved in the Fz4CRD binding site are coloured red .", "Residues without contact with Fz4CRD are coloured black .", "Grey filled boxes highlight disease-associated residues ( Figure 2—figure supplement 2 ) .", "The luciferase activities were normalized to a maximum activity value ( 100% ) for Norrin wild-type and error bars represent standard deviations ( n = 3 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06554 . 02010 . 7554/eLife . 06554 . 021Figure 6—figure supplement 1 . SPR equilibrium binding data . SPR equilibrium binding experiments using Fz4CRD as analyte and biotinylated human Norrin wild-type ( WT ) and mutants as immobilized ligands on CM5 sensor chips .", "SPR sensorgrams ( left panels ) and fitted plots of equilibrium binding response ( right panels; 1:1 Langmuir binding model ) against a series of concentrations of Fz4CRD are shown .", "The binding parameters are shown as Surface ( the amounts of biotinylated Norrin coated onto sensor chip ) , Bmax ( the maximum response ) , Kd ( binding constant ) , and RU ( response unit ) .", "N/A indicates not applicable for calculation of Kd .", "Residues associated with diseases are highlighted as filled grey boxes .", "( A ) The interaction of Norrin wild-type with Fz4CRD has a Kd of 1 . 1 μM .", "( B ) Introduction of an N-linked glycosylation site on the Fz4 binding site of Norrin results in the complete loss of Fz4CRD binding .", "( C ) Norrin mutants identified from diseases show no binding to Fz4CRD .", "( D ) Mutations designed to disrupt the hydrogen bond and salt bridge interactions of the Fz4 binding site completely abolish binding to Fz4CRD .", "( E ) Introduction of an N-linked glycosylation site on the Norrin surface locating outside the Fz4 binding site does not affect the binding affinity of Norrin to Fz4CRD .", "( F ) Norrin discriminates between different FzCRD proteins .", "Biotinylated human Norrin was immobilized onto a CM5 sensor chip to give 410 response units ( RU ) and different FzCRD proteins were used as analytes .", "The injected human Fz4CRD proteins have a concentration ranging from 2 . 3 nM to 75 μM .", "Other FzCRD concentrations ranged from 0 . 3 μM to 150 μM ( mouse Fz5CRD ) or 0 . 2 μM–100 μM ( human Fz7CRD and mouse Fz8CRD ) .", "Because of the limitations of detection , the Kd values of Fz5CRD and Fz8CRD are too low to measure accurately , resulting in variable standard deviations .", "The binding affinity of Fz7CRD to Norrin is not sufficient to calculate a Kd . DOI: http://dx . doi . org/10 . 7554/eLife . 06554 . 021 To determine the binding affinity of Norrin for different CRD of Fz receptors , we undertook a series of SPR experiments .", "The results ( Figure 6—figure supplement 1F ) show that Norrin has greatest affinity for Fz4CRD ( Kd: 1 μM ) , low affinities for Fz5CRD ( Kd: 42 μM ) and Fz8CRD ( Kd: 64 μM ) , and no binding to Fz7CRD .", "In combination , these results confirm that pairing Norrin with Fz4CRD provides selective and high affinity binding relative to interactions with other CRD of Fz receptors , in agreement with prior studies ( Xu et al . , 2004; Smallwood et al . , 2007; Ke et al . , 2013 ) .", "However , it remains to be clarified whether the low affinity interactions of Norrin with other Fz receptors can play any functional role in vivo .", "To assess our putative binding site for GAGs ( Figure 5E ) , we performed structure-guided mutagenesis and functional studies .", "Our heparin binding experiments confirmed that Norrin shows high affinity interaction with heparin ( Figure 7—figure supplement 1A ) , consistent with previous studies ( Perez-Vilar and Hill , 1997; Xu et al . , 2004; Smallwood et al . , 2007; Ohlmann et al . , 2010 ) , and further demonstrated Norrin–Fz4CRD complex binding to heparin ( Figure 7A ) .", "The Norrin triple mutation R107E/R109E/R115L ( R115L is a disease-associated mutation; Figure 2—figure supplement", "2 ) impaired heparin binding ( Figure 7B ) and abolished signalling activity ( Figure 7C ) .", "However , this mutant protein retained the ability to bind Fz4CRD ( Figure 7D ) with a 2:2 stoichiometry ( Figure 7—figure supplement 1B ) .", "Ke et al . ( 2013 ) have reported MBP-Norrin binding to the Lrp6 ectodomain fragment comprising the first two tandem β-propeller-epidermal growth factor-like domain pairs ( Lrp6P1E1P2E2; Ke et al . , 2013 ) ; we found both our wild-type and R107E/R109E/R115L mutant Norrin bind to Lrp6P1E1P2E2 ( Figure 7E , F ) .", "The Norrin K58N mutant ( a disease-associated mutation; Figure 2—figure supplement", "2 ) exhibited half of wild-type activity in our cell-based assay ( Figure 7C ) , but did not affect Fz4CRD interaction ( Figure 6—figure supplement 1C ) .", "These results are in agreement with previous functional studies ( Smallwood et al . , 2007 ) , and suggest this area is a GAG binding site rather than that , as Ke et al . ( 2013 ) proposed , residues Arg107 , Arg109 , and Arg115 are involved in Lrp5/6 binding ( Ke et al . , 2013 ) .", "HSPGs play important roles in the regulation of the Wnt signalling pathway ( Malinauskas and Jones , 2014 ) .", "Wnt signalling activity can be inhibited by treatment with exogenous heparin ( Ai et al . , 2003 ) .", "Also , Jung et al . ( 2015 ) have reported that PG545 , a heparan sulphate mimetic , can block Wnt binding to the cell surface , by competing with endogenous HSPGs , and inhibit Wnt signalling ( Jung et al . , 2015 ) .", "For Norrin mediated Wnt/β-catenin signalling , we found that SOS could inhibit activity when pre-incubated with Norrin before stimulation of reporter cells ( Figure 7—figure supplement 1D ) . 10 . 7554/eLife . 06554 . 022Figure 7 . Verification of Norrin GAG binding site . Heparin affinity chromatography of ( A ) Norrin–Fz4CRD complex and ( B ) Norrin R107E/R109E/R115L–Fz4CRD complex .", "Protein elution profiles ( left panel ) were monitored by absorbance at 280 nm ( blue curves ) for a NaCl gradient ( 0 . 25–2 M; black dashed lines ) .", "Input sample , flow-through ( green line ) and peak fractions ( red line ) were analysed on SDS-PAGE ( right panel ) .", "Norrin-Fz4CRD complex was eluted at 1 . 3 M NaCl concentration .", "( C ) Luciferase reporter assays for Norrin mutations ( coloured green ) in the GAG binding site .", "Grey filled boxes highlight disease-associated residues ( Figure 2—figure supplement 2 ) .", "( D ) SPR binding assay of Norrin R107E/R109E/R115L mutant and Fz4CRD interaction .", "Sensorgrams ( top panel ) and fitted plots of equilibrium binding response ( bottom panels ) for a series of concentrations of Fz4CRD are shown .", "( E and F )", "SPR equilibrium binding experiments of Lrp6P1E1P2E2 binding to Norrin wild-type and R107E/R109E/R115L mutant , respectively .", "Biotinylated Norrin proteins were immobilized on a CM5 chip and Lrp6P1E1P2E2 as analyte was injected over the chip .", "Sensorgrams ( top panel ) and fitted plots ( bottom panels ) for a series of concentrations of Lrp6P1E1P2E2 are presented . DOI: http://dx . doi . org/10 . 7554/eLife . 06554 . 02210 . 7554/eLife . 06554 . 023Figure 7—figure supplement 1 . Supporting experiments for GAG binding site .", "( A ) Heparin affinity chromatography of Norrin wild-type .", "Protein elution profiles ( left panel ) were monitored by absorbance at 280 nm ( blue curves ) for a NaCl gradient ( 0 . 25–2 M; black dashed lines ) .", "An input sample , flow-through ( green line ) and peak fractions ( yellow and red lines ) are analysed on SDS-PAGE ( right panel ) .", "Norrin wild-type was eluted with 1 . 6 M NaCl concentration .", "( B ) SEC-MALS experiments for Norrin R107E/R109E/R115L–Fz4CRD complex .", "The red line represents the molecular weight ( left ordinate axis ) and blue lines show the differential refractive index ( right ordinate axis ) .", "( C ) In SPR equilibrium binding experiments , biotinylated human Norrin K58N proteins were coated onto a CM5 sensor chip and a series of concentrations of Fz4CRD were injected over the chip as analyte .", "Graphs show sensorgrams ( left panels ) and fitted plot ( right panels ) .", "( D ) Luciferase reporter assay of SOS inhibition .", "The luciferase reporter activities were normalized to a maximum activity value of 100% , using Norrin wild-type as reference .", "Error bars indicate standard deviations ( n = 3 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06554 . 023 Norrin interaction with co-receptor Lrp5/6ECD ( Figure 7E ) is essential for signal activation ( Xu et al . , 2004; Ke et al . , 2013 ) .", "To identify Norrin residues potentially involved in Lrp5/6ECD binding , we assessed solvent exposure , disease-association , and lack of involvement in Fz4CRD or GAG binding ( Figure 2—figure supplement 2 ) .", "Five residues ( Lys54 , Arg90 , Arg97 , Gly112 , and Arg121 ) were highlighted by this analysis and form a continuous , positively charged , concave patch ( Figure 8A ) .", "Notably , a negatively charged region of the Lrp6ECD surface has been implicated in ligand binding ( Ahn et al . , 2011; Bourhis et al . , 2011; Chen et al . , 2011; Cheng et al . , 2011 ) .", "We therefore focused on the positively charged concave surface of Norrin as a potential Lrp5/6 binding site ( Figure 8A ) , interestingly , this putative binding site has a partially overlap , at Lys54 , with the residue suggested to be involved in Lrp5/6 interaction by Ke et al . ( 2013 ) .", "To test our proposed location for the Lrp5/6 binding site , we generated the disease-associated Norrin mutant R121W ( Arg121 is a mutational hotspot; Figure 2—figure supplement 2 ) .", "This mutation substantially impairs signalling activity ( Figure 8—figure supplement 1A ) , but retains the ability to interact with Fz4CRD ( Figure 8—figure supplement 1B ) and heparin ( Figure 8—figure supplement 1C ) .", "However , we found the R121W mutation reduced protein solubility and stability during protein production and in heparin binding assays .", "Analyses of additional Norrin mutants in biophysical and cellular assays will be required to verify the putative Lrp5/6 binding site .", "Taken together , the current data suggest three distinct and independent binding sites on Norrin for Fz4 , Lrp5/6 , and GAGs ( Figure 8B ) .", "This arrangement of binding sites likely enables Norrin to form a ternary complex . 10 . 7554/eLife . 06554 . 024Figure 8 . The potential Lrp5/6 binding site on Norrin .", "( A ) Cartoon representation of Norrin ( grey ) in complex with Fz4CRD ( cyan ) .", "Residues in the potential Lrp5/6 binding site are shown as spheres ( atom colouring: magenta , carbon; blue , nitrogen; red , oxygen ) .", "The boxes highlight residues associated with disease mutations .", "( B ) Cartoon model of Norrin showing three distinct binding sites . DOI: http://dx . doi . org/10 . 7554/eLife . 06554 . 02410 . 7554/eLife . 06554 . 025Figure 8—figure supplement 1 . Verification of Norrin potential Lrp5/6 binding site .", "( A ) Luciferase reporter assay .", "The luciferase activities were normalized to a maximum activity value ( 100% ) for Norrin wild-type and error bars represent standard deviations ( n = 3 ) .", "( B ) SPR binding assay of Norrin mutant R121W .", "Biotinylated Norrin mutant R121W was coated onto a CM5 sensor chip and a series of concentrations of Fz4CRD as analyte were injected over the chip .", "Graphs show sensorgrams ( top panels ) and fitted plot of equilibrium binding response ( bottom panels ) .", "( C ) Heparin binding assay of Norrin mutant R121W .", "Protein elution profiles ( left panel ) were monitored by absorbance at 280 nm ( blue curves ) for a NaCl gradient ( 0 . 25–2 M; black dashed lines ) .", "An input sample , flow-through ( green line ) and peak fractions ( yellow and red lines ) are analysed on SDS-PAGE ( right panel ) .", "Norrin mutant R121W was eluted at 1 . 5 M NaCl concentration .", "Notably , we found almost 50% of the Norrin mutant R121W precipitated as the NaCl concentration was reduced before injection for heparin affinity chromatography ( see ‘Materials and methods’ for detailed information ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06554 . 025 As Norrin and Wnt both trigger the canonical Wnt/β-catenin pathway , we compared their modes of action .", "Xenopus Wnt8 ( Figure 9A ) has been described as using ‘thumb’ and ‘index finger’ regions to grasp mouse Fz8CRD at two distinct sites ( Janda et al . , 2012 ) .", "In site 1 , a palmitoleoyl group ( PAM ) covalently linked to the tip of the thumb inserts into a groove in Fz8CRD , removal of this PAM moiety suppresses Wnt signalling activity ( Kakugawa et al . , 2015; Zhang et al . , 2015 ) .", "In site 2 , the index finger contacts a hydrophobic pocket .", "We superposed Norrin–Fz4CRD with Wnt8–Fz8CRD .", "There are no major structural differences between the Fz4CRD and Fz8CRD ( r . m . s . deviation of 1 . 3 Å over 110 equivalent Cα atoms; Figure 9A ) , and the structural elements that mediate site 1 PAM binding in Fz8CRD are largely conserved in Fz4CRD ( Figure 9B ) .", "The Norrin binding site on Fz4CRD ( ∼800 Å2 buried area ) overlaps with site 2 on Fz8CRD ( ∼400 Å2 buried area; Figure 8A ) , in agreement with previous mutational mapping studies ( Smallwood et al . , 2007 ) .", "The position of the Wnt8 index finger overlaps with Norrin β1 and β2 , and , unexpectedly , these β strands show some structural equivalence with Wnt8 ( Figure 9C ) .", "Site 2 Wnt8 residues are strictly conserved in all Wnts , and the apolar residues in the corresponding positions on Norrin are associated with disease mutations ( Figure 9C ) . 10 . 7554/eLife . 06554 . 026Figure 9 . Structural comparison of Norrin–Fz4CRD with Wnt8–Fz8CRD .", "( A ) Superposition of Norrin ( magenta ) –Fz4CRD ( cyan ) with Wnt8 ( green ) –Fz8CRD ( blue ) ( PDB ID: 4F0A ) .", "Disulphide bonds , N-linked glycans , and PAM ( of Wnt8 ) are shown as sticks .", "SOS is shown as grey surface .", "( B ) Comparison of site 1 ( PAM binding ) on Fz4CRD ( cyan ) and Fz8CRD ( blue ) .", "Fz4 His69 is disease associated .", "( C ) The Wnt8 index finger ( site 2; green ) structurally overlays Norrin ( β1 and β2; magenta ) .", "Norrin residues associated with diseases are boxed .", "( D–F ) , Structural comparison of Fz4CRD and Fz8CRD for ligand binding .", "Loop I-III residues for Fz4CRD and Fz8CRD are shown as sticks .", "Fz8CRD residues for Wnt8 binding ( site", "2 ) are boxed in purple .", "Fz4 disease-associated residues are boxed .", "Red arrows indicate residue substitutions between Fz4CRD and Fz8CRD .", "Fz8CRD residues Tyr151 and Asn152 are modelled as alanines ( PDB ID: 4F0A ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06554 . 026 We also used our superposition of the Fz4CRD and Fz8CRD structures ( Figure 9A ) to identify the determinants of the Norrin binding specificity for Fz4CRD ( Figure 6—figure supplement 1F ) .", "In Fz4CRD loop I ( Figure 9D ) , Asn55 is replaced by Fz8CRD Gly45 , a change that would abolish interaction with Norrin Ser34 in the complex ( Figure 5B ) .", "In Fz4CRD loop II ( Figure 9E ) , the substitution of Lys109 by Fz8CRD Asp99 would introduce an unfavorable electrostatic interaction with Norrin Asp46 ( Figure 5C ) .", "Thirdly , in Fz4CRD loop III ( Figure 9F ) , hydrogen bonds and salt bridges to Norrin would be lost on replacing Asn152 and Glu160 with Fz8CRD Gly142 and Asp150 respectively ( Figure 5D ) .", "Consistent with this analysis , these residue substitutions have been reported to affect Fz4CRD binding to Norrin ( Smallwood et al . , 2007 ) , and Fz4CRD is unique in containing this particular combination of residues ( Figure 2—figure supplement 2 ) ." ], [ "Overall , our analyses provide several advances for our understanding of Norrin and Wnt signalling .", "Firstly , our results give fresh insight into the role of HSPGs .", "HSPGs have been proposed to regulate the local distribution of ligand and receptor at the cell surface , potentially acting as an introductory agency for ligand and receptor ( Lin and Perrimon , 1999; Baeg et al . , 2001; Malinauskas et al . , 2011; Malinauskas and Jones , 2014 ) .", "We have discovered a GAG binding site that may span Norrin and Fz4CRD ( Figure 5E ) .", "Interestingly , Smallwood et al . ( 2007 ) found the binding affinity of Norrin with Fz4CRD is enhanced in the presence of heparin ( Smallwood et al . , 2007 ) .", "We propose that the extended GAG binding site may allow co-receptor HSPGs to recruit secreted Norrin for interaction with Fz4CRD and to co-localize Norrin and Fz4 receptor , similar to the role of HSPGs in Wnt signalling ( Reichsman et al . , 1996; Baeg et al . , 2001; Fuerer et al . , 2010 ) .", "For example , HSPGs have been shown to regulate the Wnt morphogenetic gradient ( Lin and Perrimon , 1999; Baeg et al . , 2004 ) .", "Also , Capurro et al . ( 2014 ) have reported that Fz4CRD binds to the GAGs of the human HSPG Glypican-3 and that these interactions are involved in Wnt signal complex formation ( Capurro et al . , 2014 ) .", "Secondly , we show the Norrin dimer binds separately to two molecules of Fz4CRD ( Figure 4 ) , in contrast to the 1:1 complex of Wnt8–Fz8CRD ( Janda et al . , 2012 ) .", "Our discovery of the Fz4 and GAG binding sites , and analysis of a potential Lrp5/6 binding region , maps out distinct binding surfaces on Norrin ( Figure 8B ) , which provide a framework in which to understand the effects of inherited mutations and probe the overall architecture of the ternary complex ( Norrin–Fz4CRD–Lrp5/6ECD ) .", "Thirdly , we determine how Norrin structurally mimics Wnt for site 2 binding surfaces on the Fz ectodomain ( Figure 9A ) .", "Interestingly , previous analyses using water-soluble ‘mini-Wnt’ proteins , which cannot contribute site 1 binding , have raised the possibility that site 2 binding to the CRD of Fz receptors can activate canonical Wnt/β-catenin signalling albeit weakly ( Janda et al . , 2012; von Maltzahn et al . , 2013 ) .", "Our findings indicate that the site 2 binding mode is central to signallosome formation for Norrin mediated signalling .", "We used SPR experiments to establish the binding affinity for Norrin–Fz4CRD complex formation .", "The Kd value of 1 . 1 μM for the interaction between Norrin and Fz4CRD we report here ( Figure 6—figure supplement 1A ) is weaker than previously published results ( Xu et al . , 2004; Ke et al . , 2013 ) .", "This discrepancy is likely due to our SPR binding assays being carried out with monomeric Fz4CRD .", "Xu et al . ( 2004 ) used an enzyme-linked immunosorbent assay to give an affinity of 3–4 nM for mouse Norrin fused with C-terminal alkaline phosphatase binding to mouse Fz4CRD dimerized by a C-terminal Fc fusion ( Xu et al . , 2004 ) .", "Ke et al . ( 2013 ) reported Kd values of 11 nM and 5 nM for the interaction between MBP-Norrin and Fc-tagged dimeric Fz4CRD using an AlphaScreen luminescence assay and biolayer interferometry , respectively ( Ke et al . , 2013 ) .", "It is noteworthy that as Fc-dimerized Fz4CRD may mimic Fz4 receptor dimerization at the cellular surface , these tighter binding affinities may be more indicative of Norrin binding in the physiologically relevant environment .", "Similarly , our Kd value of 2 . 87 μM for Norrin binding to Lrp6P1E1P2E2 in an SPR based assay ( Figure 7E ) differs from the Kd value of 0 . 45 μM reported by Ke et al . ( 2013 ) based on an homologous AlphaScreen competition assay using unlabeled MBP-Norrin against biotinylated MBP-Norrin for interaction with Lrp6P1E1P2E2 ( Ke et al . , 2013 ) .", "Our studies reported here , in combination with previous findings for Norrin and Wnt signalling , are consistent with Norrin-induced receptor clustering and signallosome formation .", "Inactive pre-dimerized Fz4 may engage with homodimeric Tspan-12 to enhance receptor clustering ( Kaykas et al . , 2004; Ke et al . , 2013 ) .", "Norrin binding generates ternary complex formation by Fz4 , Lrp5/6 and the GAGs of HSPGs to trigger signalling , which is enhanced in the presence of Tspan12 .", "In the cytoplasm , Dishevelled binds to the C-terminal tail of Fz4 and self-assembles to oligomer ( Schwarz-Romond et al . , 2007 ) , leading to Axin recruitment to the cytoplasmic domain of Lrp5/6 for phosphorylation and signallosome formation ( Bilic et al . , 2007 ) .", "Previously reported mice genetic studies have demonstrated that expression of ectopic Norrin can rescue pathological retinal vascularization ( Ohlmann et al . , 2005 , 2010 ) .", "In addition , the pathological progresses of Norrie disease and familial exudative vitreoretinopathy are highly related to age-related macular degeneration and diabetic retinopathy ( Ye et al . , 2010; Ohlmann and Tamm , 2012 ) .", "Further investigation of the therapeutic possibilities for retinal diseases has been hampered by the difficulty of producing recombinant Norrin proteins .", "In this study , we provide a method to produce fully active untagged Norrin in mammalian cells ( Figure 1 ) .", "Our recombinant Norrin opens up new avenues to explore for the treatment of genetic retinal diseases and other ophthalmic disorders .", "More generally , Norrin as a Wnt mimic , may have potential as a reagent in regenerative medicine ( Clevers et al . , 2014 ) .", "Wnt signalling is important for tissue homeostasis throughout life ( Clevers and Nusse , 2012 ) .", "Multiple Wnt extracellular antagonists function to modulate Wnt signalling ( Malinauskas and Jones , 2014 ) , these include Dickkopf and Sclerostin , which bind to Lrp5/6 , as well as Wnt inhibitory 1 and sFRPs , which sequester Wnt .", "Aberrant Wnt signalling ( insufficient or excessive ) is implicated in diseases such as neurodegeneration and tumorigenesis , respectively ( Clevers and Nusse , 2012; Anastas and Moon , 2013 ) .", "Interestingly , our Norrin mutants ( used to verify the GAG and putative Lrp5/6 binding sites ) retain Fz4CRD binding but lose the ability to activate signalling .", "These properties are similar to those of the monoclonal antibody OMP-18R5 which can bind to the CRDs of Fz1 , 2 , 5 , 7 and 8 .", "OMP-18R5 inhibits tumour growth ( Gurney et al . , 2012 ) and has just completed phase I clinical trials ( Kahn , 2014 ) .", "Engineered Norrin mutants could similarly serve as blocking agents , but with specificities tailored to target Fz4 or other individual Fz receptors ." ], [ "Synthetic complementary DNA ( cDNA ) clones ( codon-optimized for expression in mammalian cells ) of human Norrin ( UniprotKB/Swiss-prot Q00604 ) were obtained from GeneArt ( Life Technologies , UK ) .", "The cDNA templates of human receptors Fz4 ( IMAGE ID: 40082087 ) , Fz7 ( IMAGE ID: 4549389 ) , Lrp6 ( IMAGE ID: 40125687 ) , and Tspan-12 ( IMAGE ID: 5275953 ) and mouse receptors Fz5 ( IMAGE ID: 40088671 ) and Fz8 ( IMAGE ID: 8861081 ) were purchased from SourceBioScience ( UK ) .", "All expression constructs reported here are derived from the pHLsec vector backbone ( Aricescu et al . , 2006 ) .", "The human Norrin wild-type ( residues 25–133 ) construct of SUMO-Norrin ( Figure 1A ) and Norrin mutant constructs for heparin affinity binding assays were tagged N-terminally with the murine Igκ-chain secretion signal , followed by a Strep-II tag , 8xHis tag , a mammalian expression codon-optimized Saccharomyces cerevisiae SUMO ( UniprotKB/Swiss-prot Q12306; residues 2–96 ) ( Peroutka et al . , 2008 ) , and a Human Rhinovirus ( HRV ) -3C protease cleavage site .", "They were tagged C-terminally with a TETSQVAPA sequence derived from bovine rhodopsin ( Rho-1D4 ) that is recognized by the Rho-1D4 monoclonal antibody ( Molday and MacKenzie , 1983 ) .", "The construct of Norrin ( residues 25–133 ) was cloned into the pHLsec vector ( Aricescu et al . , 2006 ) in frame with a C-terminal Rho-1D4 ( Figure 1A ) .", "For large-scale protein expression , the CRD constructs for human Fz4 ( residues 42–179 ) and Fz7 ( residues 42–179 ) as well as mouse Fz5 ( residues 31–176 ) and Fz8 ( residues 30–170 ) were cloned into a modified pHLsec vector ( pHLsec-mVenus-12H ) , containing a C-terminal HRV-3C protease cleavage site followed by a linker , monoVenus ( Nagai et al . , 2002 ) , and a tandem 6×His tag .", "Human Lrp6P1E1P2E2 ( residues 1–631 ) construct was cloned into a modified vector for stable cell line generation , pNeoSec ( Zhao et al . , 2014 ) , in frame with a C-terminal 10×His tag .", "For luciferase reporter assays , the full-length constructs of the human receptors Fz4 ( residues 1–537 ) , Lrp6 ( residues 1–1613 ) , and Tspan-12 ( residues 1–305 ) were cloned into the pLEXm-1D4 vector carrying a C-terminal Rho-1D4 tag .", "Norrin wild-type and mutants for biophysical and cellular assays were obtained from GeneArt ( Life Technologies , UK ) and cloned into the pHL-Avitag3 vector encoding a C-terminal BirA recognition sequence ( Aricescu et al . , 2006 ) .", "Mutant proteins were secreted at similar levels to the wild-type proteins .", "Constructs were verified by DNA sequencing ( Source Bioscience , UK ) .", "For western blot , HEK293T ( ATCC CRL-11268 ) cells were transfected with the DNA using Lipofectamine 2000 ( Life Technologies , UK ) according to the manufacturer's instructions .", "The conditioned media were collected 2 days post transfection and were analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis ( SDS-PAGE ) gels transferred onto a nitrocellulose membrane ( GE Healthcare Life Sciences ) with Rho-1D4 monoclonal antibodies ( Flintbox , University of British Columbia , Canada ) as primary antibody and goat anti-mouse IgG-horseradish peroxidise conjugate ( Sigma ) .", "The signal was visualized by Enhanced Chemiluminescence western blotting detection kit ( ECL , GE Healthcare Life Sciences ) .", "Norrin wild-type and mutants were expressed in HEK293T cells ( Aricescu et al . , 2006 ) in the presence of 4 mM valproic acid ( Backliwal et al . , 2008 ) .", "For crystallization experiments , Fz4CRD was produced in HEK293T cells in the presence of 5 μM of the class I α-mannosidase inhibitor , kifunensine ( Chang et al . , 2007 ) .", "Norrin in complex with Fz4CRD was co-expressed in HEK293T cells in the presence of kifunensine and valproic acid .", "For all other experiments , recombinant proteins were expressed in HEK293T cells .", "The Norrin conditioned media were passed through 1D4-affinity beads covalently coupling purified Rho-1D4 antibody to CnBr-activated Sepharose 4 Fast Flow ( CnBr-1D4; GE Healthcare Life Sciences ) and eluted in 25 mM Tris , pH 7 . 5 , 0 . 5 M NaCl , 10% ( wt/vol ) Glycerol , 0 . 5% ( wt/vol ) CHAPS , 250 μM TETSQVAPA peptide ( GenScript ) .", "The eluted sample was incubated with Glutathione S-Transferase ( GST ) -tagged HRV-3C protease to remove the SUMO-tagged fusion protein .", "The cleaved Norrin was purified by CnBr-1D4 followed by SEC ( Superdex 200 10/300 GL High Performance , GE Healthcare Life Sciences ) in either 10 mM HEPES , pH 7 . 5 , 0 . 7 M NaCl , 0 . 5% ( wt/vol ) CHAPS or acetate buffer , pH 4 . 0 , 0 . 5 M NaCl , 0 . 5% ( wt/vol ) CHAPS .", "For purification of Fz4CRD , the conditioned media were dialyzed and recombinant proteins were purified by IMAC ( TALON beads , Clontech , Mountain View , CA ) .", "The purified sample was dialyzed against 25 mM Tris , pH 7 . 5 , 0 . 5 M NaCl , 10% ( wt/vol ) Glycerol and treated with GST-tagged Flavobacterium meningosepticum endoglycosidase-F1 ( Endo-F1 ) ( Chang et al . , 2007 ) and His-tagged HRV 3C protease .", "The deglycosylated and cleaved sample was further purified by IMAC and further polished by SEC ( Superdex 75 16/600 column , GE Healthcare Life Sciences ) in 10 mM HEPES , pH 7 . 5 , 0 . 15 M NaCl .", "Purification of Fz5CRD , Fz7CRD , and Fz8CRD followed the same procedure to that described above , except protein was expressed in HEK293T cells and the treatment by Endo-F1 was omitted .", "Norrin–Fz4CRD complex was isolated from dialyzed conditioned media by IMAC .", "The eluted sample was dialyzed and treated with GST-tagged HRV-3C protease and Endo-F1 .", "The deglycosylated and cleaved complex was further purified by IMAC and GST-affinity beads and subsequently isolated by SEC ( Superdex 200 16/600 column , GE Healthcare Life Sciences ) in 10 mM HEPES , pH 7 . 5 , 0 . 7 M NaCl .", "For preparation of methylated proteins , the purified sample was subject to surface lysine methylation ( Walter et al . , 2006 ) and further purified by SEC ( Superdex 200 16/600 column , GE Healthcare Life Sciences ) .", "The selenomethionine ( Se-Met ) labelled protein was prepared as described previously ( Aricescu et al . , 2006 ) .", "A stable HEK293 GnT1 ( − ) cell line ( Reeves et al . , 2002 ) for Lrp6P1E1P2E2 protein production was generated as reported previously ( Zhao et al . , 2014 ) and protein was purified following our established procedure ( Chen et al . , 2011 ) .", "Concentrated proteins ( Norrin , 5 mg/ml; Fz4CRD , 60 mg/ml; Norrin in complex with Fz4CRD including native and methylated proteins , 10–12 mg/ml ) were subjected to sitting drop vapor diffusion crystallization trials in 96-well Greiner plates consisting of 100 nl protein solution and 100 nl reservoir using a Cartesian Technologies dispensing instrument ( Walter et al . , 2005 ) .", "Crystallization plates were placed in a The Automation Partnership storage vault maintained at 294 K and imaged via a Veeco visualization system .", "Methylated Norrin–Fz4CRD complex crystallized in 0 . 1 M Bicine , pH 9 . 0 , 10% ( wt/vol ) PEG6000 , Norrin crystal form I in 0 . 1 M sodium acetate , pH 5 . 0 , 5% ( wt/vol ) PGA-LM , 30% ( wt/vol ) PEG550MME , Norrin crystal form II in 0 . 1 sodium acetate , pH 5 . 0 , 5% ( wt/vol ) PGA-LM , 4% ( wt/vol ) PEG2000MME , 24% ( wt/vol ) PEG550MME , Norrin crystal form III in 0 . 1M citrate , pH 5 . 0 , 30% ( wt/vol ) PEG6000 , Fz4CRD crystal form I in 1 . 6 M tri-sodium citrate , pH 6 . 5 , and Fz4CRD crystal form II in 0 . 1 M HEPES , pH 7 . 5 , 0 . 1 M NaCl , 1 . 6 M ammonium sulfate .", "For the Norrin–Fz4CRD–SOS complex , protein complex was mixed with 10 mM SOS ( Toronto Research Chemicals Inc . ) prior to crystallization and crystals were obtained in 0 . 1 M Tris , pH 8 . 0 , 0 . 15 M NaCl , 8% ( wt/vol ) PEG8000 .", "For cryoprotection , crystals were soaked in mother liquor supplemented with 30% ( vol/vol ) glycerol for methylated Norrin–Fz4CRD , with 20% ( vol/vol ) PEG200 and 10 mM SOS for Norrin–Fz4CRD–SOS , with 30% ( vol/vol ) PEG550MME for Norrin crystal form II , with 30% ( vol/vol ) glycerol for Norrin crystal form III , with 1 . 8 M tri-sodium citrate , pH 6 . 5 for Fz4CRD crystal form I , and with 23% ( vol/vol ) sucrose for Fz4CRD crystal form II and subsequently flash-cooled by dipping into liquid nitrogen .", "The crystals of Norrin crystal form I were frozen directly .", "Data were collected at 100 K at Diamond Light Source ( Oxfordshire , UK ) at beamlines I03 ( Norrin Se-Met ) , I04 ( methylated Norrin–Fz4CRD and Norrin crystal form II and III ) , I04-1 ( Norrin–Fz4CRD–SOS ) , and I24 ( Norrin crystal form I and Fz4CRD crystal form I and II ) .", "Diffraction data were indexed and integrated using XIA2 ( Winter , 2010 ) coupled with XDS or IMOSFLM , and scaled and merged using Aimless ( Evans and Murshudov , 2013 ) .", "A subset of 5% of randomly selected diffraction data were used for calculating Rfree ( Brunger , 1993 ) .", "The structure of Norrin crystal form I was solved using highly redundant single-wavelength anomalous dispersion data merged from four data sets and collected at the Se K absorption edge .", "HKL2MAP ( Sheldrick , 2010 ) was used to identify the Se sites , which were then fed into PHENIX AUTOSOL ( Adams et al . , 2002 ) , resulting in an interpretable density modified electron map generated by RESOLVE ( Terwilliger , 2003 ) .", "An initial model generated by BUCCANEER ( Cowtan , 2006 ) was used to solve the high-resolution native structures .", "The structure of Fz4CRD was determined by molecular replacement ( MR ) in PHASER ( McCoy , 2007 ) using mouse Fz8CRD ( PDB ID: 1IJY ) as the search model , which was modified by CHAINSAW .", "For the determination of methylated Norrin–Fz4CRD , Norrin was used as search model for MR in PHASER ( McCoy , 2007 ) to obtain the initial phases .", "The additional electron density corresponding to Fz4CRD was clearly discernible after density modification with PARROT ( Cowtan , 2010 ) .", "Subsequently , the complex structure was solved by searching for Fz4CRD with MR in PHASER ( McCoy , 2007 ) .", "All other structures were solved by MR in PHASER ( McCoy , 2007 ) using the refined Norrin and Fz4CRD structures as search models .", "The models were completed by manual rebuilding in COOT ( Emsley and Cowtan , 2004 ) and refinement in REFMAC5 ( Murshudov et al . , 1997 ) and PHENIX ( Adams et al . , 2010 ) .", "The crystallographic statistics are listed in Table 1 .", "All models were validated with MOLPROBITY ( Chen et al . , 2010 ) .", "Amino acid sequence alignments were constructed using ClustalW ( Thompson et al . , 1994 ) .", "Structure superposition was performed within the CCP4 program suite using the SSM algorithm ( Krissinel and Henrick , 2004 ) .", "Electrostatic potential calculations were generated using APBS tools ( Baker et al . , 2001 ) , surface sequence conservation was calculated using CONSURF ( Ashkenazy et al . , 2010 ) and interface areas of proteins were analyzed with the PISA web server ( Krissinel and Henrick , 2007 ) .", "High-quality images of the molecular structures were created with the PyMOL Molecular Graphics System ( Version 1 . 5 , Schrödinger , LLC ) .", "Schematic figures and other illustrations were prepared using Corel Draw ( Corel Corporation ) .", "SPR experiments were performed using a Biacore T200 machine ( GE Healthcare Life Sciences ) at 25°C in 10 mM HEPES , pH 7 . 5 , 0 . 15 M NaCl , 0 . 005% ( wt/vol ) Tween20 .", "For in vivo biotinylation ( Penalva and Keene , 2004 ) , Norrin wild-type or mutants in the pHL-Avitag3 vector ( Aricescu et al . , 2006 ) were co-transfected with a pHLsec construct of BirA-ER ( the synthetic BirA gene with a C-terminal KDEL sequence for retention in the endoplasmic reticulum ) in HEK293T cells .", "Mutant proteins were secreted at similar levels to the wild-type proteins .", "The mammalian cell secretory pathway uses stringent quality control mechanisms to ensure that secreted proteins are correctly folded ( Trombetta and Parodi , 2003 ) .", "The biotinylated Norrin variants were immobilized onto the surface of a CM5 sensor chip ( GE Healthcare Life Sciences ) on which approximately 8500 resonance units of streptavidin were coupled via primary amines .", "Fz4CRD proteins used as analytes were expressed in HEK293T cells to ensure full glycosylation and prepared as described above .", "The signal from SPR flow cells was corrected by subtraction of a blank and reference signal from a mock-coupled flow cell .", "In all analyses , the experimental trace returned to baseline line after a regeneration step with 100 mM phosphate pH 3 . 7 , 2 M NaCl , 1% ( wt/vol ) Tween20 .", "The data were fitted to a 1:1 Langmuir adsorption model ( B = BmaxC/ ( Kd + C ) , where B is the amount of bound analyte and C is the concentration of analyte in the sample ) for the calculation of dissociation constant ( Kd ) values using Biacore Evaluation software ( GE Healthcare Life Sciences ) .", "Data points correspond to the average from two independent dilution series .", "Solution scattering data were collected at beamline BM29 of the European Synchrotron Radiation Facility ( ESRF; Grenoble , France ) at 293 K within a momentum transfer range of 0 . 01 Å−1 < q < 0 . 45 Å−1 , where q = 4πsin ( θ ) /λ and 2θ is the scattering angle ( Pernot et al . , 2013 ) .", "X-ray wavelength was 0 . 995 Å and data were collected on a Pilatus 1M detector .", "Fz4CRD was measured at 1 . 47 and 3 . 10 mg/ml ( deglycosylated form ) and 0 . 97 and 1 . 45 mg/ml ( glycosylated form ) in 10 mM HEPES pH 7 . 5 , 0 . 15 M NaCl .", "Norrin was measured at 0 . 75 and 1 . 26 mg/ml in 10 mM HEPES , pH 7 . 5 , 0 . 7 M NaCl , 0 . 5% ( wt/vol ) CHAPS .", "The deglycosylated Norrin–Fz4CRD complex was measured at 1 . 02 and 2 . 14 mg/ml in 10 mM HEPES , 0 . 5 M NaCl .", "Data reduction and calculation of invariants was carried out using standard protocols implemented in the ATSAS software suite ( Petoukhov et al . , 2012 ) .", "A merged dataset was obtained by merging the low-angle part of the low-concentration dataset with the high-angle part of the high-concentration dataset .", "The Radius of gyration ( Rg ) was obtained from Guinier plot using AutoRg ( Petoukhov et al . , 2012 ) .", "The maximum dimension of the particle ( Dmax ) and Volume Porod ( Vp [nm3] ) were calculated by GNOM ( Svergun , 1992 ) .", "Molecular weights were obtained by", "( a ) comparison with the reference bovine serum albumin ( BSA ) and", "( b ) dividing the Porod Volume by 1 . 66 ( Rambo and Tainer , 2011 ) .", "Theoretical X-ray scattering patterns of structural models were calculated and fitted to experimental X-ray scattering curves using the program FoXS ( Schneidman-Duhovny et al . , 2010 ) .", "The Norrin , Fz4CRD and the Norrin–Fz4CRD complex solution structures were modeled starting from their respective crystal structures .", "Complex glycan structures and missing regions of N- and C-termini were added using the program Modeller ( Eswar et al . , 2003 ) .", "All-atom simulations , and calculation and fitting of scattering patterns of Norrin , Fz4CRD and the Norrin–Fz4CRD complex were performed using the automated AllosMod-FoXS procedure ( Guttman et al . , 2013 ) .", "SEC-MALS experiments were performed by using SEC on an analytical Superdex S200 10/300 GL column ( GE Healthcare Life Sciences ) connected to online static light-scattering ( DAWN HELEOS II , Wyatt Technology , Santa Barbara , CA ) , differential refractive index ( Optilab rEX , Wyatt Technology , Santa Barbara , CA ) and Agilent 1200 UV ( Agilent Technologies , Santa Clara , CA ) detectors .", "Purified sample ( FzCRD proteins at 50 μM or Norrin–Fz4CRD complex at 25 μM ) was injected into a column equilibrated in 10 mM HEPES , pH 7 . 5 , 0 . 15 mM NaCl .", "Molecular mass determination was performed using an adapted RI increment value ( dn/dc standard value; 0 . 186 ml/g ) to account for the glycosylation state .", "The theoretical molecular weight was predicated from amino acid sequence plus 2 . 35 kDa per N-linked glycosylation site for full glycosylated protein produced from HEK293T cells or 203 Da per site for deglycosylated protein produced from HEK293T cells in the presence of kifunensine ( Chang et al . , 2007 ) with limited glycosylation and treated with Endo-F1 .", "Data were analyzed using the ASTRA software package ( Wyatt Technology , Santa Barbara , CA ) .", "The stable HEK293STF cell lines ( Xu et al . , 2004 ) carrying the Super Top Flash firefly luciferase reporter were split into 96-well plates and transfected 24 hr later with 200 ng DNA per well using Lipofectamine 2000 ( Life Technologies , UK ) according to the manufacturer's instructions .", "For assessment of interface mutants used in SPR experiments , the DNA mix contained 80 ng Norrin plasmid , 40 ng each of Fz4 and Lrp6 plasmids , 20 ng each of Tspan-12 and constitutive Renilla luciferase plasmids ( pRL-TK , Promega , Madison , WI ) .", "The firefly and Renilla luciferase activities were measured 48 hr later with Dual-Glo luciferase reporter assay system ( Promega , Madison , WI ) using an Ascent Lunimoskan luminometer ( Labsystems ) .", "For evaluation of recombinant Norrin and SOS inhibition , the DNA mix ( 80 ng pLEXm plasmid , 40 ng each of Fz4 and Lrp6 plasmids , 20 ng each of Tspan-12 and pRL-TK plasmids ) was used for transfection .", "Cells were stimulated 6 hr post transfection with 9 μg/ml Norrin , 9 μg/ml Norrin preincubated with 2 mM SOS for 15 min , or control 9 μg/ml Fetal Calf Serum ( FCS ) .", "The Dual-Glo luciferase reporter assays were performed 48 hr later .", "The firefly luciferase activity was normalized to Renilla luciferase activity ( relative light unit , RLU ) .", "Luciferase reporter assays were performed 3 times in triplicate .", "Protein samples produced in HEK293T cells were freshly purified by SEC and then adjusted in 50 mM Tris , pH 7 . 5 , 0 . 25 M NaCl .", "Purified protein ( 0 . 5 mg ) was loaded onto a 1 ml HiTrap heparin HP column ( GE Healthcare Life Sciences ) equilibrated in 20 mM Tris , pH 7 . 5 , 0 . 25 M NaCl and eluted with a linear NaCl gradient to 20 mM Tris , pH 7 . 5 , 2 M NaCl , 5% ( wt/vol ) glycerol over 10 column volumes .", "Notably , we found that Norrin–Fz4CRD complex tends to partially disassemble ( Fz4CRD detected in flow-through; Figure 7A ) during sample preparation for the heparin binding assay ( NaCl concentration was reduced from 0 . 5M to 0 . 25M ) ." ] ]

[ "Wnt signalling regulates multiple processes including angiogenesis , inflammation , and tumorigenesis .", "Norrin ( Norrie Disease Protein ) is a cystine-knot like growth factor .", "Although unrelated to Wnt , Norrin activates the Wnt/β-catenin pathway .", "Signal complex formation involves Frizzled4 ( Fz4 ) , low-density lipoprotein receptor related protein 5/6 ( Lrp5/6 ) , Tetraspanin-12 and glycosaminoglycans ( GAGs ) .", "Here , we report crystallographic and small-angle X-ray scattering analyses of Norrin in complex with Fz4 cysteine-rich domain ( Fz4CRD ) , of this complex bound with GAG analogues , and of unliganded Norrin and Fz4CRD .", "Our structural , biophysical and cellular data , map Fz4 and putative Lrp5/6 binding sites to distinct patches on Norrin , and reveal a GAG binding site spanning Norrin and Fz4CRD .", "These results explain numerous disease-associated mutations .", "Comparison with the Xenopus Wnt8–mouse Fz8CRD complex reveals Norrin mimics Wnt for Frizzled recognition .", "The production and characterization of wild-type and mutant Norrins reported here open new avenues for the development of therapeutics to combat abnormal Norrin/Wnt signalling ." ]

[ "The cells within an animal need to be able to communicate with each other to coordinate many complex processes in the body , such as the formation of tissues and organs .", "One way in which the cells can communicate is through a pathway called Wnt signalling .", "Generally , one cell releases a protein called Wnt , which binds to a receptor protein called Frizzled that sits on the surface of the same or another cell .", "This activates a series of events in the cells that can change the activity of particular genes .", "Wnt signalling has many roles in animals , and defects in it can contribute to cancer and other devastating diseases .", "Another protein called Norrin can also activate Wnt signalling by binding to Frizzled and another receptor protein called Lrp5/6 .", "This group or ‘complex’ also includes molecules called glycosaminoglycans .", "In humans , mutations in the gene that encodes Norrin can cause a disease in which blood vessels in the eye fail to form correctly , which can result in blindness .", "However , it is not clear how Norrin activates Wnt signalling .", "Chang et al . developed a method to produce large quantities of Norrin protein to allow them to study the structure of the protein .", "Then , a technique called X-ray crystallography was used to reveal the three-dimensional structure of Norrin when it is bound to Frizzled .", "The model reveals that a pair of Norrin proteins form a complex with two Frizzled proteins and highlights particular areas of the Norrin protein that interact with Frizzled .", "Molecules of glycosaminoglycan bind to a site in the complex that spans both Norrin and Frizzled .", "The model also predicts that other areas of the Norrin protein may be involved in binding Lrp5/6 .", "Chang et al . compared the model to the structure of a Wnt protein bound to Frizzled , which revealed that Norrin and Wnt show some fundamental similarities in the way they bind to Frizzled .", "These findings move us closer to defining the essential features of the protein complexes that modify Wnt signalling , and may aid the development of new therapies for diseases that affect the development of the eye ." ]

[ "Introduction", "Results and discussion", "Materials and methods" ]

[ "chromosomes and gene expression", "short report" ]

[ [ "The CRISPR-Cas9 system is a versatile genome-editing tool that enables the introduction of site-specific genetic modifications ( Jinek et al . , 2012 ) .", "In its most widespread variant a programmable chimeric single guide RNA ( sgRNA ) directs the Cas9 nuclease to the genomic region of interest , where it generates a site-specific DNA double-strand break ( DSB ) ( Mali et al . , 2013 ) .", "In mammalian cells the repair of DSBs by different end-joining ( EJ ) pathways , such as classical non-homologous end joining ( c-NHEJ ) , alternative non-homologous end-joining ( a-NHEJ ) , or single-strand annealing ( SSA ) often leads to the formation of insertions or deletions ( indels ) ( Shalem et al . , 2014; Ceccaldi et al . , 2016 ) .", "Alternatively , when a donor template is provided , mammalian cells can also resolve DSBs via homology-directed repair ( HDR ) mechanisms , such as the classical homologous recombination ( HR ) pathway ( Mao et al . , 2008 ) and the Fanconi Anemia ( FA ) repair pathway ( Richardson , 2017 ) .", "While the formation of indels allows the elimination of gene function , repair from an ectopic donor oliogonucleotide ( oligo ) via HDR mechanisms enables the introduction of DNA modifications with single base pair precision ( van den Bosch et al . , 2002 ) .", "Therapeutic applications of CRISPR-Cas9 generally require the precise correction of pathogenic mutations using donor templates .", "However , DSBs introduced in mammalian cells are predominantly repaired by error-prone EJ pathways .", "As the resulting indels inhibit the CRISPR-Cas9 complex from retargeting the locus , error-prone repair indirectly competes with HDR , and therefore reduces the rates of precise correction from donor templates .", "Furthermore , if the targeted allele is a hypomorph with residual gene function , the generated indels can further worsen the clinical phenotype of the disease ( Chu et al . , 2015 ) .", "In recent years , several attempts have therefore been made to enhance HDR-mediated correction of CRISPR-Cas9-induced DSBs from donor oligos .", "Based on the knowledge that HDR pathways are primarily active during the S/G2 phase of the cell cycle , cells have been synchronized prior to CRISPR-Cas9 delivery ( Lin et al . , 2014a ) , and Cas9 expression has been limited to the S/G2/M phase of the cell cycle ( Gutschner et al . , 2016; Howden et al . , 2016 ) .", "Other studies have increased HDR by chemically modulating the EJ and HDR pathways ( Chu et al . , 2015; Maruyama et al . , 2015; Yu et al . , 2015; Song et al . , 2016 ) , and by rationally designing DNA repair templates with optimal homology arm lengths ( Richardson et al . , 2016 ) .", "In addition , it has been proposed that the availability of the DNA repair template might present a rate-limiting factor for HDR , and that enhancing the local concentration of donor oligos could increase the correction rates ( Ruff et al . , 2014; Carlson-Stevermer et al . , 2017 ) .", "Based on this hypothesis , we here generated and tested novel CRISPR-Cas9 variants , in which the DNA repair template is covalently conjugated to Cas9 ( Figure 1 ) ." ], [ "To be able to measure HDR efficiencies of novel CRISPR-Cas9 variants in a rapid and high-throughput manner , we first generated a fluorescent reporter system ( Figure 2a–b ) .", "In brief , the reporter cassette was stably integrated in HEK293T cells , and expresses a green fluorescent protein ( GFP ) that is preceded by an inactive mutant version of a red fluorescent protein ( mutRFP ) .", "While precise correction of the mutation via HDR from donor templates leads to re-activation of RFP activity , the generation of frame shifts via error-prone EJ pathways leads to loss of GFP activity ( Figure 2a–b ) .", "The correction and indel formation events can be visualized by fluorescence imaging and quantified by FACS ( Figure 2c–d ) .", "To test the functionality of the reporter system , and to determine the optimal length of DNA repair templates , we first transfected Cas9-sgRNA ribonucleoprotein ( RNP ) complexes and single stranded ( ss ) oligo repair templates of different lengths ( Figure 3—figure supplement 1a ) .", "In line with previous studies ( Zuo et al . , 2017 ) , we found that maximal DSB correction rates are reached with ss-donor oligos of approximately 80 bases .", "We therefore continued our study with 81-nucleotide ( 81-mers ) ss-donor oligos but also included 65-nucleotide ( 65-mers ) oligos , as we reasoned that if repair templates are brought in proximity to DSBs also shorter homology arms could be sufficient for HDR .", "In order to link the donor oligos to the Cas9 protein , we used the SNAP-tag technology , which allows covalent binding of O6-benzylguanine ( BG ) -labeled molecules to SNAP-tag fusion proteins ( Keppler et al . , 2003 ) .", "To generate O6-benzylguanine ( BG ) -linked DNA repair templates , we first coupled amine-modified oligos to commercially available amine-reactive BG building blocks ( Figure 3a , Figure 3—figure supplement 1c ) .", "The BG-linked oligos were further separated from unreacted oligos by HPLC ( Figure 3b ) and analyzed by liquid chromatography-mass spectrometry ( LC-MS ) to confirm their purity ( Figure 3—figure supplement 1d ) .", "Next , we produced recombinant Cas9 proteins with a SNAP-tag fused to the C-terminus ( Figure 3c , d ) .", "The fusion proteins were then complexed with the BG-coupled oligos , and covalent binding was confirmed by SDS-PAGE ( Figure 3e–h ) .", "The protein-oligo conjugate was mixed with in vitro transcribed sgRNAs targeting the mutRFP locus ( Figure 3—figure supplement 1g , h ) , finally generating the Cas9 ribonucleoprotein-DNA ( RNPD ) complex .", "To test if linking the donor oligo to Cas9 changes the ratio between indel formation and correction from the repair template , we used our reporter system to compare Streptococcus pyogenes Cas9 ( SpCas9 ) complexes with linked repair oligos ( Figure 4b ) to the control SpCas9 complexes with unlinked repair oligos ( SpCas9-SNAP with unlabeled oligos ) .", "Notably , the correction efficiency ( percentage of corrections in edited cells ) with bound complexes was significantly enhanced , from 2 . 1% to 22 . 5% with the 65-mers and from 8 . 9% to 25 . 7% with the 81-mers ( Figure 4a , Figure 4—figure supplement 1a , b ) .", "In comparison to unbound complexes this represented 11- and 3-fold increases , respectively .", "To further test our hypothesis that spatial and temporal co-localization of repair templates and Cas9 enhances correction rates , we next developed an independent approach where the donor oligo is not bound to the Cas9 complex that induces the DSB , but to a second catalytically inactive Cas9 complex that binds in close proximity to the DSB ( RNP-RNPD system ) .", "To avoid the interchange of sgRNAs between both complexes , we designed a two-component system in which the DSB is induced by SpCas9 , and the repair template is linked to a catalytically inactive Staphylococcus aureus ( Sa ) dCas9 ( Figure 4c , d ) .", "We co-transfected both complexes into the reporter cell line , and quantified correction and indel formation rates .", "Notably , the correction efficiency increased from 4 . 1% to 29 . 9% with 65-mers , and from 11 . 6% to 32 . 3% with 81-mers ( Figure 4e , Figure 4—figure supplement 1c , d ) , confirming our previous results with the RNPD system .", "In vivo , the delivery efficiency of RNPs and oligos is generally lower than in vitro .", "Thus , if the repair template is not bound to Cas9 , there is a substantial probability that only one of the two components would be delivered into the cell .", "In addition , at lower transfection efficiencies fewer repair templates are present in the nucleus , potentially decreasing HDR rates .", "As we presumed that linking the donor oligo to Cas9 should largely alleviate these limitations , we investigated whether the repair efficiency with template-conjugated Cas9 is affected when complexes are transfected at 5-fold lower concentrations .", "Importantly , although under these conditions the correction efficiencies were generally lower with both coupled and uncoupled Cas9 complexes , the difference between the two systems was however even more pronounced .", "Compared to the uncoupled RNP complex , the RNPD system yielded 20-fold and a 4-fold increases in repair efficiency with 65-mer and 81-mer repair template oligos , respectively ( Figure 4f , Figure 4—figure supplement 1e , f ) .", "Similarly , the two-component RNP-RNPD system led to a 21-fold increase with 65-mers and a 6-fold increase with 81-mers ( Figure 4f , Figure 4—figure supplement 1e , f ) .", "Taken together , our results suggest that linking the repair template to the Cas9 complex leads to improved correction efficiency compared to the unlinked control CRISPR-Cas9 system , and that this effect is even more pronounced when CRISPR-Cas9 components are delivered at lower concentrations .", "Next , we aimed to gain mechanistic insight into the processes that lead to enhanced correction rates when the donor oligo is linked to the Cas9 complex .", "We first assessed if the BG-labelling of the donor oligo itself already influences the correction efficiencies , and compared the correction rates of wild-type SpCas9 lacking the SNAP-tag together with either unlabelled oligo or BG-labelled oligo .", "While the correction rates were enhanced when the donor oligo was labelled with BG , the increase was several fold lower compared to the system where the oligo was conjugated to the Cas9 RNP complex ( Figure 4—figure supplement 2a–c ) .", "We then investigated if the observed improvement in correction efficiency is due to the donor oligo being brought in close proximity to the DSB , or if it is sufficient to direct the donor oligo to the nucleoplasm .", "We therefore again employed the two-component system with DSB inducing SpCas9 and catalytically inactive SadCas9 conjugated to the donor oligo .", "While in one group SadCas9 was complexed with a sgRNA that directs it in close proximity to the DSB , in the other group the sgRNA was omitted and SadCas9 was therefore only directed into the nucleus .", "Importantly , our results demonstrated that adding the sgRNA did not further enhance correction rates , suggesting that the increase of donor oligo concentration in the nucleoplasm was sufficient to fully account for the positive effect of the Cas9-donor oligo conjugation ( Figure 4g , Figure 4—figure supplement 2d , e ) .", "In line with these observations , a number of previous studies demonstrated that exogenous DNA transport into the nucleus is one of the major barriers to effective gene delivery ( Subramanian et al . , 1999; Zanta et al . , 1999; Ludtke et al . , 1999; Aronsohn and Hughes , 1998 ) .", "To validate our results from the HEK293T reporter cells , we next tested our approach at different endogenous genomic loci and in different cell types .", "We first targeted the human beta globin ( HBB ) locus in the K562 cell line , and analyzed correction and editing frequencies using next generation sequencing ( NGS ) .", "The mean correction efficiency with the RNPD system was 19 . 6% , which represented a 17-fold increase compared to the control RNP system ( Figure 5a , Supplementary file 2 ) .", "Next we targeted the Rosa26 and proprotein convertase subtilisin/kexin type 9 ( Pcsk9 ) locus in mouse embryonic stem cells ( mESCs ) .", "Again , the mean correction efficiencies of RNPD systems were significantly increased , to 18 . 6% at the Rosa26 locus and 23 . 2% at the Pcsk9 locus ( Figure 5b , c , Supplementary file 2 ) .", "In comparison to the uncoupled RNP complexes , this represented 2- and 6-fold increases , respectively .", "In the previous experiments the RNPD system was always compared to Cas9 SNAP-tag fusion proteins with uncoupled donor oligos .", "To also directly compare the engineered RNPD system to the classical CRISPR-Cas9 system , we performed experiments where we used wild-type Cas9 with the uncoupled donor oligos as a control .", "We first targeted the fluorescent reporter locus and analyzed it by NGS .", "We found that while the mean percentage of corrected loci increased from 0 . 8% with the classical Cas9 system to 4 . 9% with the RNPD system , the number of incorrectly edited loci slightly decreased from 12 . 6% to 9 . 3% ( Figure 6a , Supplementary file 2 , 3 , 4 ) .", "This corresponds to a 7-fold increase in correction efficiency ( Figure 6b ) .", "In addition , the analysis of three computationally predicted off-target sites ( Lin et al . , 2014b; Cradick et al . , 2014 ) of the reporter locus , suggests that the risk for generating off-target mutations is not enhanced with the RNPD system ( Figure 6c , Supplementary file 2 , 3 , 4 ) .", "In the next step we also targeted and analyzed the endogenous loci HBB , empty spiracles homeobox 1 ( EMX1 ) , and C-X-C chemokine receptor type 4 ( CXCR4 ) in HEK293T cells .", "NGS analysis revealed that in all three loci the mean correction efficiency of the RNPD system was markedly increased to: 34 , 4% at the HBB locus , 28 . 6% at the EMX1 locus and 33 . 1% at the CXCR4 locus ( Figure 6d , e , f , Supplementary file 2 , 3 , 4 ) .", "Compared to the classical CRISPR-Cas9 system this represents a 20-fold , a 10-fold , and a 24-fold increase , respectively ( Figure 6d , e , f ) .", "Direct delivery of Cas9 RNP complexes into tissues promises great potential for therapeutic applications .", "Compared to genetically encoded systems , RNPs avoid the danger of genomic integration , and due to their limited lifetime , the risk of off-target activities is low ( Kim et al . , 2014 ) .", "In addition , procedures for large-scale production of recombinant proteins for clinical use are well established , and several recently developed protocols enable in vivo delivery of Cas9 RNP complexes in animal models ( Wang et al . , 2016; Zuris et al . , 2015; Staahl et al . , 2017; Lee et al . , 2017 ) .", "Here , we present a method where we enhance correction efficiency of Cas9-induced DSBs by conjugating the donor oligo to the Cas9 complex .", "Our data suggests that the increase in HDR efficiency is caused by enhanced nuclear concentration of the repair template .", "Unlike previous approaches that increase HDR rates by chemically modulating DNA repair pathways , our approach does not alter endogenous cellular processes , thus reducing risk of potential negative side effects .", "In addition , covalent linkage of the repair template to the Cas9 RNP complex also addresses another central challenge of in vivo gene editing therapies – namely that simultaneous delivery of the RNP complex and the repair template needs to be ensured .", "Taken together , we suggest that covalently linking the DNA repair template to the Cas9 RNP complex is poised to further drive the CRISPR/Cas technology towards clinical translation ." ], [ "All plasmids used in this study ( listed in Supplementary file 1-Supplementary Table 6 ) have been deposited for the TULIPs system , along with maps and sequences , in Addgene .", "Cloning of pNS19-LV-mutRFP-2A-GFP: pEGIP ( addgene plasmid #26777 ) was mutagenized using QuikChange Lightning Multi Site-Directed Mutagenesis Kit ( Agilent Technologies ) to destroy the start codon of eGFP .", "Next the vector was linearized with BamHI and In-Fusion HD Cloning Plus CE ( Takara ) was used to insert the mutRFP-2A gBlocks Gene Fragment ( Integrated DNA Technologies ) .", "Cloning of pNS20-SpCas9-SNAP: pMJ922-SpyCas9-GFP bacterial expression vector was a kind gift from Prof . Martin Jinek .", "GFP was digested using BamHI and KpnI , and SNAPtag-NLS gBlocks ( Integrated DNA Technologies ) were integrated using In-Fusion HD Cloning Plus CE ( Takara ) .", "Cloning of pNS38-SadCas9-SNAP: pAD-SaCas9-GFP was generated by replacing the SpCas9 coding sequence in pMJ922 with SaCas9 sequence using Gibson cloning ( Keppler et al . , 2003 ) .", "QuikChange Lightning Multi Site-Directed Mutagenesis Kit ( Agilent Technologies ) was used to remove the stop codon and to introduce the D10A and N580A mutations into the SaCas9 ( SadCas9 ) gene .", "Subsequently , GFP was cut out using BamHI and KpnI , and replaced by a SNAP-tag-NLS gBlock ( Integrated DNA Technologies ) using In-Fusion HD Cloning Plus CE ( Takara ) .", "Plasmid pMJ806 was a gift from Jennifer Doudna ( Addgene plasmid # 39312 ) ( Jinek et al . , 2012 ) .", "Synthetic oligonucleotides with a 5′-Amino Modifier C6 functional group ( 100 μM ) ( Integrated DNA Technologies ) were incubated with benzylguanine-GLA-NHS ( 1 mM ) ( NEB ) and Hepes pH8 . 5 ( 200mM ) for 60 min at 30°C .", "Coupling reactions were performed in following ratios: 30:1 , 60:1 and 100:1 BG-GLA-NHS: amino modified oligo .", "After the coupling reaction all oligos were purified by ethanol precipitation .", "Repair oligo sequences can be found in Supplementary file 1-Supplementary Table", "4 . The benzylguanine ( BG ) coupled reactions were run on 20% polyacrylamide TBE gel containing 8M urea at 200 V for 60 min .", "The gel was stained for 30 min in 1x TBE containing Sybr Gold ( Invitrogen ) , and imaged with a UV transilluminator ( Biorad ) .", "Benzylguanine coupled oligos were purified on an Agilent 1200 series preparative HPLC fitted with a Waters XBridge Oligonucleotide BEH C18 column , 10 × 50 mm , 2 . 5 μm at 65°C using a gradient of 5–25% buffer B over 8 min , flow rate = 5 ml min-1 .", "Buffer A was 0 . 1 M triethylammonium acetate , pH 8 . 0 .", "Buffer B was methanol .", "Fractions were pooled , dried in a speedvac and dissolved in H2O .", "Analysis of the purified BG-oligonucleotide was conducted on an Agilent 1200/6130 LC-MS system fitted with a Waters Acquity UPLC OST C18 column ( 2 . 1 × 50 mm , 1 . 7 μm ) at 65°C , with a gradient of 5–35% buffer B in 14 min with a flowrate of 0 . 3 mL min−1 .", "Buffer A was aqueous hexafluoroisopropanol ( 0 . 4 M ) containing triethylamine ( 15 mM ) .", "Buffer B was methanol .", "Snap-tagged Streptococcus pyogenes Cas9 ( SpCas9-SNAP ) , Staphylococcus aureus dCas9 ( SadCas9-SNAP ) and Wild Type Streptococcus pyogenes Cas9 ( SpCas9 WT ) proteins were expressed in Escherichia coli BL21 ( DE3 ) Rosetta 2 ( Novagen ) fused to an N-terminal fusion protein containing a hexahistidine affinity tag , the maltose binding protein ( MBP ) polypeptide sequence , and the tobacco etch virus ( TEV ) protease cleavage site .", "The cells were lysed in 20 mM Tris pH 8 . 0 , 500 mM NaCl , 5 mM Imidazole pH 8 . 0 .", "Clarified lysate was applied to a 10 ml Ni-NTA ( Qiagen ) affinity chromatography column .", "The column was washed by increasing the imidazole concentration to 10 mM and bound protein was eluted in 20 mM Tris pH 8 . 0 , 250 mM NaCl , 100 mM Imidazole pH 8 . 0 .", "To remove the His6-MBP affinity tag , the eluted protein was incubated overnight in the presence of TEV protease .", "The cleaved protein was further applied to a heparin column ( HiTrap Heparin HP , GE Healthcare ) and eluted with a linear gradient of 0 . 1–1 . 0 KCl .", "The eluted protein was further purified by size exclusion chromatography using a Superdex 200 16/600 ( GE Healthcare ) equilibrated in 20 mM HEPES pH 7 . 5 , 500 mM KCl .", "Repair oligo templates coupled to BG were incubated with Cas9-SNAP proteins on the same day when the transfection is performed .", "BG-coupled oligos ( 2 . 2 pmols ) were mixed with either SpCas9-SNAP or SadCas9-SNAP ( 2 . 2 pmols ) and incubated for 60 min at 30°C .", "The negative controls ( wild-type Cas9 +BG oligo or Cas9-SNAP + unlabeled oligo ) were treated in the same way .", "For confirming successful labeling of the Cas9-SNAP proteins with the BG-coupled oligonucleotides , BG-coupled and uncoupled oligonucleotides were mixed with either SpCas9-SNAP , SadCas9-SNAP or only the Cas9-SNAP proteins alone , reactions were incubated for one hour at 30°C .", "For the SNAP-Vista Green ( NEB ) substrate , the protein was incubated for 30 min on 30°C in the dark .", "After incubation , reactions ( 300 ng ) were loaded on 6% SDS-PAGE gel and run at 80V for 160 min .", "Gels that were containing BG-Vista Green ( NEB , SNAP-Vista Green ) , were imaged prior to silver staining .", "The green fluorescence signal of the SNAP-tag was detected with a UV transilluminator ( Biorad ) .", "Subsequently , silver staining was completed using the Pierce Silver Stain Kit ( Thermo Scientific ) according to manufacturer instructions , and imaged with a UV transilluminator ( Biorad ) .", "sgRNAs were generated from DNA templates using the T7 RNA Polymerase ( Roche ) in vitro transcription ( IVT ) kit .", "In short , sgRNA specific primers that also contain the T7 sequence were annealed with a common reverse primer that contains the sequence of the sgRNA scaffold ( final concentrations 10 μM ) .", "DNA was purified with the QIAquick purification ( Qiagen ) kit and eluted in DEPC-treated water .", "PCR products were run on agarose to estimate concentration and to confirm amplicon size .", "In vitro transcription was performed at 37°C overnight .", "For purification , DNase I was added to the sgRNAs and incubated for 15 min at 37°C , and subsequently ethanol precipitation was performed overnight at −20°C .", "The sgRNAs were then further purified using RNA Clean and Concentrators ( Zymo Research ) .", "Before use , all sgRNAs were checked on denaturing 2% MOPS gels .", "Complete sequences for all sgRNA protospacers , IVT primers and crRNAs can be found in Supplementary file 1-Supplementary Table 1 , 2 and 3 , respectively .", "HEK293T were PEI transfected with following plasmids: pNS19-LV-mutRFP-2A-eGFP , Pax2 and VSV-G .", "After 12 hr , the supernatant was discarded and changed to DMEM plus 10% FBS .", "24 and 72 hr post-transfection , the media was collected and filtered through 0 . 45 μm filter and centrifuged at 20 000 G for 2:00 hr at 4°C .", "The pellet was then resuspended in 1 ml of DMEM and stored at −80°C .", "HEK293T cells were transduced with a lentiviral vector carrying the fluorescent reporter construct .", "Serial virus dilutions were used to isolate clonal populations using Puromycine selection ( 2 μg/ml ) for 2 weeks .", "HEK293T cells were obtained from ATCC and verified mycoplasma free ( GATC Biotech ) .", "The HEK293T reporter line was maintained in DMEM with GlutaMax ( Gibco ) .", "Media was supplemented with 10% FBS ( Sigma ) , and 100 μg/mL Penicillin-Streptomycin ( Gibco ) .", "K562 cells were obtained from Sigma Aldrich , verified mycoplasma free and were maintained in RPMI 1640 medium with GlutaMax .", "Additional the medium supplemented with 10% FBS , and 100 μg/mL Penicillin-Streptomycin .", "Cells were passaged three times per week .", "Cells were grown at 37°C in a humidified 5% CO2 environment .", "WT E14 mESC line ( ATCC CRL-1821 ) was cultured in Dulbecco’s Modified Eagle Media ( DMEM ) ( Sigma-Aldrich ) , containing 15% of fetal bovine serum ( FBS; Life Technologies ) , 100 U/mL LIF ( Millipore ) , 0 . 1 mM 2-ß-mercaptoethanol ( Life Technologies ) and 1% Penicillin/Streptomycin ( Gibco ) , on 0 . 2% gelatin-coated support in absence of feeder cells .", "The culture medium was changed daily .", "Cells were grown at 37°C in 8% CO2 .", "HEK293T cells were seeded in 24-well plates at 120 . 000–140 . 000 cells per well , 1 day prior to transfection .", "K562 cells were 6 hr prior to transfection distributed in 24 well plates at a density of 220 . 000–240 . 000 cells per well .", "On the day of transfection , RNP and RNPD complexes ( 2 . 2 pmols ) were complexed with sgRNA ( 3 . 88 pmols ) in Opti-MEM ( Invitrogen ) and briefly vortexed , followed by adding 3 μl the Lipofectamine 2000 reagent ( Invitrogen ) with Opti-MEM .", "The resulting mixture was incubated for 15 min at room temperature to allow lipid particle formation .", "After 15 min of incubation at room temperature , the mixture was dropped slowly into the well .", "One day post-transfection , cells were transferred to an 10 cm dish .", "mESCs were transfected into 6-well plate using Lipofectamine 2000 .", "Cells were plated 24 hr before transfection at a density of 20 , 000 cells/cm2 per well and cultured in culture medium without streptomycin and penicillin .", "The medium was changed to mESC culture medium 8 hr after transfection .", "Cells were collected 48 hr post-transfection .", "For flow cytometry analysis , HEK293T reporter cells were analysed 5 days after transfection .", "Cells were trypsinized with TrypLE Express Enzym ( Gibco ) , and resuspended in FACS buffer containing PBS/1% FBS/1% EDTA .", "Sytox Red was added for the exclusion of dead cells .", "Data were acquired on a BD LSR Fortessa cell analyser ( Becton-Dickinson ) and were further analysed using FlowJo software ( FlowJo 10 . 2 ) .", "In all experiments , a minimum of 200 . 000 cells were analysed .", "Gating strategy: Forward versus side scatter ( FSC-A vs SSC-A ) gating was used to identify cells of interest .", "Doublets were excluded using the forward scatter height versus forward scatter area density plot ( FSC-H vs . FSC-A ) .", "Live cells were gated based on Sytox-Red-negative staining .", "Live-gated cells were further used to quantify the percentage of eGFP negative and turboRFP positive populations .", "Correction efficiency ( % ) or ( percentage of corrections in edited cells ) was culculated as 100 * ( eGFP/turboRFP double positive population / ( eGFP/turboRFP double negative population + eGFP/turboRFP double positive population ) ) .", "Transfected cells were collected by trypsinisation and were washed with PBS .", "PBS was discarded and DNA extraction was preformed using DNeasy Blood and Tissue kit ( Qiagen ) following manufacturer’s protocol .", "The PCR amplicons flanking the targeted site were generated using NEBNext High-Fidelity 2X PCR Master Mix ( NEB ) , primers that were used are listed in Supplementary file 1-Supplementary Table", "5 . PCR cycling conditions used were as follows: 1 × 98°C for 3 min; 27 × 95°C for 15 s , 65°C for 15 s , 72°C for 30 s; 1 × 72°C for 5 min .", "Annealing temperature was optimized for each primer set to ensure that a single amplicon was produced .", "PCR amplicons were purified by solid phase reversible immobilization ( SPRI ) bead cleanup using Agencourt AMPure XP reagent ( #A63881 , Beckmann-Coulter , Indianapolis , IN , USA ) , per the manufacturer’s instructions .", "For the generation of the pooled sequencing libraries , the TruSeq ( Illumina ) Index Adaptor Sequences were added at the second amplification step .", "The resulting Illumina libraries with Index Adaptors were purified with AMPure XP reagent .", "Quality control for the final library was performed using the High sensitivity D1000 ScreenTape at Agilent 2200 TapeStation .", "The libraries were sequenced using an Illumina MiSeq sequencer ( MiSeq Reagent Kit v2 ( 15M , 500 cycle kit ) or MiSeq Reagent Micro Kit v2 ( 4M , 500 cycle kit ) , Illumina , San Diego , CA , USA ) .", "Sequences were received in the format of demultiplexed FASTQ files produced by Illumina's bcl2fastq software ( v2 . 19 . 0 ) .", "Reads were merged with Pear v0 . 9 . 8 ( Zhang et al . , 2014 ) and merged reads mapped to the amplicon sequences with BWA-MEM v0 . 7 . 13-r1126 ( Li , 2010 ) .", "Unmerged reads were discarded .", "Sequence analysis was performed in R using CrispRVariants v1 . 9 . 0 ( Lindsay et al . , 2016 ) .", "Variants within the protospacer +PAM region were analysed .", "Reads that align linearly and do not match the guide sequence are considered ‘Edited’ .", "Percentage of edited alleles is calculated as 100* Edited reads/Total reads ( excluding non-linear alignments ) .", "Correction efficiency is 100* Perfectly corrected/Edited reads .", "The Scripts for mapping sequencing data , counting mutations and generating plots are available at https://github . com/HLindsay/Savic_CRISPR_HDR ( Lindsay , 2018; copy archived at https://github . com/elifesciences-publications/Savic_CRISPR_HDR ) Fastq files have been uploaded to ArrayExpress ( Brazma et al . , 2003 ) , the accession number is E-MTAB-6808 .", "HEK293T reporter cells were imaged 7 days after transfection .", "Transfected cells were grown on Poly-L-lysine coated 8-well glass chamber slides ( Vitaris ) to 80–90% confluence .", "Hoechst 33342 ( Thermo Scientific , Pierce ) was added in the cell culture media to a final concentration of 0 . 1 μg/ml , and cells were incubated for 10 min at 37°C , 5% CO2 , prior to the image session .", "Confocal imaging was performed using a Leica DMI8-CS ( ScopeM ) with a sCMOS camera ( Hamamatsu Orca Flash 4 . 0 ) .", "The laser unit for confocal acquisition ( AOBS system ) contains 458 , 477 , 488 , 496 , 514 nm ( Argon laser ) , 405 nm , 561 nm , 633 nm .", "Images were acquired using Leica LAS X SP8 Version 1 . 0 software , through using a 20 × 0 . 75 NA HC PLAN APO CS2 objective .", "Imaging conditions and intensity scales were matched for images presented together .", "Images were analysed using the Leica LAS AF ( Lite ) software version 3 . 3 .", "Confocal images were processed using ImageJ software ( Version 1 . 51 n ) .", "Statistical analyses were conducted using Graphpad’s Prism7 software .", "A Mann-Whitney T-test was conducted for two-sample analyses ( P value style: 0 . 1234 ( ns ) , 0 . 0332 ( * ) , 0 . 0021 ( ** ) ) .", "All values are shown as mean ± s . e . m of biological replicates .", "The number of biological replicates for each experiment was detailed in the Figure Legends .", "Numerical data and the exact p values for all graphs have been included in the Source data files .", "The data that support the findings of this study are available within the paper , Supplementary files , Source data and NGS Fastq files have been uploaded to ArrayExpress ." ] ]

[ "The CRISPR-Cas9 targeted nuclease technology allows the insertion of genetic modifications with single base-pair precision .", "The preference of mammalian cells to repair Cas9-induced DNA double-strand breaks via error-prone end-joining pathways rather than via homology-directed repair mechanisms , however , leads to relatively low rates of precise editing from donor DNA .", "Here we show that spatial and temporal co-localization of the donor template and Cas9 via covalent linkage increases the correction rates up to 24-fold , and demonstrate that the effect is mainly caused by an increase of donor template concentration in the nucleus .", "Enhanced correction rates were observed in multiple cell types and on different genomic loci , suggesting that covalently linking the donor template to the Cas9 complex provides advantages for clinical applications where high-fidelity repair is desired ." ]

[ "Genome editing allows scientists to change an organism’s genetic information by adding , replacing or removing sections of its DNA sequence .", "The CRISPR-Cas9 system is a genome-editing tool that has had a large impact on biological research in recent years , and also shows promise for the treatment of patients with genetic disorders .", "The tool works as follows: a small piece of RNA ( a close cousin to DNA ) is used to guide an enzyme called the Cas9 endonuclease to the desired region of the genome .", "Then , like a pair of molecular scissors , the enzyme cuts the DNA , breaking both strands of its double helix .", "The cell naturally starts to repair the damaged DNA , and one way to do this is to use another similar piece of intact DNA as a template .", "Scientists can exploit this repair mechanism ( known as homology-directed repair ) by giving the cell extra DNA that carries their desired sequence change , with the hope that the cell will use it as a template and edit its own genome in precisely the same way .", "However , it turns out that mammalian cells rarely use the template DNA to repair the damage .", "Instead , mammals tend to fix double-stranded breaks in DNA by simply joining the broken ends together , a method that is prone to errors .", "To overcome this specific issue , Savic , Ringnalda et al . tested the effect of physically linking the template DNA to the Cas9 enzyme , so that the DNA was already nearby when the enzyme made the cut .", "Experiments with human cells confirmed that this new approach increased the frequency of homology-directed repair up to 24-fold compared to leaving the enzyme and the template DNA separate .", "Improving the CRISPR-Cas9 system in this manner makes it more likely that genome editing may one day become a routine treatment for patients with genetic disorders .", "But first , more preclinical studies are needed to assess the safety of the CRISPR-Cas9 technology for gene editing in patients ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "chromosomes and gene expression", "computational and systems biology", "tools and resources" ]

[ [ "Systematic perturbation of gene function in eukaryotic cells using arrayed ( well-by-well ) reagents is a powerful technique that has been used to successfully assay many fundamental biological questions such as proliferation , protein secretion , morphology , organelle maintenance , viral entry , synthetic lethality , and other topics ( Mohr et al . , 2014 ) .", "An alternative approach , widely used in mammalian cells , is pooled screening that uses limited titers of integrating lentiviral vectors carrying a perturbative DNA sequence such that each cell receives one integrating virus .", "In pooled screens , the perturbing DNA reagent serves as the tag in subsequent sequencing ( Berns et al . , 2004; Moffat et al . , 2006; Brummelkamp and Bernards , 2003 ) .", "A key benefit of this approach is that pool size can be extremely large , allowing high reagent multiplicity and thus increased screen quality .", "The pooled approach in mammalian cells has been used extensively to perform RNAi and more recently single guide RNA ( sgRNA ) screens using CRISPR/Cas9 ( Shalem et al . , 2014; Wang et al . , 2015; Hart et al . , 2015 ) .", "Genetic loss-of-function arrayed RNAi screens in Drosophila cell lines have provided insight into genes regulating various biological processes ( Boutros et al . , 2004; Björklund et al . , 2006; Kiger et al . , 2003; D'Ambrosio and Vale , 2010; Bard et al . , 2006; Guo et al . , 2008; Hao et al . , 2008; Housden et al . , 2015 ) .", "However , this approach has drawbacks that limit resolution , including off-target effects and incomplete loss-of-function due to RNAi , and the high cost of reagent multiplicity and replication due to the arrayed format .", "Pooled CRISPR may address the major drawbacks: CRISPR generates complete loss-of-function alleles and causes fewer off-target effects on average ( Morgens et al . , 2016; Evers et al . , 2016 ) , and the pooled format allows greater multiplicity and replication for unit cost .", "Approximately half of the genes in Drosophila , arguably the best characterized multicellular genetic model system , lack functional characterization ( Ewen-Campen et al . , 2017 ) , so the need to develop orthogonal screening approaches is clear .", "By comparing the abundance of guides present in actively growing populations of cells at different time points during growth , CRISPR screens provide a relative measurement of cell doubling , but because the cause of proliferation reduction is unclear from the screen alone , the screens are said to identify genes necessary for optimal fitness rather than essential genes ( Hart et al . , 2015 ) .", "Essential genes , those absolutely required for cell doubling , are , by definition , a subset of fitness genes .", "Here , we introduce a new method to deliver pooled DNA libraries stably into cell lines .", "We use this technology to conduct a genome-wide CRISPR screen for optimal fitness in Drosophila cells and identify 1235 genes essential for fitness , 303 of which are uncharacterized in Drosophila .", "Moreover , we show that the system can be used in combination with drug perturbation to identify genes that when knocked out buffer cells against the drug or act synergistically with it .", "The method should be amenable to adapting any pooled DNA library screening approach to Drosophila or other invertebrate cell lines , such as shRNA knockdown ( Berns et al . , 2004 ) or CRISPR activation/inhibition ." ], [ "Pooled mammalian cell-line screens use lentiviral vectors to deliver highly complex libraries of DNA reagents .", "However , the use of lentiviral vectors in insect cells is extremely inefficient ( unpublished observations ) possibly due to toxicity ( Qin et al . , 2010 ) .", "An important advantage of library delivery using lentiviral transduction is that each sequence integrates into a transcriptionally active site in the host chromosome where it is expressed and remains in the genome as a detectable barcode such that enrichment or depletion of each sequence can be monitored by massively parallel ( next-generation ) sequencing .", "As an alternative strategy , we assessed the efficiency of phiC31 site-specific recombination mediated by plasmid transfection .", "Methods for recombination in cell lines were recently developed ( Manivannan and Simcox , 2016; Neumüller et al . , 2012; Cherbas et al . , 2015 ) , but the quantitative efficiency of integration has not been reported .", "Efficiency is critical for pooled screening applications as it must reach a threshold above which generating and maintaining >1000X representation of a library of tens of thousands of elements becomes technically and cost-feasible ( Kampmann et al . , 2014 ) .", "We note that a previous study attempted to use pooled transient transfection for barcoded delivery of a DNA library in Drosophila cells but failed to identify any essential genes using the system , most likely due to the lack of a mechanism for retention of the DNA reagents in the cells through extended passaging ( Bassett et al . , 2015 ) .", "To test phiC31 integration efficiency in Drosophila cells , a Drosophila S2R + cell line derivative , PT5 , harboring a mobilized MiMIC transposon containing attP sites flanking mCherry ( Neumüller et al . , 2012 ) was transfected with a plasmid containing attB flanking a GFP-2A-Puro ( res ) cassette , which we termed ‘pLib6 . 4’ , along with a phiC31 helper plasmid ( Figure 1A ) .", "The population was then passaged for two months to dilute unintegrated DNA .", "Importantly , no selection reagent was added during these passages in order to monitor the efficiency of integration rather than the added efficiencies of integration and selection .", "Integration efficiency , inferred through flow cytometry ( Figure 1B ) , suggested that phiC31-mediated cassette exchange occurred in ~20% of the cells ( even without accounting for incomplete transfection ) , 123-fold more than the background illegitimate recombination rate observed without phiC31 ( Figure 1B ) .", "Interestingly , pLib6 . 4 , which contains separated attB sites flanking the DNA sequence to be integrated , allowed ~40 fold greater integration efficiency than traditional Drosophila attB40 vectors such as pCa4B ( Markstein et al . , 2008 ) , in which the attB sites are adjacent and require integration of the entire plasmid ( data not shown ) .", "Growth in puromycin enriched for integrants ( Figure 1B ) .", "We next adapted the platform to CRISPR-Cas9 knockout screening .", "First , we generated PT5 S2R + cells stably expressing metallothionein-driven SpCas9 ( ‘PT5/Cas9’ ) .", "In combination with an sgRNA targeting the nonessential gene Dredd , PT5/Cas9 cells without induction were capable of editing nearly 50% of Dredd alleles , which was higher than that achieved by repeated rounds of transient transfection with a SpCas9 expression plasmid ( Figure 1C ) and not improved by copper induction ( Figure 1—figure supplement 1 ) .", "In a pilot test of a pooled screening system , we transfected cells with a pool of sgRNAs in pLib6 . 4 and monitored sgRNA abundance following passaging , reasoning that sgRNAs targeting genes required for optimal fitness would be lost while genes whose presence or absence has no effect on cell growth would be retained ( Figure 1D ) .", "After 60 days ( roughly 60 cell doublings ) , the pools were significantly depleted of some sgRNAs targeting the essential genes Rho1 and Diap1 relative to those targeting genes predicted to have non-essential functions ( Figure 1E ) .", "Monitoring Rho1 or Diap1-targeted sgRNAs in the screen pools 30 , 45 , or 60 days post-transfection showed that 45 days of passaging is optimal ( Figure 1F ) .", "A principle concern of transfection-mediated pooled screening is the potential for low signal-to-noise due to multiple sgRNA delivery to the same cell shortly after transfection , but the subsequent loss of all but one sgRNA at the end of the assay .", "To determine whether the signal-to-noise ratio could be improved by reducing transfection multiplicity , we developed in parallel an inducible Cas9 expression system in S2R+/PT5 cells using intein-Cas9 ( Davis et al . , 2015 ) coupled with inducible expression in order to withhold Cas9 activity until sgRNAs have integrated ( Figure 1G , Figure 1—figure supplement 2 ) .", "Surprisingly , a comparison of dropout efficiencies between the inducible and constitutive Cas9 platforms showed more selective reduction of Rho1 or Diap1 sgRNAs with constitutive rather than inducible Cas9 , most likely due to the lower overall editing efficiency from intein-Cas9 as previously reported ( Liu et al . , 2016 ) ( Figure 1G , Figure 1—figure supplement 2B , C ) .", "From these results , we conclude that the use of phiC31 integration into the PT5/Cas9 cells is suitable for scalable perturbation screening using a pooled sgRNA library in Drosophila cells .", "To construct a genome-wide sgRNA knockout library for Drosophila , we pre-computed sgRNAs for the first half of the coding region of all genes ( Ren et al . , 2013 ) , applied efficiency and frame-shift filters previously shown to correlate with reagent success ( Housden et al . , 2015; Bae et al . , 2014 ) , and ranked the remaining designs based on the uniqueness of the seed region sequence ( 12 bp downstream of the PAM ) and the number of potential off-target sites .", "The top ranked 6–8 sgRNAs per gene ( 85 , 558 in total ) were chosen and synthesized together on a microarray chip with non-targeting and intergenic negative controls , harvested by PCR , and cloned into pLib6 . 4 using published methods ( Gilbert et al . , 2014 ) .", "We used a barcode strategy to separate the full library into three focused library pools so that we could perform focused or genome-wide screens .", "Each focused subset of the library targets a unique set of experimental genes as well as a common set of controls ( Figure 2A; Supplementary file 1 ) .", "To identify fitness genes under basal growth conditions , we transfected cells with pLib6 . 4 containing the library of sgRNAs along with phiC31 helper plasmid and passaged the cells every 5 days for 45 days .", "We transfected ~1500 cells per sgRNA and carried >1500 cells per sgRNA per passage to maintain the diversity of the original library .", "Each experimental library was in-line barcoded using specific primers and sequenced together using next-generation sequencing ( Figure 1—figure supplement 3 ) .", "To determine reagent correlation , the three separated sgRNA pools were each transfected and cells passaged independently and common controls were compared ( Figure 2A; Supplementary file 1 ) .", "Comparing sgRNAs prior to transfection versus 45 days after transfection yielded high reagent correlations between the same sgRNAs as demonstrated for Rho1 or intergenic guides , for which different sequences produced a highly reproducible range of varied dropout efficiencies ( Figure 2B; Supplementary file 1 ) .", "We used MAGeCK ( Li et al . , 2014 ) maximum likelihood estimation ( MLE ) to compute fitness Z-scores for each gene from the log2 fold-changes of individual sgRNAs , which yielded good gene-level correlation between sequential biological replicates ( Figure 2C; Pearson’s r = 0 . 65 ) .", "Because all true fitness genes must be expressed as mRNA , fitness scores were compared with gene expression from S2R+ cells ( Celniker et al . , 2009 ) as an orthogonal validation of the CRISPR results ( Figure 2D ) .", "Fitness genes were highly enriched for genes with any expression level of RPKM > 1 ( Figure 2D ) .", "By ranking genes by fitness score , we calculated a rank-wise false-discovery rate ( FDR , defined as cumulative distribution of false-positive/[true positive + false positive] ) and set a cutoff at 5% , identifying 1235 fitness genes ( Figure 2E ) .", "The current state-of-the-art for Drosophila cell-based screens is arrayed RNAi ( Mohr et al . , 2014 ) .", "We re-analyzed a genome-wide RNAi viability screen in S2R+ cells ( Boutros et al . , 2004 ) and removed double-stranded RNA designs with predicted off-targets , which significantly lowered the FDR of the RNAi screen ( see Materials and methods ) .", "Nevertheless , compared with pooled CRISPR screen results , the re-analyzed RNAi-based fitness gene set still had a far higher FDR , allowing the discovery of only 145 fitness genes at an FDR of 5% or incurring 44% FDR with a fitness gene set of 1235 genes ( Figure 2E ) .", "Among genes with an RPKM > 1 , RNAi was more likely to identify highly expressed genes , whereas this bias was less prominent in the CRISPR screen ( Figure 2—figure supplement 1 ) .", "We used receiver-operating characteristic ( ROC ) curves to determine screen sensitivity for conserved , large macromolecular complexes .", "Whereas CRISPR and RNAi were both able to detect cytoplasmic ribosomal or proteasomal genes , CRISPR was better able than RNAi to identify significant fractions of the mitochondrial ribosome , aminoacyl-tRNA ligases , RNA polymerase II , basal transcription factors , RNA exosome , or the replication fork at low FDR ( Figure 2F ) .", "As a control , neither method detected peroxisome genes , consistent with the observation that cell lines lacking peroxisomes grow normally ( Goldfischer et al . , 1973 ) ( data not shown ) ( Figure 2F ) .", "Interestingly , a human CRISPR-RNAi screen comparison also found a similar inability of RNAi to detect genes of the mitochondrial ribosome whereas it exceeded CRISPR sensitivity in detecting genes of the cytoplasmic ribosome , possibly due to differences in mRNA stability ( Hart et al . , 2015 ) .", "A technical comparison of CRISPR screens in humans and fly cells indicated that the method performs similarly in both systems .", "First , the fly screen had similar sensitivity ( true-positive rate at 5% FDR ) to genome-wide screens in human cell-lines performed with similar reagent number ( GeCKO v2 screens ) when compared against top RNAi hits ( Sanjana et al . , 2014 ) ( Figure 2G ) .", "By comparing sgRNAs that dropped out versus those that failed to dropout for known essential genes , we were able to compute a probability-based position matrix for optimal sgRNA design in Drosophila ( Figure 2H ) .", "The position matrix shows complementary nucleotide biases at 15/21 positions and is broadly consistent with human CRISPR screens ( Doench et al . , 2016 ) .", "Specifically , out of the first position of the PAM sequence and the 8 bp seed region before the PAM sequence , 5 of 9 positions are fully consistent while 2 of 9 position are partially consistent with the Doench score ( Doench et al . , 2016 ) ( Figure 2H ) .", "The analyses support an underlying mechanistic unity of targeting by Cas9 and NHEJ repair between human and fly cells .", "Copy number has been shown to correlate with the CRISPR viability score in some mammalian CRISPR screens , presumably due to induction of greater DNA damage foci for copy-number-amplified ( CNA ) loci , creating spurious fitness calls ( Meyers et al . , 2017 ) .", "By contrast , we find that CRISPR in Drosophila shows no detectable bias towards CNA genes for 97% of the genome ( Figure 2—figure supplement 1B ) .", "Interestingly , genes with extreme CNA of 8 or more copies ( 3% of genes ) exhibited a bias of ~1 . 8 fold in the CRISPR screen , but the magnitude of this bias was similar to that seen in an RNAi screen for the same genes ( Figure 2—figure supplement 1B ) , suggesting that CNA genes in Drosophila cells are enriched for fitness essentiality .", "Finally , copy-number correction had no detectable effect on FDR , true-positive rate , or enrichment of major macromolecular complexes ( not shown ) .", "The analysis shows that Drosophila CRISPR screens do not have detectable copy-number bias .", "We next analyzed fitness genes in Drosophila S2R+ cells .", "We first performed gene ontology enrichment on CRISPR screen hits at 5% FDR .", "The gene set is enriched for essential processes such as translation , transcription , splicing , etc . and depletion for processes such as chitin metabolism that are necessary in whole flies but not cells ( Figure 3A ) .", "Next , we compared CRISPR hits to fly genes with lethal alleles annotated in FlyBase after removing genes for which no allele has yet been constructed .", "At 5% FDR , ~17% of cell-essential genes overlapped with whole-fly essential genes ( Figure 3B ) .", "This overlap is in a similar range as knockout mouse viability compared with mouse cell-line fitness essentiality ( where the overlap is ~27% ) ( Bartha et al . , 2018 ) , and may represent different genetic requirements of whole animals versus cell lines or of germline versus somatic cells ( Garcia-Bellido and Robbins , 1983 ) .", "A high-resolution fitness map in fly cells now allows us to compare fitness genes among species .", "The fly fitness gene set partially but significantly overlaps with characterized fitness gene sets from yeast and human cells ( Figure 3C ) .", "Moreover , an unbiased comparison of gene ontology terms between fly and human fitness genes displays a high degree of correlation ( Figure 3D; Pearson’s r = 0 . 56 ) .", "Despite significant overlap between fly and human cell-line fitness genes and conservation of gene ontology terms enriched in both screens , many fly fitness genes map to human genes that were not identified in human CRISPR screens .", "We hypothesized that fly CRISPR screens identify essential genes in Drosophila cells but not in human cells due to paralog redundancy .", "To test this hypothesis , we inferred putative paralog relationships for moderately or highly conserved orthologs using the DRSC Integrative Ortholog Prediction Tool ( DIOPT ) ( Hu et al . , 2011 ) and retrieved fitness scores for the corresponding human gene using genome-wide human cell-line CRISPR data ( Hart et al . , 2015; Lenoir et al . , 2018 ) ( Figure 3E ) .", "Further , to account for the possibility that paralogs may not be expressed , we only analyzed genes that were expressed as mRNA in the human cell-line ( Lenoir et al . , 2018 ) ( Figure 2E ) .", "Using this framework , 35% of conserved human genes contain a fly-to-human paralog , and 407 fly fitness genes ( at 5% FDR ) are the orthologs of human genes containing at least one paralog .", "In the human cell-line CRISPR screen , we noted a significant bias against detecting genes that are conserved in flies and have a paralog in the human genome , and this bias was dramatically elevated for the human orthologs of fly fitness genes ( Figure 3F ) .", "Furthermore , this paralog effect extended to a larger dataset generated by the cancer Dependency Map project , in which CRISPR screens were conducted in 436 human cell-lines .", "By defining a fitness gene as any gene with a CERES score less than 0 . 8 , we sorted human orthologs of fly fitness by the number of cell-lines out of 436 in which they displayed a fitness defect and according to their paralog relationship between flies and humans ( Figure 3G ) .", "The result again showed a bias against detecting genes with fly-to-human paralogs in the panel of CRISPR screens ( Figure 3H ) .", "Thus , we propose that functional redundancy among closely related genes buffers each of them and makes them invisible in viability screens , and that Drosophila cells may be more appropriate for screening genes that expanded during the vertebrate lineage .", "Although the Drosophila fitness genes we identified are enriched for characterized phenotypes and publication count relative to all genes ( data not shown ) , phenotypes have yet to be described for 303 of them .", "Among these 303 genes , 251 have human orthologs ( Supplementary file 2 ) .", "Thus , further studies of these conserved genes are likely to provide new insights into conserved , cell essential processes not yet studied in flies .", "Also of interest are fly-specific fitness genes , as they present a paradox and may reveal novel species-specific biology or overlooked structural/functional analogs without sequence orthology and may have potential as targets for new insecticides .", "At 5% FDR , we obtained 62 fitness genes with no sequence similarity outside of flies using DIOPT , and phenotype information exists regarding 25 of these ( Supplementary file 2 ) .", "In confirmation of our methods , these included known cell-essential divergent genes such as ver and HipHop , which encode components of the telomerin complex , the putative functional analog of mammalian telomerase ( Raffa et al . , 2011; 2010 ) , and Kmn1 , Kmn2 and Spc105R , whose gene products may be structural anologs of Ndc80 , and Mis12 complex components that interact with centromeric DNA , a proposed driver of speciation ( Schittenhelm et al . , 2007; 2009; Henikoff et al . , 2001 ) , as well as several chromatin-interacting proteins ( Supplementary file 2 ) .", "Characterization of the remaining conserved and non-conserved genes is likely to bring new insights into essential gene functions .", "We next asked whether our CRISPR screening platform could be used to identify genes acting in signaling pathways that regulate cell growth and proliferation ( Friedman and Perrimon , 2007 ) .", "To do this , we performed positive selection screens in the presence of trametinib ( tra ) , an inhibitor of the Ras/ERK/ETS pathway , or rapamycin ( RAP ) , an inhibitor of the PI3K/mTor pathway , with the aim of uncovering known and novel compensatory mechanisms or synergizing loss-of-function mutations .", "Both pro-survival pathways have been extensively mapped through loss-of-function studies in fly tissues and cell-lines ( Friedman and Perrimon , 2007 ) ( Figure 4A ) .", "For these experiments , we first transfected cells with Group 1 and Group 2 sublibraries targeting a total of 3974 genes ( Figure 2A ) .", "The gene set interrogated comprises kinases , phosphatases , the fly ortholog of FDA-approved drugs ( Housden et al . , 2017 ) , and fly-to-human paralogs ( Figure 2A ) .", "The cell pools were passaged for 15 days to allow sgRNA integration , subjected to passaging for an additional 30 in sub-lethal doses of tra or RAP , and then re-sequenced ( Figure 2B ) .", "The effect of each drug was carefully monitored by periodically counting cells during the screen to confirm the effect on cell doubling rate ( Figure 4C ) .", "We observed highly context-specific modes of resistance to each drug .", "As an illustration , sgRNAs against aop , a transcriptional repressor of the Ras/ERK/ETS pathway ( Lai and Rubin , 1992 ) , or the putative intracellular co-factor for rapamycin , FK506-bp2 ( Thomson and Johnson , 2010 ) , conferred resistance specifically in the context of tra or RAP , respectively ( Figure 4D ) .", "We focused on other such genes for which multiple sgRNAs provide a survival benefit or synergistic lethality in the context of drug treatment using maximum likelihood estimate ( Li et al . , 2014 ) ( MLE ) ( Figure 4E; Supplementary file 3 ) .", "Additional established context-specific pathway negative regulators emerged: PTP-ER , which inactivates Erk ( rl ) ( Karim and Rubin , 1999 ) and Pi3K21B and Pten , which antagonize the activity of Pi3K92E ( Weinkove et al . , 1999; Huang et al . , 1999; Goberdhan et al . , 1999 ) .", "The analysis also identified several pathway positive regulators as synthetic lethal ( Figure 4E , and mapped into pathway diagram , Figure 4A ) , cross-pathway synergy , and several novel candidate pathway regulators ( Supplementary file 3 ) .", "Gene ontology showed similar but non-overlapping categories were detected as synergistically fitness-compromising with each drug ( Figure 4E ) .", "Moreover , by using differential CRISPR score to map physical protein-protein interaction ( PPI ) networks , both distinct and overlapping PPI relationships emerged ( Figure 4F ) .", "For instance , a network involved in photoreceptor cell differentiation scored most strongly as synergistically lethal with tra , possibly because photoreceptor differentiation has been a workhorse phenotype for the characterization of mutants in the Ras/ERK/ETS pathway ( Nagaraj and Banerjee , 2004 ) .", "Similarly , the top scoring PPI network for RAP-synergy was one involved in the positive regulation of cell-size , a key morphological consequence of upgregulating the PI3K/mTOR pathway ( Fingar and Blenis , 2004 ) .", "Interestingly , similar PPI networks involved in mitosis was identified in both screens ( Figure 4F ) .", "Taken together , these results demonstrate that CRISPR screening in Drosophila cells can reveal context-specific compensatory mechanisms or synergy relevant to major signaling pathways ." ], [ "CRISPR screening technologies have illuminated the functions of uncharacterized genes and provided a straightforward , cost-effective pipeline to evaluate gene function in different contexts ( Doench , 2018 ) .", "However , the benefits of this approach have been inaccessible for Drosophila and other insects because the delivery of highly multiplexed DNA libraries has thus far required lentiviral transduction , a process that fails to produce transformed cells ( unpublished ) .", "Here , we show that multiplexed DNA delivery by transfection followed by site-specific recombination is an effective alternative strategy .", "We use this technique to deliver a genome-wide library of sgRNA expression cassettes and perform CRISPR knockout fitness screens in Drosophila S2R+ cells , identifying 1235 fitness genes at 5% FDR .", "Of note , our CRISPR screens were conducted after approximately 45 doublings ( basal essentiality ) or 30 doublings ( context-specific screens ) , while most mammalian screens have been conducted with fewer than 30 doublings .", "This could possibly due to efficiency differences of the CRISPR systems encoded in the two systems or because of the high ploidy of S2R+ cells .", "Since our timing optimization data used only two sgRNAs ( Figure 1F ) , we do not know how the set of fitness genes would change in screens conducted with fewer doublings .", "A practical use for viability screens is examining context-specific growth requirements .", "The Drosophila CRIPSR knockout system identified mutants conferring drug resistance or synergy , and should thus be suitable for many future context-specific fitness experiments examining gene-drug/nutrient or gene-gene interactions .", "The introduction of systematic knockdown and knockout approaches have greatly reduced false-positive assertions in human cell-line loss-of-function studies , but equally important is knowing what genes are missed by these approaches and providing alternative screening strategies that can target them .", "Of critical importance are paralogous genes with redundant functions , i . e . , the presence of one paralog buffers against the loss of another ( Ewen-Campen et al . , 2017 ) .", "Although relevant to human disease ( Dickerson and Robertson , 2012 ) , these genes are ‘invisible’ in singe-gene screens and dramatically reduce search space in gene-by-gene screens ( Ewen-Campen et al . , 2017 ) .", "When we compared human and fly CRISPR screens , human CRISPR screens were less able to detect the fly orthologs of fitness essential genes when those genes had paralogs relative to the fly genome .", "This supports the use of parallel Drosophila and human screens as one approach to offset genetic redundancy .", "In addition to CRISPR knockout , the strategy we report can be used with numerous other high-throughput screening modalities which were previously not possible in Drosophila , including CRISPR activation , CRIPSR inhibition , CRISPR base-editing , shRNA knockdown , cDNA overexpression , perturb-SEQ , and combinatorial approaches for multigene suppression/activation ( Doench , 2018; Najm et al . , 2018; Shen et al . , 2017 ) .", "Finally , the methods and constructs we employ are likely to be directly transferable to the large number of existing cultured cell-lines derived from other insects such as mosquitos , where they could be used to characterize viral propagation mechanisms in the vectors of human pathogens such as Dengue or Zika ." ], [ "pBS130 , phiC31 integrase under control of the HSP70 promoter , was obtained from Addgene .", "Transient Cas9 expression used pl018 ( Housden et al . , 2015 ) containing Drosophila-optimized Cas9 under the strong Actin promoter .", "Cas9 from pl018 followed by the BGH terminator from pcDNA3 . 1 ( Invitrogen ) were cloned into the SpeI/NotI site of pMK33 ( Koelle et al . , 1991 ) to generate pMK33/Cas9 .", "Human codon-optimized intein-Cas9_S219-3XFLAG ( Davis et al . , 2015 ) , a kind gift of D . Lui , was amplified by PCR and cloned into pMK33 to generate pMK33/intein-Cas9_S219-3XFLAG .", "pLib6 . 4 was generated by using standard cloning methods as follows .", "First , the Drosophila U6:2 and Act5C promoter cassette from pl018 ( Housden et al . , 2015 ) was amplified by PCR using primers containing the 5’ attB40 site and inserted into pUC19 using Gibson assembly .", "Next , GFP-T2A-Puro from pAc-STABLE2 ( González et al . , 2011 ) was amplified with primers containing the 3’ attB40 and introduced by ligation into an engineered site in the resulting vector and verified by Sanger sequencing .", "For minipool experiments in Figure 1 , sgRNAs were cloned individually using standard methods into the BpiI/BbsI site of pLib6 . 4 and verified by Sanger sequencing and then mixed .", "Sequencing reactions were carried out with an ABI3730xl DNA analyzer at the DNA Resource Core of Dana-Farber/Harvard Cancer Center ( funded in part by NCI Cancer Center support grant 2P30CA006516-48 ) .", "S2R+ derivative PT5 ( NPT005 ) was obtained from the Drosophila RNAi Screening Center ( Neumüller et al . , 2012 ) and grown in Schneider’s medium ( Thermo Fisher Scientific ) containing 10% heat-inactivated FBS .", "PT5 cells were transfected with pMK33/Cas9 or pMK33/intein-Cas9_S219-3XFLAG and selected over a period of two months in 200 ng/uL Hygromycin B ( Calbiochem ) .", "The resulting PT5/Cas9 cells were maintained in Hygromycin until CRISPR library transfection .", "pMK33/Cas9 ( accession # EvNO00483429 ) , pMK33/intein-Cas9_S219-3XFLAG ( accession # EvNO00483430 ) , and pLib6 . 4 ( accession # EvNO00483431 ) are available through DF/HCC DNA Resource Core ( https://plasmid . med . harvard . edu/ ) .", "In order to allow focused sublibrary screening as in Figure 3 , sgRNA library was encoded in three separable sublibraries with common controls ( Suppl . Figure 2A ) .", "For gene-targeted sgRNAs , Group 1 targets kinases and phosphatases as assigned using GLAD ( Hu et al . , 2015 ) and FDA-approved drug-targets ( Housden et al . , 2017 ) .", "Group 2 targets fly-to-human paralogs , identified by using ‘moderate’ or ‘high’ orthology assignment according to DIOPT ( Hu et al . , 2011 ) , not already present in Group 1 .", "Group 3 targets all other remaining genes .", "Library synthesis was performed by CustomArray using published methods ( Gilbert et al . , 2014; Shalem et al . , 2014 ) .", "Briefly , CRISPR sgRNAs were encoded within a 110-nt single-stranded DNA oligo containing unique library-specific barcode sequences and flanked by BpiI/BbsI sites .", "15-cycles of PCR using Phusion Polymerase ( New England Biolabs ) were used to amplify each sub-library , and then BpiI ( Fermentas ) was added to the amplicons .", "A non-denaturing 20% TBE gel ( Thermo ) was used to purify the resulting 23-mer fragment and eluted overnight using the crush-soak method .", "The concentration of ligatable fragments in each preparation was empirically optimized by test ligations in pLib6 . 4 .", "Optimized concentrations were used in ligations with BpiI-digested pLib6 . 4 and then electroporated into Ecloni 10GF’ electrocompetent cells ( Lucigen ) at a yield of 20–50 times diversity for each library , generating dense colonies on ~60 , 150 mm LB-carbenicillin plates , which were grown for 18 hr at 37°C and harvested by scraping into LB medium followed by mixing suspended colonies by extensive vortexing .", "Glycerol was added to 50% and the libraries were flash frozen in 1 mL aliquots .", "Each sublibrary was prepared by pooled minipreps and eluted in buffer EB ( Qiagen ) .", "Libraries are available at DRSC/TRiP Functional Genomics Resources ( https://fgr . hms . harvard . edu/crispr-cell-screening-reagents ) .", "pBS130 and pLib6 . 4 containing CRISPR sublibraries were mixed and co-transfected 1:1 into PT5/Cas9 cells using Effectene with the manufacturer’s recommended protocol ( Qiagen ) .", "For each transfection , cells were first grown until just confluent on T75 flasks for 2–3 days .", "Doubling was monitored at this passage and determined to be approximately 1/day .", "Then cells were removed by forceful tapping and replated at 3 × 106 per well into 6-well dishes .", "The number of dishes required reflected the library size and accounted for incomplete transfection efficiency to give at least 1500 cells/sgRNA ( for reference , a full-genome screen required all wells of eight 6-well dishes ) .", "t = 0 used plasmid readcount values .", "Transfection efficiency and integration efficiency were monitored periodically after transfection for the first twelve days using flow cytometry ( BD LSRII ) .", "Flow cytometry was performed at the Harvard Medical School Department of Immunology Flow Core .", "Each 1 . 5 wells of cells was transferred to one 10 cm dish and passaged in the presence of 5 ug/mL puromycin and cells were allowed to become confluent over 4–6 days .", "Next , the pool was contracted by two-fold , and 2 × 107 cells from each of two plates was combined into a single 10 cm dish .", "2 × 107 cells were passaged every 5 days until downstream processing at the indicated time .", "These steps during early phases of selection ensured no loss of diversity due to variable transfection efficiency .", "To determine partially inhibitory dose of trametinib ( Selleck ) or rapamycin ( Tocris ) , the effect of varying doses of each drug was first measured in a 4 day treatment to PT5/Cas9 cells by Cell Titer Glo assay ( Promega ) , using manufacturer’s recommended protocol ( not shown ) .", "From this data , 50 nM trametinib or 2 nM rapamycin was chosen as partially perturbative concentrations for focused library screens ( Figure 3 ) .", "To verify that drug treatment decreased doubling rate during the screen , cell counts were performed periodically using a hemocytometer ( Figure 3B ) .", "Genomic DNA was prepared using the Zymo miniprep kit .", "Assuming DNA content in S2R + cells was 4N , each cell contains ~0 . 6 pg of DNA .", "To maintain diversity , all PCRs were conducted from ~5 , 000 cells per sgRNA per condition .", "Library amplification was performed using a two-step procedure ( illustrated in Figure 1—figure supplement 3 ) .", "First , in-line barcoded inside primers ( PCR1F x PCR1R ) were used to amplify the library in 23 cycles such that the resulting amplicon had the following sequence: 1/2Read1- ( N ) nANNEALINGSEQUENCE-sgRNA-tracrRNA .", "Primer sequences conformed to: 5’- CTTTCCCTACACGACGCTCTTCCGATCT ( N ) n ( B ) 6 gttttcctcaatacttcGTTCg-3’ ( where N = any nucleotide; n = variable number between 1–9; and B = barcode nucleotide ) and 5’-TTTGTGTTTTTAGAATATAGAATTGCATGCTGggtacctc-3’ .", "Next , common outside primers were used to amplify these amplicons with an additional 11 cycles such that the resulting amplicons had the following sequence: P5-Read1- ( N ) n-ANNEALINGSEQUENCE-sgRNA-tracrRNA-P7 .", "All sequences are provided in Supplementary file 1 .", "Following second amplification , amplicons were gel purified and concentration was determined using Qubit dsDNA HS Assay Kit ( Thermo ) .", "Amplicons were pooled according to concentration of sgRNAs per unit volume .", "Pooled barcoded amplicons were subjected to sequencing using the NextSeq500 1 × 75 SE platform ( Illumina ) .", "Sequencing was performed at the CCIB DNA Core Facility at Massachusetts General Hospital ( Cambridge , MA ) or the Harvard Medical School Biopolymers Facility ( Boston , MA ) .", "De-multiplexing of the library was performed using TagDust ( Lassmann et al . , 2009 ) and all downstream analysis was performed within MAGeCK ( Li et al . , 2014 ) with the following experiment-specific changes:", "1 ) Readcount files were stripped of sgRNAs below the 10th percentile lowest initial readcounts for each sublibrary before processing in MAGeCK MLE;", "2 ) we performed 1000 iterations for all Z-score assignments .", "For drug selection experiments , several context-nonspecific sgRNAs providing survival benefit to cells under normal growth conditions were removed due to wide variation of these sgRNAs upon selection .", "For ROC curves , major eukaryotic essential complex components for Drosophila were from KEGG ( http://www . genome . jp/ ) .", "For gene ontology enrichment analysis , annotation file for Drosophila genes was retrieved from NCBI ( ftp://ftp . ncbi . nlm . nih . gov/gene/DATA/gene2go . gz ) .", "Hypergeometric analysis was done to calculate the enrichment P value using in-house program written in JAVA .", "Ortholog assignment as well as fly-to-human protein similarity score were obtained using DIOPT v 6 . 0 . 2 using ‘moderate’ or ‘high’ confidence calls ( Hu et al . , 2011 ) .", "RNAseq analysis in this paper used the webtools DGET ( http://www . flyrnai . org/tools/dget/web ) or CellExpress ( http://www . flyrnai . org/cellexpress ) , which used RNAseq expression data from modENCODE ( Celniker et al . , 2009 ) .", "For re-analysis of genome-wide arrayed RNAi viability experiment , we re-examined all dsRNA amplicon targets reported in the earlier screen ( Boutros et al . , 2004 ) using FlyBase v 6 .", "All amplicons with greater than one unique target were discarded ( removing 7818 dsRNAs and retaining 13 , 488 ) .", "Z-score was then re-calculated using the original methods ( Boutros et al . , 2004 ) .", "Gene ontology analysis in Figures 3D and 4E was performed using PantherDB ( Mi et al . , 2013 ) .", "In Figure 3D , terms were restricted to those with greater than or equal to 50 members .", "Complex enrichment analysis in Figure 4F used COMPLEAT ( Vinayagam et al . , 2013 ) .", "In Figure 4F , Z-scores in Supplementary file 3 were multiplied by −1 and top three non-redundant complexes from COMPLEAT with a minimum number of 6 members are reported .", "Readcount files for CRISPR analysis compatible with MAGeCK are provided as Supplementary file 4–15 and Supplementary file 3 .", "pMK33/Cas9 , pMK33/intein-Cas9_S219-3XFLAG , and pLib6 . 4 .", "are available through Harvard PlasmID Database ( http://plasmid . med . harvard . edu ) .", "The three CRISPR sublibraries used in this study are available through DRSC/TRiP Functional Genomics Resources ( https://fgr . hms . harvard . edu/ ) ." ] ]

[ "Genome-wide screens in Drosophila cells have offered numerous insights into gene function , yet a major limitation has been the inability to stably deliver large multiplexed DNA libraries to cultured cells allowing barcoded pooled screens .", "Here , we developed a site-specific integration strategy for library delivery and performed a genome-wide CRISPR knockout screen in Drosophila S2R+ cells .", "Under basal growth conditions , 1235 genes were essential for cell fitness at a false-discovery rate of 5% , representing the highest-resolution fitness gene set yet assembled for Drosophila , including 407 genes which likely duplicated along the vertebrate lineage and whose orthologs were underrepresented in human CRISPR screens .", "We additionally performed context-specific fitness screens for resistance to or synergy with trametinib , a Ras/ERK/ETS inhibitor , or rapamycin , an mTOR inhibitor , and identified key regulators of each pathway .", "The results present a novel , scalable , and versatile platform for functional genomic screens in invertebrate cells ." ]

[ "Genes are made up of DNA and carry the instructions necessary to build an organism .", "Humans have over 20 , 000 genes , while other animals , such as fruit flies , have about 14 , 000 .", "An ongoing challenge in biology is to identify the role of every gene in the human body .", "Since most of them are conserved in the fruit fly , this insect is one of the most extensively studied organisms .", "Scientists often use a technique called CRISPR to edit genes .", "It enables researchers to modify DNA sequences to selectively alter the purpose of a gene or even turn it off to find out what it does .", "CRISPR requires a guide molecule ( for example , sgRNAs ) , which leads the system to a particular DNA sequence to start the process .", "Often , researchers create many sgRNAs and deliver them to a large pool of cells with the help of viruses , so that each cell gets a different sgRNA that mutates a different gene .", "When the cells are then treated with a specific drug , the composition of the sgRNAs in the pool changes , depending on which genes are needed to withstand the drug , and which genes – when turned-off – create cells that are resistant to the drug .", "Although thousands of mutant flies have been created to investigate how a deactivated or faulty gene can affect the health and behavior of the fly , we still lack meaningful information on about half of their genes .", "This is partly because the viruses used to deliver sgRNAs in mammals do not work in fly cells .", "Here , Viswanatha et al . developed a simple protocol to generate cell pools of CRISPR mutants , which uses a new strategy that uses bacteria to deliver DNA to fly cells .", "This allowed to identify over 1 , 000 genes necessary for cells to multiply properly , many of which had not been studied before .", "The technique was also used in combination with drugs to examine the interactions between genes and drugs – an approach that could be further adapted to examine interactions between genes and nutrients , or between genes .", "This new approach will open doors to systematically uncover the purpose of every gene in the fly .", "A better understanding of what genes do could help to identify potential genetic weaknesses in certain types of cancer or other diseases , which may lead to the development of more effective treatments .", "Moreover , the method is likely to work in other insects , for example , mosquitos , where it may uncover new genes involved in mosquito-borne diseases such as malaria or Zika virus ." ]

[ "Introduction", "Results and discussion", "Materials and methods" ]

[ "biochemistry and chemical biology", "structural biology and molecular biophysics" ]

[ [ "The evolution of more than 500 human protein kinases from a few protein kinases in unicellular organisms allowed for the development of complexity via differential regulation ( Manning et al . , 2002 ) .", "Such regulation can be achieved by autophosphorylation or interactions with other domains or binding partners .", "While many of the signaling cascades and their in vivo biological effectors have been well characterized , and a wealth of structural information is available ( Johnson et al . , 1996; Huse and Kuriyan , 2002; Manning et al . , 2002; Tyson et al . , 2003 ) , the molecular mechanism whereby kinase activity is modulated is a topic of controversial debate ( Oliver et al . , 2006; Dodson et al . , 2013 ) .", "Here we investigate two fundamentally distinct regulation mechanisms by characterizing autophosphorylation of Aurora A as well as activation by TPX2 ( Targeting Protein for Xklp2 ) .", "Aurora A , a Ser/Thr kinase , is a key regulator of mitotic events , including mitotic entry ( Macurek et al . , 2008; Seki et al . , 2008 ) , centrosome maturation ( Glover et al . , 1995; Hannak et al . , 2001; Toji et al . , 2004; Abe et al . , 2006; Mori et al . , 2007 ) , and spindle formation ( Giet et al . , 2002; Kapitein et al . , 2005; Tsai and Zheng , 2005; Koffa et al . , 2006; Venoux et al . , 2008; Wong et al . , 2008; Zhang et al . , 2008 ) .", "Aurora A depletion leads to cell cycle arrest , while overexpression has been found in many cancer cell lines ( Kallioniemi et al . , 1994; Sen et al . , 1997; Zhou et al . , 1998; Jeng et al . , 2004 ) .", "Therefore extensive interest has been recently directed towards Aurora A for anti-cancer drug development ( Aliagas-Martin et al . , 2009; Bebbington et al . , 2009; Cheok et al . , 2010 ) .", "TPX2 recruits Aurora A to the spindle microtubules , an event that is essential in spindle formation ( Kufer et al . , 2002; Giubettini et al . , 2011 ) .", "Autophosphorylation of T288 in the activation loop increases the catalytic activity of Aurora A ( Walter et al . , 2000; Littlepage et al . , 2002 ) .", "Intramolecular autophosphorylation has recently been suggested for Aurora A and Chk2 based on indirect kinetic measurements ( Dodson et al . , 2013 ) adding to the controversy by disagreeing with the intermolecular mechanism proposed for other protein kinases ( Oliver et al . , 2006 , 2007; Pike et al . , 2008; Lee et al . , 2009 ) .", "A second puzzling result has also been reported recently .", "It was shown that in vivo during mitosis , TPX2-bound Aurora A at the spindle microtubules is dephosphorylated at the crucial T288 ( Toya et al . , 2011 ) .", "Since T288-dephosphorylated Aurora A exhibits very low kinase activity , a second kinase-independent function of Aurora A was postulated ( Littlepage , 2002 ) .", "There is evidence suggesting that TPX2 also plays an active role in upregulating Aurora A activity , however the interplay between the two distinct activation mechanisms , phosphorylation and TPX2-binding , is not well understood ( Kufer et al . , 2002; Carmena and Earnshaw , 2003; Eyers and Maller , 2003; Eyers et al . , 2003; Kufer et al . , 2003; Trieselmann et al . , 2003; Tsai et al . , 2003; Bayliss et al . , 2004; Brunet et al . , 2004; Eyers and Maller , 2004; Ozlu et al . , 2005; Tsai and Zheng , 2005; Anderson et al . , 2007 ) .", "Here we address both controversies by directly measuring autophosporylation and by characterizing the molecular mechanism of Aurora A regulation by TPX2 ." ], [ "While it is generally accepted that phosphorylation of a Ser/Thr in the activation loop activates Ser/Thr kinases , this regulation has not been characterized quantitatively .", "Part of the difficulty consists in obtaining a dephosphorylated protein , since Escherichia coli-produced kinases are heavily phosphorylated due to autophosphorylation and phosphorylation by E . coli kinases during expression ( Enami and Ishihama , 1984 ) ( Figure 1A ) . 10 . 7554/eLife . 02667 . 003Figure 1 . TPX21−45 drastically accelerates the kinetics of the dephosphorylated form of Aurora A kinase .", "( A ) Mass spectrometry data of heavily phosphorylated ( P ) and dephosphorylated ( deP ) Aurora A . The dephosphorylated protein was obtained after treatment of heavily phosphorylated , Escherichia coli-produced Aurora A with λ-protein phosphatase ( λPP ) .", "( B ) AP phosphorylation by dephosphorylated ( , 0 . 01 ± 0 . 005 s−1 ) or T288V mutant Aurora A ( , 0 . 05 ± 0 . 002 s−1 ) is increased by up to 50-fold ( , 0 . 5 ± 0 . 1 s−1 ) and 25-fold ( , 1 . 2 ± 0 . 1 s−1 ) , respectively , in the presence of TPX21−45 .", "This rate is comparable to the kinetics of phosphorylated Aurora A in the absence of TPX21−45 ( , 1 . 0 ± 0 . 2 s−1 ) .", "Phosphorylated Aurora A shows up to a twofold increase in AP kinetics in the presence of TPX21−45 ( , 2 . 3 ± 0 . 2 s−1 ) .", "Reactions are carried out in the presence of 1 μM protein , 50 μM TPX21−45 , 5 mM ATP , and 1 mM AP in assay buffer ( 20 mM TrisHCl , 200 mM NaCl , 20 mM MgCl2 , 3% ( vol/vol ) glycerol , 1 mM TCEP , pH 7 . 50 ) at 25°C .", "Phosphorylated peptide production was monitored by reverse phase-high performance liquid chromatography ( RP-HPLC ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 02667 . 00310 . 7554/eLife . 02667 . 004Figure 1—figure supplement 1 . Kinase assays were conducted under saturating conditions of peptide and ATP . To ensure peptide and nucleotide would not be rate-limiting , ( A ) the KM for AP was determined to be 116 ± 70 μM for phosphorylated Aurora A ( left ) and 320 ± 150 μM for dephosphorylated-mimic , T288V mutant Aurora A ( right ) and ( B ) the kinetics of 10 μM Aurora A T288V were monitored under 1 mM AP at 5 mM ATP ( , 0 . 0054 s−1 ) and 15 mM ATP ( , 0 . 0055 s−1 ) as well as 2 mM AP and 15 mM ATP ( , 0 . 0056 s−1 ) .", "In each case , the kinetics of AP phosphorylation were identical within experimental error .", "Since the KM for ATP for WT Aurora A is about 10 μM ( Kelly et al . , 2011 ) , all following kinetic reactions were run at 5 mM ATP ( 20 mM TrisHCl , 200 mM NaCl , 20 mM MgCl2 , 3% [vol/vol] glycerol , 1 mM TCEP , pH 7 . 50 ) at 25°C .", "Phosphorylated peptide production was monitored by reverse phase-high performance liquid chromatography ( RP-HPLC ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 02667 . 00410 . 7554/eLife . 02667 . 005Figure 1—figure supplement 2 . Aurora A exhibits the same activity towards AP whether the protein is phosphorylated on multiple sites or singly phosphorylated on T288 . The rates are 0 . 93 s−1 and 0 . 97 s−1 , respectively .", "To obtain singly phosphorylated Aurora A ( 1P_AurA ) , dephosphorylated protein was autophosphorylated in the presence of ATP and a final concentration of 1 μM of this protein was used for the assay described here .", "Aurora A phosphorylated on multiple sites ( mP_AurA ) was obtained through classic expression in Escherichia coli cells ( see Figure 1A ) .", "Reactions were carried out in the presence of 5 mM ATP and 1 mM AP in assay buffer at 25°C . DOI: http://dx . doi . org/10 . 7554/eLife . 02667 . 00510 . 7554/eLife . 02667 . 006Figure 1—figure supplement 3 . Aurora A kinase autophosphorylation and substrate phosphorylation were simultaneously followed either in the absence or presence of TPX21−45 . In the experiments aimed at measuring activity of AP phosphorylation of dephosphorylated Aurora A ( Figure 1B ) , autophosphorylation can occur during the time-course of the reaction .", "Therefore autophosphorylation of 1 μM Aurora A in the presence of 1 mM AP and 5 mM ATP and in the absence or presence of 50 μM TPX21−45 was monitored simultaneously with AP phosphorylation .", "Densitometry analysis ( left ) of raw Western blot data ( right ) is shown .", "To account for Aurora A's dynamic range , time points up to 300 s were diluted 50× and the rest of the time points were diluted 225× .", "The amount of phosphorylated protein made during the reaction accounts for only 10% of the detected rate acceleration of AP phosphorylation in the presence of TPX2 . DOI: http://dx . doi . org/10 . 7554/eLife . 02667 . 00610 . 7554/eLife . 02667 . 007Figure 1—figure supplement 4 . TPX21−45 drastically accelerates the kinetics of the dephosphorylated-like Aurora A species irrespective of the nature of the peptide used . TPX2 increases the kinetics of the dephosphorylated-protein mimic ( T288V mutant ) towards the peptides AP ( APSSRRTTLCGTL ) ( left ) , Kemptide ( LRRASLG ) ( middle ) , and Lats2373−387 ( ATLARRDSLQKPGLE ) ( right ) between 20- and 30-fold .", "Lats2 is an Aurora A substrate important in centrosome maturation , and Kemptide is a synthetic construct generally used as a substrate of cAMP-dependent protein kinase A ( PKA ) , a protein closely related to Aurora A . Reactions are carried out in the presence of 5 mM ATP and 1 mM peptide in assay buffer at 25°C .", "A longer TPX2 variant ( TPX21−147 ) was used as control to ensure full capture of TPX2 activity by the shorter variant ( TPX21−45 ) .", "P: phosphorylated . DOI: http://dx . doi . org/10 . 7554/eLife . 02667 . 00710 . 7554/eLife . 02667 . 008Figure 1—figure supplement 5 . Representative RP-HPLC time traces during AP phosphorylation . These traces ( A ) show well resolved non-phosphorylated and phosphorylated peptide peaks at different reaction time points .", "( B ) Screenshot of time traces for AP_T287E ( left ) and AP_T288E ( right ) phosphorylation show that Aurora A selectively phosphorylates AP on T288 .", "Assays were carried out in the presence of 1 μM phosphorylated Aurora A , 1 mM AP or 1 mM AP_T287E or AP_T288E , 5 mM ATP , at 25°C in kinase assay buffer ( 20 mM TrisHCl , 200 mM NaCl , 20 mM MgCl2 , 3% glycerol , 1 mM TCEP , pH 7 . 50 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 02667 . 00810 . 7554/eLife . 02667 . 009Figure 1—figure supplement 6 . Dose-dependence of the concentration of TPX21−147 on the phosphorylation kinetics of AP by Aurora A T288V . The calculated KA = 1 . 0 ± 0 . 5 μM compared well with the KD = 1 . 1 ± 0 . 1 μM obtained from ITC data ( Figure 7A ) .", "Assays were carried out in the presence of 1 μM Aurora A T288V , 1 mM AP , increasing concentrations of TPX21−147 , 5 mM ATP , at 25°C in kinase assay buffer ( 20 mM TrisHCl , 200 mM NaCl , 20 mM MgCl2 , 3% glycerol , 1 mM TCEP , pH 7 . 50 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 02667 . 009 Aurora A was co-expressed with the generic Ser/Thr/Tyr phosphatase lambda ( λPP ) and treated again with λPP after purification to ensure complete dephosphorylation ( Figure 1A ) .", "We then measured the enzymatic activity of phosphorylated versus dephosphorylated Aurora A towards AP , a peptide encompassing residues 281–293 of the kinase's activation segment .", "The experiments were conducted under saturating conditions of peptide , ATP , and Mg2+ , and we ensured that the singly and heavily phosphorylated kinases exhibited similar kinetics ( Figure 1—figure supplement 1 and 2 ) .", "Phosphorylated Aurora A catalyzes AP phosphorylation 100-fold faster than the dephosphorylated kinase ( 1 . 0 ± 0 . 2 s−1 vs 0 . 01 ± 0 . 005 s—1; Figure 1B ) .", "Since dephosphorylated Aurora A can also autophosphorylate during this experiment , we not only quantified autophosphorylation during the reaction time frame ( Figure 1–figure supplement 3 ) , but we also designed a second experiment that eliminates this competing reaction by mutating T288 to V . This mutant shows comparable low activity to the dephosphorylated kinase ( kcatAP = 0 . 05 ± 0 . 002 s−1 ) serving as a control for quantifying the rate acceleration provided by T288 phosphorylation .", "Does TPX2 activate Aurora A to the same extent as phosphorylation or are both activation mechanisms additive ?", "We used TPX21−45 in our studies since this fragment was shown to be sufficient for kinase activation ( Bayliss et al . , 2003; Brunet et al . , 2004 ) .", "Our data suggest the first scenario since AP phosphorylation is stimulated by 50- and 25-fold by TPX21−45 for the dephosphorylated Aurora A and T288V Aurora A , respectively ( Figure 1B ) .", "The rates of dephosphorylated Aurora A plus TPX21−45 are comparable to that of phosphorylated Aurora A alone .", "Addition of TPX2 to phosphorylated Aurora A results in only a twofold increase in kcat .", "This effect is independent of the nature of the peptide used ( Figure 1–figure supplement 4 ) .", "Having shown kinetically that TPX2 is sufficient to activate dephosphorylated Aurora A similarly to T288 phosphorylation , we next studied the underlying molecular mechanism .", "We first solved the crystal structure of dephosphorylated Aurora A in the absence and presence of TPX21−45 and bound to an ATP-mimic ( β , γ-methyleneadenosine 5′ triphosphate , AMPPCP ) .", "The AMPPCP-bound , dephosphorylated Aurora A is monomeric and in an inactive conformation , similar to previously solved structures of the same protein bound to adenosine ( PDB ID 1MUO [Cheetham et al . , 2002] ) or AMPPNP ( PDB ID 2C6D [Heron et al . , 2006] ) .", "In contrast , the TPX2-bound Aurora A structure reveals a dimer made by two molecules of Aurora A , TPX2 , and AMPPCP in the asymmetric unit ( Figure 2A , Table 1 ) . 10 . 7554/eLife . 02667 . 010Figure 2 . Dephosphorylated Aurora A adopts an active conformation in the presence of TPX21−45 . ( A ) Dephosphorylated Aurora A + TPX21−45 + AMPPCP and ( B ) superposition of Aurora A moieties .", "( C ) A detailed view of structural elements that define an active Aurora A kinase: the nucleotide binding region ( top inset ) and the regulatory spine ( bottom inset ) .", "Dephosphorylated ( deP ) Aurora A in the presence of TPX2 ( red , PDB ID 4C3P ) superposes very well to the phosphorylated ( P ) Aurora A either in the absence ( orange , PDB ID 1OL7 ) or presence of TPX2 ( yellow , PDB ID 1OL5 ) .", "For comparison , dephosphorylated Aurora A alone ( light blue , PDB ID 4C3R ) shows the characteristic features of an inactive kinase . DOI: http://dx . doi . org/10 . 7554/eLife . 02667 . 01010 . 7554/eLife . 02667 . 011Table 1 . Data collection and refinement statisticsDOI: http://dx . doi . org/10 . 7554/eLife . 02667 . 011deP Aurora A + AMPPCPdeP Aurora A + AMPPCP + TPX2Data collection Space groupP 61 2 2P 21 21 21 Cell dimensions a , b , c ( Å ) 83 . 47 , 83 . 47 , 172 . 6349 . 93 , 86 . 72 , 153 . 55 α , β , γ ( ° ) 90 , 90 , 12090 , 90 , 90 Resolution ( Å ) 86 . 3–2 . 79 ( 2 . 87–2 . 79 ) 86 . 7–2 . 69 ( 2 . 76–2 . 69 ) Rmerge0 . 08 ( 2 . 08 ) 0 . 26 ( 3 . 51 ) I/σ19 . 8 ( 1 . 8 ) 8 . 0 ( 2 . 3 ) Completeness ( % ) 100 ( 100 ) 100 ( 100 ) Redundancy15 . 7 ( 16 . 7 ) 6 . 9 ( 7 . 2 ) Refinement Resolution ( Å ) 55 . 4–2 . 79 ( 2 . 87–2 . 79 ) 47 . 5–2 . 69 ( 2 . 76–2 . 69 ) No .", "reflections845918104 Rwork/Rfree0 . 221/0 . 306 ( 0 . 327/0 . 451 ) 0 . 201/0 . 289 ( 0 . 284/0 . 400 ) No .", "atoms Protein20744574 Ligand/ion3167 Water021 B-factors Protein100 . 354 . 6 Ligand/ion109 . 162 . 5 WaterNA43 . 3 R . m . s deviations Bond lengths ( Å ) 0 . 0100 . 011 Bond angles ( ° ) 1 . 541 . 53PDB ID4C3R4C3PValues in parentheses correspond to the highest-resolution shell . deP: dephosphorylated; PBD , Protein Data Bank .", "Since the classic bilobal fold of protein kinases is by now well known from many elegant structural studies ( Cheetham , 2002; Bayliss et al . , 2003 ) , and our structure in the absence of TPX2 does not provide new information , we will only discuss novel insights gained from the TPX2-bound dephosphorylated Aurora A dimer .", "Superposition of the Aurora A monomers shows that they are similar , but not identical within the heterodimer , with implications for autophosphorylation discussed below ( Figure 2B ) .", "Interestingly , both monomers are in an active conformation despite being dephosphorylated ( Figure 2C ) , a feature that has never been seen before in Aurora kinases .", "Comparison of dephosphorylated Aurora A in the absence or presence of TPX2 reveals subtle but significant interactions by which TPX2 stabilizes the active form of the kinase ( Figure 2C ) .", "First , binding of TPX2 causes a slight rotation of the αC-helix towards the catalytic center , thus allowing for the conserved , stabilizing E181-K162 ion pair to form .", "The αC-helix rotation also results in movement of F275 of the conserved DFG motif from the DFG-out into the DFG-in position ( Martin et al . , 2012 ) ( Figure 2C , bottom inset ) .", "This positions the catalytic D274 in the correct orientation to carry out phosphoryl transfer .", "Second , movement of F275 initiates a cascade of side chain interactions that result in the completion of the regulatory spine originating from the αF-helix ( R-spine; Figure 2C , bottom inset ) .", "Identification of the completed R-spine , a hallmark of an active kinase , is based on the Local Spatial Patterns ( LSP ) alignment , a bioinformatics tool developed by the Taylor laboratory ( Kornev et al . , 2008 ) .", "From these structural features it appears that Aurora A , despite being dephosphorylated , is in an active conformation when bound to TPX2 .", "We note that Bayliss et al . ( 2003 ) propose that the structure of phosphorylated Aurora A represents a partially active state because the authors interpret the activation segment to be in an inactive conformation as defined by the exposure of pThr288 to the solvent ( Bayliss et al . , 2003 ) .", "We would rather interpret that structure as an active state based on all hallmarks for active kinases ( Kornev et al . , 2008 ) , in agreement with our activity data ( Figure 1B ) .", "The final signatures for an active state Ser/Thr kinase involve changes around the activation segment .", "Formation of the β6/β9 antiparallel β sheets , that are not present in the inactive kinase , prime the MgATP for catalysis together with the other conformational changes described above .", "Finally , the correct positioning of D256 for activating the hydroxyl of the substrate in active Aurora A is achieved by a hydrogen bond network to T292 , which in turn H-bonds to K258 with distances shown in Figure 3A .", "All these features are fully conserved among eukaryotic Ser/Thr kinases ( Nolen and Taylor , 2004 ) . 10 . 7554/eLife . 02667 . 012Figure 3 . TPX2-bound domain-swapped Aurora A captures an enzyme–substrate complex .", "( A ) Left: in the presence of TPX21−45 , the N-terminal ( β6 and β9 sheets ) anchor point of the activation loop is present in both monomers whereas the C-terminal H-bond contacts typical for a fully active kinase ( between D256/K258 and T292-OH ) are only visible for the enzyme monomer in red , which we therefore define as the enzyme molecule .", "Right: for comparison , in the absence of TPX21−45 , the N- and C-terminal anchor points are not present and the protein is in an inactive state .", "Interactions that further stabilize the swapped dimer ( W313-P297/P298 and R371-E299 ) are shown in the bottom inset , highlighting that these intermolecular interactions ( left ) are identical to the corresponding intramolecular interactions ( right ) .", "( B ) The loop spanning residues 283–288 in monomer II , for which there was too weak electron density , was remodeled using the software Modeller and biased molecular dynamics .", "The loop can be arranged by TMD so that the distance between T288 of monomer II and γ-phosphate of AMPPCP of monomer I is compatible with phosphoryl transfer . DOI: http://dx . doi . org/10 . 7554/eLife . 02667 . 01210 . 7554/eLife . 02667 . 013Figure 3—figure supplement 1 . Comparison of the dimeric Aurora A + TPX2 structure with other domain-swapped Ser/Thr kinases . The activation loop and αEF-helix of one of the monomers nestle between the αF- and αG-helices of the other monomer in the dimer structures .", "Shown are SLK phosphorylated or non-phosphorylated ( PDB IDs 2JFL and 2J51 , respectively , both bound to triazole inhibitor DKI ) , Chk2 ( PDB ID 2CN5 bound to ADP ) , LOK ( PDB ID 2 J7T bound to SU11274 ) , DAPK3 ( PDB ID 2J90 bound to pyridone 6 ) , p70S6K1 ( PDB ID 3A60 bound to staurosporine ) , and OSR1 ( PDB ID 3DAK bound to AMPPNP ) .", "One monomer is shown in surface representation and the other in ribbon representation with bound nucleotides shown as sticks ( pink ) .", "Angles between the monomers and dimeric interface contacts vary significantly . DOI: http://dx . doi . org/10 . 7554/eLife . 02667 . 013 Curiously , the last two hallmarks of the active state are created by a swap of the activation segments of Aurora A resulting in a dimer conformation that has not been previously reported for this protein .", "In the C-lobe , W313 that would typically interact with P297 and P298 in monomeric Aurora A , now interacts with the identical Pro residues of the other Aurora A monomer .", "Lastly , the E299–R371 salt bridge is also domain swapped ( Figure 3A ) .", "Since autophosphorylation is considered a crucial regulation mechanism for protein kinases , the biological meaning of such a dimer structure becomes immediately apparent .", "While trans-activation through domain-swapped dimers seen in X-ray structures has been proposed for a number of kinases ( Figure 3–figure supplement 1 ) ( Oliver et al . , 2006 , 2007; Pike et al . , 2008; Lee et al . , 2009 ) , a recent report on Aurora A and Chk2 suggesting a strict intramolecular autophosphorylation of the activation segment for both enzymes has fueled controversy on this topic ( Dodson et al . , 2013 ) .", "Another puzzle is the fact that the activation-segment swapped dimers in the literature ( Figure 3–figure supplement 1 ) show both monomers to be either in the inactive or active kinase conformations , and a number of these structures are either bound to an inhibitor or already phosphorylated .", "In contrast we find an asymmetric dimer within the unit cell with monomer I showing complete electron density for the activation segment while monomer II is missing residues 283–288 ( Figure 2B , red and blue , respectively ) .", "In addition , only monomer I has the perfect geometry of the conserved hydrogen bond network found in a catalytically prone kinase between residues D256 , K258 , and T292 ( Figure 3A ) .", "Therefore we speculated that monomer I may be the enzyme molecule recognizing monomer II as its substrate .", "As a first ( and only crude ) test of this hypothesis , we used targeted MD ( TMD ) simulations to model the target hydroxyl for autophosphorylation of the domain-swapped activation segment towards the γ-phosphate of AMPPCP ( Figure 3B ) to address the question whether it is even physically possible that the hydroxyl can approach the γ-phosphate of AMPPCP .", "Only T288 of monomer II ( proposed as the substrate molecule above ) with a more flexible activation loop segment can be rearranged at a close distance to the γ-phosphate of AMPPCP without displacing the AMPPCP from its original position ( Figure 3B ) .", "Neither the crystallographic dimer nor the TMD simulations are compelling evidence for intermolecular autophosphorylation within such a swapped dimer .", "To answer the first obvious question whether a swapped dimer exists in solution , we performed small-angle X-ray scattering ( SAXS ) and sedimentation velocity analytical ultracentrifugation ( AUC ) experiments on dephosphorylated Aurora A in the absence and presence of TPX2 ( Figure 4 ) .", "A significant amount of dimer was detected for Aurora A and the relative concentration was independent of the presence of TPX2 .", "Importantly , the fact that separate peaks for the dimer and monomer were observed in a sedimentation velocity run reveals a slow interconversion rate between dimer and monomer .", "Fitting of the SAXS data required inclusion of 9% dimer of the shape of our X-ray structure , supporting the notion that the swapped dimer seen in X-ray crystallography exists in solution in agreement with the AUC data .", "Protein solubility prohibited determination of the KD , but from both the concentration dependence observed in AUC as well as SAXS experiments one can estimate that the KD is above 300 μM . 10 . 7554/eLife . 02667 . 014Figure 4 . TPX2-bound domain-swapped Aurora A forms a stable dimer in solution .", "( A ) Sedimentation velocity analytical ultracentrifugation data show discrete peaks for monomer and dimer .", "TPX21−25 does not increase the percentage of Aurora A dimer in solution .", "It is unclear why there is an increased dimer concentration for the kinase-dead Aurora A D274A mutant .", "( B ) Small-angle X-ray scattering ( SAXS ) data show an increase in dimer concentration with increased Aurora A amounts .", "All data were collected in the presence of 500 μM AMPPCP in kinase assay buffer .", "deP , dephosphorylated . DOI: http://dx . doi . org/10 . 7554/eLife . 02667 . 014 The existence of the dimer in solution is essential but not sufficient for an intermolecular autophosphorylation mechanism .", "To directly investigate the mechanism , functional assays were performed .", "First , we designed two Aurora A constructs that differed in activity and length .", "D274A is a kinase-impaired version ( Wan et al . , 2008 ) that is unable to autophosphorylate within our reaction time frame ( Figure 5A , middle panel ) .", "Since the N- and C-terminal truncations do not affect activity ( Figure 5–figure supplement 1 ) , this trick allowed for easy simultaneous detection of wild type and kinase-impaired Aurora A . While Aurora A125−392 D274A cannot phosphorylate itself , equal concentrations of dephosphorylated wild type Aurora A122−403 and Aurora A125−392 D274A lead to comparable phosphorylation rates for both proteins , demonstrating an intermolecular autophosphorylation mechanism for Aurora A ( Figure 5A , right panel ) . 10 . 7554/eLife . 02667 . 015Figure 5 . Mechanism of autophosphorylation .", "( A ) The kinetics of autophosphorylation was monitored by SDS-PAGE and Western blot of 25 μM Aurora A122−403 WT or 25 μM Aurora A125−392 D274A .", "WT Aurora A can phosphorylate catalytically dead D274A Aurora A intermolecularly .", "To account for Aurora A's dynamic range , time points up to 300 s were diluted 50-fold and the rest of the time points were diluted 225-fold .", "( B ) Dilution to 1 μM protein from a stock solution of 200 μM Aurora A ± TPX2 shows much faster autophosphorylation kinetics than from a lower concentrated stock solution ( 20 μM ± TPX2 ) revealing that autophosphorylation occurs within the long-lived dimer .", "All experiments were carried out at 25°C in kinase assay buffer in the presence of 5 mM ATP . DOI: http://dx . doi . org/10 . 7554/eLife . 02667 . 01510 . 7554/eLife . 02667 . 016Figure 5—figure supplement 1 . Dephosphorylated Aurora A125−392 can autophosphorylate as efficiently as Aurora A122−403 . SDS-PAGE and Western blot of 25 μM Aurora A122−403 WT or 25 μM Aurora A125−392 show that both proteins can autophosphorylate to similar extents , suggesting that impaired kinetics in Figure 5A are due to the D274A mutation and not the length of the protein construct .", "To account for Aurora A's dynamic range , time points up to 300 s were diluted 50× and the rest of the time points were diluted 225× .", "All experiments were carried out as described in Figure 5A .", "deP: dephosphorylated; P: phosphorylated . DOI: http://dx . doi . org/10 . 7554/eLife . 02667 . 016 The fact that the dimer is long lived as identified by AUC triggered a second independent test of the functional relevance of this swapped dimer .", "The rate of autophosphorylation was measured for samples of 1 μM Aurora A prepared by dilution from 200 μM and 20 μM stock solutions .", "Although the final protein concentration in both samples is the same , much faster autophosphorylation was detected for the sample diluted from the more highly concentrated stock solution ( Figure 5A ) .", "Since the latter sample contains a higher concentration of this slowly dissociating dimer , this experiment directly demonstrates dimer-dependent autophosphorylation .", "TPX2 strongly accelerates autophosphorylation , in agreement with previous reports ( Eyers et al . , 2003 ) , and as seen for peptide phosphorylation ( Figure 1B and Figure 1–figure supplement 4 ) .", "Notably , this intermolecular autophosphorylation again proceeds via the long-lived swapped dimer ( Figure 5B ) .", "We note that although necessary , the dimer is not sufficient for autophosphorylation , because the catalytically impaired D274A mutant shows a high percentage of dimer ( Figure 4A ) .", "Clearly , TPX2 triggers a conformational change that results in a catalytically active dimer .", "As the third line of evidence , we aimed at designing a mutation that would weaken the swapped dimer formation and therefore autophosphorylation without compromising the kinase activity of the phosphorylated monomer towards peptides .", "Realization of this thought experiment is challenging because most of the intermolecular interactions for the dimer are present as corresponding intramolecular contacts in the monomer ( Figure 3A ) .", "We rationalized that a C290A mutation could work because C290 of one monomer contacts Y334 of the αG-helix of the other monomer , while in phosphorylated monomeric Aurora A , C290 contacts K143 , W277 , L289 , and G291 ( Figure 6A , B ) .", "The C290A mutation indeed results in a primarily monomeric form ( Figure 6C ) that also has severely impaired autophosphorylation activity ( Figure 6D ) .", "However , once phosphorylated , C290A has nearly normal catalytic activity towards the AP substrate ( Figure 6E ) , buttressing the functional role of the swapped WT dimer for autophosphorylation .", "Particularly striking is the observation that in a 1:1 mixture of dephosphorylated WT and C290A mutant , WT autophosphorylation precedes C290A phosphorylation in the early reaction time course ( Figure 6D , right and inset ) but phosphorylation kinetics are identical at later time points .", "Such kinetic behavior is expected for a model of initial autophosphorylation between two dephosphorylated molecules within the swapped dimer and the subsequent taking over of intermolecular autophosphorylation by newly phosphorylated enzyme molecules .", "This latter reaction is much faster , suggesting atomistic differences in comparison to the swapped dimer reaction .", "The C290A mutant is incapable of forming a hybrid swapped dimer between one molecule each of dephosphorylated C290A and WT protein , explaining the lag in its phosphorylation kinetics relative to WT . 10 . 7554/eLife . 02667 . 017Figure 6 . A mutant at the dimer interface ( C290A ) disrupts the swapped-dimer formation and autophosphorylation without affecting the activity of the phosphorylated Aurora A C290A monomer .", "( A ) C290 of monomer I ( light pink spheres ) packs against Y334 of the αG-helix of monomer II .", "Other residues within a 4 . 5 Å radius of C290 in monomer I are shown as red spheres .", "( B ) In the monomeric , phosphorylated Aurora A ( PDB ID 1OL7 ) , C290 ( light orange ) does not contact the αG-helix ( contact residues within a 4 . 5 Å radius are shown as orange spheres ) .", "( C ) Sedimentation velocity analytical ultracentrifugation of 100 μM dephosphorylated ( deP ) Aurora A C290A + 500 μM AMPPCP in kinase assay buffer shows that this protein is predominately monomeric in solution .", "( D ) The kinetics of autophosphorylation was monitored by SDS-PAGE and Western blot of 25 μM Aurora A122−403 WT or 25 μM Aurora A125−392 C290A as described in Figure 5 .", "C290A mutant has impaired autophosphorylation , but is readily phosphorylated by WT Aurora A . ( E ) Activity of the phosphorylated ( P ) , monomeric C290A towards AP peptide ( 0 . 7 ± 0 . 1 s−1 ) is comparable to that of the WT protein ( 1 . 0 ± 0 . 2 s−1 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 02667 . 017 TPX21−43 had been identified as essential for Aurora A activation and protection from PP1 , PP2A , or λPP-directed dephosphorylation ( Eyers et al . , 2003; Tsai et al . , 2003; Satinover et al . , 2004 ) .", "This finding was further substantiated by an X-ray structure of TPX21−43 bound to phosphorylated Aurora A ( Bayliss et al . , 2003 ) ( Figure 7–figure supplement 1 ) .", "In this structure , TPX26−23 was seen in an extended conformation , whereas TPX230−43 formed a regular helix that was proposed to be crucial in protecting Aurora A from phosphatase-mediated T288 dephosphorylation .", "Surprisingly , in our dimer structure , we could only visualize TPX24/6−20/22 ( Figure 2A , B and Figure 7–figure supplement 1 ) , and the previously seen helical part was completely missing .", "To investigate this unexpected result , we designed and functionally characterized the interplay between two peptides , TPX21−25 and TPX225−45 , and Aurora A . First , isothermal titration calorimetry ( ITC ) showed that TPX21−25 bound to Aurora A with the same affinity as longer versions ( TPX21−147 or TPX21−45 ) and did not discriminate between the phosphorylated and dephosphorylated states of the protein ( Figure 7A ) .", "On the other hand , no signal was detected for TPX225−45 with Aurora A in ITC .", "Second , TPX21−25 binding could trigger an increase in activity of dephosphorylated Aurora A towards peptides and autophosphorylation ( data not shown ) .", "In contrast , TPX225−45 had no effect on Aurora A activity .", "Third , TPX21−25 could protect Aurora A from λPP-directed dephosphorylation as well as TPX21−45 , whereas no protection was found for TPX225−45 ( Figure 7B ) .", "These functional data suggest that the first 25 amino acids of TPX2 are primarily responsible for both activation of the enzyme and protection from dephosphorylation . 10 . 7554/eLife . 02667 . 018Figure 7 . The N-terminal half of TPX21−25 is the minimal region needed for binding to Aurora A .", "( A ) Isothermal titration calorimetry ( ITC ) measurements conducted with various TPX2 constructs show that TPX2 binds with similar affinity to either the phosphorylated ( P ) or the dephosphorylated ( deP ) Aurora A and that the minimal length required for binding encompasses the first 25 residues of TPX2 .", "( B ) At the functional level , TPX21−25 can protect Aurora A from λ protein phosphatase ( λPP ) -directed dephosphorylation to the same extent as TPX21−45 . DOI: http://dx . doi . org/10 . 7554/eLife . 02667 . 01810 . 7554/eLife . 02667 . 019Figure 7—figure supplement 1 . The first 25 amino acids of TPX2 bind similarly to either dephosphorylated or phosphorylated Aurora A . Superposition of TPX2 from the dephosphorylated Aurora A ( red ) + TPX2 ( light pink ) and the phosphorylated Aurora A ( not shown ) + TPX2 ( magenta ) ( PDB ID 1OL5 ) shows that the N-terminal half of TPX2 binds similarly to both proteins whereas the C-terminal half of TPX2 only binds to phosphorylated Aurora A that is monomeric in the X-ray structure , but not to the dimeric , dephosphorylated Aurora A . The dotted line represents missing electron density for residues TPX223−29 . DOI: http://dx . doi . org/10 . 7554/eLife . 02667 . 01910 . 7554/eLife . 02667 . 020Figure 7—figure supplement 2 . Two conserved tyrosines of TPX2 nestled inside a hydrophobic pocket in Aurora A trigger allosteric activation .", "( A ) Y8 and Y10 of TPX2 make extensive contacts with residues lining αB- and αC-helices in Aurora A . ( B ) Superposition of TPX2 bound to Aurora A with the C-terminal tails of several AGC kinases reveals equivalent positioning of Phe of the FxxF hydrophobic motifs in the conserved kinases’ hydrophobic pockets .", "Right: a zoom into this highly conserved protein/protein interaction motif used for allosteric regulation .", "We note that while for most AGC kinases this interaction occurs via its own C-terminal tails , in the evolutionarily younger Aurora A kinase , this regulation is mediated by interaction with a second binding partner , TPX2 ( Davis et al . , 2008 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 02667 . 020 The functional role for the first 25 residues in TPX2 makes sense from a comparison to the regulation of other human protein kinases ( Gold et al . , 2006; Kannan et al . , 2007; Keshwani et al . , 2012; Arencibia et al . , 2013 ) .", "This specific interaction between a hydrophobic groove at the junction of the αB/αC-helices in the N-lobe of kinases and a short sequence called ‘the hydrophobic motif’ ( Tyr/Phe-X ( X ) -Tyr/Phe ) from either another partner or from its own C-terminal tail , seems to be a conserved regulatory mechanism across the AGC family ( Figure 7–figure supplement 2 ) ( Zhang et al . , 1994 ) .", "For CMGC kinases , the hydrophobic groove is occupied by activating cyclin proteins , as originally reported for cyclin binding to the N-lobe of Cdk2 which leads to reorientation of the activation loop and αC-helix into an active conformation ( Jeffrey et al . , 1995 ) similar to the effect of TPX2 on Aurora A . In MAPK , activity is increased by binding of the proteins’ own C-terminal tails into this hydrophobic groove ( Baumli et al . , 2008 ) .", "In the tyrosine kinase ( TK ) family , an N-terminal fragment that precedes the kinase domain of c-KIT , MET , and Ephrin nestles inside the hydrophobic pocket , and autoinhibits the kinases ( Chan et al . , 2003; Mol et al . , 2003 , 2004; Davis et al . , 2008; Eathiraj et al . , 2011 ) .", "In the EGFR members of the TK family , this hydrophobic motif is used as a docking point for kinase activation through dimerization ( Zhang et al . , 2006; Jura et al . , 2011 ) .", "This striking conservation of a very specific recognition mechanism across evolutionarily divergent kinase families suggests that Y8 and Y10 of TPX2 nestled inside the hydrophobic αB/αC pocket in Aurora A are likely the key triggers for kinase activation ( Figure 7–figure supplement 2 ) .", "In this work , we characterized two distinct molecular activation mechanisms of Aurora A: autophosphorylation and allosteric activation through TPX2 binding .", "Because of the controversy about the autophosphorylation mechanism of protein kinases ( Oliver et al . , 2006 , 2007; Pike et al . , 2008; Lee et al . , 2009; Lochhead , 2009; Dodson et al . , 2013; Hu et al . , 2013 ) , we felt the need for multiple lines of evidence .", "Our aim was to determine the structure of a dephosphorylated enzyme/substrate complex ‘ready for autophosphorylation’ .", "The swapped dimer is indeed asymmetric , with one monomer playing the role of the enzyme and the other that of the substrate .", "In the substrate molecule , the hydroxyl group of T288 is in principle capable of reaching the γ-phosphate of AMPPCP bound to the enzyme monomer as shown by TMD simulations .", "Domain-swapped dimers have been solved for a number of protein kinases and questioned for their relevance or crystal artifacts ( Dodson et al . , 2013 ) .", "To address such a critique directly , we showed a long-lived dimer in solution .", "Importantly , using three biochemical tricks of", "( i ) a mixture between wild type and dead mutant and", "( ii ) a serial dilution , and", "( iii ) a C290A mutant with severely impaired dimer formation and autophosporylation but nearly normal peptide phosphorylation activity in its phosphorylated monomeric state , we could directly demonstrate an intermolecular autophosphorylation mechanism within this long-lived dimer .", "Our combined biochemical , thermodynamic , structural , and computational data resolve the controversy about the molecular mechanism ( s ) of autophosphorylation in Aurora A by directly measuring the process of autophosphorylation and linking it to a long-lived functional dimer of dephosphorylated Aurora A . This is in sharp contrast to the mechanism of intramolecular autophosphorylation put forward by Dodson et al . ( 2013 ) based on measuring the kinetics of peptide phosphorylation using an automated microchip assay as indirect readout , and not by monitoring autophosphorylation .", "While the experiments in Dodson et al . ( 2013 ) are conceptually correct , there are a number of errors in the experimental setup and data analysis .", "Michaelis–Menten equations applied to analyze all data cannot be used because Michaelis–Menten conditions are not met , since the peptide concentration of 3 μM is not much higher than the enzyme concentrations used .", "Competition of free enzyme for the peptide versus another enzyme molecule is not included in the scheme .", "The peptide concentration is far below its KM and ATP hydrolysis is faster under these conditions than peptide phosphorylation producing ADP concentrations that cause significant and time-dependent enzyme inhibition ( DK , unpublished ) .", "Finally , knowledge of the slow dissociation of the dimer and its consequences for the measured kinetics ( Figure 5B ) exposes another source for incorrect data interpretation since the experiments in Dodson et al . ( 2013 ) were started by serial dilutions from a high enzyme stock solution .", "The ongoing discussions on a large collection of functional , structural , and computational data on autophosphorylation in protein kinases underscore both the evolution of differential regulation mechanisms and the difficulty of elucidating these complex biological mechanisms .", "Our findings may have more general implications for the family of eukaryotic Ser/Thr kinases .", "A number of swapped dimer structures have been solved from this enzyme class , and while Dodson et al . ( 2013 ) argued that these dimers are crystallographic artifacts ( such as Chk2 ) , our correlation between a domain-swapped dimer and its functional relevance would instead suggest that the reported structures are mechanistically meaningful .", "For direct evidence the functional relevance of these dimer structures would need to be assessed , as has been reported by elegant experiments for Chk2 ( Oliver et al . , 2006; Pike et al . , 2008 ) .", "Interestingly , most of the known swapped dimers sit in an inactive conformation ( using all structural hallmarks for an active kinase discussed in Figure 2 ) ( Figure 3–figure supplement 1 ) .", "However , a few dimers capture all active-state structural signatures ( Figure 3–figure supplement 1 , LOK and Chk2 [Oliver et al . , 2006; Pike et al . , 2008] ) .", "All previously reported Aurora A dimers are in inactive conformations ( PDB IDs 2BMC [Fancelli et al . , 2005] , 3DJ5 , and 3DJ6 ) , while in our new structure both Aurora A molecules are in the active conformation with one monomer serving as the enzyme molecule and the other as the substrate .", "The functional effect of autophosphorylation is a 100-fold increase in the catalytic activity of Aurora A . While this catalytic boost is comparable to other Ser/Thr kinases ( Hagopian et al . , 2001; Prowse and Lew , 2001; Adams , 2003 ) , the more surprising result has been our finding that TPX2 binding activates dephosphorylated Aurora A to similar levels .", "This is again in contrast to previous reports of additive effects from the two distinct activation mechanisms ( Dodson and Bayliss , 2012 ) .", "Our results point to a classic allosteric regulation mechanism where either phosphorylation in the activation loop or TPX2 binding in the conserved remote hydrophobic groove shifts the equilibrium far towards the active state .", "While the work here has only been concerned with in vitro experiments , it may provide insight into the regulatory roles of Aurora A in the cellular context .", "Immunofluorescence data have shown that the centrosome-associated Aurora A pool is mainly phosphorylated , whereas the spindle-associated and TPX2-bound Aurora A is dephosphorylated ( Tyler et al . , 2007; Scutt et al . , 2009; Sloane et al . , 2010; Zeng et al . , 2010 ) .", "In parallel , a recent study on the Caenorhabditis elegans homologue of Aurora A kinase , AIR-1 , showed that spindle-microtubule associated Aurora A was not phosphorylated and could nonetheless carry on centrosome-independent microtubule formation ( Toya et al . , 2011 ) .", "In light of our new findings that TPX2 fully activates dephosphorylated Aurora A , the previous in vivo experiments can be re-interpreted as kinase-activity dependent functions of spindle-microtubule associated Aurora A , and not a kinase-independent function ( Toya et al . , 2011 ) .", "Our in vitro data together with previous in vivo results suggest that nature has evolved two distinct regulation mechanisms for Aurora A in different locations within the cell: autophosphorylation as activation in the centrosomes to promote phosphorylation of downstream targets , and TPX2-mediated activation at the spindle microtubules promoting Aurora A activity to another subset of downstream targets .", "This hypothesis is supported by the differential timing of Aurora A and TPX2 availability .", "Aurora A kinase levels are available as early as S-phase and peak in the G2 phase ( Dutertre et al . , 2002; Liu and Ruderman , 2006 ) .", "On the other hand , TPX2 levels peak in the prometaphase/spindle formation stage that follows the G2 phase ( Gruss et al . , 2002; Eckerdt and Maller , 2008; Macurek et al . , 2008 ) .", "Our findings help shed light on an elegant strategy for fine-tuning cellular kinetics that provides more complex regulation in higher organisms .", "We finally want to raise the question whether Aurora A autophosphorylation is physiologically relevant in cells or whether phosphorylation-mediated activation is primarily accomplished by upstream kinases .", "While we do not have a conclusive answer to this challenging question , which has been tackled for a number of other protein kinases , we discuss several conjectures .", "Although the upstream PAK1 kinase can phosphorylate Aurora A at T288 , autophosphorylation appears to be the essential mode of activation because PAK1 inhibition does not abolish cell division but Aurora A inhibitors do ( Zhao et al . , 2005; Liu and Ruderman , 2006; Kelly et al . , 2011 ) .", "Are the in vitro autophosphorylation kinetics reported here compatible with the known phosphorylation kinetics of Aurora A during the cell cycle ?", "Our data imply that the initial rate of Aurora A autophosphorylation is very slow because this reaction occurs via the long-lived swapped dimer between two dephosphoryated Aurora A molecules .", "Further autophosphorylation displays a strong sigmoidal character revealing much faster kinetics of Aurora A phosphorylation by an already phosphorylated Aurora A molecule ( Figures 5 and 6 ) .", "In light of the increased local concentration of Aurora A in the centrosomes ( Glover et al . , 1995 ) ( although the exact concentration is not known ) , our measured autophosphorylation kinetics is qualitatively in line with the progression of Aurora A T288 phosphorylation during the 3–4 h of G2/M duration in HeLa cells at 37°C ( Crosio et al . , 2002; Littlepage and Ruderman , 2002; Ohashi et al . , 2006 ) ." ], [ "TEV-cleavable , His6-tagged Aurora A kinase , either long ( 122–403 ) or short ( 125–392 ) constructs , were cloned into pET28a and expressed in Rosetta 2 ( DE3 ) E . coli cells ( Stratagene ) for 13–15 h at 21°C .", "Cells were centrifuged at 5000 rpm for 15 min , resuspended in buffer A , and sonicated in the presence of EDTA-free protease inhibitor cocktail and DNAse for 4 min ( 20 s on , 20 s off , 3 . 0 V ) .", "Lysates thus obtained were filtered using a 0 . 22 μm filtering unit and passed through a NiNTA column .", "The protein was eluted at 20% buffer B and Aurora A kinase fractions were pooled and TEV-cleaved overnight at 4°C in a 5 kDa dialysis cassette that was exchanged against buffer C . Cleaved Aurora A was passed through another nickel column to remove any uncleaved reactants and His6-TEV-protease , and then purified to homogeneity through a 26/60 S200 size exclusion column .", "Protein thus produced was aliquoted and flash-frozen before being stored at −80°C and used for kinase assays .", "Mutant Aurora A122−403 T288V was also purified the same way .", "The phosphorylation of all Aurora A samples including mutant forms used here were quantitatively confirmed by mass spectrometry ( MS ) .", "Dephosphorylated Aurora A kinase was obtained through a λPP co-expression system .", "Codon-optimized Aurora A122−403 in pET28a and untagged λPP in T7-7 plasmid were co-transformed in BL21 ( DE3 ) cells and spread on Kan/Amp 2× YT plates .", "The most robust colony was used for a 2× YT pre-culture and later on to inoculate a 1 L culture to an OD of 0 . 2 .", "Cells were induced with 0 . 6 mM IPTG for 5 h at 37°C .", "It was noticed that although Aurora A could grow reasonably well in LB media , λPP could not; hence , the choice of 2× YT media for all co-expression needs .", "Purification involved the NiNTA column , followed by overnight TEV cleavage and GST-λPP treatment , in tandem NiNTA-GST columns and finally a 26/60 S200 size exclusion column .", "MS was used to confirm that Aurora A kinase was completely dephosphorylated .", "At the end of the purification , Aurora A was dialyzed against buffer C , flash-frozen with liquid nitrogen into 1 mL aliquots and stored at −80°C .", "The buffers used were:Buffer A: 50 mM TrisHCl ( pH 8 . 0 ) , 300 mM NaCl , 40 mM imidazole , 20 mM MgCl2 , 10% ( vol/vol ) glycerolBuffer B: 50 mM TrisHCl ( pH 8 . 0 ) , 300 mM NaCl , 500 mM imidazole , 20 mM MgCl2 , 10% ( vol/vol ) glycerolBuffer C: 20 mM TrisHCl ( pH 7 . 0 ) , 200 mM NaCl , 20 mM MgCl2 , 5 mM TCEP , 10% ( vol/vol ) glycerolBuffer D: 135 mM NaCl , 3 mM KCl , 8 mM Na2HPO4 , 1 . 5 mM KH2PO4 , 5 mM TCEP , 10% ( vol/vol ) glycerol , pH 7 . 40Buffer E: 135 mM NaCl , 3 mM KCl , 8 mM Na2HPO4 , 1 . 5 mM KH2PO4 , 5 mM TCEP , 10% ( vol/vol ) glycerol , 10 mM glutathione , pH 7 . 40 Typical yields were 8–10 mg of phosphorylated Aurora A and 45–50 mg of dephosphorylated Aurora A ( expressed in the presence of λPP ) per liter of E . coli culture .", "The LCMS system consisted of an Agilent 1200 series HPLC connected to an Agilent series 6520 ESI Q-TOF .", "Protein samples ( 10 µM ) dissolved in a 5% acetonitrile–0 . 1% formic acid buffer were separated on a C18 Poroshell 300SB column ( 1 mm × 75 mm × 5 µm ) at 0 . 5 mL min−1 using a linear gradient of 5–70% acetonitrile in 0 . 1% formic acid .", "MS data were collected up to 3000 m/z and raw spectra were deconvoluted using the maximum entropy algorithm of Agilent Masshunter version B . 03 . 01 software .", "External mass calibration was performed using a mixture of purine ( 121 m/z ) and HP-0921 ( 922 m/z ) immediately prior to measuring protein samples .", "Aurora A , either phosphorylated/dephosphorylated wild type or mutant protein , was mixed with either AP ( APSSRRTTLCGTL ) , Kemptide ( LRRASLG ) , or Lats2 ( ATLARRDSLQKPGLE ) , in the absence or presence of 50 μM TPX2 in kinase buffer ( 20 mM TrisHCl , 200 mM NaCl , 3% [vol/vol] glycerol , 20 mM MgCl2 , 1 mM TCEP , pH 7 . 50 ) .", "These substrates comprise the consensus sequence for Aurora A ( [R/K/N]-R-X-[S/T]-B where B is any hydrophobic residue with the exception of Pro ) ( Ferrari et al . , 2005; Ohashi et al . , 2006; Sardon et al . , 2010 ) .", "Peptides were ordered through Genscript .", "The reaction was initiated with the addition of 5 mM ATP .", "Then 5 μl time points were collected , resuspended in 10 μl 6% ( vol/vol ) trichloroacetic acid ( in water ) to quench the reaction , and neutralized with 50 μl 100 mM KH2PO4 , pH 8 . 0 to provide the appropriate pH for nucleotide separation .", "The mixture was then passed through a 0 . 22 μm SpinX column to remove any protein precipitation .", "Reverse phase-high performance liquid chromatography ( RP-HPLC ) and an ACE 5 C18-AR , 100 Å pore size column , were used to separate nucleotides as well as peptides .", "For nucleotide runs , 2 μl of the aforementioned mixture was sufficient for analysis , whereas for the peptide runs the optimal injection volume was 20 μl .", "Nucleotide runs were routinely performed to ensure no unproductive hydrolysis was occurring during the experiment .", "An isocratic elution run in 100 mM KH2PO4 , pH 6 . 0 was performed for this purpose .", "For the peptide runs , a gradient of 0–30% of elution buffer lasting 10 min at 0 . 4 mL/min was sufficient to separate phosphorylated from non-phosphorylated species .", "The running buffer was 0 . 1% TFA ( vol/vol ) in water , while the elution buffer was 100% acetonitrile .", "Representative peptide RP-HPLC traces are shown in Figure 1–figure supplement 5 .", "Lastly , to ensure full saturation of Aurora A by TPX2 and test these proteins were well behaved , a dose-dependence curve of the effect of TPX2 on Aurora A as shown in Figure 1–figure supplement 6 was obtained .", "All titrations were carried out using Nano ITC ( TA Instruments ) and analyzed via the NanoAnalyze software using the independent fit model .", "Injectant was added in 1 μl volume , every 180 s , with a constant stirring speed at 350 rpm and at 25°C .", "Prior to ITC titration , both protein and peptide were dialyzed/resuspended in 20 mM TrisHCl , 200 mM NaCl , 3% ( vol/vol ) glycerol , 1 mM TCEP , pH 7 . 50 .", "The concentrations used for each of the runs in Figure 7A were: 35 μM dephosphorylated ( deP ) A122−403 + 280 μM TPX21−147 , 48 μM deP A122−403 + 680 μM TPX21−45 , 13 μM deP A122−403 + 250 μM TPX21−25 , 18 μM deP A122−403 + 250 μM TPX225−45 , 20 μM phosphorylated ( P ) A122−403 + 280 μM TPX21−147 , 90 μM P A122−403 + 940 μM TPX21−45 , 20 μM P A122−403 + 300 μM TPX21−25 , and 18 μM P A122−403 + 300 μM TPX225−45 .", "Crystals of dephosphorylated Aurora A122−403 in complex with AMPPCP and TPX21−45 were grown at 18°C by vapor diffusion and the hanging drop method .", "A 2:1 ratio of protein mixture:mother liquor was obtained by combining 300 μM ( 10 mg/ml ) deP Aurora A122−403 + 1 . 5 mM AMPPCP + 300 μM TPX21−45 with 0 . 2 M lithium sulfate monohydrate , 0 . 1 M BisTris , pH 5 . 5 , 25% PEG3350 .", "Similarly , crystals of dephosphorylated Aurora A122−403 in complex with AMPPCP were obtained by mixing a 2:1 ratio of 570 μM ( 18 mg/ml ) deP Aurora A122−403 + 1 mM AMPPCP with mother liquor ( 0 . 2 M ammonium sulfate , 0 . 2 M TrisHCl , pH 7 . 50 , 30% ( wt/vol ) PEG3350 ) .", "These latter crystals were also grown at 18°C by vapor diffusion and the hanging drop method .", "The protein , peptide , and nucleotide were originally stored in 20 mM TrisHCl , 200 mM NaCl , 10% ( vol/vol ) glycerol , 20 mM MgCl2 , 1 mM TCEP , pH 7 . 50 .", "Diffraction data were collected at 100 K at Advanced Light Source ( Lawrence Berkeley National Laboratory ) beamlines ( 8 . 2 . 1 and 8 . 2 . 2 ) .", "The details of data collections are listed in Table 1 .", "Data were processed with the automated data reduction program Xia2 ( Winter , 2010 ) that is part of the CCP4 suite ( Winn et al . , 2011 ) and uses iMOSFLM ( Battye et al . , 2011 ) for integration and Scala ( Evans , 2006 ) for scaling .", "Initial phases were obtained by molecular replacement ( CCP4 program Molrep [Vagin , 1997] ) by using an Aurora A kinase structure ( PDB ID 1MQ4 ) as a search model .", "The refinement was carried out with REFMAC5 ( Murshudov et al . , 2011 ) and phenix . refine ( Adams et al . , 2010 ) , followed by manual rebuilding in Coot ( Emsley and Cowtan , 2004; Emsley et al . , 2010 ) .", "Sedimentation velocity runs were performed on a Beckman Optima XL-A Analytical Ultracentrifuge at 50 , 000 rpm and 18°C ( same as crystallization temperature ) .", "Sedimentation of 100 μM deP Aurora A122−403 ( or 150 μM deP Aurora A122−403 , or 160 μM Aurora A125−392 D274A , or 100 μM Aurora A125−392 C290A ) + 500 μM AMPPCP and/or 100 μM TPX21−25 was followed at three different wavelengths ( 285 nm , 290 nm , and 295 nm ) .", "Data were analyzed using the SEDFIT software ( Schuck , 2000; Dam and Schuck , 2004 ) and the continuous size-distribution option .", "All SAXS experiments were done on a BioSAXS-1000 system at Brown University , Providence , RI , USA ( camera length 480 . 3 mm , Pilatus 100 K detector ) .", "SAXS data were recorded for Aurora A at concentrations between 0 . 33 and 6 . 6 mg/ml at 20°C with 1 mg/mL TPX21−25 each .", "The momentum transfer axis ( s = 4πsinθ/λ , where 2θ represents the scattering vector s and λ = 1 . 54187 nm ) was calibrated by using silver behenate as standard .", "The experiment time was between 15 min and 6 h per sample , depending on the protein concentration .", "Data reduction of the raw image files and conversion into scattering curves was done with the SAXSLab software ( Rigaku ) .", "The SAXS curves were further processed ( buffer subtraction , correction for unbound TPX2 ) with the program Primus ( Konarev et al . , 2003 ) .", "We used calculated SAXS curves ( program Crysol [Svergun , 1995] ) from the X-ray structures of this study as reference for the monomeric and dimeric state .", "The amount of dimers was calculated by using a script based on the least squares method calculations .", "The crystal structure of dephosphorylated Aurora A bound to AMPPCP and TPX2 ( PDB ID 4C3P ) was used as the starting point for building the model presented in Figure 3B .", "The electron density for the amino acids in the region 283–288 of monomer II was not distinguishable from noise .", "We used the tools in the software package Modeller 9 . 11 ( Eswar et al . , 2006 ) to model the missing residues .", "The lowest energy model was then used as the starting point for a molecular dynamics simulation run , in which the distance between the oxygen in the sidechain hydroxyl group of T288 and the γ-phosphate of the AMPPCP moiety bound to monomer I was reduced to 3 Å .", "To achieve this , the structure was parameterized with the CHARMM 22-protein all-atom force field with the CMAP backbone energy correction ( MacKerell et al . , 1998 , 2001 ) .", "The system was solvated in a rectangular box with TIP3 water molecules and neutralized with NaCl counterions .", "The final simulation box contained approximately 65 , 000 atoms .", "Periodic boundary conditions were applied to the simulation box .", "After energy minimization , the simulation box was gradually heated to 300 K with a time step of 1 fs while gradually reducing positional restraints in an MD simulation of 2 ns with the software NAMD 2 . 8 ( Phillips et al . , 2005 ) .", "The system was then equilibrated for 10 ns in the NPT ensemble ( T = 300 K , p = 1 . 01325 bar ) with the software NAMD , using the Langevin dynamics method for controlling temperature , and the combined Langevin piston Nose–Hoover method for equilibrating pressure ( Martyna et al . , 1994; Feller et al . , 1995 ) .", "We then used the software GROMACS 4 . 5 . 5 ( Hess et al . , 2008 ) with the steered molecular dynamics functionality as implemented in the extension PLUMED 1 . 3 ( Bonomi et al . , 2009 ) to progressively reduce the distance between the hydroxyl group of T288 and the γ-phosphate of the AMPPCP moiety bound to monomer I . This distance was reduced to 3 Å within a simulation run of 2 ns ." ] ]

[ "We elucidate the molecular mechanisms of two distinct activation strategies ( autophosphorylation and TPX2-mediated activation ) in human Aurora A kinase .", "Classic allosteric activation is in play where either activation loop phosphorylation or TPX2 binding to a conserved hydrophobic groove shifts the equilibrium far towards the active conformation .", "We resolve the controversy about the mechanism of autophosphorylation by demonstrating intermolecular autophosphorylation in a long-lived dimer by combining X-ray crystallography with functional assays .", "We then address the allosteric activation by TPX2 through activity assays and the crystal structure of a domain-swapped dimer of dephosphorylated Aurora A and TPX21−25 .", "While autophosphorylation is the key regulatory mechanism in the centrosomes in the early stages of mitosis , allosteric activation by TPX2 of dephosphorylated Aurora A could be at play in the spindle microtubules .", "The mechanistic insights into autophosphorylation and allosteric activation by TPX2 binding proposed here , may have implications for understanding regulation of other protein kinases ." ]

[ "The kinase , Aurora A , is a human protein that is needed for cells to divide normally .", "Kinases are enzymes that control other proteins by adding phosphate groups to these proteins; however , like other kinases , Aurora A must first be activated or ‘switched on’ before it can do this .", "Aurora A kinase can be switched on in two ways: by having a phosphate group added to its ‘activation loop’; or by binding to another protein called TPX2 .", "Also like other kinases , Aurora A can self-activate , but the details of this process are not understood .", "Does a single Aurora A kinase add a phosphate group to its own activation loop , or does one Aurora A kinase activate a second ?", "Furthermore , it is not clear how binding to TPX2 can activate an Aurora A kinase without adding a phosphate group to the activation loop .", "Zorba , Buosi et al . now show that Aurora A kinases that have been activated in different ways—via the addition of a phosphate group or binding to TPX2—are equally good at adding phosphate groups to other proteins .", "Zorba , Buosi et al . also worked out the three-dimensional shapes of the kinases activated in these two ways—since many proteins change shape when they are switched on—and found that they were also the same .", "Finally , it was shown that self-activation involves two Aurora A kinases binding to each other , and one kinase adding a phosphate group to the other , rather than a single kinase adding a phosphate group to itself .", "Since other protein kinases can be activated in similar ways to Aurora A , the findings of Zorba , Buosi et al . might also help us to understand how other protein kinases can be switched ‘on’ or ‘off’ .", "And , as mutations in Aurora A have been linked to the development of cancer , uncovering how this kinase is controlled could help efforts to design new drugs to treat this disease ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "developmental biology", "tools and resources" ]

[ [ "Morphogenesis , or the origin of biological form , is one of the oldest and most enduring problems in biology .", "Embryonic tissues change their size and shape during development through patterned cell activities controlled by intricate physico-chemical mechanisms ( Day and Lawrence , 2000; Heisenberg and Bellaïche , 2013; Keller , 2013 , 2012; Lecuit and Mahadevan , 2017; LeGoff and Lecuit , 2015 ) .", "Developmental processes have been explained traditionally in terms of genes and gene regulatory networks , and a major challenge is to understand how the genetic and molecular information is ultimately translated into cellular activities like proliferation , death , change of shape and movement .", "Therefore , detailed descriptions of cell lineages and behaviors can provide a firm ground for studying morphogenesis from a bottom-up cellular perspective ( Buckingham and Meilhac , 2011; Kretzschmar and Watt , 2012; Schnabel et al . , 1997; Spanjaard and Junker , 2017; Sulston et al . , 1983 ) .", "We have focused here on the crustacean Parhyale hawaiensis that satisfies a number of appealing biological and technical requirements for multi-level studies of appendage ( limb ) morphogenesis ( Stamataki and Pavlopoulos , 2016 ) .", "Parhyale is a direct developer; its body plan is specified during the 10 days of embryogenesis when imaging is readily possible ( Browne et al . , 2005 ) .", "Each embryo develops a variety of specialized appendages along the anterior-posterior axis that differ in size , shape and pattern ( Martin et al . , 2016; Pavlopoulos et al . , 2009; Wolff and Scholtz , 2008 ) .", "Parhyale eggs have good size and optical properties for microscopic live imaging at cellular resolution; the eggshell is transparent and embryos are 500 µm long with low autofluorescence and light scattering .", "Several functional genetic approaches , embryological treatments and genomic resources also allow diverse experimental manipulations in Parhyale ( Kao et al . , 2016 ) .", "Previous reports have used transmitted light and fluorescence time-lapse microscopy to live image early processes like gastrulation and germband formation during the first couple days of Parhyale development ( Alwes et al . , 2011; Chaw and Patel , 2012; Hannibal et al . , 2012 ) .", "However , for a comprehensive coverage of Parhyale limb formation , embryos need to be imaged from multiple angular viewpoints from day 3 to day 8 of embryogenesis ( Browne et al . , 2005 ) .", "We demonstrate here that transgenic embryos with fluorescently labeled nuclei can be imaged routinely for several consecutive days using Light-sheet Fluorescence Microscopy ( LSFM ) .", "LSFM is an ideal technology for studying how cells form tissues and organs in intact developing embryos ( Huisken et al . , 2004; Keller et al . , 2008; Truong et al . , 2011 ) .", "It enables biologists to capture fast and dynamic processes at very high spatiotemporal resolution , over long periods of time , and with minimal bleaching and photo-damage ( Combs and Shroff , 2017; Huisken and Stainier , 2009; Khairy and Keller , 2011; Schmied et al . , 2014; Weber et al . , 2014 ) .", "In addition , samples can be optically sectioned from multiple angles ( multi-view LSFM ) that can be combined computationally to reconstruct the entire specimen with more isotropic resolution ( Chhetri et al . , 2015; Krzic et al . , 2012; Schmid et al . , 2013; Swoger et al . , 2007; Tomer et al . , 2012; Wu et al . , 2016; Wu et al . , 2013 ) .", "Although the amount and type of data generated by multi-view LSFM raise several challenges for image analysis , many of them have been efficiently addressed .", "Software solutions exist for registration of acquired views , fusion of raw views ( z-stacks ) into a single output z-stack , and visualization of the raw and fused images ( Chhetri et al . , 2015; Ingaramo et al . , 2014; Pietzsch et al . , 2015; Preibisch et al . , 2014; Preibisch et al . , 2010; Rubio-Guivernau et al . , 2012; Wu et al . , 2016 ) .", "These processes should be repeated for hundreds or thousands of time-points to generate a 4D representation of the embryo as it develops over time ( Amat et al . , 2015; Schmied et al . , 2014; Schmied et al . , 2016 ) .", "Automated approaches for cell segmentation and tracking have also been developed ( Amat et al . , 2014; Du et al . , 2014; Dufour et al . , 2017; Faure et al . , 2016; Schiegg et al . , 2015; Stegmaier et al . , 2016; Ulman et al . , 2017 ) , however they do not yet reach the precision required for unsupervised extraction of cell lineages .", "To address this issue , we describe here the Massive Multi-view Tracker ( MaMuT ) software that allows visualization , annotation , and accurate lineage reconstruction of large multi-dimensional microscopy data .", "We quantitatively analyzed Parhyale LSFM datasets with MaMuT to understand the cellular basis of arthropod limb morphogenesis .", "As revealed by lineage tracing experiments in the leading arthropod model Drosophila melanogaster , the leg and wing primordia become progressively subdivided into distinct cell populations ( called compartments when lineage-restricted ) along the anterior-posterior ( AP ) and dorsal-ventral ( DV ) axes ( Dahmann et al . , 2011; Garcia-Bellido et al . , 1973; Steiner , 1976 ) .", "Tissue subdivisions acquire distinct cell fates driven by domain-specific expression of patterning genes ( called selectors if lineally inherited ) , as well as by the localized induction of signaling molecules at compartment boundaries ( organizers ) that control patterning and growth of developing organs ( García-Bellido , 1975; Lawrence and Struhl , 1996; Mann and Carroll , 2002; Restrepo et al . , 2014 ) .", "Besides regionalization mechanisms , oriented cell divisions have been implicated as a general mechanism in shaping the Drosophila wing and other growing organs ( Baena-López et al . , 2005; Legoff et al . , 2013; Mao et al . , 2013 ) .", "Other mechanisms like differential cell proliferation and cell rearrangement could also play a role in the formation of limb buds and their elongation along the proximal-distal ( PD ) axis .", "So far , these processes have not been possible to live image and quantify in direct developing arthropod limbs .", "Our understanding of cell dynamics shaping arthropod limbs has relied exclusively on studies of the indirectly developing Drosophila limbs ( primarily the wing disc ) using clonal analysis and lineage tracing across fixed specimens ( Baena-López et al . , 2005; González-Gaitán et al . , 1994; Resino et al . , 2002; Weigmann and Cohen , 1999; Worley et al . , 2013 ) and recent improvements in imaging discs in vivo and ex vivo ( Dye et al . , 2017; Heemskerk et al . , 2014; Legoff et al . , 2013; Mao et al . , 2013; Strassburger et al . , 2017; Tsao et al . , 2016; Zartman et al . , 2013 ) .", "By tracking all constituent cells in direct developing Parhyale limbs , we identified the lineage restrictions and morphogenetic cellular behaviors operating during limb bud formation and elongation , and compared these to Drosophila and other arthropod and vertebrate paradigms .", "We validated our cellular models of morphogenesis by studying the expression of developmental regulatory genes implicated in limb patterning and growth ." ], [ "Three-day old transgenic embryos with fluorescently labeled nuclei were mounted for LSFM in low melting agarose with scattered fluorescent beads .", "Several parameters were optimized to cover all stages of Parhyale appendage development at single-cell resolution with adequate temporal sampling for accurate cell tracking ( see Materials and methods ) .", "A typical 4 to 5 day long recording was composed of more than 1 million images resulting in >7 TB datasets .", "The relatively slow tempo of Parhyale development enabled imaging of each embryo from multiple highly overlapping views with minimal displacement of nuclei between views acquired in each time-point ( Figure 1A ) .", "As detailed in Materials and methods , development of the entire embryo was reconstructed using the Fiji ( Fiji Is Just ImageJ ) biological image analysis platform ( Schindelin et al . , 2012 ) according to the following steps: ( 1 ) image file preprocessing , ( 2 ) bead-based spatial registration of views in each time-point , ( 3 ) fusion by multi-view deconvolution , ( 4 ) bead-based temporal registration across time-points , ( 5 ) computation of temporally registered fused volumes , and ( 6 ) 4D rendering of the spatiotemporally registered fused data ( Preibisch et al . , 2014; Preibisch et al . , 2010; Schmied et al . , 2014 ) .", "This processing resulted in almost isotropic resolution of fused volumes ( Figure 1B ) and was used for visualization of Parhyale embryogenesis with cellular resolution ( Figure 1C–K and Figure 1—video 1 ) .", "Segment formation and maturation in Parhyale occurred sequentially in AP progression ( Figure 1—video 2 ) .", "Appendage morphogenesis involved patterning , growth and differentiation of ectodermal cells organized in an epithelial monolayer that gave rise to the appendage epidermis .", "In our LSFM recordings , we were particularly interested in imaging the limbs in the anterior thorax of Parhyale embryos that were specified at about 3 . 5 days after egg-lay ( AEL ) at 25˚C .", "Over the next 4 days , limb buds bulged out ventrally , elongated along their PD axis and became progressively segmented until they acquired their definite morphology at around 8 days AEL ( Figure 1C–K and Figure 1—video 2 ) .", "During germband formation , the ectoderm contributing to the posterior head and the trunk became organized in a stereotyped grid-like pattern with ordered AP rows and DV columns of cells ( Figure 2A–A’’ ) ( Browne et al . , 2005; Dohle et al . , 2004; Dohle and Scholtz , 1988; Gerberding et al . , 2002; Scholtz , 1990; Wolff and Gerberding , 2015 ) .", "Each row of cells corresponded to one parasegment , which is the unit of early metameric organization in Parhyale embryos , like in Drosophila and other arthropods ( Hejnol and Scholtz , 2004; Scholtz et al . , 1994 ) .", "Two rounds of longitudinally-oriented cell divisions in each formed parasegmental row ( Figure 2B–D’ ) , together with the progressive addition of new parasegments at the posterior end , led to embryo axial elongation ( Figure 1C–H ) .", "Subsequent divisions of ectodermal cells had a more complex pattern disrupting the regularity of the grid and contributing to the transition from parasegmental to segmental body organization and the evagination of paired appendages in each segment .", "Appendage buds appeared successively from the head region backwards ( Figure 1D–H ) and started lengthening ( Figure 1F–K ) and differentiating along their PD axis ( Figure 1G–K ) .", "At the end of the imaging period , morphogenesis appeared nearly complete .", "Thus , multi-view LSFM imaging captured the entire gamut of differential appendage morphogenetic events along the body axis of the Parhyale embryo in a single time-lapse experiment .", "To examine the cellular basis of morphogenesis , we developed a novel Fiji plugin to extract cell lineages from multi-view and multi-terabyte datasets .", "This tool was dubbed MaMuT for Massive Multi-view Tracker ( Figure", "3 ) and is a hybrid and extension of two existing Fiji plugins: the BigDataViewer visualization engine ( Pietzsch et al . , 2015 ) and the TrackMate annotation engine ( Tinevez et al . , 2017 ) .", "MaMuT can be installed through the Fiji updater and is tightly integrated with the other Fiji plugins for LSFM data processing .", "The source code for MaMuT is available on GitHub ( Tinevez et al . , 2018; copy archived at https://github . com/elifesciences-publications/MaMuT ) and detailed tutorials and training datasets can be found at http://imagej . net/MaMuT .", "MaMuT is an interactive , user-friendly tool for visualization , annotation , tracking and lineage reconstruction of large multi-dimensional microscopy data ( Figure 3 and Figure 3—figure supplement 1 ) .", "It is a versatile platform that can be used either for manual or semi-automated tracking of selected populations of cells of interest , or for visualization and editing of fully automated computational predictions for systems-wide lineage reconstructions .", "MaMuT can handle multiple data sources but was developed primarily to enable the analysis of LSFM datasets .", "Its unique feature is the ability to annotate image volumes synergistically from all available input views ( detailed in Materials and methods ) .", "This functionality of MaMuT allowed us to identify and track all constituent cells in developing limbs continuously from the early germband stages until the later stages of 3D organ outgrowth , when the information from multiple views was required for full reconstructions .", "We deployed the manual version of MaMuT to extract the lineage of one Parhyale thoracic limb .", "By convention , Parhyale parasegments are identified by ascending indices E1 , E2 , E3 etc . , the AP rows of ectodermal cells in each parasegment by the letters a , b , c and d , and the DV columns of cells in each parasegment by numbers ( Figure 2 ) .", "In accordance with previous studies in malacostracan crustaceans and other arthropods , our reconstructions demonstrated that each Parhyale thoracic limb consisted of cells from two neighboring parasegments ( Browne et al . , 2005; Dohle et al . , 2004; Dohle and Scholtz , 1988; Hejnol and Scholtz , 2004; Scholtz , 1990; Scholtz et al . , 1994; Wolff and Scholtz , 2008 ) .", "The T2 limb ( referred to as limb#1 ) that we analyzed in-depth ( Figure 4A–E ) developed from rows b , c and d of the E4 parasegment and from rows a and b of the following E5 parasegment ( Figure 4F–J’ ) .", "Cells that arose from rows c , d and a occupied the entire length of the limb and body wall parts of the T2 segment , while rows b contributed only to the proximal limb and intersegmental territories ( Figure 4—figure supplement 1K–O’ ) .", "Cells in medial columns 1 and 2 gave rise to the nervous system and sternites and were not considered in this study .", "The more lateral columns 3 to 9 gave rise to the forming limb ( Figure 4—figure supplement 1F–J ) .", "We fully tracked 34 founder cells constituting the limb#1 primordium over 50 hr of development , giving rise to a total of 361 epidermal cells ( Figure 5 and Figure 5—video 1 ) .", "We started tracking each of these 34 cells as they were born during transition from the 2-row to the 4-row-parasegment ( Figure 5A–C ) , and then continuously during the subsequent rounds of divisions , referred to as differential divisions ( DDs ) ( Figure 5D ) .", "The number of DDs observed during these 50 hr varied dramatically between cells from just 1 DD in the slowest dividing lateral cells of the primordium ( cells E4b8 , E5a9/b9 ) to 5 DDs in the fastest dividing central cells ( cells E4c3-c6 and E4d3-d6 ) .", "Although the clonal composition of crustacean appendages had been described previously with lipophilic dye injections ( Wolff and Scholtz , 2008 ) , the reconstruction presented here is the most comprehensive lineage tree for any developing arthropod limb published to date ( Figure 5—figure supplement 1 ) .", "We first asked whether these complete reconstructions could reveal any lineage-based subdivisions in the developing limb#1 .", "The AP restriction at the border of neighboring parasegments at the 1-row stage has been revealed in Parhyale and other embryos by embryological descriptions , lineage tracing and expression studies for the engrailed ( en ) gene that marks the posterior compartment ( Browne et al . , 2005; Dohle et al . , 2004; Dohle and Scholtz , 1988; Hejnol and Scholtz , 2004; Patel et al . , 1989; Scholtz , 1990; Scholtz et al . , 1994 ) .", "In agreement with this AP restriction , during limb specification and outgrowth there was a straight clonal boundary running between the anterior cells derived from the E4b , c and d rows and the posterior cells derived from the E5a and b rows ( Figure 4F–J’ and Figure 4—figure supplement 1K–O’ ) .", "After the well-known AP boundary , we sought to identify any subdivision along the DV axis .", "Compartments were classically discovered by clonal analysis using mitotic recombination .", "In our reconstructions , we could generate clones digitally from arbitrary cells at different stages of development .", "We reasoned that we could reveal the timing and position of any heritable DV restriction by piecing together correctly all founder cells of dorsal or ventral identity in a way that the two polyclones ( i . e . compartments ) would stay separate and form a lasting straight interface between them .", "This analysis suggested that there is indeed a DV separation that took place at the 4-row-parasegment .", "The DV boundary ran between the E4b and c rows anteriorly , between the E5a and b rows posteriorly , and between cells E4c4-c5 , E4d3-d4 and E5a4-a5 medially ( Figure 4F ) .", "Throughout limb#1 development , the dorsal and ventral cells formed a sharp boundary between themselves extending along the PD axis ( Figure 4F–J’ ) .", "To investigate the stereotypy of the AP and DV separation across Parhyale limbs , we analyzed a second , independently imaged and reconstructed T2 limb ( referred to as limb#2 ) from a different embryo ( Figure 4—figure supplement 2A–D ) .", "Four identical compartments ( anterior-dorsal , anterior-ventral , posterior-dorsal and posterior-ventral ) could be derived in this independent reconstruction with straight boundaries and no cell mixing between neighboring compartments ( Figure 4—figure supplement 2E–H’ ) .", "These results suggested that in silico studies of comprehensive and accurate lineages can provide novel insights into clonal subdivisions in species where sophisticated genetic methodologies for lineage tracing are not implemented yet .", "The first T2 limb ( limb#1 ) was lineaged with the new MaMuT software from a multi-view acquisition of an embryo imaged at 26˚C ( Figure 4 ) , while the second T2 limb ( limb#2 ) was lineaged with the previously developed SIMI°BioCell software ( Hejnol and Scholtz , 2004; Schnabel et al . , 1997 ) from a single-view of another embryo imaged at 29–30˚C ( Figure 4—figure supplement 2 ) .", "Analysis of the birth sequence of the founder cells in the two reconstructed T2 limbs largely confirmed that the second mitotic wave creating the 4-row-parasegment propagated from anterior to posterior rows and from medial to lateral columns ( Figure 5A–C and Figure 5—source data 1 ) .", "For example , division of the ab cells in parasegment E4 had already progressed to column 5 or even more laterally before ab3 divided in the next posterior parasegment E5 .", "However , we also found two notable deviations from this general pattern .", "First , as previously noted ( Scholtz , 1990 ) , division of the posterior cd cells within the 2-row-parasegment was slightly more advanced temporally compared to their anterior ab sister cells ( Figure 5A–C ) .", "Second , the temporal sequence of divisions , which gave rise to a stereotyped number and spatial arrangement of the 34 founder cells in each primordium , exhibited a certain degree of variability between the two analyzed limbs; for example , division of the E4cd8/9 cells preceded division of E4cd7 in limb#1 but not in lim#2 , whereas division of the E4cd6/7 cells preceded division of E4cd5 in limb#2 but not in limb#1 ( Figure 5A–B ) .", "We then examined the increase in cell number over time in the two limbs during the analyzed stages of limb outgrowth .", "The embryo with limb#2 imaged at higher temperature exhibited a faster growth rate compared to the embryo with limb#1 ( Figure 5D ) .", "Yet , it was possible to register the two growth curves during the period when all cells were tracked faithfully by applying a linear temporal rescaling factor of 1 . 6 , effectively correcting for the temperature-induced change in growth ( Figure 5D ) .", "After this temporal alignment , the increase in cell number was very similar between developing limbs , up to 35 hr after the first tracked division .", "The matching curves demonstrated that cell numbers were highly reproducible between developing limbs after aligning them temporally and allowed their pairwise quantitative comparison ( see next section ) .", "Beyond this time-point , it was not possible to track all cells in the outgrowing limb#2 due to their increasing higher density , the deterioration of the fluorescence signal along the detection axis and the lack of the multi-view information for lineaging this limb .", "Limb bud formation entailed the remodeling of the flat epithelium into a 3D bulge ( Figure 4A–C and Figure 4—figure supplement 2A–C ) .", "At the cellular level , the first step in this transformation was the rise of few cells at the intersection of the four compartments above the level of the germband at around 96 hr AEL ( Figure 4G , G’ and Figure 4—figure supplement 2F , F’ ) .", "Within the following 3 hr , this initial phase was followed by a large-scale elevation of most cells in the dorsal compartment .", "As this elevation continued , the medial ventral cells folded and became apposed to the medial dorsal cells forming the convex surface of the limb bud ( Figure 4H , H’ and Figure 4—figure supplement 2G , G’ ) .", "The intersection of the AP and DV boundaries was at the tip of the limb bud and persisted in this position throughout subsequent elongation ( Figure 4H–J’ and Figure 4—figure supplement 2G–H’ ) .", "From 103 hr AEL onwards , a second element appeared bulging distally off the original bud in limb#1 ( Figure 4I , I’ ) .", "The limb elongated as a convoluted rather than straight cylinder and acquired progressively an S-shape ( Figure 4J , J’ ) .", "Two cell behaviors implicated in organ morphogenesis were readily quantifiable in our nuclear trackings: the pattern of cell proliferation and the orientation of cell divisions .", "These cell activities have been traditionally inferred from the distribution , size and shape of somatic clones induced in developing tissues ( Baena-López et al . , 2005; González-Gaitán et al . , 1994; Mao et al . , 2013; Resino et al . , 2002; Weigmann and Cohen , 1999; Worley et al . , 2013 ) .", "This approach could be also adapted here by generating in silico clones ( Figure 6—figure supplement 1 ) .", "Yet , the MaMuT reconstructions enabled us to enrich the lineage information with rigorous quantitative analyses of the rate and orientation of mitotic divisions for all tracked nuclei .", "First , we calculated the cell cycle length ( CCL ) , i . e . the branch length for every constituent cell in the lineage of limb#1 ( Figure 6A–D and Figure 6—figure supplement 2 ) .", "This quantification revealed a striking difference in CCL between central cells that were dividing faster than their neighbors in the periphery of the primordium ( average CCL 7 . 1–8 . 5 hr versus 8 . 5–16 . 4 hr , respectively ) .", "This difference started from early primordium specification at the 4-row-parasegment ( Figure 6E ) , but became most pronounced during the global elevation of the limb bud ( Figure 4F ) , suggesting a causal association between spatially controlled cell proliferation and initiation of limb outgrowth ( see Discussion ) .", "During subsequent elongation stages , a high concentration of fast dividing cells was located at the intersection of the four presumptive compartments , resembling a growth zone at the distal tip of the growing appendage ( Figure 6G , H ) .", "Another row of faster dividing cells was localized in the anterior cells abutting the AP boundary ( Figure 6H , H’ ) .", "To explore the levels of variability in the pattern of cell divisions , we performed a hierarchical clustering of the founder cells within each of the two analyzed T2 limbs based on a lineage distance metric computed from the division patterns exhibited by the 34 cells ( see Material and methods ) .", "This analysis revealed very similar profiles in the two limbs , as well as their average , with their cells forming three clusters ( Figure 7A–C and Figure 7—source data 1 ) : the first cluster contained cells E4c3-c7 and E4d3-d7 displaying the fastest proliferation rates and giving rise to most of the limb structures; the second cluster contained the majority of E4 and E5 b cells corresponding to the slowest dividing cells of ventral fate and contributing to the proximal limb and intersegmental territories; the third cluster contained the remaining cells exhibiting mixed division patterns , including most of the posterior E5a cells and the more lateral E4c and d cells .", "This clustering suggested that a common set of patterning mechanisms operates across T2 limbs specifying the distinct properties of these groups of cells .", "At the same time , the linkages and distances of cells within each cluster varied from identical ( e . g . E4b3/b4 ) to very different ( e . g . E4d4/d5 ) between limbs , revealing a certain degree of flexibility in the behaviors exhibited by homologous cells in a limb-specific manner .", "Extra support for this interpretation came from plotting the distribution of the lineage distances between founder cells across the two limbs .", "Pairwise comparisons revealed low distances between the 34 homologous cells in limb#1 and limb#2 with a median difference of 19 . 3% ( Figure 7D ) .", "Thus , homologous cells exhibited similar but not identical division patterns across limbs .", "The distribution of these distances between homologous cells was significantly shifted towards lower values relative to pairwise comparisons between non-homologous cells across the two limbs ( Figure 7D and Figure 7—source data 1 ) .", "Next , we looked for any biases in the orientation of mitotic divisions that could be associated with limb morphogenesis ( Figure 8A–E ) .", "All early divisions in the limb#1 primordium were parallel to the AP axis confirming the strict longitudinal orientation of row divisions ( Figure 8F ) .", "Cell divisions acquired a more heterogeneous pattern after the 4-row-parasegment ( Figure 8G ) .", "An increasing number of mitotic spindles aligned progressively along the PD axis during limb bud formation ( Figure 8H ) and elongation ( Figure 8I , J ) .", "Collectively , the information extracted from our spatiotemporally resolved lineage trees strongly suggested that Parhyale limb outgrowth is driven by at least two patterned cell behaviors: the differential rates of cell proliferation and the orderly arrangement of mitotic spindles .", "To understand the cellular basis of the establishment of positional values along the PD axis , we followed the fate of cells during T2 limb#1 segmentation .", "Segmentation involved the progressive subdivision of the elongating PD axis into an increasing number of elements ( Figure 9A–L ) .", "We tracked neighboring cells in rows E4c ( cells E4c5-c8 , not shown ) and E5a ( cells E5a5-a8 , shown in Figure 9A–F ) from 84 to 151 hr AEL .", "These cells were ideal for reconstructing the PD axis at single-cell resolution because they mostly divided proximodistally forming elongated thin clones ( Figure 6—figure supplement 1 ) .", "This analysis showed that the cells that gave rise to the proximal , medial and distal limb segments occupied distinct mediolateral positions in the germband grid at the 4-row-parasegment ( Figure 9M ) and distinct PD positions in the early limb bud ( Figure 9N ) .", "When the limb bud split into two elements , the proximal element gave rise to the proximal limb segments coxa , basis and ischium , while the distal element gave rise to the distal limb segments merus , carpus , propodus , and dactylus ( Figure 9O–R ) .", "The cells forming the distal segments originated as a disc of cells centered at the intersection of the four compartments with contributions from the E4c4-c6 , E4d3-d6 and E5a3-a6 sublineages ( Figure 9—figure supplement 1 ) .", "During the subsequent elongation stages , distal cells kept separate from more proximal cells at the prospective ischium/merus joint , suggesting that limb segments may pose secondary lineage restrictions along the PD axis ( Figure 9—figure supplement", "1 ) ( Milán and Cohen , 2000 ) .", "This first ischium/merus subdivision ( Figure 9O ) was followed by the basis/ischium subdivision ( Figure 9P ) , the propodus/dactylus , carpus/propodus and coxa/basis subdivisions ( Figure 9Q ) , and the carpus/merus subdivision ( Figure 9R ) .", "To test our cellular models and make a first link between expression of limb patterning genes and morphogenetic cell behaviors , we analyzed by in situ hybridization the expression of the Parhyale decapentaplegic ( Ph-dpp ) gene that encodes a Bone Morphogenetic Protein 2/4 signaling molecule ( Figure 10—figure supplement 1G ) .", "In Drosophila , Dpp signaling controls dorsal cell fate in the leg and growth via cell proliferation in the wing ( Barrio and Milán , 2017; Bosch et al . , 2017; Brook and Cohen , 1996; Matsuda and Affolter , 2017; Rogulja and Irvine , 2005; Svendsen et al . , 2015 ) .", "Therefore , probing Ph-dpp expression in Parhyale limb buds could provide a direct test for our cell-based predictions regarding the DV lineage restriction and the differential cell proliferation rates in the limb primordium .", "Analysis of embryos 84–96 hr AEL revealed alternating regions of high/moderate and low/no Ph-dpp expression in the anterior thoracic region ( Figure 10A , A’and Figure 10—figure supplement 1A , A’ ) .", "We used MaMuT to annotate both the gene expression and identity of cells in stained T2 limbs at cellular resolution .", "Acknowledging that the graded Ph-dpp expression obscured the precise limits of its expression , this analysis suggested that the region of high/moderate Ph-dpp expression was localized to rows E4c , E4d and E5a that mostly contribute to the presumptive dorsal compartment , while low/no Ph-dpp expression could be detected in the prospective ventral rows E4b anteriorly and E5b posteriorly ( Figure 10A’’ ) .", "Furthermore , Ph-dpp expression faded in the medial ( prospective ventral ) columns and the border between high/moderate and low/no expressing cells was located in descendent cells from column 4 as also predicted by our in silico cellular analysis ( Figure 10A–A’’ ) .", "In embryos 96–108 hr AEL , the domain of strong Ph-dpp expression was more localized in the row of anterior-dorsal cells abutting the AP boundary ( Figure 10D–D’’ and Figure 10—figure supplement 1D , D’ ) .", "To get an insight into the downstream effects of Dpp signaling in the Parhyale limb , we also analyzed expression of the Tbx6/Dorsocross ( Doc ) gene ( Figure 10—figure supplement 1H ) that responds to high levels of Dpp signaling in the dorsal region of the Drosophila embryo and leg disc ( Svendsen et al . , 2015 ) .", "Expression of the single Doc gene identified in Parhyale ( Ph-Doc ) was detected in a subset of the Ph-dpp-expressing cells at 84–96 hr AEL ( Figure 10B–B’’ and Figure 10—figure supplement 1B , B’ ) , while 12 hr later the two genes exhibited essentially identical strong expression in the cells abutting the AP boundary ( Figure 10E–E’’ and Figure 10—figure supplement 1E , E’ ) .", "In both stages analyzed , cells expressing Ph-dpp and Ph-Doc also exhibited the highest rates of cell proliferation ( compare Figure 10D’’ , E’’ with Figure 6G , H ) providing strong correlative evidence for a morphogen-dependent control of Parhyale limb growth .", "As a last validation of our cellular models , we probed the expression of the Parhyale H15 ( Ph-H15 ) gene during early limb formation ( Figure 10—figure supplement 1H ) .", "In Drosophila and other arthropods studied , the Tbx20 genes H15/midline act antagonistically to dorsal selector genes and control ventral cell fate in developing legs ( Janssen et al . , 2008; Svendsen et al . , 2015 ) .", "Our model for the timing and position of limb DV compartmentalization predicted that Ph-H15 would come up in the b cells from the 4-row-parasegment stage onwards .", "In agreement with these predictions , in situ hybridization analyses detected the Ph-H15 transcripts specifically in the b row cells .", "Furthermore , expression initiated shortly after the ab cells divided longitudinally into the a and b daughter cells in each forming 4-row-parasegment ( Figure 10C–C’’ and Figure 10—figure supplement 1C , C’ ) .", "Although Ph-H15 was first activated in all b cells , during later divisions Ph-H15 expression faded in the more medial columns ( Figure 10C–C’’ ) and persisted only in the ventral limb cells close to the body wall ( Figure 10F–F’’ and Figure 10—figure supplement 1F , F’ ) .", "All these results demonstrated how the reconstruction of cell lineages and behaviors can provide solid predictions and powerful contexts to study the expression and function of associated genes ." ], [ "The LSFM technology is empowering biologists to image developmental processes with unprecedented spatiotemporal resolution .", "Together with MaMuT-based lineaging and tracking , various experimental designs can be addressed ranging from analyzing a small subset of objects in the imaged volume to systems-wide analyses of all constituent parts .", "The lineage reconstructions presented in this article were generated manually and required 2 to 3 months for each limb .", "More generally , manual lineaging efforts can take anything between few days to several months depending on the number of tracked cells , the complexity of the imaged tissue of interest , the duration of the tracked process , the quality of the image dataset , and the desired accuracy and completeness of the reconstructed lineages .", "The main advantage of manual tracking by experts is that the extracted lineage is more likely error-free compared to results of automated trackers that must be manually proofread before any meaningful analysis can be attempted .", "In addition to allowing reliable biological insights , manually generated lineages serve as important ‘ground truth’ datasets for the application of machine learning based automated tracking solutions ( Ulman et al . , 2017 ) .", "Acknowledging that fully manual tracking is a laborious and repetitive task that may be impractical for large-scale comparative lineaging approaches , the latest MaMuT architecture offers , in addition to manual tracking , two functionalities for automated tracking:", "( i ) a semi-automated option where individual nuclei can be selected by the user and tracked computationally over time , and", "( ii ) the option to import into MaMuT fully automated annotations generated by the Tracking with Gaussian Mixture Models ( TGMM ) software ( Amat et al . , 2014 ) , which is currently one of the most accurate and computationally efficient methods for segmentation and tracking of fluorescently labeled nuclei .", "After the import , MaMuT can be used to manually proofread and correct the results of the automated tracking pipeline .", "However , we also note that the graph data structure in MaMuT can handle efficiently up to about a hundred thousand annotations .", "This number is well within the realm of manually generated annotations , but is normally exceeded by large-scale fully automated lineaging engines like TGMM .", "As a trade-off until this constraint is addressed in the future , we also provide users the option to crop the imported TGMM annotations in space and/or in time to make them compatible with MaMuT .", "The crustacean Parhyale is already an attractive new model for developmental genetic and functional genomic studies ( Kao et al . , 2016; Liubicich et al . , 2009; Martin et al . , 2016; Pavlopoulos et al . , 2009; Stamataki and Pavlopoulos , 2016 ) .", "By extending here the experimental toolkit with multi-view LSFM and cellular reconstructions with MaMuT , it is feasible to study gene expression and function in the context of single-cell resolution fate maps .", "Especially when it comes to appendage development , the Parhyale body plan provides exceptional material to probe the molecular and cellular basis of tissue patterning , growth and differentiation during normal embryogenesis and post-embryonic regeneration ( Alwes et al . , 2016; Konstantinides and Averof , 2014 ) .", "The tempo and mode of development has also important ramifications for Parhyale imaging and tracking .", "The relatively slow tempo of development enables us to image embryos at a very high spatial resolution through the acquisition of multiple and highly overlapping views without compromising the temporal resolution .", "Parhyale can match the spatiotemporal resolution of Drosophila or zebrafish LSFM datasets , even when access to highest-speed instruments is not available .", "Due to the optical clarity of the embryo and positioning of the appendages on the surface of the developing embryo , all constituent cells can be followed for quantitative analyses .", "Finally , the stereotyped and ordered organization of the Parhyale ectoderm will allow to identify homologous cells and compare lineages , cell behaviors and associated genes between serially homologous structures in the same embryo , across embryos and even across malacostracan crustaceans ( Browne et al . , 2005; Dohle et al . , 2004; Dohle and Scholtz , 1988; Gerberding et al . , 2002; Hejnol and Scholtz , 2004; Scholtz , 1990; Scholtz et al . , 1994; Wolff and Gerberding , 2015; Wolff and Scholtz , 2002; 2008 ) .", "The combination of multi-view light-sheet imaging and tracking has enabled a detailed analysis of the dynamics of all constituent cells in an outgrowing and elongating animal limb .", "So far , these descriptions have been only partly available for Drosophila limbs that are derived and not representative for many insects , much less arthropods in general , in two very important respects .", "First , limb specification , patterning , growth and differentiation take place at distinct developmental stages during embryonic , larval and pupal development .", "On the contrary , all these processes come about during embryogenesis in most other arthropods , including Parhyale .", "In addition to these heterochronic shifts , limb patterning mechanisms in Drosophila operate in the flat imaginal disc epithelia , rather than the 3D epithelial outgrowths observed in Parhyale that are typical for most other arthropod limbs .", "Classical lineaging experiments revealed that tissue compartmentalizations in the Drosophila wing and leg primordia take place along the AP axis during early embryogenesis and along the DV axis during larval development ( Garcia-Bellido et al . , 1973; Steiner , 1976 ) .", "Our understanding of the AP and DV organization in other arthropod limbs has relied so far entirely on gene expression studies .", "Expression of segment polarity genes , like en and wingless ( wg ) , has demonstrated that the AP separation is conserved across arthropods and takes place during segmentation ( Angelini and Kaufman , 2005; Damen , 2007 ) .", "In Parhyale , the AP compartment boundary is established at the 1-row stage at the interface of neighboring parasegments ( Browne et al . , 2005; Dohle and Scholtz , 1988; Hejnol and Scholtz , 2004; Scholtz et al . , 1994 ) .", "With the exception of descriptive gene expression studies ( Angelini and Kaufman , 2005; Damen , 2007; Janssen et al . , 2008 ) , the mechanism , timing and position of the DV separation in arthropod limbs has remained unexplored at the cellular level due to the lack of lineage tracing methodologies .", "Even in Drosophila , it is not entirely clear yet whether DV separation in the leg disc relies on heritable or non-heritable subdivisions or a combination of both mechanisms ( Brook and Cohen , 1996; Steiner , 1976; Svendsen et al . , 2015 ) .", "By analyzing the dynamics of digital clones in reconstructed T2 limbs , we have been able to explore the cellular basis of limb patterning in Parhyale .", "This approach first confirmed the position and timing of the known AP compartment boundary , and then revealed a putative heritable subdivision along the DV axis from the 4-row-parasegment stage onwards .", "Interestingly , expression of the Distal-less gene , which is an early marker of limb specification , is first detected at the 4-row-parasegment in the d3/d4 cells located at the intersection of the AP and DV boundaries ( Browne et al . , 2005; Hejnol and Scholtz , 2004 ) .", "This intersection also marks the tip of the forming limb throughout epithelial remodeling and outgrowth .", "Thus , the Parhyale limb appears to perfectly conform to Meihardt’s boundary model ( Meinhardt , 1983 ) .", "This model postulates that a secondary developmental field , i . e . the PD axis of a limb that is specified during embryogenesis de novo relative to the main AP and DV body axes , initiates and is patterned around the intersection of the AP and DV compartment boundaries .", "The inference of the four constituent compartments provided a powerful framework to interpret the cell behaviors during limb development both in a qualitative and quantitative manner .", "This analysis strongly suggested that a combination of cellular mechanisms is at work to remodel the embryonic epithelium during limb outgrowth .", "First , there was a significant difference in cell proliferation rates between the center ( faster dividing ) and the periphery ( slower dividing ) of the limb primordium from early specification until limb bud formation .", "Such a growth-based morphogenesis model has been the dominant hypothesis for almost 50 years to explain the outgrowth of the vertebrate limb ( Ede and Law , 1969; Hornbruch and Wolpert , 1970; Morishita and Iwasa , 2008; Searls and Janners , 1971 ) – oriented cell motion and division were also recently involved ( Boehm et al . , 2010; Wyngaarden et al . , 2010 ) - but has never been implicated as the driving mechanism behind arthropod limb evagination .", "Limb bud formation can be reduced by inhibiting cell proliferation pharmacologically , as has been demonstrated in larvae of another crustacean with direct developing limbs , the brine shrimp Artemia ( Freeman et al . , 1992 ) .", "Second , limb elongation was tightly associated - and presumably effected - by two patterned cell behaviors:", "i ) increased cell proliferation at the tip of the limb resembling a putative growth zone which generates many of the new cells necessary for limb outgrowth , and", "ii ) strong bias in the orientation of mitotic divisions parallel to the PD axis of growth .", "Third , the different PD domains of the Parhyale limb could be traced back to distinct mediolateral positions in the early germband stage .", "During limb bud formation and elongation , there was a transition and refinement of these positional values along the PD axis .", "Fourth , besides the early AP and DV lineage restrictions , we observed a secondary PD separation between neighboring segments during limb segmentation .", "Overall , our approach demonstrates that the comprehensive fine-scale reconstruction of a developmental process can shed light into functionally interdependent patterning mechanisms operating across multiple scales .", "In the Drosophila leg disc , the Dpp and Wg ligands are induced at the AP boundary in the dorsal and ventral cells , respectively .", "Dpp and Wg create a concentration gradient with the highest overlap in their expression in the center of the disc and cooperate in the establishment of concentric domains of gene expression of a set of limb gap genes that pattern the PD axis ( Estella et al . , 2012 ) .", "Dpp and Wg signaling also act antagonistically to control dorsal and ventral cell fate through regulation of the downstream selector T-box genes optomotor blind/Doc dorsally and H15/midline ventrally ( Svendsen et al . , 2015 ) .", "The PD expression of the limb gap genes is conserved in arthropods , including Parhyale ( Angelini and Kaufman , 2005; Browne et al . , 2005; Prpic and Telford , 2008 ) .", "Our analysis of dpp , Doc and H15 expression in a crustacean species also suggests conserved roles for these genes in dorsal and ventral cell fate specification , and provides extra independent support for a compartment-based mechanism to pattern the DV axis of arthropod limbs .", "Wg expression is currently not known in Parhyale .", "If it is expressed in a complementary pattern to Ph-dpp in the prospective ventral territory , it could point to a similar logic for patterning the limb PD axis like in Drosophila .", "In fact , our reconstructions have suggested that the distal DV margin ( that in this scenario would experience the highest levels of Dpp and Wg signaling ) is located between descendent cells from columns 4 and 5 .", "These are indeed the cells that contribute to the distal-most limb segments .", "Although the function of the Dpp morphogen gradient in patterning the Drosophila limbs is well understood , its role in promoting growth is still controversial ( Akiyama and Gibson , 2015; Barrio and Milán , 2017; Bosch et al . , 2017; Harmansa et al . , 2015; Matsuda and Affolter , 2017; Restrepo et al . , 2014; Rogulja and Irvine , 2005 ) .", "The anterior-dorsal cells expressing Ph-dpp and Ph-Doc were among the fastest dividing cells in the center of the limb primordium .", "Later , strong expression of Ph-dpp and Ph-Doc resolved into a row of cells abutting the AP compartment boundary .", "Again , these cells displayed some of the highest proliferation rates quantified during limb outgrowth , suggesting a Dpp-dependent control of Parhyale limb growth .", "We anticipate that the LSFM imaging and tracking approaches described here , together with the recent application of CRSIPR/Cas-based methodologies for genome editing ( Kao et al . , 2016 ) will provide excellent tools to further explore how morphogens like Dpp regulate growth and form at cellular resolution ." ], [ "Parhyale hawaiensis ( Dana , 1853 ) rearing , embryo collection , microinjection and generation of transgenic lines were carried out as previously described ( Kontarakis and Pavlopoulos , 2014 ) .", "To fluorescently label the chromatin in transgenic Parhyale , we fused the coding sequences of the Drosophila histone H2B and the mRFPruby monomeric Red Fluorescent Protein and placed them under control of a strong Parhyale heat-inducible promoter ( Pavlopoulos et al . , 2009 ) .", "H2B was amplified from genomic DNA with primers Dmel_H2B_F_NcoI ( 5’-TTAACCATGGCTCCGAAAACTAGTGGAAAG-3’ ) and Dmel_H2B_R_XhoI ( 5’-ACTTCTCGAGTTTAGAGCTGGTGTACTTGG-3’ ) , and mRFPruby was amplified from plasmid pH2B-mRFPruby ( Fischer et al . , 2006 ) with primers mRFPruby_F_XhoI ( 5’-ACAACTCGAGATGGGCAAGCTTACC-3’ ) and mRFPruby_R_PspMOI ( 5’-TATTGGGCCCTTAGGATCCAGCGCCTGTGC-3’ ) .", "The NcoI/XhoI-digested H2B and XhoI/PspOMI-digested mRFPruby fragments were cloned in a triple-fragment ligation into NcoI/NotI-digested vector pSL-PhHS>DsRed , placing H2B-mRFPruby under control of the PhHS promoter ( Pavlopoulos et al . , 2009 ) .", "The PhHS>H2B-mRFPruby-SV40polyA cassette was then excised as an AscI fragment and cloned into the AscI-digested pMinos{3xP3>EGFP} vector ( Pavlopoulos and Averof , 2005; Pavlopoulos et al . , 2004 ) , generating plasmid pMi{3xP3>EGFP; PhHS>H2B-mRFPruby} .", "Three independent transgenic lines were established with this construct for heat-inducible expression of H2B-mRFPruby .", "The most strongly expressing line was selected for all applications .", "In this line , nuclear H2B-mRFPruby fluorescence plateaued about 12 hr after heat-shock and high levels of fluorescence persisted for at least 24 hr post heat-shock labeling chromatin in all cells throughout the cell cycle .", "Standard procedures for multi-view LSFM recordings of Parhyale embryogenesis were established after imaging several dozen embryos individually in pilot experiments , first on a Zeiss prototype and , later on , on the commercial Zeiss Lightsheet Z . 1 microscope .", "Several parameters described below were optimized to ensure that the two embryos used for lineage reconstruction", "( i ) survived the recording process and hatched into juveniles without any morphological abnormalities , and", "( ii ) were imaged with the appropriate spatiotemporal resolution and signal-to-noise ratio for accurate and comprehensive cell tracking in developing appendages .", "To prepare embryos for LSFM imaging , 2 . 5 day old transgenic embryos ( early germband stage; S11 according to ( Browne et al . , 2005 ) ) were heat-shocked for 1 hr at 37˚C .", "About 12 hr later ( stage S13 ) , they were mounted individually in a cylinder of 1% low melting agarose ( SeaPlaque , Lonza ) inside a glass capillary ( #701902 , Brand GmbH ) with their AP axis aligned parallel to the capillary .", "A 1:4000 dilution of red fluorescent beads ( #F-Y050 microspheres , Estapor Merck ) were included in the agarose as fiducial markers for multi-view reconstruction .", "During imaging , the embedded embryo was extruded from the capillary into the chamber filled with artificial seawater supplemented with antibiotics and antimycotics ( FASWA; ( Kontarakis and Pavlopoulos , 2014 ) ) .", "The FASWA in the chamber was replaced every 12 hr after each heat shock ( see below ) .", "The Zeiss Lightsheet Z . 1 microscope was equipped with a 20x/1 . 0 Plan Apochromat immersion detection objective and two 10x/0 . 2 air illumination objectives producing two light-sheets 5 . 1 µm thick at the waist and 10 . 2 µm thick at the edges of a 488 µm x 488 µm field of view .", "We started imaging Parhyale embryogenesis from three angles/views ( the ventral side and the two ventral-lateral sides 45˚ apart from ventral view ) during 3 to 4 . 5 days AEL to avoid photo-damaging the dorsal thin extra-embryonic tissue , and continued imaging from five views ( adding the two lateral sides 90˚ apart from ventral view ) during 4 . 5 to 8 days AEL .", "A multi-view acquisition was made every 7 . 5 min at 26˚C .", "The H2B-mRFPruby fluorescence levels were replenished regularly every 12 hr by raising the temperature in the chamber from 26˚C to 37˚C and heat-shocking the embryo for 1 hr .", "Each view ( z-stack ) was composed of 250 16-bit frames with voxel size 0 . 254 µm x 0 . 254 µm x 1 µm .", "Each 1920x1920 pixel frame was acquired using two pivoting light-sheets to achieve a more homogeneous illumination and reduced image distortions caused by light scattering and absorption across the field of view .", "Each optical slice was acquired with a 561 nm laser and exposure time of 50 msec .", "With these conditions , Parhyale embryos , like the one bearing the T2 limb#1 analyzed in detail with MaMuT , were imaged routinely for a minimum of 4 days or even up to hatching .", "After hatching , the morphology of imaged specimen was compared between the left and the right side , as well as to its non-imaged siblings , to confirm that no obvious developmental or morphological abnormalities were detected .", "The embryo bearing the T2 limb#2 was imaged on a Zeiss LSFM prototype ( Preibisch et al . , 2010 ) that offered single-sided illumination and single-sided detection with a 40x/0 . 8 immersion objective .", "One side of this embryo was imaged from 3 views 40˚ apart ( ventral , ventral-left and left ) every 7 . 5 min over a period of 66 hr .", "Each view was composed of 150 frames ( 1388 × 1080 pixels ) with voxel size 0 . 366 µm x 0 . 366 µm x 2 µm .", "The embryo was imaged at 29–30˚C and was heat-shocked for 1 hr twice a day by perfusing warm FASWA at 37 ˚C .", "Cell tracking was carried out with the SIMI°BioCell software ( Hejnol and Scholtz , 2004; Schnabel et al . , 1997 ) on a single view , the ventral-left view , of this dataset .", "Lineage reconstruction of limb#2 with SIMI°BioCell was complete up to about 22 hr of imaging time ( 35 hr when scaled to the growth rate of limb#1 ) .", "After this time-point , an increasing number of cells in limb#2 , in particular the descendant cells from the medial columns , became intractable .", "Parhyale LSFM acquisitions typically resulted in 192 time-points/240K images/1 . 7 TB of raw data per day .", "Image processing was carried out on a MS Windows 7 Professional 64-bit workstation with 2 Intel Xeon E5-2687W processors , 256 GB RAM ( 16 X DIMMs 16384 MB 1600 MHz ECC DDR3 ) , 4 . 8 TB hard disk space ( 2 × 480 GB and 6 × 960 GB Crucial M500 SATA 6 Gb/s SSD ) , 2 NVIDIA Quadro K4000 graphics cards ( 3 GB GDDR5 ) .", "The workstation was connected through a 10 GB network interface to a MS Windows 2008 Server with 2 Intel Xeon E5-2680 processors , 196 GB RAM ( 24 X DIMM 8192 MB 1600 MHz ECC DDR3 ) and 144 TB hard disk space ( 36 X Seagate Constellation ES . 3 4000 GB 7200 RPM 128 MB Cache SAS 6 . 0 Gb/s ) .", "All major LSFM image data processing steps were done with software modules available through the Multiview Reconstruction Fiji plugin ( http://imagej . net/Multiview-Reconstruction ) according to the following steps: MaMuT was developed as a tool for cell lineaging in multi-view LSFM image volumes by enabling to track objects synergistically from all available views .", "This functionality has a number of advantages .", "Raw views do not have to be fused into a single volume , which is computationally by far the most demanding step ( Preibisch et al . , 2014 ) .", "The users also preserve the original redundancy of the data , which in many cases like in Parhyale allows capturing cells from two or more neighboring views that can be interpreted independently for a more accurate analysis .", "Finally , MaMuT allows users to analyze sub-optimal datasets that cannot be fused properly or may create fusion artifacts .", "Of course , combining the raw views with a high-quality fused volume is the best available option , especially when handling complex datasets with high cell densities .", "While offering multi-view tracking , MaMuT delivers also other important functionalities .", "First , it is a turnkey software solution with a convenient interface for interactive exploration , annotation and curation of image data .", "Any image acquired by any microscopy modality that can be opened in Fiji can be also imported into MaMuT .", "Second , MaMuT offers a highly responsive and interactive navigation through multi-terabyte datasets .", "Individual z-stacks representing different views , channels and time-points of a multi-dimensional dataset can be displayed independently or in combinations in multiple synced Viewer windows .", "Third , objects of interest like cells and nuclei ( spots ) can be selected synergistically from all available Viewers and followed over time to reconstruct their trajectories ( tracks ) and lineage information .", "Fourth , the created spots and tracks can be visualized and edited interactively in the Viewers and the TrackScheme lineage browser , and animated in the 3D Viewer .", "For visual interpretation of the data , annotations can be colored based on the primary lineage information or derived numerical parameters .", "Fifth , lineages can be reconstructed in a manual , semi-automated or fully automated manner followed by manual curation if necessary .", "Sixth , all spot and track information can be exported from MaMuT to other interfaces for more specialized analyses .", "Seventh , decentralized annotation by multiple users has been made possible by also developing a web service for remote access to large image volumes stored online .", "Following on the tradition of the Fiji community for open-source distribution of biological image analysis software , MaMuT is provided freely and openly to the community , it is extensively documented and can be customized by other users .", "In practical terms , for lineaging purposes , the Parhyale multi-view LSFM raw views were registered spatiotemporally and the image data together with the registration parameters were converted into the custom HDF5/XML file formats utilized by the BigDataViewer and MaMuT Fiji plugins .", "The MaMuT reconstruction of the Parhyale T2 limb described in this article required about 10 weeks of dedicated manual cell tracking by an experienced annotator .", "The raw image data were displayed in Viewer windows and each z-stack was visualized in any desired color and brightness , scale ( zoom ) , translation ( position ) and rotation ( orientation ) .", "All Viewer windows were synced based on the calculated registration parameters and shared a common physical coordinate system; upon selecting an object of interest ( spot ) in one Viewer , the same spot was identified and displayed in all other windows , and its x , y , z position was mapped onto this common physical space .", "To guarantee the accuracy of our lineage reconstructions , the center of each tracked nucleus was verified in at least two neighboring views and by slicing the data orthogonally in separate Viewer windows .", "The nuclei contributing to the T2 limb of interest were identified in the first time-point and tracked manually every five time-points except during mitosis , in which case we also tracked one time-point before and one after segregation of the daughter chromosomes during anaphase/telophase .", "The reconstructed trajectories and lineages were also displayed in two additional synced windows , the TrackScheme and the 3D Viewer .", "The TrackScheme lineage browser and editor displayed the reconstructed cell lineage tree with tracked nuclei represented as nodes connected by edges over time and cell divisions depicted as split branches in the tree .", "The 3D Viewer window displayed interactive animations of the spots depicted as spheres and their tracks over time .", "The spots and the tracks in the Viewer , TrackScheme and 3D Viewer windows could be color-coded by lineage , position and other numerical features to assist visual analysis and interpretation of the data .", "In addition , all these windows were synced to simultaneously highlight active spots of interest at the selected time-point , greatly facilitating the cell lineaging process .", "For comparative purposes , each reconstructed lineage tree was defined as a set of division times .", "For example , let’s consider a lineage tree L that starts with cell d .", "Cell d divides at time t0 giving rise to the two daughter cells d1 and d2 .", "Then d1 divides at time t1 giving rise to daughter cells d11 and d12 .", "Finally , d12 divides at time t12 giving the daughter cells d121 and d122 .", "In this scenario , we define L as L={t0 , t1 , t12} .", "Let’s now consider two lineage trees Lx andLy , where x and y refer to the founder cells whose lineage trees are under comparison ( e . g . x corresponds to E4c5 cell from limb#1 and y to E5b6 cell from limb#2 ) .", "In order to be comparable , these two lineage trees need to be registered temporally .", "In our study , we performed a linear rescaling by an empirically determined factor of 1 . 6 to match the increase in cell number between limb#1 and limb#2 that were imaged at different temperatures and exhibited different growth rates .", "We then defined Δ ( Lx , Ly ) as the distance between the two registered lineage trees .", "This distance takes into consideration two metrics , the difference in the timing of divisions and the difference in the number of divisions between the two lineages , and is computed in the following way:ΔLx , Ly= δtLx , Ly/nt+ δn ( Lx , Ly ) /nn2 In this equation , δtLx , Ly is the difference in the timing of divisions and δn ( Lx , Ly ) is the difference in the number of division between the two lineages .", "nt and nn are used to normalize the two metrics so that their values are comparable .", "They are defined as the maximum values observed for δtLx , Ly and δn ( Lx , Ly ) in a given run of pairwise comparisons , i . e . they are the maximum values obtained in the 34 × 34 comparisons to calculate the distances between the 34 founder cells within limb#1 or within limb#2 or between limb#1 and limb#2 .", "δn is computed as the absolute value of the difference between the respective numbers of divisions in the two lineage trees:δnLx , Ly=Card ( Lx-Card ( Ly ) | To calculate δt , we first paired the division times between the two lineage trees .", "For such a pairing P={ ( tix , tjy ) | tix∈Lx , tjy∈Ly} the difference in division times δt ( P ) is computed as follows:δt ( P ) =1Card ( P ) ∑ ( tix , tjy ) ∈P|tix− tjy| The pairing P⋆ that minimizes δt is used to compute the temporal distance between the lineage trees .", "Let ℘ be the set of all possible pairings , then P⋆ is defined as followed:P⋆=argminP∈P δt ( P ) We then define δt as δt=δt ( P⋆ ) .", "Once we computed all the pairwise distances between lineages of the cells under comparison , hierarchical clustering was performed using Ward’s method .", "For the hierarchical clustering in the average Parhyale T2 limb , we combined for each founder cell the information from the two limbs .", "The average lineage tree L12x of lineage trees L1x={t11x , t12x , t13x} and L2x={t21x , t22x , t23x , t24x} , where x corresponds to the founder cell x with lineage trees L1x in limb#1 and L2x in limb#2 , is defined as L12x=L1x∪L2x={t11x , t12x , t13x , t21x , t22x , t23x , t24x} .", "The computation of the pairwise distance Δ between average lineage trees was then performed as described above .", "Parhyale decapentaplegic ( Ph-dpp ) , Dorsocross ( Ph-Doc ) , engrailed-2 ( Ph-en2 ) and H15 ( Ph-H15 ) genes were identified by BLAST analysis against the Parhyale transcriptome and genome ( Kao et al . , 2016 ) using the protein sequence of Drosophila orthologs as queries .", "Sequence accession numbers are KY696711 for Ph-dpp , KY696712 for Ph-Doc , and KY696713 for Ph-en2 .", "Phylogenetic tree construction was performed with RAxML using the WAG + G model from MAFFT multiple sequence alignments trimmed with trimAl ( Stamatakis , 2014 ) .", "In situ hybridizations were carried out as previously described ( Rehm et al . , 2009 ) .", "Stained samples were imaged on a Zeiss 880 confocal microscope using the Plan-Apochromat 10x/0 . 45 and 20x/0 . 8 objectives .", "Images were processed using Fiji and Photoshop CS6 ( Adobe Systems Inc ) .", "For color overlays , the brightfield image of the Ph-dpp , Ph-Doc or Ph-H15 BCIP/NBT staining was inverted , false-colored green and merged with the fluorescent signal of the Ph-en2 FastRed staining in magenta and the nuclear DAPI signal in blue .", "In order to map gene expression patterns onto cell lineages , the z-stacks from imaged fixed specimens were imported into MaMuT and the manually reconstructed nuclei and annotated gene expression patterns were compared with the corresponding stages of the live imaged and lineaged embryos .", "This analysis was performed with single-cell accuracy thanks to the well characterized and invariant patterns of cell division across Parhyale embryos , the orderly arrangement of cells in the earlier stages analyzed , and the easily identifiable straight boundary between anterior and posterior cells in the later stages analyzed ." ] ]

[ "During development , coordinated cell behaviors orchestrate tissue and organ morphogenesis .", "Detailed descriptions of cell lineages and behaviors provide a powerful framework to elucidate the mechanisms of morphogenesis .", "To study the cellular basis of limb development , we imaged transgenic fluorescently-labeled embryos from the crustacean Parhyale hawaiensis with multi-view light-sheet microscopy at high spatiotemporal resolution over several days of embryogenesis .", "The cell lineage of outgrowing thoracic limbs was reconstructed at single-cell resolution with new software called Massive Multi-view Tracker ( MaMuT ) .", "In silico clonal analyses suggested that the early limb primordium becomes subdivided into anterior-posterior and dorsal-ventral compartments whose boundaries intersect at the distal tip of the growing limb .", "Limb-bud formation is associated with spatial modulation of cell proliferation , while limb elongation is also driven by preferential orientation of cell divisions along the proximal-distal growth axis .", "Cellular reconstructions were predictive of the expression patterns of limb development genes including the BMP morphogen Decapentaplegic ." ]

[ "During early life , animals develop from a single fertilized egg cell to hundreds , millions or even trillions of cells .", "These cells specialize to do different tasks; forming different tissues and organs like muscle , skin , lungs and liver .", "For more than a century , scientists have strived to understand the details of how animal cells become different and specialize , and have created many new techniques and technologies to help them achieve this goal .", "Limbs – such as arms , legs and wings – form from small lumps of cells called limb buds .", "Scientists use the shrimp-like crustacean , Parhyale hawaiensis , to study development , including limb growth .", "This species is useful because it is easy to grow , manipulate and observe its developing young in the laboratory .", "Understanding how its limbs develop offers important new insights into how limbs develop in other animals too .", "Wolff , Tinevez , Pietzsch et al . have now combined advanced microscopy with custom computer software , called Massive Multi-view Tracker ( MaMuT ) to investigate this .", "As limbs develop in Parhyale , the MaMuT software tracks how cells behave , and how they are organized .", "This analysis revealed that for cells to produce a limb bud , they need to split at an early stage into separate groups .", "These groups are organized along two body axes , one that goes from head to tail , and one that runs from back to belly .", "The limb grows perpendicular to these main body axes , along a new ‘proximal-distal’ axis that goes from nearest to furthest from the body .", "Wolff et al . found that the cells that contribute to the extremities of the limb divide faster than the ones that stay closer to the body .", "Finally , the results show that when cells in a limb divide , they mostly divide along the proximal-distal axis , producing one cell that is further from the body than the other .", "These cell activities may help limbs to get longer as they grow .", "Notably , the groups of cells seen by Wolff et al . were expressing genes that had previously been identified in developing limbs .", "This helps to validate the new results and to identify which active genes control the behaviors of the analyzed cells .", "These findings reveal new ways to study animal development .", "This approach could have many research uses and may help to link the mechanisms of cell biology to their effects .", "It could also contribute to new understanding of developmental and genetic conditions that affect human health ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "developmental biology", "cell biology" ]

[ [ "Cilia and flagella are highly conserved organelles present at the surface of eukaryotic cells .", "They play major physiological roles in animals such as cell or fluid mobility , signaling during development and cellular homeostasis .", "The importance of cilia and flagella is highlighted by the discovery of human diseases , classified as ciliopathies , that are associated with defects in cilia structure and/or function ( Badano et al . , 2006; Baker and Beales , 2009; Brown and Witman , 2014 ) .", "Cilia and flagella are templated from the basal body , derived from the mother centriole , from which the microtubule based axoneme is assembled .", "At the base of the cilium , a specific compartment , the transition zone ( TZ ) plays a critical role in cilium assembly and function .", "Many genes responsible for cilia associated diseases such as the Meckel syndrome ( MKS ) , Joubert syndrome or nephronophthisis ( NPHP ) are caused by defects in proteins of the TZ ( Czarnecki and Shah , 2012 ) .", "The TZ functions as a ciliary gate by sorting selected components in and out of the cilium , thus controlling the specific composition of the ciliary compartment ( Garcia and Reiter , 2016; Reiter et al . , 2012; Gonçalves and Pelletier , 2017 ) .", "TZ assembly starts during the first steps of cilia formation , when the mother centriole associates with cytoplasmic vesicles before docking to the plasma membrane .", "Assembly of TZ proteins is spatiotemporally controlled and requires , in most organisms , at least three different protein modules namely MKS , NPHP and CEP290 .", "Extensive genetic , biochemical studies and super resolution microscopy analysis helped to establish a hierarchy of these components and a structural view of the TZ architecture ( Williams et al . , 2011; Garcia-Gonzalo et al . , 2011; Sang et al . , 2011; Chih et al . , 2012; Gupta et al . , 2015; Garcia and Reiter , 2016; Reiter et al . , 2012; Gonçalves and Pelletier , 2017 ) .", "Although largely conserved from worms to mammals , all TZ proteins are not conserved in all ciliated species and variations exist between model organisms .", "For example , the NPHP module , present in mammals , worms and protozoa , is not conserved in flies , whereas CEP290 and several members of the MKS module are conserved in most organisms ( Basiri et al . , 2014; Pratt et al . , 2016; Vieillard et al . , 2016 ) .", "In addition to the core TZ components , several others have been identified but their precise relationships with the core known TZ components are not understood .", "Among these other proteins , Chibby ( Cby ) , a conserved TZ component in vertebrates and flies , is required for cilia function both in mammals and Drosophila ( Burke et al . , 2014; Voronina et al . , 2009; Enjolras et al . , 2012; Vieillard et al . , 2016 ) .", "In vertebrates , CBY has been shown to interact with several basal body ( BB ) associated proteins , such as ODF2 or CEP164 ( Lee et al . , 2014; Siller et al . , 2017; Burke et al . , 2014; Steere et al . , 2012; Chang et al . , 2013 ) and more recently DZIP1L , DZIP1 and FAM92a or b proteins ( Wang et al . , 2018; Li et al . , 2016b; Breslow et al . , 2017 ) .", "However , the functional integration of CBY and these interactors in TZ assembly is unclear and some of those , such as Cep164 cannot likely sustain Cby function in Drosophila , as Cep164 does not seem to be expressed in Drosophila testes ( Flybase ) .", "We show here that the unique Drosophila orthologs Dzip1 ( CG13617 ) and Fam92 ( CG6405 ) of respectively , vertebrate DZIP1 or DZIP1L and FAM92a or b , interact and cooperate with Cby in flies .", "We demonstrate that all three proteins form a strictly ordered functional module , and cooperate in building the TZ in the two Drosophila ciliated tissues , with Dzip1 acting upstream of Fam92 and Cby .", "While our observations establish that Dzip1 and Fam92 localization at the TZ relies on Cep290 , they reveal that Dzip1 and Fam92 exert a negative regulatory feedback loop by restraining Cep290 localization to the ciliary base .", "Last , our work reveals remarkable differences in the role of Dzip1 and Fam92 in regulating basal bodies ( BB ) docking between the two Drosophila ciliated tissues .", "Whereas , loss of Dzip1 or Fam92 does not affect basal body docking in sensory cilia , it impairs BB-membrane growth and attachment in spermatocytes .", "As a consequence , we observed aberrant and premature elongation of the axoneme before completion of meiosis , highlighting a primary role of the BB-membrane associated compartment for regulating axonemal microtubule elongation in Drosophila spermatocytes .", "These aberrant elongations mostly affect the daughter centrioles , revealing functional differences of the mother and daughter centrioles in Drosophila spermatocytes ." ], [ "We initially identified mouse CBY1 interactors by LAP-Tag affinity purification using IMCD3 cells ( murine Inner Medullary Collecting Duct cells ) stably expressing CBY1 fused to the EGFP-TEV-S peptide tag ( see Materials and methods section ) .", "This EGFP-TEV-S tag in N-terminus allows to detect the protein in cells and its purification by a two-step affinity procedure ( Torres et al . , 2009 ) .", "Analysis of LAP-CBY1 complex revealed the presence of DZIP1 and FAM92 among 22 identified proteins ( see full list in Supplementary file 1 ) , in agreement with recently published data ( Li et al . , 2016b; Breslow et al . , 2018; Wang et al . , 2018 ) .", "DZIP1/1L and FAM92a/b each show a unique ortholog gene in Drosophila , CG13617 and CG6405 respectively ( Figure 1—figure supplement 1A ) , but are absent , like CBY1 , from the C . elegans genome .", "Hereafter , we name CG13617 and CG6405 as Dzip1 and Fam92 , respectively .", "By co-immunoprecipitating Cby-GFP and HA-Dzip1 or HA-Fam92 , we demonstrate that Drosophila Dzip1 and Fam92 , each interact with Cby ( Figure 1—figure supplement 1B–C ) .", "Dzip1 or Fam92 do not apparently interact with each other in these co-IP experiments ( Figure 1—figure supplement 1D ) .", "However , when all three proteins are expressed together , immunoprecipitation of GFP-Dzip1 pulls down both Cby and Fam92 , suggesting that all three proteins are present in one complex when co-expressed in mammalian cells ( Figure 1—figure supplement 1D ) .", "To identify the subcellular localization of Drosophila Dzip1 and Fam92 , we generated transgenic flies expressing Dzip1-GFP or Fam92-GFP under the control of their respective promoters ( Figure 1—figure supplement 2 ) .", "We determined that both dzip1 and fam92 are exclusively expressed in the two kinds of Drosophila ciliated cell types , namely type I sensory neurons and male germ cells ( Figures 1 and 2 ) .", "Type I sensory neurons comprise chordotonal ( Ch ) and external sensory ( ES ) neurons , which harbor motile and immotile cilia respectively ( Figure 1A ) ( Gogendeau and Basto , 2010; Jana et al . , 2016 ) .", "Each sensory neuron is enclosed in several support cells forming the sensory organ or scolopidia .", "Dzip1 and Fam92 decorate the base of the cilia at the tip of the sensory dendrites ( labeled with 22C10 ) ( Figure 1B , arrows ) .", "By performing , super-resolution 3D structured-illumination microscopy ( 3D-SIM ) , we confirmed that Dzip1 and Fam92 co-localize with Cby in sensory neurons ( Figure 1C ) , demonstrating their restricted localization at the ciliary transition zone .", "In the male germline , Dzip1 and Fam92 appear first in spermatocytes ( Figure 2 ) at the distal end of centrioles .", "In spermatocytes , centrioles have a specific behavior as both mother and daughter centrioles of each pair elongate and dock to the plasma membrane .", "All four basal bodies ( BB ) extend a TZ , also described as a primary cilium-like structure ( Figure 2A ) ( Pasmans and Tates , 1971; Riparbelli et al . , 2012; Gottardo et al . , 2013; Vieillard et al . , 2016 ) .", "Subsequently , during meiosis , all four basal bodies are engulfed inside the cytoplasm , together with the primary like cilium which hence creates a ciliary cap that ensheaths each basal body distal end .", "After meiosis and at the onset of axoneme elongation , the ciliary cap is remodeled and a distinct domain , the ring centriole , appears at its base ( Vieillard et al . , 2016; Basiri et al . , 2014; Fabian and Brill , 2012 ) .", "The axoneme grows inside the ciliary cap , which concomitantly migrates , extruding the nascent axoneme out in the cytoplasm ( Figure 2A ) ( Pasmans and Tates , 1971; Riparbelli et al . , 2013; Gottardo et al . , 2013 ) .", "We observed that Dzip1 , Fam92 and Cby all localize at the centriolar distal ends in early spermatocytes ( Figure 2B–C ) .", "During centriolar/BB maturation , as the ciliary cap grows , Dzip1 and Fam92 localizations are extended and overlap with Cby , as revealed by 3D-SIM observations ( Figure 2D ) .", "In spermatids , when the TZ migrates away from the basal body , we observed that Dzip1 and Fam92 strongly accumulate with Cby at the ring centriole ( Figure 2B–C , arrows ) .", "Together , these results strongly indicate that Dzip1/Fam92/Cby interact at the ciliary transition zone .", "To determine the roles of the Dzip1/Fam92 module in TZ assembly or function , we generated two deletion alleles of dzip1 or fam92 ( Figure 1—figure supplement 2 and Materials and method section ) .", "In the dzip11 allele , the 65 N-ter codons ( including start codon ) were deleted and replaced by a 3 kb insertion including the mini-white gene ( Figure 1—figure supplement 2 ) .", "In fam921 , exon two and part of exon three were removed ( 294 bp , Figure 1—figure supplement 2 ) , leading to the deletion of 81 aa and a frameshift with several stop codons in the remaining downstream sequence , leaving only 47 aa of the WT protein and 39 amino acids of the −1 frameshifted open reading frame .", "dzip11 and fam921 flies are viable but show typical behavioral defects associated with ciliary dysfunction in Drosophila .", "Fly geotaxis response ( Figure 3A ) was monitored by bang assay ( Enjolras et al . , 2012 ) .", "Whereas most of the control flies reach and stay at the top of the tube 30 s after the bang , no dzip11 flies reached the top of the tube ( Video 1 ) .", "This phenotype was worsened in dzip11/Df as the flies not only fail to climb , but are completely immotile with held up wings ( Video 2 ) , indicating that dzip11 is likely not a complete null allele .", "Both dzip11 and dzip11/Df behavioral phenotypes were fully rescued by one copy of dzip1::GFP ( not shown ) .", "In contrast , a few percentage ( 17% ) of fam921 flies could reach the top of the tube ( Figure 3A ) and no differences could be observed when comparing fam921 flies and fam921 over its cognate deficiency ( fam921/Df ) , indicating that fam921 is likely a null allele .", "The fam92::GFP rescue construct partially restores the climbing behavior of the fam921 mutant flies .", "These observations reveal a graded requirement for Fam92 and Dzip1 in Drosophila geotaxis response and indicate a regulatory role of this complex in sensory cilia assembly or function .", "To determine the impact of Dzip1 or Fam92 loss on cilia assembly , we performed ultra-structural analysis by transmission electron microscopy ( EM ) .", "Strikingly , we observed that cilia were essentially absent in dzip11 antennal chordotonal organs , but were still present in fam921 antennae ( Figure 3B and F ) , in agreement with their milder geotaxis defect behavior .", "In fam921 flies , 25% of scolopidia ( total observed n = 54 ) showed reduced cilia number ( one or no cilia ) .", "In addition , defects in axonemal structure were occasionally observed , such as lack of microtubule doublets ( Figure 3C , asterisks , 6% ) , excess accumulation of material beneath the ciliary membrane ( arrows ) and deformation of the membrane ( Figure 3C , upper panel , dot , 4% ) .", "Most TZ sections showed normal ultrastructure , but a few ( 6% ) showed incomplete radial symmetry and accumulating material as observed for cilia .", "Linkers connecting the axoneme to the membrane could still be observed ( Figure 3C , arrowhead , lower panel ) .", "Transition zones were completely disorganized in dzip11 antennae as revealed by serial-cross and longitudinal sections ( Figure 3D–E ) and no more axoneme-to-membrane linkers could be observed .", "Basal bodies were normally present at the dendrite distal tip ( Figure 3D , lower panel ) , but we observed a rapid disorganization of the axoneme , with its complete abrogation a few microns above the basal body ( Figure 3D–E ) .", "Hence , these observations demonstrate that loss of Dzip1 or Fam92 affects TZ assembly required for sensory cilia formation .", "To address the function of Dzip1 and Fam92 in sperm flagellum assembly , we first investigated male fertility .", "We observed a strong reduction of the fertility of fam921 males compared to controls .", "This defect is restored by expressing Fam92-GFP ( Figure 4A ) .", "Because dzip11 mutant flies are severely uncoordinated , their fertility could not be tested .", "However , dzip11 testes showed a marked dispersion of the nuclei along the cysts and , as a consequence , altered migration of sperm individualization complexes ( Figure 4B and Figure 4—figure supplement 1A ) .", "Nuclear dispersion is also observed , to a lesser extent , in fam921 testes ( Figure 4B and Figure 4—figure supplement 1A ) .", "One possible origin of nuclei dispersion is defective axonemal elongation ( Vieillard et al . , 2016; Soulavie et al . , 2014 ) and EM analysis confirmed that axonemes were affected in fam921 cells ( Figure 4D , 125/448 broken axonemes , 25/448 missing central pair , 52/448 missing axoneme ) .", "As well , in dzip11 or dzip11/Df mutant testes , marked axonemal defects could be observed .", "In dzip11 testes , we observed 6/126 broken axonemes , 1/126 missing central pair , 1/126 missing axonemes ( Figure 4C ) .", "More severe defects were observed in very young dzip11/Df spermatid cysts , with up to 60% of spermatids with missing or broken axonemes ( Figure 4—figure supplement 1B–C ) .", "However , in older cysts , the number of spermatids/cyst was much reduced compared to control ( mean = 44 spermatids/cyst compared to control = 63 . 4 ) , but almost all remaining elongated spermatids showed normal axonemes ( Figure 4—figure supplement 1B ) .", "These observations suggest that young spermatids with severe axonemal defects fail to elongate and are not observed in cross sections of older spermatid cysts .", "Taken together , these results demonstrate that Fam92 and Dzip1 are necessary at the ciliary TZ for cilia formation both in sensory neurons and male germ cells .", "To determine the functional hierarchy between Dzip1 , Fam92 and Cby , we analyzed their respective localization in dzip11 or fam921 or previously described cby1 mutants ( Figure 5 and Figure 5—figure supplement 1 ) .", "Cby-Tom is almost completely lost from ciliary TZ in dzip11/Df sensory neurons ( Figure 5—figure supplement 1A ) .", "In spermatocytes , Cby-Tom signal is strongly reduced at the tip of centrioles ( Figure 5A , D ) but , in rare occasions , expanded at one centriole of the pair ( asterisk , 2% ) .", "The amount of Fam92-GFP is severely reduced in dzip11/Df sensory cilia base ( Figure 5—figure supplement 1B ) and in spermatocytes ( Figure 5B , D , arrows ) .", "Altogether , these observations indicate that Dzip1 is required at the basal body/TZ to recruit or stabilize both Cby and Fam92 .", "In fam921 mutant flies , Dzip1-GFP is slightly reduced at the distal tip of basal bodies in sensory cilia ( Figure 5—figure supplement 1C ) .", "In fam921 spermatocytes , Dzip1-GFP domain is sometimes expanded ( 8 . 3% of cases , Figure 5D ) , but the overall Dzip1-GFP expression is also slightly reduced ( Figure 5C , arrows , 5D ) .", "Cby-Tom is completely lost in both fam921 ciliated tissues ( Figure 5A , arrowheads and Figure 5—figure supplement 1A ) .", "Hence , Fam92 is required to recruit Cby , but not Dzip1 , at the TZ .", "As well , in cby1 tissues , Fam92-GFP completely disappears from basal bodies ( Figure 5B , arrowhead and Figure 5—figure supplement 1B ) , indicating that Cby and Fam92 stabilize each other at the basal body .", "In cby1 mutant spermatocytes , an extended domain of Dzip1-GFP is occasionally observed ( Figure 5C , arrows , 4 . 5% , 5D ) .", "However , no overall difference in Dzip1-GFP intensity was observed in cby1 mutant testes ( Figure 5C–D ) or antennae ( not shown ) compared to controls .", "These observations establish a functional hierarchy for Dzip1 , Fam92 and Cby: Dzip1 is required to recruit or stabilize both Fam92 and Cby at centrioles , but does not depend on Fam92 or Cby for its targeting to centrioles , the latter only restricting Dzip1 to the proximal TZ .", "Conversely , Fam92 and Cby mutually depend on each other to localize at the TZ .", "To understand how Dzip1 and Fam92 organize the ciliary base , we investigated their functional relationships with other core TZ components .", "We first investigated the behavior of Mks1 , a component of the core conserved MKS complex ( Weatherbee et al . , 2009 ) .", "In Drosophila , Mks1 is required to assemble the MKS complex , but removal of MKS components leads to only very mild defects of cilia assembly ( Vieillard et al . , 2016; Pratt et al . , 2016 ) .", "In dzip11/Df and fam921 mutant flies , Mks1-GFP is lost or reduced at the TZ in both ciliated tissues ( Figure 6A , arrows , and Figure 6—figure supplement 1A ) .", "Dzip1 and Fam92 are hence important for the recruitment or stabilization of the MKS module at the TZ .", "Cep290 is a core conserved TZ component which plays a prominent role in TZ assembly in many organisms , including Drosophila ( Craige et al . , 2010; Rachel et al . , 2015; Li et al . , 2016a; Basiri et al . , 2014 ) .", "In flies , Cep290 is located at the base of the TZ in sensory neurons and is a critical component of the migrating ring centriole during spermatogenesis ( Basiri et al . , 2014 ) .", "In the absence of Dzip1 , besides a small but significant reduction of Cep290-GFP on spermatocyte centrioles , we observed striking Cep290-GFP expanded domains , both in spermatocytes and in Ch neurons ( Figure 6A–B , arrows ) .", "Around 13% of centrioles in spermatocytes and a majority in antennae showed an expanded Cep290-GFP domain ( Figure 6A–B ) .", "In fam921 mutants , we also observed a few Cep290-GFP expanded domains in spermatocytes and antennae , but with no significant difference in overall Cep290-GFP intensity ( Figure 6A–B , arrows ) .", "To further understand the relationships between Cep290 , Dzip1 and Fam92 , we took advantage of a strong cep290 hypomorphic mutant , cep2900153-G4 .", "This mutant shows severe uncoordination and completely disorganized spermatid cysts with dispersed nuclei ( Figure 6—figure supplement 2A ) , which is a consequence of the severe axonemal elongation defects observed in this mutant ( not shown ) .", "The phenotypes were completely rescued by two copies of cep290::GFP ( Figure 6—figure supplement 2A–B ) .", "We observed that in cep2900153-G4 flies , Dzip1 and Fam92 are strongly reduced or lost at the TZ , both in spermatocytes ( Figure 6C ) and in sensory neurons ( Figure 6—figure supplement 1B ) .", "These observations demonstrate that Cep290 is required during TZ assembly to recruit Dzip1 and Fam92 , which in turn restrict Cep290 scaffolding to the proximal part of the TZ .", "Defects in BB anchoring to the plasma membrane in spermatocytes lead to aberrant growth of axonemal microtubules ( Vieillard et al . , 2016 ) .", "We used the specific axonemal marker CG6652-GFP that only labels axonemal microtubules in flies ( Figure 6—figure supplement 2C–D ) .", "With this marker , we observed aberrant growth of axonemal microtubules in spermatocytes , with graded severities increasing from fam921 , dzip11 to cep2900153-G4 mutant flies ( Figure 7A–B ) .", "30% of centrioles showed CG6652 extensions in fam921 testes , 34% in dzip11/Df and 76% in cep2900153-G4 .", "More strikingly , whereas 68% of the centriole pairs present extensions from both centrioles in cep2900153-G4 spermatocytes , we observed that in fam921 testes , 96% of the centriole pairs present microtubule extensions on only one centriole .", "This asymmetric penetrance of the phenotype is related to centriole age .", "Indeed , among 24 centriole pairs for which mother and daughter identities could be unequivocally assigned , 19 daughter centrioles ( 79% ) and only five mother centrioles ( 21% ) were affected .", "As well , in dzip11/Df testes , among 40 centriole pairs , we found 30 daughter centrioles ( 75% ) and 10 mother centrioles ( 25% ) affected by the absence of Dzip1 ( Figure 7A–B ) .", "We analyzed the ultrastructure of the centrioles and primary like cilium in the initial stages of elongation ( Figure 7C ) .", "We observed irregular distal end ( asterisk ) of centrioles in dzip11 testes at young spermatocytes stages before docking ( upper panel ) .", "Whereas centrioles dock in polar spermatocytes before reaching their full size in control testes , undocked centrioles were observed in dzip11 .", "Either both centrioles of the pair were undocked or partially docked ( n = 2 , lower panel ) ( Figure 7C , arrowhead ) or only one centriole of the pair , the mother , was docked ( n = 3 ) ( Figure 7C , arrow ) .", "These results show that Dzip1 is required to cap the centriole distal end and foster its anchoring to the plasma membrane .", "As well , we observed undocked centrioles in fam921 mutant spermatocytes , with irregular distal end ( asterisk ) or with microtubules extending from the distal end ( Figure 7C , arrow ) , illustrating the role of Fam92 in controlling centriolar distal growth and docking to the plasma membrane .", "Altogether , these observations demonstrate that Dzip1 and Fam92 are required for the proper distal elongation of basal bodies and their membrane anchoring in Drosophila spermatocytes .", "This docking is required to regulate TZ elongation ( Figure 7D ) .", "In addition , our results show that in male germ cells , although all centrioles have the capacity to generate a ciliary cap , a functional asymmetry of the mother and the daughter centrioles is revealed by their ability to dock to the plasma membrane in absence of key TZ proteins ." ], [ "In vertebrates , two orthologs have been described for Dzip1 and Fam92 and three for Cby .", "DZIP1 , DZIP1L , FAM92a and b also interact with CBY1 in vertebrates ( Wang et al . , 2018; Breslow et al . , 2017; Ye et al . , 2014 ) indicating a conservation of the interacting capacities of the family members during evolution .", "The precise hierarchy between members of the complex has not been established in mammals , but depletion of CBY1 prevents the recruitment of FAM92a/b to the centriole ( Li et al . , 2016b ) and DZIP1L was shown to act upstream of CBY1 ( Wang et al . , 2018; Keller et al . , 2009 ) .", "In vertebrates , mutations in members of this module lead to ciliogenesis defects of various severities and with different phenotypic outcomes ( Wolff et al . , 2004; Tay et al . , 2010; Glazer et al . , 2010; Li et al . , 2016b; Breslow et al . , 2017 ) Dzip1 or Dzip1L KO mice die during embryogenesis ( Wang et al . , 2018; Wang et al . , 2013 ) , whereas Fam92a-/- mice show skeletal defects ( Schrauwen et al . , 2019 ) and Cby1 KO mice exhibit differentiation defects of motile ciliated epithelia ( Burke et al . , 2014; Voronina et al . , 2009 ) .", "This phenotypic variability is not expected if all three proteins only act together in a functional module at the TZ , as demonstrated by our work in Drosophila .", "This suggests that the different mouse paralogs of Cby and Fam92 may have acquired specialized ciliogenic functions in mouse .", "However , we also observed that dzip11 and fam921 mutant phenotypes show small differences indicating specific functions of each proteins .", "For instance , in both ciliated tissues , dzip11 hypomorphic mutant phenotype is more severe than fam921 , suggesting that Dzip1 has additional functions that are not solely mediated by Fam92 .", "Despite the conserved role of Dzip1 , Cby and Fam92 in TZ and cilia assembly from Drosophila to humans , no homologs could be detected in the genomes of C . elegans .", "This could be linked to the diversification of cilia function , with both motile and immotile cilia being present from Drosophila to humans , in contrast to C . elegans , where only one subtype of cilia that are immotile is found .", "Another possible explanation could be that DZIP1/FAM92/CBY are associated with specific signaling or developmental functions in animals that still need to be understood .", "This work emphasizes the essential role of Dzip1 and Fam92 in building the ciliary transition zone in the two types of ciliated tissues of Drosophila .", "Strikingly , it also reveals tissue specific function of these proteins in priming basal body/membrane docking in Drosophila testes .", "This reveals intrinsic differences in the mechanisms that link basal body to membranes in Drosophila ciliated tissues .", "In mammals , basal body docking requires transition fibers built from the distal appendages on the mother centriole prior to docking ( Wei et al . , 2015 ) .", "In Drosophila , distal appendages have not been observed on centrioles , but structures similar to transition fibers are described at the base of sensory cilia ( Ma and Jarman , 2011; Vieillard et al . , 2016 ) , whereas only scarce links could be observed in male germ cells ( Gottardo et al . , 2018; Roque et al . , 2018 ) .", "These differences could explain why destabilization of the TZ leads to basal body anchoring defects in spermatocytes , but not in sensory neurons .", "This structural characteristic of the spermatocyte TZ is likely to be related to its specific functional properties .", "Indeed , whereas the TZ is stably built at the ciliary base in sensory neurons , it shows a dynamic behavior during sperm flagella extension , separating from the basal body and migrating along the growing end of the axoneme ( Basiri et al . , 2014; Avidor-Reiss et al . , 2017; Baker et al . , 2004; Wei et al . , 2008 ) .", "In addition , in spermatocytes , basal bodies have a dynamic behavior , being first docked at the plasma membrane and next internalized during meiosis ( Pasmans and Tates , 1971; Fabian and Brill , 2012 ) .", "This could induce mechanical constraints on basal bodies that would increase their sensitivity to TZ disruption .", "In agreement with this hypothesis , when TZ maturation was challenged by modulating membrane phospholipids ( Gupta et al . , 2018 ) , BB were released from the plasma membrane during meiosis , but their initial docking was not impaired .", "However , we observed by EM , that BB fail to initially dock in significant occurrences ( 8 among 13 ) in dzip11 and fam921 mutant spermatocytes , indicating that Dzip1 and Fam92 are at least involved in the initial steps of BB docking .", "Previous observations of another strong hypomorphic cep290mecH allele showed docked basal bodies in spermatocytes and spermatids ( Basiri et al . , 2014 ) .", "In cep2900153-G4 mutant , we did not quantify the number of docked versus undocked basal bodies in spermatocytes , but we observed up to 76% of aberrant axonemal growth , suggesting that basal body to membrane attachment is compromised in this mutant .", "Differences in the organization of the ciliary base associated with variations in the distribution and function of several centriolar and TZ proteins have been documented in Drosophila ciliated cells ( Jana et al . , 2018 ) .", "However , none of these identified differences help to explain the behavioral properties of BB docking and TZ dynamics that we have identified in the two ciliated Drosophila tissues .", "Hence , additional screens for specific factors of basal-body docking or TZ assembly either in sensory neurons or male germ cells need to be designed .", "In all our observations , there is a striking phenotypic difference between the mother and daughter centrioles in spermatocytes .", "In all mutants examined ( i . e . dzip1 , fam92 and cby ) we observed a more penetrant defect on the daughter centriole than the mother .", "Thus , although the 2 centrioles of each pair are able to form cilia , the daughter centriole appears more sensitive to transition zone perturbations .", "We do not have molecular explanations for these intrinsic differences of mother and daughter centrioles in spermatocytes .", "In Drosophila sensory neurons , centrobin plays a critical role in maintaining the daughter centriole fate , precluding its capacity to build a cilium ( Gottardo et al . , 2015 ) .", "In sperm cells , centrobin is required for the formation of the C tubule ( Reina et al . , 2018 ) , which also plays a critical role in TZ assembly ( Gottardo et al . , 2018 ) .", "However , in spermatocytes centrobin is equally distributed at the base of both the mother and daughter centrioles and does not show a functional asymmetry .", "Recently , a transient microtubule based structure that anchors the base of the mother centriole on one of the centriole pair at the onset of meiosis was identified , but its function is unknown ( Riparbelli et al . , 2018 ) .", "This structure could stabilize the mother centriole and favor its attachment to the membrane , but the function of this microtubule rootlet in Drosophila spermatocytes needs further investigations .", "The intrinsic difference of the mother versus daughter centrioles could also be related to the timing of centriole docking during spermatogenesis ( Gottardo et al . , 2018 ) , where the mother centriole was shown to dock first .", "This timing difference would be sufficient to better stabilize the TZ of the mother centriole , and hence explain the phenotypic differences observed in our study .", "There are other situations in the animal kingdom were both mother and daughter centrioles build a cilium .", "Among the best studied are the bi-flagellated Chlamydomonas and the peculiar case of multiple ciliated epithelia , where numerous de novo centrioles are assembled just at the onset of ciliogenesis .", "Interestingly , in mammals , CBY1 was shown to play a critical role in multiple ciliated cells to allow proper docking of the multiple basal bodies to the plasma membrane ( Burke et al . , 2014 ) .", "It is tempting to speculate that the increased susceptibility of multiple ciliated cells to CBY1 loss , is related to a particular status of these de novo centrioles , as we observed for daughter centrioles in male germ cells .", "More work will be needed to understand the molecular determinants of the mother versus daughter centriolar functional asymmetry in Drosophila male germ cells .", "We observed that defects of TZ assembly and/or basal body docking lead to aberrant elongation of axonemal microtubules as revealed by the specific Drosophila axonemal marker CG6652-GFP .", "These abnormal elongation defects appear only in late G2 phase , just at the onset of meiosis , indicating that specific signals , yet to be identified , enable centrioles to start axonemal elongation at the onset of meiosis .", "The membrane cap restricts this capacity by inhibiting axonemal growth until the round spermatid stage , where a second signal turns on axonemal elongation and TZ migration .", "Among the candidate proteins recruited by the membrane cap which may coordinate axonemal assembly are microtubule depolymerizing kinesins as previously proposed ( Vieillard et al . , 2016 ) .", "This membrane cap could also be involved in stabilizing centriolar capping proteins such as CP110 or CEP97 , which in mammals need to be removed from centrioles to allow ciliary assembly ( Spektor et al . , 2007 ) .", "However , there are no clear evidence in Drosophila that these proteins need to or are specifically removed before axonemal elongation ( Galletta et al . , 2016; Franz et al . , 2013; Delgehyr et al . , 2012 ) .", "Our observations indicate that regulation of axonemal assembly and cell cycle regulation are tightly linked in these dividing cells , but the effectors of this control still need to be identified .", "In conclusion , our work demonstrates the critical role of the conserved Dzip1/Fam92/Cby module downstream of Cep290 in initiating the assembly of the ciliary transition zone in flies .", "It also reveals key tissue specific differences in basal body docking pathways in Drosophila ." ], [ "CBY1 coding sequence was PCR amplified from mouse ependymal primary cell cDNA with primers F-CBY1 and R-CBY1 .", "PCR product was cloned into pDONR223 ( Invitrogen ) and then subsequently Gateway cloned into the pG-LAP3 vector ( Torres et al . , 2009; gift from P . Jackson , Addgene#79704 ) .", "This vector contains the double EGFP-TEV-S peptide tag in N- terminus allowing a two step affinity purification .", "Coding sequences of Drosophila cby , fam92/CG6405 and dzip1/CG13617 were obtained by PCR on cDNA from testis .", "Cby cDNA was cloned in pEGFP-N1 ( Clontech ) in frame with GFP ( primers F- CbyGFP/R-CbyGFP ) and in pCDNA3 . 1-HA ( gift from S . Khochbin , Institut Albert Bonniot , Grenoble ; primers F-HACby/R-HACby ) .", "cDNA of fam92/CG6405 and dzip1/CG13617 were cloned in pCDNA3 . 1-HA with primers F-Fam92HA/R-Fam92HA and F-Dzip1HA/R-Dzip1HA , respectively .", "pCDNA3 . 1-GFP , GFP-Fam92 and GFP-Dzip1 were obtained by replacement of the HA tag with EGFP .", "V5-Fam92 was obtained by replacement of the HA tag with V5 tag .", "Drosophila reporter gene construct of dzip1/CG13617 was obtained by cloning 1 . 6 kb upstream regulatory sequences and the entire coding sequence ( primers F-Dzip1GFPrep/R-Dzip1GFPrep ) in frame with GFP of pJT61 , a pattB plasmid with an extra EGFP-6xMycTag-SV40polyA cassette ( Vieillard et al . , 2016 ) .", "The 4 kb fragment of fam92/CG6405 including 1 . 4 kb of upstream regulatory sequences , the entire coding sequence fused to the multipurpose tag cassette including GFP and the 3'UTR was obtained by PCR with primer F-Fam92GFPrep/R-Fam92GFPrep on fosmid from the FlyFos library ( Sarov et al . , 2016 ) .", "The resulting constructs were integrated in the 53B2 VK00018 landing site on the second chromosome by PhiC31 integrase ( BestGene ) .", "All transgenic lines were obtained from BestGene Inc .", "The dzip11 allele ( CG13617 ) was generated by CRISPR/Cas9 induced homologous directed repair ( Gratz et al . , 2015 ) .", "Two gRNA were selected using the http://crispor . tefor . net/crispor . py website: 5’- CCCGTTTCACGGACCATCTG CGG −3’ and 5’-GTTTCCAGCACTGTGCCCAG TGG −3’ ( protospacer adjacent motifs are underlined ) .", "Oligos were phosphorylated by T4PNK ( New England Biolabs , Inc ) and annealed .", "Double-stranded 5’ gRNA and 3’ gRNA were cloned in the BbsI site of pBFv-U6 . 2 and pBFv-U6 . 2B vectors , respectively ( Kondo and Ueda , 2013 ) .", "5’ gRNA was further subcloned in the EcoRI–NotI sites of pBFv-U6 . 2B to express the two gRNAs from one vector .", "The 5’ and 3’ homology arms ( 1 . 7 kb and 2 . 3 kb respectively ) were amplified by PCR ( primers F-5’armDzip1/R-5’armDzip1 and F-3’armDzip1/R-3’armDzip1 ) and cloned , respectively , into the pJT38 plasmid ( pRK2 plasmid [Huang et al . , 2008] , with an attB cassette ) using Gibson Assembly Master Mix ( New England Biolabs , Inc ) .", "The two vectors ( gRNAs and homology arms ) were injected into vasa::Cas9 embryos .", "Flies were crossed to w; TM2 , e Ubx/TM6B , e Hu Tb virgin females and the offspring were screened for red-eyed-flies .", "Homologous recombination was checked by PCR .", "The fam921 allele ( CG6405 ) was generated by CRISPR/Cas9 induced deletion .", "Two gRNA were selected as before: 5’- CATAAGACCTTGCAGATATC GGG −3’ and 5’- GGCTGTCATAGCGCGGGATA AGG −3’ ( protospacer adjacent motifs are underlined ) .", "Double-stranded phosphorylated 5’ gRNA and 3’ gRNA were cloned into the pBFv-U6 . 2B plasmid .", "The vector was integrated into the 89E11 VK00027 landing site on the third chromosome by PhiC31 integrase ( BestGene ) .", "Surviving G0 males were individually crossed to y2 v1 virgins .", "A single male transformant from each cross was mated to y2 cho2 v1; Pr Dr/TM6C , Sb Tb virgins .", "Males carrying a U6-double gRNA transgene were crossed to nos-Cas9 females to obtain male founder animals .", "Each male founder was crossed to three virgin females y2 cho2 v1 ; Sco/CyO .", "Deletion in the fam92 locus was characterized by PCR with primers F-Fam92KO/R-Fam92KO on genomic DNA from the resultant offspring .", "Flies showing a 294 bp deletion in the fam92 locus were selected for further studies .", "All reagents for cell culture were purchased from Thermo Fisher Scientific .", "IMCD3 cells ( murine Inner Medullary Collecting Duct cells , ATCC CRL-2123 a gift from A . Benmerah , Institut Imagine , Paris ) were cultured in DMEM/HAM'S F12 medium supplemented with 10% FBS , 1X non-essential amino acids , 100 U/ml penicillin-streptomycin and 100 µg/ml hygromycin .", "COS-7 cells ( ATCC CRL-1651 ) or HEK293 ( ATCC CRL-1573 ) were maintained in DMEM medium containing 10% FBS , 1X non-essential amino acids and 100 U/ml penicillin-streptomycin .", "Cells were tested negative for mycoplasma .", "Transfections were performed using jetPRIME ( Polyplus transfection ) according to the manufacturer’s instructions .", "IMCD3 cell lines expressing LAP-CBY1 or control LAP-GFP were created using the Flp-In system kit ( Invitrogen ) and established method ( Torres et al . , 2009 ) .", "Two rounds of purification of CBY1-protein complexes were performed .", "Cells were seeded in 35 15 cm dishes and grown to confluence before serum starvation for 24 hr to induce primary cilia formation .", "Cells were washed once with ice-cold PBS1X and purification was performed as described previously ( Nachury , 2008 ) .", "Briefly , cells were collected by scraping and resuspended in 11 ml lysis buffer containing HEPES pH7 . 5 50 mM , EGTA pH8 1 mM , MgCl2 1 mM , KCl 300 mM , Glycerol 10% , NP-40 0 . 31% and 1X protease inhibitor cocktail ( Roche ) .", "CBY1-complexes were purified using GFP-TRAP ( Chromotek ) .", "S-Tag-CBY1 was cleaved off GFP beads by TEV cleavage and the eluate was further purified on S-protein agarose ( Merck ) and eluted in 100 μl 2X Laemmli Sample Buffer .", "For protein identification , samples were separated by 10% SDS-PAGE .", "Gels were stained with Coomassie and cut into four slices .", "Each gel slice was washed , reduced with 10 mM DTT , alkylated with 55 mM iodoacetamide , and subjected to in-gel trypsin digestion overnight ( Trypsin Protease , MS Grade Pierce , Thermo Fisher Scientific ) .", "The extracted tryptic peptides were cleaned up using OMIX C18 100 µl pipette tips ( Agilent ) , and lyophilized before being reconstituted for the LC-MS/MS analysis .", "The peptides were separated using an Eksigent Ultra nano-LC HPLC system coupled with an AB Sciex Triple TOF 5600 mass spectrometer .", "The LC separations were performed using a Discovery Bio Wide Pore HPLC column ( C18 , 3 µm , 100 × 5 mm ) .", "The mobile phases used were 0 . 1% formic acid in water ( A ) and 100% acetonitrile with 0 . 1% formic acid .", "The gradient elution steps were performed with a flow rate of 5 µl/min as follows: 0–40% B for 106 min , 40–80% for 5 min , and then 80% B for an additional 5 min .", "All data were acquired using Analyst software ( AB Sciex ) in the data dependent mode .", "Peptide profiling was performed using a mass range of 350–1600 Da , followed by a MS/MS product ion scan from 100 to 1500 Da .", "For each survey MS1 scan ( accumulation time of 250 msec ) , MS/MS spectra ( accumulation time of 75 msec per MS/MS ) were obtained for the 30 most abundant precursor ions .", "The protein identification was performed with the ProteinPilot Software 5 . 0 ( AB Sciex ) .", "The MS/MS spectra obtained were searched against the mouse UniProt Proteome database ( release 2015_09 with 46470 proteins ) .", "The search parameters for tryptic cleavage and accuracy are built-in functions of the software .", "The other data analysis parameters were as follows: sample type: identification; Cys-alkylation: Iodoacetamide; Digestion: Trypsin; Instrument: TripleTOF 5600; Special factor: gel based ID , and biological modifications; Species: Homo sapiens; Search effort: Thorough ID .", "Proteins comprising one or more peptides with a high confidence score ( 95% ) and a low false discovery rate ( FDR ) ( estimated local FDR of 1% ) were considered positively identified .", "Co-IP assays were performed using transfected COS-7 cells or HEK293 cells , harvested in ice-cold PBS1X .", "Cell pellet was resuspended in either lysis buffer ( NaCl 150 mM , Tris-HCl pH 7 . 2 50 mM , NP-40 1% , Desoxycholate Na 1% ) or milder lysis buffer ( NaCl 150 mM , Tris-HCl pH 7 . 2 50 mM , NP-40 0 . 5% , glycerol 10% ) with 1X protease inhibitor cocktail ( Roche ) and incubated for 1 hr at 4°C under agitation .", "Cell lysates were cleared by centrifugation at 15 000 g for 20 min at 4°C .", "Supernatants were incubated with either GFP-TRAP ( Chromotek ) or HA-coupled beads ( Sigma ) as indicated for 1 or 2 hr at 4°C .", "The beads were collected and washed five times with 1 ml of ice-cold lysis buffer before SDS-PAGE .", "Eluates were loaded on 10 or 12% SDS-PAGE .", "After migration , proteins were transferred onto a P 0 . 45 µm PVDF or 0 . 45 µm nitrocellulose Amersham Hybond membranes ( GE Healthcare ) and immunoblotted with according antibodies .", "IP were repeated three times with anti-GFP for Cby-GFP/HA-Fam92 and once each ( anti-HA or anti-GFP ) for Cby-GFP/HA-Dzip1 and IP was performed once with anti-GFP for GFP-Dzip1/V5-Fam92/HA-Cby .", "Primary antibodies used for western blotting were the following: rat anti-HA ( 1:5000; Roche ) , rabbit anti-GFP ( 1:10000; Abcam ) and mouse anti-V5 ( 1:5000; Invitrogen ) .", "HRP-conjugated secondary antibodies were the following: goat anti-rabbit ( 1:10000; Biorad ) , goat anti-mouse ( 1:3000; Biorad ) and goat anti-rat ( 1:20000; Sigma ) .", "Membranes were visualized using ECL prime from GE Healthcare .", "The cby1 mutant and cby::Tomato transgene were previously described ( Enjolras et al . , 2012 ) .", "The cep290::GFP transgene was a gift from T . Avidor- Reiss ( University of Toledo , USA; Blachon et al . , 2009; Basiri et al . , 2014 ) .", "CG6652::GFP , mks1::GFP and unc::mKate transgenes have been described in Vieillard et al . ( 2016 ) .", "sas4s2214 mutant flies were kindly provided by R . Basto ( Basto et al . , 2006 ) .", "Flies were raised on standard media between 21°C and 25°C .", "Df ( 3R ) Exel8178 ( dzip1 deficiency ) , Df ( 2L ) Exel6033 ( fam92 deficiency ) were obtained from the Bloomington Stock Center and uncover the 95F8-−96A6 and 33E4-−33F2 cytological interval respectively .", "cep2900153-G4 ( Bloomington Drosophila Stock Center ) harbors a piggy-bac insertion in the beginning of exon 11 .", "Testes from young adult flies or pupae were dissected in PBS1X , fixed 15 min in PBS1X/PFA 4% and either whole mounted or squashed between coverslip and slide and frozen in liquid nitrogen .", "Coverslip was removed and slides were soaked 2 min in ethanol 100% at −20°C .", "Testes were treated 15 min in PBS1X/Triton 0 . 1% ( PBT ) and blocked 2 hr in PBT/BSA 3%/NGS 5% .", "Primary antibodies were incubated in blocking buffer overnight at 4°C .", "Samples were then washed in PBS1X and incubated 2 hr in secondary antibodies diluted in PBS1X .", "Slides were washed in PBS1X and rinsed in ultrapure water .", "Slides were mounted using Vectashield containing Hoescht 1:1000 .", "Antennae were processed as previously described ( Vieillard et al . , 2015 ) .", "Briefly , Drosophila heads from 38 to 45 hr pupae were dissected in PBS1X , fixed 1 hr in PBS1X/PFA 4% and washed in PBS1X .", "Antennae were permeabilized 1 hr in PBS1X/Triton 0 . 3% , blocked 1 hr in PBS1X/Triton 0 . 3%/BSA 3%/NGS 5% and incubated in primary antibodies diluted in blocking solution for 48 hr at 4°C .", "Samples were washed three times in PBS1X and incubated in secondary antibodies diluted in PBS1X for 48 hr at 4°C .", "Antennae were washed three times in PBS1X and mounted in Vectashield .", "For the whole mount staining of antennae , Drosophila heads from 38 to 45 hr pupae were dissected in PBS1X/0 . 3% Triton X-100 and fixed in in PBS1X/0 . 3% Triton X-100/4% PFA for 1 hr at RT .", "After a few rinses , samples were incubated 1 hr at RT in PBS1X/0 . 3% Triton X-100/BSA 0 . 1% and then in phalloidin FluoProbes 505 or 547 ( Interchim ) diluted 1:200 in PBS1X/0 . 3% Triton X-100/BSA 0 . 1% at 4°C for 48 hr .", "The samples were washed 3 times for 15 min in PBS1X and mounted in Vectashield .", "All fluorescent observations were performed on at least five different pairs of testes or antennae .", "Quantifications were performed on at least three independent experiments .", "Most slides were imaged using either the IX83 microscope from Olympus equipped with an iXon Ultra 888 EMCDD camera from Andor and the IQ3 software from Andor .", "The PlanApo N Apochromat 60 × 1 . 42 NA objective from Olympus was used for all acquisitions .", "Some slides were imaged using an SP5X confocal laser scanning microscope ( Leica Biosystems ) equipped with the Application Suite software ( Leica Biosystems ) .", "An HCX Plan-Apochromat CS 63 × 1 . 4 NA objective ( Leica Biosystems ) was used for all acquisitions .", "All images were processed with ImageJ .", "Figures were created with Adobe photoshop .", "Only contrasts and offset were adjusted .", "Testes or antennae were squashed on 12 diameter round coverslip with a 44 × 60 mm overlaying coverslip .", "Immunofluorescence protocols were the same as above using PFA .", "Images were acquired using the Elyra PS . 1 system from Zeiss ( Carl Zeiss , AG , Jena ) equipped with a PCO edge 5 . 5 camera and the ZEN 2012 SP2 software ( black edition ) .", "The objective used for all acquisitions is a Plan-apochromat 63 × 1 . 4 NA .", "The antibodies used were the following: mouse anti-Futsch/22C10 ( 1:250; DSHB = 22C10 ) , mouse anti-γ-Tubulin ( 1:500; Sigma ) , rabbit anti-GFP ( 1:1000; Abcam ) , guinea pig and rat anti-Asterless ( 1:50000; gift from C . Rogers , University of Arizona , Tucson , USA; Klebba et al . , 2013; McLamarrah et al . , 2018 ) .", "The following secondary antibodies were used ( all at 1:1000 dilution ) : goat anti-mouse Alexa 594 , goat anti-rabbit Alexa 488 or Alexa 647 , goat anti-guinea pig Alexa 488 or Alexa 594 , goat anti-rat Alexa 488 or Alexa 555 or Alexa 647 ( Invitrogen ) and donkey anti-guinea pig Alexa 647 ( Jackson Immuno Research ) .", "Bang assays were performed as previously described ( Enjolras et al . , 2012 ) .", "Approximately 20 staged female flies were placed in graduated tubes ( 8 cm ) and banged on the table at t = 0 .", "The % of flies that reached each quarter of the tube was counted after 30 s .", "three different batchs were quantified for each genotype .", "Quantifications of mother versus daughter centrioles were based on the principle that the daughter centriole , being nucleated from the mother centriole , is orthogonally positioned on the lateral wall of the mother centriole .", "Quantifications of TZ protein intensities was performed using ImageJ and by measuring the sum of pixel intensity in a defined region encompassing the centrioles .", "Background intensity was measured by measuring the sum of pixel intensity in an area close to and devoid of centrioles and then subtracted to TZ protein intensities .", "Results of fluorescence intensity quantifications are represented as scatter plots with the mean and SD on all figures .", "Statistical significance was determined by two-tailed unpaired parametric Student’s t test ( Figure 4A , fertility assay; Figure 5D , Cby-Tom; Figure 6A , Cep290-GFP in fam921 ) or non-parametric Mann-Withney’s test when variances were not comparable ( all other intensity quantifications of Figure 5D and Figure 6A–C ) .", "Results of phenotypic proportions are represented as contingency bar graph and statistical significance was determined by two-tailed Chi-square ( Figures 5D and 6A ) .", "( Prism six software; ns , p>0 . 05; * , p≤0 . 05; ** , p≤0 . 01; *** , p≤0 . 001; **** , p≤0 . 0001 ) .", "Samples were processed as previously described ( Enjolras et al . , 2012; Vieillard et al . , 2015 ) .", "Observations were performed on at least two independent tissue samples ." ] ]

[ "Cilia and flagella are conserved eukaryotic organelles essential for cellular signaling and motility .", "Cilia dysfunctions cause life-threatening ciliopathies , many of which are due to defects in the transition zone ( TZ ) , a complex structure of the ciliary base .", "Therefore , understanding TZ assembly , which relies on ordered interactions of multiprotein modules , is of critical importance .", "Here , we show that Drosophila Dzip1 and Fam92 form a functional module which constrains the conserved core TZ protein , Cep290 , to the ciliary base .", "We identify cell type specific roles of this functional module in two different tissues .", "While it is required for TZ assembly in all Drosophila ciliated cells , it also regulates basal-body growth and docking to the plasma membrane during spermatogenesis .", "We therefore demonstrate a novel regulatory role for Dzip1 and Fam92 in mediating membrane/basal-body interactions and show that these interactions exhibit cell type specific functions in basal-body maturation and TZ organization ." ]

[ "Many animal cells have hair-like structures called cilia on their surface , which help them to sense and interact with their surroundings .", "The cilia are supported by protein filaments and must assemble correctly because faulty cilia can lead to several life-threatening diseases .", "Problems in an area at the base of the cilia , known as the ‘transition zone’ , account for the most severe forms of these diseases in humans .", "The transition zone is responsible for selecting which proteins are allowed in and out of the cilia .", "The transition zone itself is made up of many proteins that work together to determine the cilia composition .", "But not all of these proteins are known , and it is unclear how those that are known affect cilia structure .", "One protein found in transition zones of several animals , including fruit flies and mice , is called Cby .", "Lapart et al . set out to understand which other proteins interact with Cby in fruit flies to better understand what this protein does in the transition zone .", "A series of experiments showed that Cby interacts with two proteins called Dzip1 and Fam92 to regulate the assembly of transition zones .", "Together these three proteins constrain a core component of the transition zone , a fourth protein called Cep290 , to the base of the cilia .", "Fruit flies only have cilia on cells in their sensory organs and testes and , in both types of tissue , cilia could only form properly when Dzip1 and Fam92 were present .", "Lapart et al . also showed that , in the fruit fly testes , Dzip1 and Fam92 helped to anchor the newly forming cilia to the cell surface .", "This anchoring role was particularly important for the fruit flies’ sperm to grow their characteristic whip-like tails , which are a specialized type of cilia that allow sperm cells to move .", "Overall , the findings show how some transition zone proteins work together and that they can have different effects in different tissues .", "Understanding the mechanisms behind healthy cilia assembly will likely be key to tackling cilia-related diseases ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "microbiology and infectious disease" ]

[ [ "Mutation is the raw material of evolution .", "As a result , all organisms must strike a balance between allowing enough random mutations for selection to act upon , and the fact that most of these mutations are likely to be deleterious and must be purged from the population .", "This is particularly true for pathogenic microbes that take part in Red Queen conflicts with their hosts and require a continuous supply of mutations to remain competitive .", "In this case , increased pressure may exist to maintain an elevated mutation rate to increase the rate of adaptation .", "Increases in mutation rate may also serve to accelerate adaptation when microbes are introduced into new environments or encounter novel stresses , such as antimicrobial therapy .", "Adaptive variation in mutation rate is common in bacteria .", "One example is the Long Term Evolution Experiment ( LTEE ) , where defects in DNA repair emerged and the resulting hypermutator phenotype swept the population in six out of twelve E . coli lines ( Tenaillon et al . , 2016 ) .", "Mutator alleles likely emerge frequently but are typically purged from the population because individual mutations are more likely to be deleterious than adaptive; as a result hypermutators generally produce less fit offspring than non-hypermutators .", "However , occasionally a sufficiently beneficial mutation is potentiated by the presence of the hypermutator and the hypermutator allele is able to hitchhike to higher frequency within the population .", "This is more likely to occur in populations at local evolutionary minima , where many different large-effect adaptive mutations are possible .", "For example , hypermutator phenotypes are common in isolates of Pseudomonas aeruginosa growing within the lungs of patients with Cystic Fibrosis , where environmental conditions are constantly changing ( Oliver et al . , 2000 ) .", "This changing environment causes an ongoing need to adapt , and increases the likelihood of hypermutators emerging by increasing the target size for adaptive variation .", "However , once beneficial mutations have fixed , and organisms have adapted to their new environment , antimutator suppressor alleles can emerge to reduce the mutation rate or defective mutator alleles can even be replaced by functional alleles through horizontal gene transfer ( Wielgoss et al . , 2013; Denamur et al . , 2000 ) .", "This balance is likely critical to evolution in microbes as they face diverse stresses and environmental changes , and as a result over 1% of natural bacterial isolates display hypermutator phenotypes ( LeClerc et al . , 1996 ) .", "Even more extreme cases also exist , like in Mycoplasma pneumoniae and M . genitalium , where the mismatch repair machinery has been completely lost ( Himmelreich et al . , 1997 ) .", "In fact , many mutator phenotypes are the result of defects in mismatch repair , commonly referred to as MMR .", "In bacteria , MMR requires the MutHLS system , where MutS binds to mismatches and recruits MutL , which subsequently activates MutH to excise the mispair ( Su and Modrich , 1986; Au et al . , 1992; Grilley et al . , 1989; Cox et al . , 1972 ) .", "In eukaryotes , both the MutS and MutL families have expanded , yielding MutS homolog ( MSH ) ( Reenan and Kolodner , 1992 ) and MutL homolog ( MLH ) families ( Strand et al . , 1993 ) .", "Heterodimers of various MSH family members participate in mismatch repair , but Msh2 in particular is a core component of the majority of mismatch repair pathways in yeast ( Harfe and Jinks-Robertson , 2000 ) .", "As a result , defects in MMR , and MSH2 mutations in particular , result in elevated rates of simple mismatches , but also dramatically elevated rates of repeat tract instability ( Strand et al . , 1993 ) , which are associated with hereditary colon cancer in humans .", "MMR also plays a role in rejecting heteroduplexes of divergent sequences during both mitosis ( Datta et al . , 1997 ) and meiosis ( Alani et al . , 1994 ) , meaning that loss of the complex can lower species boundaries and allow increased recombination between divergent chromosomes ( Alani et al . , 1994; Hunter et al . , 1996; Rayssiguier et al . , 1989 ) .", "In contrast to bacteria , substantially less is known about hypermutators in natural populations of eukaryotes .", "Evolutionary theory predicts that the presence of sex abrogates selection for hypermutator strains by eliminating genetic linkage between the mutator allele and the associated beneficial mutations it hitchhikes upon to high frequency ( Tenaillon et al . , 2000 ) .", "Studies of allele incompatibility within the model fungus Saccharomyces cerevisiae have supported this traditional paradigm .", "Incompatible alleles of PMS1 and MLH1 exist within the population , such that a cross of two strains with wildtype mutation frequency can give rise to progeny with elevated mutation rates ( Heck et al . , 2006; Bui et al . , 2015 ) .", "However , the homozygous incompatible arrangement was not found among any of the original 65 strains examined , and was only identified within one clinical isolate of 1010 global diverse yeast isolates ( Bui et al . , 2017 ) and four clinical strains out of 93 sequenced in a separate study ( Skelly et al . , 2017 ) .", "This frequency is far below that of the individual alleles in the population , and in each case observed suppressors had arisen to restore a wildtype mutation rate despite the incompatibility .", "In contrast , eukaryotic mutators can adapt and thrive in clonally expanding populations such as in human tumor environments ( Bielas et al . , 2006 ) .", "Recent studies have begun to challenge this dogma in eukaryotic microorganisms as well , with over 50% of clinical C . glabrata isolates harboring loss of function mutations in MSH2 , resulting in loss of mismatch repair function ( Healey et al . , 2016 ) .", "Two groups also recently independently identified hypermutators in the fungal pathogen Cryptococcus neoformans .", "In the first case , long branch lengths were observed in genome sequencing of serial isolates from a recurrent infections that are correlated with nonsense mutations in MSH2 , MSH5 , and RAD5 Rhodes et al . , 2017 . In the second case , two of eleven clinical isolates exhibited elevated mutation rate that is linked to either an MSH2 nonsense mutation or multiple MSH2 missense mutations ( Boyce et al . , 2017 ) .", "In previous work we also identified a clade of candidate hypermutators within the Pacific Northwest Cryptococcus deuterogattii outbreak that contain a coding single base deletion within the critical mismatch repair component MSH2 ( Billmyre et al . , 2014 ) .", "Cryptococcus deuterogattii , is a basidiomycete human fungal pathogen previously known as Cryptococcus gattii VGII and recently elevated to species level ( Hagen et al . , 2015 ) , although this decision is disputed by a portion of the community ( Kwon-Chung et al . , 2017; Hagen et al . , 2017 ) .", "Unlike the other species of the Cryptococcus pathogenic species complex that infect immunocompromised hosts , C . deuterogattii is characterized by its ability to cause infection in otherwise healthy hosts ( Springer et al . , 2012 ) and by loss of the RNAi pathway ( Feretzaki et al . , 2016; D'Souza et al . , 2011 ) .", "C . deuterogattii is responsible for an ongoing outbreak in the Pacific Northwest region of the United States and Canada , which began in the late 1990's ( Kidd et al . , 2004 ) .", "This outbreak was originally analyzed using Multi-locus sequence typing ( MLST ) and shown to be comprised of three clonal expansions , denoted VGIIa , VGIIb , and VGIIc ( Fraser et al . , 2005; Byrnes et al . , 2010 ) .", "These three subtypes differ in terms of virulence and total number of cases , with VGIIa responsible for the majority of infections .", "More recently , whole genome sequencing studies of these subpopulations showed that the clonal subtypes appeared to have different proximal geographic origins , and most interestingly , that VGIIa has three very closely related isolates , here referred to as VGIIa-like , that were considerably less virulent and differed in isolation location or date from the outbreak ( Billmyre et al . , 2014 ) .", "We identified a candidate potential large effect mutation shared by these three diminished virulence isolates that resulted in a predicted non-functional Msh2 .", "Here we show that these VGIIa-like isolates exhibit a bona fide hypermutator phenotype .", "Furthermore , we show that homopolymer runs are particularly unstable , and that these runs are common within the coding regions of genes in the Cryptococcus genome .", "We predict that this results in dramatic phenotypic diversity from inactivation and possibly also activation or reactivation of genes .", "Finally , we show that the mutant msh2 allele is not directly responsible for the decrease in virulence of the VGIIa-like isolates , nor does it appear to have directly played a role in the evolution of virulence in the VGIIa clonal outbreak .", "Rather , it appears to represent a parallel route of adaptation to a new environment in a successful lineage isolated from two continents over two decades and from both the environment and patient samples .", "This work suggests that hypermutator phenotypes are not limited to prokaryotes , but may represent a frequent avenue to evolutionary change and phenotypic diversity in eukaryotic microbes as well ." ], [ "Because Msh2 is a critical component of the mismatch repair complex , we next tested whether the VGIIa-like strains displayed an increased mutation rate .", "We utilized a standard fluctuation assay approach to determine the rate of 5-FOA resistance , as previously applied in Cryptococcus ( Magditch et al . , 2012 ) .", "The resistance rate was increased in the hypermutator lineage between 4 . 4-fold at the minimum ( CBS7750 ) and 6 . 6-fold at the maximum ( ICB107 ) compared to the type strain VGIIa R265 ( Figure 2A ) .", "The majority of 5-FOA resistance in Cryptococcus neoformans is acquired through mutation of URA5 ( Kwon-Chung et al . , 1992 ) ; the URA5 locus was PCR amplified and sequenced in independently derived 5-FOA resistant isolates to determine the genetic basis of resistance .", "Mutations were characterized as either substitutions , or indels of either single or multiple bases ( Figure 2B ) .", "The majority of resistant isolates were the result of substitutions , and the mutation profile was similar between the hypermutator VGIIa-like strains and the non-hypermutator VGIIa strain EJB17 ( EJB17 vs NIH444: p=1 . 00 , EJB17 vs CBS7750 p=0 . 60 , EJB17 vs ICB107 p=1 . 00 , Fisher's Exact Test ) .", "Next , the whole genome data was analyzed to ascertain whether the similarity in mutation spectrum observed in the URA5 locus was recapitulated at multiple loci or if the URA5 locus had unique properties .", "To do this , we focused on two relatively long branches within the C . deuterogattii phylogeny: the branch separating the outgroup VGIIb strain R272 from the VGIIa clonal cluster ( 57 , 373 variants ) and the private ICB107 variants , which represented the longest available hypermutator branch ( 719 variants ) .", "The frequency of individual SNPs was examined first .", "While a high rate of transitions as compared to transversions was maintained in the hypermutator branch , there was a slight reduction in the frequency of A->G and T->C mutations relative to C->T and G->A mutations ( Figure 3A ) .", "However , much more striking was a dramatic increase in indels within homopolymer runs ( Figure 3B ) .", "While shifts in homopolymer runs in the R272 branch account for only 0 . 84% of the variants , in ICB107 they account for 45 . 3% , exceeding even the proportion of SNPs .", "By looking further at the context of the homopolymer run mutations within the ICB107 branch , it is apparent that longer base runs appear to be less stable than shorter runs , as approximately 24% of the mutations occur within the context of a nine base homopolymer run ( Figure 3C ) .", "This peak is likely a function of both the decreased stability of longer runs and the low quantity of longer homopolymer runs in the genome .", "Many of these longer runs occur within intergenic regions in the genome , but a substantial portion of the coding genes in C . deuterogattii contain at least one longer coding homopolymer run ( Figure 3C ) .", "The longest homopolymer run in the URA5 locus contains only four bases , which is predicted to be both relatively stable and among the coding genes with relatively shorter homopolymer runs ( Figure 3C ) .", "In contrast , the FRR1 gene that encodes the FKBP12 homolog responsible for the antifungal action of both FK506 and rapamycin has a run of seven cytosines ( 7C ) within its coding region .", "This places it both within the range of commonly mutated homopolymers in the ICB107 branch , and in the upper half of homopolymer containing genes in C . deuterogattii , suggesting that FRR1 is an appropriate locus to test the effect of mismatch repair mutants on homopolymers containing genes ( Figure 3C ) .", "The FRR1 locus was utilized to further assess whether VGIIa-like strains were hypermutators at more than one locus and to test the hypothesis that homopolymer runs are particularly unstable in these isolates .", "The protein product of FRR1 , FKBP12 , binds to either FK506 or rapamycin to form a protein-drug complex that inhibits calcineurin or TOR , respectively .", "By selecting with both drugs at the same time at the non-permissive temperature of 37°C , loss of function mutations in FRR1 are selected as the only single step mutation conferring resistance to both drugs .", "Here we utilized a semi-quantitative assay to identify gross differences in mutation rate whereby independent colonies were grown in liquid culture and then swabbed onto quadrants of a selective plate .", "On FK506/rapamycin media , the wildtype strains produced a small number of resistant colonies ( Figure 4A ) .", "In contrast , the NIH444 hypermutator strain produced prolific FK506/rapamycin resistant colonies .", "Surprisingly , the CBS7750 hypermutator was completely resistant to both drugs .", "Upon examining the FRR1 locus in the whole genome sequence data we discovered that there was already a single base deletion within the 7C homopolymer run in this strain .", "This may represent an unselected drug resistance phenotype potentiated by the msh2 defect .", "We attempted to use this assay for the ICB107 strain as well but were unsuccessful , likely as a result of its inability to grow at the higher growth temperature necessary for this assay that is based upon calcineurin-dependent growth at 37°C ( Figure 7B ) .", "Next a standard fluctuation assay approach was employed to quantify the mutation rate difference between the VGIIa and VGIIa-like hypermutators .", "There was a greater than 120-fold increase in mutation rate to FK506/rapamycin resistance in NIH444 compared to R265 , in contrast with the maximum of 6 . 6-fold increase observed for 5-FOA resistance ( Figure 4C ) .", "As before , we selected independent resistant colonies , PCR amplified , and sequenced the FRR1 gene to determine the mechanism of resistance .", "All resistance was explained by mutations within FRR1 as expected , but while R265 still primarily acquired resistance through substitutions , NIH444 now almost exclusively underwent single base additions or single base deletions , all within the 7C homopolymer run ( R265 vs NIH444 , p=0 . 0003 , Fisher's Exact Test ) .", "These indels resulted in nonsense mutations and resistance to both FK506 and rapamycin .", "Once we established a bona fide hypermutator phenotype in the VGIIa-like strains , we sought to verify that this phenotype was linked to the msh2 del131 allele .", "We crossed the NIH444 strain to a very closely related R265a congenic strain recently generated by serial backcrossing ( Zhu et al . , 2013 ) .", "C . deuterogattii matings are less fertile than C . neoformans , but we successfully dissected 14 viable spores from this cross .", "We typed these strains for the MSH2 allele by sequencing and observed 1:1 segregation of the MSH2 and the msh2 del131 alleles .", "The spores were then tested for rate of FK506/rapamycin resistance using the same semi-quantitative swabbing assay described above ( Figure 5A ) .", "While five of the msh2 del131 strains displayed the predicted hypermutator phenotype , surprisingly two of the spores ( #4 and 5 ) appeared wildtype despite inheritance of a defective copy of MSH2 .", "Mating in Cryptococcus has previously been demonstrated to produce phenotypic plasticity through the generation of aneuploid progeny at a high rate ( Ni et al . , 2013 ) .", "These aneuploids often display defects in growth at high temperature; thus , we tested whether the high growth temperature of 37°C employed in our FK506/rapamycin resistance assay may have masked the ability of these meiotic progeny to develop drug resistance .", "Congruent with this hypothesis , both spore products #4 and #5 demonstrated growth defects at 37°C and 39°C ( Figure 5C ) .", "Whole genome sequencing confirmed that both segregants carried an extra copy of one chromosome , suggesting that a 1N + 1 aneuploidy was causing temperature sensitivity ( Figure 5B ) .", "In fact , all of the progeny from this mating had an unusually high rate of aneuploidy with 8 of the 14 progeny aneuploid for at least one scaffold/chromosome ( Figure 5—source data 1 ) .", "By passaging all of the spores on YPD at 37°C we were able to restore the ability to grow at high temperatures in all of the progeny after four to nine passages after which all no longer demonstrated a high temperature growth defect ( Figure 5C ) .", "As a result the passaged derivatives of isolates #4 and #5 now demonstrated a hypermutator phenotype as expected ( Figure 5D ) and all 14 progeny produced the results predicted through linkage of the msh2 defect to the hypermutator phenotype ( Figure 5—figure supplement 1 ) .", "Further , whole genome typing of the 14 meiotic progeny demonstrated that sufficient recombination had occurred to establish linkage .", "All 14 progeny demonstrated unique SNP profiles and only one SNP demonstrated inheritance congruent with the hypermutator phenotype: a SNP on scaffold 3 that was linked to the msh2 del131 indel ( ~175 kb ) ( Figure 5E ) .", "As a final verification of phenotypic linkage , we constructed two independent deletions of the MSH2 gene in the R265 VGIIa background via biolistic transformation and homologous recombination replacing the MSH2 ORF with a neomycin resistance cassette .", "A complete deletion of MSH2 resulted in the same hypermutator phenotype as the msh2 del131 allele , providing further evidence that the hypermutator phenotype was linked to the loss of function in MSH2 ( Figure 5F ) .", "A fluctuation assay was used to determine the mutation rate for resistance to FK506 and rapamycin and demonstrated that the null mutants did not have an elevated mutation rate in comparison to the NIH444 strain ( Figure 5G ) .", "This suggests that suppressors have not arisen on the NIH444 background to moderate the effects of the msh2 del131 allele .", "During growth of the msh2Δ::NEO strains a spontaneous ade2 mutant was fortuitously identified based on its classic red pigmentation ( Figure 6A ) .", "We verified that this was the result of a defect in adenine biosynthesis as this strain grows on YNB supplemented with adenine , but not YNB media ( Figure 6B ) .", "As expected , sequencing of the ADE2 locus revealed that the ade- phenotype was linked to a single base mutation that results in a His->Arg amino acid change ( Figure 6C ) .", "The red pigment produced by ade2 mutants is a toxic intermediate in adenine synthesis that accumulates in the vacuoles of mutant cells .", "As a result , these mutants have a growth defect , and suppressor mutations can readily be isolated that eliminate production of this toxic intermediate and now produce white colonies ( Figure 6C ) .", "We isolated two red and two white derivative colonies and sequenced the ADE2 locus .", "While the red colonies retained the causative substitution , one of the white colonies retained it and the other had undergone reversion at this site back to the functional nucleotide ( Figure 6C ) .", "We confirmed that the revertant isolate was no longer an adenine auxotroph and that the second white isolate was still auxotrophic , despite the lack of pigmentation ( Figure 6D ) .", "This is likely the result of a second mutation upstream in the adenine biosynthetic pathway , resulting in further inactivation and loss of the ability to produce the toxic red intermediate .", "We tested a number of independent white reverted colonies for the ability to grow on media lacking adenine and observed that direct reversion to functional adenine biosynthesis was more common than additional inactivating mutations , with 37/42 ( 88% ) white colonies demonstrating adenine prototrophy ( Figure 6E ) .", "Taken together , these results suggest that hypermutation allows both frequent inactivation of pathways , and also the means to either adapt to the consequences or to simply directly revert the original mutation .", "In the context of antifungal drug action or activity , this phenotypic switching could be highly important .", "We previously demonstrated that the VGIIa-like mutants exhibit diminished virulence in a nasal instillation model of virulence ( Fraser et al . , 2005; Byrnes et al . , 2010 ) .", "Here , we tested whether loss of Msh2 function was directly responsible for the decrease in virulence but observed no difference in virulence between R265 and two independent de novo deletions of msh2 ( Figure 7A ) .", "This suggested that the defects in murine virulence in the VGIIa-like isolates could be the result of multiple independent deleterious mutations unique to each lineage .", "We next tested the ability to grow at higher temperature , a critical virulence factor , in the VGIIa-like isolates .", "All three isolates showed defects in high temperature growth , varying from minor defects in NIH444 , the isolate with the shortest branch to R265 , to moderate defects in CBS7750 , the isolate with an intermediate length branch , and finally severe defects in ICB107 , the isolate with the longest branch affected by the hypermutator ( Figure 7B ) .", "This suggests that instead of an immediate change in virulence , defects in mismatch repair may instead result in loss of virulence over time as mutations accumulate in critical pathways .", "Thus , rather than the virulence difference being explained by a single change or set of changes shared by all three VGIIa-like strains , instead they may all be avirulent for their own unique reasons .", "Our data suggests that over time a high mutation rate comes at a cost to organisms .", "We next addressed the early stages of hypermutator emergence and the direct effects of Msh2 inactivation to address if temporary benefits may be conferred .", "To this end , independent msh2 de novo deletions were employed in competition experiments with wildtype R265 .", "Briefly , liquid YPD cultures were mixed in 50:50 ratios of wildtype R265 and a neomycin resistant msh2 mutant or a random insertion of the neomycin resistance cassette as a control .", "After 48 hr incubation , co-cultures were spread onto YPD plates and then colonies were picked and restruck to media containing neomycin to determine the percentage of colonies derived from each original strain .", "If mutations are neutral , the expectation is that the 50:50 ratio will be maintained .", "Instead , growth defects were observed for the msh2 mutants at either 30°C or 37°C , suggesting that the msh2 mutation is deleterious under rich growth conditions ( Figure 7C ) .", "Notably , in three of the 24 replicates at 30°C or 37°C the msh2 mutant was able to grow moderately better than the wildtype parent strain .", "This may indicate that in these individual experimental replicates the hypermutator allowed a beneficial mutation and hitchhiked to higher frequency .", "However , under highly stressful conditions , such as exposure to the drugs FK506/rapamycin at 37°C , the hypermutator was highly advantageous .", "In multiple replicates the hypermutator rapidly acquired resistance and overtook the entire co-culture .", "This suggests that mutator alleles can be advantageous when a population faces an evolutionary landscape where large effect beneficial mutations exist .", "In contrast , mutators are deleterious in landscapes where few large effect mutations can provide advantages .", "Antifungal treatment and transitions from environment to host are likely to provide opportunities for adaptive mutation and favor mutator alleles .", "Historically , C . deuterogattii has been thought of as a tropical and subtropical pathogen .", "As a result , the origin of the VGIIa outbreak in the Pacific Northwest , a non-tropical environment , was surprising .", "In addition , ( 1 ) the South American origin of the ICB107 strain ( within the proposed cradle of the C . deuterogattii species [Hagen et al . , 2013] ) , ( 2 ) the isolation of NIH444 in Seattle near the outbreak origin , and ( 3 ) the diminished virulence of the VGIIa-like subclade ( Fraser et al . , 2005; Byrnes et al . , 2010 ) , all combined to suggest that the VGIIa-like group might have been the immediate precursors to the clonal VGIIa outbreak cluster ( Billmyre et al . , 2014; Engelthaler et al . , 2014 ) .", "Identification of the hypermutator phenotype further suggested that the defect in Msh2 , carried by the older VGIIa-like group , may have played a role in adaptation to the climate of the Pacific Northwest and may have even potentiated the increase in virulence .", "To test this , a maximum parsimony phylogeny of the VGIIa-like and VGIIa strains was constructed , including additional sequences for a related South American VGII clade not included in our previous analysis ( Engelthaler et al . , 2014 ) and using the VGIIb R272 strain as an outgroup ( Figure 8 ) .", "As described above , the msh2 mutation can allow restoration of function mutations at the exact mutation site that are indistinguishable from the original sequence ( Figure 6 ) .", "Likewise , mating could also reintroduce a functional MSH2 allele .", "For these reasons , we tested for the possible presence and impact of the past msh2 del131 mutator allele throughout the phylogeny by examining the imputed mutation spectrum .", "As discussed above ( Figure 3 ) , branches with defects in mismatch repair show an increased frequency of shifts in homopolymer runs .", "Increases in homopolymer run shifts were observed only on the most proximal branch ancestral to the VGIIa-like mutator lineage .", "This suggests that the mutator phenotype is congruent with the presence of the allele throughout the VGIIa-like phylogeny , and that the VGIIa group did not experience a transient period of msh2 mediated hypermutation , followed by repair or mating-mediated replacement .", "Instead , the VGIIa-like lineage may represent a unique pathway to adaptation distinct from that followed by the VGIIa group that resulted in diminished rather than enhanced virulence ." ], [ "In this study we have identified and characterized a successful lineage of eukaryotic hypermutators .", "Elevated mutations rates are a common adaptive mechanism in bacteria , but are typically thought of as transient states that allow beneficial mutations in the short term but are selected against in the long term ( Wielgoss et al . , 2013 ) .", "Bacteria solve this problem through horizontal gene transfer of genes from the mismatch repair pathway , allowing the initial beneficial mutation to be separated from the deleterious mutator allele ( Denamur et al . , 2000 ) .", "In contrast , few cases have been identified in natural isolates of eukaryotes , suggesting that variation in mutation rate may play a less substantial role than in bacteria .", "Recent studies have identified exceptions to this rule .", "In S . cerevisiae , incompatible alleles of PMS1 and MLH1 result in elevated mutation rates if present in combination ( Heck et al . , 2006; Bui et al . , 2015 ) .", "However , in the rare cases where the incompatible arrangement is found in nature , additional suppressors have arisen to restore wildtype mutation rate , suggesting that hypermutator phenotypes may not be tolerated over long periods of time ( Bui et al . , 2017; Skelly et al . , 2017 ) .", "In addition , a recent study of Candida glabrata has demonstrated that a substantial proportion of clinical isolates ( >50% ) carry nonsynonymous mutations in the MSH2 gene , some causing elevated mutation rates similar to the null phenotype and others with intermediate changes in mutation rate ( Healey et al . , 2016 ) .", "The authors correlate the mismatch repair defects with multi-drug resistance .", "A second study with a different sample cohort confirmed the presence of MSH2 mutations , but concluded that drug resistance was better correlated with drug exposure than with mismatch repair defects ( Dellière et al . , 2016 ) .", "In contrast with the studies in S . cerevisiae and C . glabrata , here we identified a group of viable hypermutator strains resulting from a nonsense mutation in MSH2 .", "These strains were isolated over a period of fifteen years from two different continents and include both clinical and environmental strains .", "Increases in mutation rate were similar , although slightly lower in magnitude , to those observed previously in the model yeast S . cerevisiae , where msh2 mutations increase resistance rate to Canavanine by 40 fold compared to 6 . 8 fold for 5-FOA here , or reversion of a 7xT run in a HOM3 mutant by 662 fold compared to 120 fold for 7xC in FRR1 here ( Marsischky et al . , 1996 ) .", "The mutation spectrum was also similar , as homopolymer run indels were responsible almost exclusively for mutations within run-containing genes in msh2 mutants , but not in wildtype strains as previously reported in S . cerevisiae ( Tran et al . , 1997 ) .", "The msh2 del131 allele carried by these strains appears to result in a complete loss of function , rather than simply reducing the efficiency of mismatch repair .", "This is distinct from the mutators identified in C . glabrata , and suggests that the VGIIa-like lineage is a successful and relatively long-lived hypermutator lineage , capable of both disseminating over a large area and persisting in the environment .", "Cryptococcus deuterogattii may also represent an intermediate between mutator-rich C . glabrata and mutator-poor S . cerevisiae .", "C . deuterogatttii is not an obligate pathogen and can cycle between an environmental lifestyle and an infectious lifestyle as an ‘accidental pathogen’ ( Springer et al . , 2012 ) .", "Elevated mutation rates may be selected for by Red Queen interactions with a host , meaning that pathogens like C . glabrata that grow only in association with their hosts would experience higher mutation rates than facultative pathogens like C . deuterogattii , or effectively nonpathogenic yeasts like S . cerevisiae .", "In fact , a putative recurrent infection of Cryptococcus neoformans was recently demonstrated to contain nonsense mutations in MSH2 , MSH5 , and RAD5 , predicted to result in a mutator phenotype ( Rhodes et al . , 2017 ) .", "In addition , a second recent study also identified two other hypermutator strains from clinical isolates of C . neoformans as well ( Boyce et al . , 2017 ) .", "Boyce et al . went on to examine changes in mutation rate and spectrum with similar results to those presented here , although with a much larger induction of mutation rate in URA5 than reported here ( around 200 fold vs 6 . 8 fold ) .", "This could reflect differences between DNA repair in C . neoformans and C . deuterogattii .", "They also observed changes in stability of homopolymer runs , similar to those described here but in a second homopolymer-containing locus .", "Interestingly , this study also observed loss of ability to grow at high temperatures in one of three replicates of msh2 mutants in an experimental evolution assay , like that observed here in the VGIIa-like hypermutator clade .", "Notable as well is that hypermutators in C . neoformans have thus far been observed only in clinical isolates , even in studies that included matched environmental sample populations like Boyce et al . This provides further evidence that microbial lifestyle may be affecting the frequency of mutator strains , with pathogens being more likely to evolve elevated mutation rate , perhaps in response to drug or host challenges .", "An alternate hypothesis is that ploidy may play a role in the success of mutator strains .", "C . deuterogattii , C . neoformans , and C . glabrata exist primarily as haploids in nature , while S . cerevisiae is predominantly a diploid .", "Diploid strains are buffered from the effects of loss of function mutations like those observed in homopolymer runs in this study , which may reduce the supply of large effect beneficial mutations for mutator alleles to hitchhike upon in diploids .", "However , past work in S . cerevisiae suggests that diploids adapt more quickly in msh2 mutant backgrounds , rather than less , suggesting that ploidy may play the opposite role , at least in S . cerevisiae ( Thompson et al . , 2006 ) .", "Evolutionary theory predicts that the admixture provided by sex in a population nullifies the ability of mutator alleles to hitchhike to high frequency ( Tenaillon et al . , 2000 ) .", "Unlike obligately sexual animals , or asexual bacteria , fungi can reproduce both sexually and asexually , with the frequency of sex varying substantially between different species .", "We previously described C . deuterogattii as a species characterized by long periods of mitotic clonal expansion and only intermittent sexual crosses ( Billmyre et al . , 2014 ) .", "A contibuting factor is likely the highly biased mating type distribution in C . deuterogattii , as the vast majority of C . deuterogattii isolates are MATα , with only a handful of MATa isolates described globally ( Byrnes et al . , 2010 ) .", "In Cryptococcus deneoformans , which shares a similar biased mating type distribution , this has resulted in the development of a unisexual α-α sexual cycle , dispensing with the obligate need for a MATa partner ( Lin et al . , 2005 ) .", "This unisexual cycle can result in both de novo variation , but also recombination and admixture at similar levels to that observed in typical bisexual crosses ( Ni et al . , 2013; Lin et al . , 2005; Sun et al . , 2014 ) .", "However , no laboratory unisexual cycle has been observed in C . deuterogattii to this point .", "Consequently , as strains are introduced to new locales , they may need to survive and adapt via mitotic growth without sexual crosses for long periods of time .", "This could elevate linkage between mutator alleles and the beneficial mutations they elicit , but also eliminate the ability to separate those beneficial mutations from additional deleterious alleles .", "We also showed that hypermutators can allow both inactivating mutations in genes , and reversion of those mutations to the wildtype .", "Mismatch repair-defective mutator strains are characterized by particularly high rates of slippage within homopolymer runs , and these occur at run lengths that are common within fungal genomes .", "This may indicate that eukaryotes may harbor contingency loci featuring homopolymer runs , like those observed in bacteria ( Moxon et al . , 2006 ) .", "Fungal contingency loci could be critical for responses to antifungal agents , both in the environment and the clinical setting .", "In addition , variation could be important in host-pathogen interactions as well .", "For example , in S . cerevisiae , tandem repeats are enriched within cell surface genes , which could enable alteration of antigenic diversity during pathogenic interactions ( Verstrepen et al . , 2005 ) .", "While these loci are unstable even in wildtype populations , defects in the MMR pathway could enhance this instability and result in increased diversity .", "In C . neoformans , a mutator phenotype has been previously described in a lab-passaged strain notable for frequent mutations in the RAM pathway , which enables a dimorphic transition between pseudohyphal and yeast phase growth , although the molecular basis of the elevated mutation rate is unknown ( Magditch et al . , 2012 ) .", "This transition is highly important for survival in the face of environmental threats such as amoeba , but compromises pathways responsible for high temperature growth , suggesting that oscillation between RAM+ and RAM- states may allow populations to survive as conditions change .", "In addition , we provided evidence that the VGIIa-like lineage was not a subvirulent progenitor of the Pacific Northwest outbreak , in contrast with previous hypotheses from two groups ( Billmyre et al . , 2014; Engelthaler et al . , 2014 ) .", "Rather , the VGIIa-like lineage is derived and may represent either an alternative pathway of adaptation or potentially an accelerated stage of the evolutionary trajectory of the outbreak .", "The ability to grow at high temperatures is a critical component of virulence for fungal pathogens .", "However , many pathogenic fungi are thought to be ‘accidental’ pathogens where virulence factors are selected for by other environmental factors .", "In this model , thermotolerance could be selected by high ambient temperatures in a non-host environment ( Casadevall et al . , 2003 ) .", "Moving from a tropical/sub-tropical environment in South America to the more temperate climate of the Pacific Northwest may relieve this high temperature selection .", "Consequently , the loss of ability to grow under thermal stress observed in the VGIIa-like strains may be adaptive for the majority of their growth conditions in the environment , but also render them relatively avirulent in mammals .", "This could suggest that as the VGIIa outbreak strains continue to adapt to the Pacific Northwest , they will gradually lose virulence potential , like the VGIIa-like lineage , making the outbreak self-limiting over time .", "Alternatively , the defects could simply reflect a decrease in viability caused by a long period of growth with an elevated mutation rate , resulting in mutational meltdown .", "The most severe temperature defect was observed in ICB107 , a strain isolated from a patient in Brazil and also the strain with the largest number of mutations separating it from the VGIIa group , which would support the second hypothesis .", "These strains may have been able to persist in the Pacific Northwest in spite of the high mutation load because of a bottleneck effect during their introduction .", "Our original hypothesis was that the common variants that differentiated the VGIIa group from the progenitor of the VGIIa-like mutator group would explain the decline in virulence observed in the VGIIa-like strains ( Billmyre et al . , 2014; Byrnes et al . , 2010 ) .", "However , the msh2 mutation , the only predicted large effect mutation , has no obvious immediate effect on virulence in a murine inhalation model .", "This is interesting , at least in part , because msh2 mutants of C . neoformans were previously reported to exhibit a modest growth advantage in mouse lungs in pooled signature tagged mutant experiments ( Liu et al . , 2008 ) .", "This suggests that the mutator strains are better able to adapt to growth in the mouse lung , but not in a way that correlates directly with virulence in a dissemination and survival model of virulence .", "Boyce et al . obtained similar results in C . neoformans to those reported here , with msh2Δ and mlh1Δ mutants appearing neutral in a murine infection model , and pms1Δ mutants even relatively less virulent ( Boyce et al . , 2017 ) .", "In addition , our results suggest that loss of virulence in the VGIIa-like strains may be a consequence of independent private mutations , i . e . each VGIIa-like strain is avirulent for its own reason .", "In previous work , CBS7750 and ICB107 have the most substantial virulence defects , while NIH444 has a more modest defect ( Byrnes et al . , 2010 ) .", "This is congruent with the fact that NIH444 has the shortest divergence from the VGIIa clonal group , while CBS7750 and ICB107 have much longer branches , and is also congruent with the high temperature growth defects we observed .", "Finally , we demonstrated that mutation spectrum analysis could be utilized within a phylogeny to infer the presence of a hypermutator allele through an increase in the proportion of indels within homopolymer runs .", "In bacteria , the hypermutator state is often transient and wildtype mismatch repair is often restored via horizontal gene transfer ( Denamur et al . , 2000 ) .", "This paradigm could also operate in fungi , with sexual recombination replacing direct DNA transfer .", "However , these events are difficult to detect if the hypermutator allele is purged from the population by selection after it is separated from its linked beneficial allele .", "The widespread availability of whole genome sequencing could now allow changes in mutation spectrum to be used to detect episodic hypermutation throughout entire populations of microbes .", "We suspect that these episodes are common throughout the evolution of eukaryotic microbes and may be even more common among pathogenic microbes , reflecting their natural history as well as the result of Red Queen host-pathogen conflicts ." ], [ "Strains used in this study are listed in Supplementary file", "1 . Primers used are listed in Supplementary file", "2 . Strains were routinely grown on YPD media at 30°C and maintained in permanent glycerol stocks at −80°C .", "Strains marked with neomycin resistance were grown on YPD supplemented with G418 .", "To conduct crosses and isolate spores , NIH444 was cocultured with the R265a congenic strain ( Zhu et al . , 2013 ) on solid V8 pH = 5 . 0 medium in the dark for eight weeks .", "Basidiospores were isolated using a microdissection microscope equipped with a 25 μm needle ( Cora Styles Needles ‘N Blocks , Dissection Needle Kit ) as previously described ( Hsueh et al . , 2006 ) . Approximately eight week old A/J mice were anesthetized with phenobarbital via intraperitoneal injection and then were infected with 5 × 104 cells from strains R265 , RBB17 , and RBB18 by intranasal inhalation . Ten animals per group were infected . Mice were monitored daily for signs of cryptococcal infection and sacrificed when exhibiting signs of clinical distress . The animal study was conducted in the Division of Laboratory Animal Resources ( DLAR ) facilities at Duke University Medical Center ( DUMC ) . All of the animal work was performed according to the guidelines of NIH and Duke University Institutional Animal Care and Use Committee ( IACUC ) under protocol number A245-13-09 . Deletions of the MSH2 gene were constructed using a standard overlap PCR approach as previously described ( Davidson et al . , 2000 ) . Briefly , 1 kb flanking regions of genomic DNA were amplified from both the 5' and 3' regions flanking the MSH2 open reading frame in R265 and the selectable marker for NEO resistance was amplified from plasmid pJAF1 . An overlap PCR was carried out to generate a full-length deletion cassette and R265 was transformed using biolistic transformation . Gene replacement was confirmed using in-gene , 5' junction , 3' junction , and spanning PCRs . Two independent transformations of independent overnight cultures of R265 were carried out to isolate independent mutants . The R265 NEO resistance marked strain was constructed by transforming XbaI digested pJAF1 plasmid into wild-type R265 by biolistics . Transformants were selected for on YPD containing neomycin . Transformants were checked for tandem insertions using primers JOHE40500/JOHE40501 and a strain with only a single insertion was chosen and employed for the competition assay . Fluctuation assays were performed on either synthetic medium containing 5-FOA ( 1 g/L ) at 30°C or YPD supplemented with rapamycin ( 1 μg/mL ) and FK506 ( 1 μg/mL ) at 37°C . For each strain tested , ten independent 5 mL YPD liquid cultures were grown overnight at 30°C . Cultures were then split and either spread directly on selective media or diluted and spread on solid YPD to determine colony forming units . Resistant colonies and total colonies were counted and mutation rate was calculated using the Maximum Likelihood method as implemented via the FALCOR calculator ( Hall et al . , 2009 ) . Competition assays were carried out by growing independent liquid cultures overnight in 5 mL YPD . Cultures were then counted using a hemocytometer and 500 , 000 cells each of a tester neomycin resistant strain and a wildtype R265 culture were mixed in a 5 mL liquid YPD culture . Co-cultures were grown for 48 hr and then spread onto a YPD plate , such that individual colonies could be isolated . All colonies were picked ( up to 60 and at least 5 ) and restruck to neomycin media to determine the proportion of colony forming units derived from each of the original strains in the competition . DNA was isolated with the CTAB isolation protocol as previously described ( Pitkin et al . , 1996 ) . Library construction and genome sequencing were carried out at the University of North Carolina Next Generation Sequencing Facility . Paired-end libraries with approximately 300-base inserts were constructed and 100 base reads were generated A number of genome sequences were previously available ( Billmyre et al . , 2014; Engelthaler et al . , 2014 ) . The remaining were generated here and are deposited on the SRA under project accession no . PRJNA387047 . All sequences were mapped to the V2 R265 reference genome ( Farrer et al . , 2015 ) . Alignment was performed using BWA MEM with default settings ( Li and Durbin , 2009 ) . Further processing was carried out using the Genome Analysis Toolkit ( GATK ) version 3 . 8 ( McKenna et al . , 2010 ) , including SAMtools ( Li et al . , 2009 ) and Picard . SNPs and indels were called with the HaplotypeCaller from GATK , using the haploid setting . GATK's VariantFiltration module was used to hard filter variants as suggested using the following expressions: ‘QD <2 . 0 || FS >60 . 0 || MQ <40 . 0 \" for SNPs and ‘QD <2 . 0 || FS >200 . 0’ for indels .", "GATK's VariantAnnotator was used to define the homopolymer context of indels .", "VCFtools was used to filter sites with missing data and extract private variants ( Danecek et al . , 2011 ) .", "Variants resulting from erroneous calls in repetitive regions were manually removed .", "SnpEff was employed to determine the predicted impact of mutations ( Cingolani et al . , 2012 ) .", "Maximum parsimony phylogenies were constructed using MEGA6 ( Tamura et al . , 2013 ) to analyze SNP matrices extracted from VCF format using a custom Perl script ( Source code file 1 ) .", "SNPs and indels were placed on branches where they were predicted to change states using VCFtools ( Danecek et al . , 2011 ) to extract the variants supporting each node ." ] ]

[ "Pathogenic microbes confront an evolutionary conflict between the pressure to maintain genome stability and the need to adapt to mounting external stresses .", "Bacteria often respond with elevated mutation rates , but little evidence exists of stable eukaryotic hypermutators in nature .", "Whole genome resequencing of the human fungal pathogen Cryptococcus deuterogattii identified an outbreak lineage characterized by a nonsense mutation in the mismatch repair component MSH2 .", "This defect results in a moderate mutation rate increase in typical genes , and a larger increase in genes containing homopolymer runs .", "This allows facile inactivation of genes with coding homopolymer runs including FRR1 , which encodes the target of the immunosuppresive antifungal drugs FK506 and rapamycin .", "Our study identifies a eukaryotic hypermutator lineage spread over two continents and suggests that pathogenic eukaryotic microbes may experience similar selection pressures on mutation rate as bacterial pathogens , particularly during long periods of clonal growth or while expanding into new environments ." ]

[ "As humans , we often think of genetic mutations as being bad .", "Over the past several decades we have seen health warnings issued on a variety of environmental exposures , from cigarettes to tanning beds , and with good reason because they cause mutations .", "For multicellular organisms like humans , these mutations are strongly associated with cancer .", "But in bacteria , this is not true .", "In fact , the rate at which mutations occur sometimes increases to help bacteria cope with stressful environments .", "Unlike bacteria , humans are eukaryotes – the name given to organisms whose cells contain different compartments separated by membranes , such as the nucleus of the cell .", "For years , we have assumed that eukaryotic microbes , like fungi and parasites , act more like humans than like bacteria because work in budding yeast ( another eukaryote ) has suggested this to be the case .", "However , recent work in disease-causing fungi has shown that , much like bacteria , elevated mutation rates may help them to respond to stress .", "This could also enable fungi to become resistant to drugs used to treat fungal infections .", "Cryptococcus deuterogattii is a fungus that causes human diseases including meningoencephalitis and a lung infection called pulmonary cryptococcosis .", "An ongoing outbreak of the fungus began in the Pacific Northwest of Canada in the late 1990s and emerged in the United States in 2006/2007 .", "Among isolates closely related to those fungi causing the outbreak , three were found that appear to have a specific mutation in their DNA mismatch repair pathway , meaning that they may also experience a higher mutation rate .", "These strains are also less able to cause disease than others .", "Billmyre et al . now demonstrate experimentally that all three isolates have a specific DNA mismatch repair defect , and show that these fungi experience elevated mutation rates , resulting in what is known as a hypermutator state .", "Furthermore , whole genome sequencing and phylogenetic analysis showed that these hypermutator strains are derived from the outbreak-causing fungi , and that their reduced ability to cause disease is likely a result of accumulating mutations and the loss of the ability to grow at the higher temperatures found in the human body .", "Fungal infections are difficult to treat , in part because there are a limited number of available drugs .", "Elevated mutation rates will likely increase how often and how rapidly fungi develop resistance to these drugs .", "Understanding how commonly fungi exhibit a hypermutator state that could impact the development of drug resistance will therefore be important for treating patients with fungal infections , which account for millions of infections and hundreds of thousands of deaths annually worldwide ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "immunology and inflammation" ]

[ [ "To generate mature T cells that express functional and self-tolerant T cell receptors ( TCRs ) , T cells undergo elaborate developmental selection and maturation processes within the thymus ( Starr et al . , 2003 ) .", "Signals that result from successful TCR β-chain rearrangement and a pre-TCR formation drive the most immature CD4 and CD8 double negative ( DN ) thymocytes to become CD4 and CD8 double positive ( DP ) , known as β-selection ( Mallick et al . , 1993 ) .", "Following TCR α-chain rearrangement and mature TCR expression , DP thymocytes undergo positive selection using their newly assembled TCRs to recognize self peptide-major histocompatibility ( pMHC ) proteins expressed by the cTECs ( cortical thymic epithelial cells ) .", "A small number of DP thymocytes successfully undergo positive selection and ultimately mature into CD4 or CD8 single positive ( SP ) cells ( Scollay and Shortman , 1985; Petrie et al . , 1990; Starr et al . , 2003 ) .", "TCR-mediated signals that determine thymocyte development are only partially understood .", "Following positive selection in the thymic cortex , positively selected thymocytes migrate into the medulla and mature to ensure their functional competency to react to cognate antigens and to survive in the periphery .", "Fully mature SP thymocytes become competent to proliferate , when stimulated via their TCR , in contrast to DP or semi-mature SP thymocytes that are subject to apoptosis ( Hogquist et al . , 2005; Takada et al . , 2011 ) .", "Moreover , post-positive selection DP and SP thymocytes increase their IL-7R expression to promote their survival ( Van De Wiele et al . , 2004; Yu et al . , 2006 ) .", "As SP thymocytes mature , they decrease expression of CD69 and CD24 and concurrently increase expression of CD62L and Qa2 ( Vernachio et al . , 1989; Ramsdell et al . , 1991 ) .", "Therefore , stages of CD4SP thymocyte maturation can be further defined as ‘semi-mature’ ( CD69/CD24hi CD62L/Qa2low ) and ‘mature’ ( CD69/CD24low CD62L/Qa2hi ) states .", "Only functionally mature T cells exit the thymus , which is enabled , in part , by the marked increase in expression of egress receptors , including sphingosine 1 phosphate receptor 1 ( S1P1 ) ( Allende et al . , 2004; Matloubian et al . , 2004; Weinreich and Hogquist , 2008 ) .", "The transcription factor Kruppel Lung Factor 2 ( KLF2 ) controls expression of S1P1 and CD62L ( Carlson et al . , 2006; Weinreich and Hogquist , 2008 ) , thereby governing egress of T cells from the thymus and facilitating entry to the lymph nodes .", "However , it is not clear which signaling pathways control maturation of SP thymocytes after selection and promote egress by increasing KLF2 .", "The actin cytoskeleton and proteins that regulate it are involved in almost all aspects of T cell biology .", "The cytoskeleton , composed of actin filaments , microtubules , and intermediate filaments , provides mechanical support for organization of cellular structure .", "Reorganization of the cytoskeleton generates dynamic changes in cell shape during cell–cell interaction and migration .", "TCR engagement drastically induces changes in actin cytoskeletal architecture and reorganizes it .", "Actin cytoskeletal reorganization influences T cell signaling leading to T cell activation .", "For instance , TCR-mediated actin polymerization is required to form extensive contacts between T cells and antigen presenting cells ( APCs ) , organize gross cell polarity , and stabilize signaling complexes acting as a scaffold for further assembly ( Wülfing and Davis , 1998; Kaizuka et al . , 2007; Babich et al . , 2012 ) .", "However , the mechanism that links the actin cytoskeleton to T cell signaling is not well known .", "Rho family GTPases such as Rac and Cdc42 regulate cytoskeletal reorganization , microtubule dynamics , and cell polarity in many cell types , including T cells ( Zhao and Manser , 2005 ) .", "Activation of Rho family GTPases is tightly regulated by guanine nucleotide exchange factors ( GEFs ) and GTPase-activating proteins ( GAPs ) ( Tybulewicz and Henderson , 2009 ) .", "Vav family proteins ( Vav1 , Vav2 and Vav3 ) are GEFs for Rac and Cdc42 ( Tybulewicz , 2005 ) .", "Rac , Cdc42 , and Vav play crucial roles in T cell development and activation .", "For example , mice lack both isoforms of Rac1 and Rac2 or mice deficient in Vav1 show defects in pre-TCR β-selection and positive selection and T cell activation ( Turner et al . , 1997; Fujikawa et al . , 2003; Guo et al . , 2008; Dumont et al . , 2009; Tybulewicz and Henderson , 2009 ) .", "Cdc42-deficient mice display impaired positive selection and homeostasis of T cells ( Guo et al . , 2010 , 2011 ) .", "Rac and Cdc42 exert their functions by binding and activating to a large collection of their effector molecules .", "However , it is not known which protein among many effector molecules of Rac and Cdc42 is required to mediate their defined functions in T cells .", "The p21-activated kinases ( PAKs ) , effector molecules of Rac and Cdc42 , participate in diverse cellular signaling pathways including cell spreading , adhesion , migration , activation , proliferation , and survival ( Manser et al . , 1994; Bokoch , 2003 ) .", "PAKs are serine/threonine kinases that phosphorylate multiple substrates , including those that are involved in cytoskeletal reorganization , cell proliferation , and survival .", "The group I PAK family consists of three kinases ( Pak1 , Pak2 and Pak3 ) ( Bokoch , 2003; Kelly and Chernoff , 2012 ) .", "Pak2 is the major PAK expressed in T cells ( Chu et al . , 2004 ) and has been implicated in T cell function ( Bubeck Wardenburg et al . , 1998; Yablonski et al . , 1998; Phee et al . , 2005 ) .", "However , the in vivo role of Pak2 in T cells is not known due to embryonic lethality of Pak2 knock-out ( KO ) mice ( Hofmann et al . , 2004; Kelly and Chernoff , 2012 ) .", "Given that Pak2 is one of the well-described effector molecules of Rac and Cdc42 , it has long been speculated that Pak2 may mediate the function of Rac and Cdc42 in T cell development and activation .", "However , considering the fact that Rac and Cdc42 have many effector molecules and may utilize a combination of effectors to mediate a defined cellular function , it was unclear whether Pak2 is the effector molecule that mediates the function of Rac and Cdc42 in T cell development and activation .", "In this study , we show for the first time that Pak2 is required for the development , maturation , and timed egress of thymocytes .", "Lineage-specific deletion of Pak2 at an early T cell developmental stage using the Lck-Cre impaired pre-TCR β-selection and positive selection similar to the mice that lack Rac1/2 or Vav1 .", "Surprisingly , deletion of Pak2 at a later developmental stage using the Cd4-Cre severely inhibited functional maturation of CD4SP thymocytes , including impaired expression of S1P1 accompanied by reduced expression of its transcription factor , KLF2 .", "TCR-mediated signaling was greatly decreased in the absence of Pak2 , including Nur77 induction and S6 phosphorylation , leading to decreased proliferation .", "Mechanistically , Pak2 deficiency impaired actin cytoskeletal remodeling and activation of PLCγ1 and Erk1/2 triggered by plate-bound TCR stimulation .", "These findings reveal a critical function of Pak2 as an essential regulator that drives the actin cytoskeleton-dependent signaling for normal thymocyte development and maturation ." ], [ "To examine the role of Pak2 in vivo , we generated Pak2−/− mice using a conventional knock-out ( KO ) strategy .", "However , Pak2 deficiency resulted in lethality at embryonic day 8 ( Hofmann et al . , 2004; Arias-Romero and Chernoff , 2008; Kelly and Chernoff , 2012 ) .", "To circumvent this problem , we generated conditional Pak2 KO mice by flanking exon 2 of Pak2 with loxP sites ( Kosoff et al . , 2013 ) and introduced , by breeding , two different Cre transgenes , Lck-Cre ( Hennet et al . , 1995 ) and Cd4-Cre ( Lee et al . , 2001 ) , in which the Cre recombinase is expressed under the promoters of T cell-specific Lck or Cd4 genes .", "The Cre genes in these mice are expressed at different developmental stages .", "The Lck-Cre transgene is expressed during the DN2 ( CD4-CD8-double negative 2 ) stage ( Hennet et al . , 1995 ) , while the Cd4-Cre transgene is expressed later at the DP stage ( Lee et al . , 2001 ) .", "We confirmed that over 95% of Pak2 protein was deleted in DP thymocytes from Pak2F/F;Lck-Cre or Pak2F/F;Cd4-Cre mice ( Figure 1A–C ) . 10 . 7554/eLife . 02270 . 003Figure 1 . T cell lymphopenia in T cell-specific Pak2-deficient mice .", "( A ) Western blot analysis using anti-Pak2 antiserum and cell lysates from the thymus of Pak2F/F ( WT ) , and Pak2F/F;Cd4-Cre ( KO ) mice .", "Anti-Erk1/2 antibody was used as loading control .", "Shown are representative of two independent experiments .", "( B ) Quantitative PCR analysis of Pak2 mRNA expression in DP , semi-mature and mature CD4SP thymocytes .", "Shown are Pak2 mRNA levels from DP , semi-mature and mature CD4SP thymocytes relative to Pak2F/F DP thymocytes ( error bars; SD ) .", "Data are representative of two independent experiments .", "( C ) Western blot analysis using anti-PAK2 and cell lysates from the thymi of Pak2F/F ( WT ) , Pak2F/+;Lck-Cre ( HET ) and Pak2F/F;Lck-Cre ( KO ) mice .", "( D ) Representative flow cytometry analyses of CD4 and CD8 expression on lymphocytes from spleens ( n = 5 ) , peripheral lymph nodes ( pLn; axillary , brachial and inguinal lymph nodes , n = 4 ) , and mesenteric lymph nodes ( mLn , n = 4 ) from Pak2F/F ( WT ) and Pak2F/F;Lck-Cre ( KO ) mice .", "Numbers in each quadrant represent the percentage of cells in the indicated quadrant .", "( E ) Flow cytometry analyses of CD62L and CD44 on CD4+ T cells from Pak2F/F ( WT ) and Pak2F/F;Lck-Cre ( KO ) mice .", "( F ) Quantification of cell numbers of different lymphocyte subsets from Pak2F/F and Pak2F/F;Lck-Cre mice .", "Error bars: SD ( spleen [n = 5 mice per genotype] , pLn [n = 4 mice per genotype] , and mLn [n = 4 mice per genotype]; blood [n = 2 mice per genotype] ) .", "* , 0 . 01<p<0 . 05; ** , 0 . 001<p<0 . 01; *** , 0 . 0001<p<0 . 001; **** , p<0 . 0001 ( unpaired two-tailed Student’s t test ) .", "( G ) Representative flow cytometry analyses of CD4 and CD8 expression on lymphocytes from spleen , pLn , mLn , and blood from Pak2F/F ( WT ) and Pak2F/F;Cd4-Cre ( KO ) mice .", "Spleen ( n = 4 mice ) , pLn ( n = 4 mice ) , mLN ( n = 3 ) and blood ( n = 2 mice ) .", "( H ) Flow cytometry analyses of CD62L and CD44 within CD4+ T cells from Pak2F/F and Pak2F/F;Cd4-Cre mice .", "( I ) Absence of naïve ( CD62Lhi CD44low ) CD4 or CD8 T cells generated from Pak2F/F;Cd4-Cre donor bone marrow cells in 1:1 mixed bone marrow chimeras .", "Data shown are representative of five bone marrow chimeras .", "( J ) Quantification of cell numbers of different subsets from Pak2F/F and Pak2F/F;Cd4-Cre mice .", "Error bars: SD ( spleen [n = 4 mice per genotype] , pLn [n = 4 mice per genotype] , mLn [n = 3 mice per genotype] , blood [n = 2 mice per genotype] ) .", "* , 0 . 01<p<0 . 05; ** , 0 . 001<p<0 . 01; *** , 0 . 0001<p<0 . 001; **** , p<0 . 0001 ( unpaired two-tailed Student's t test ) .", "See Figure 1—figure supplements 1 and 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 02270 . 00310 . 7554/eLife . 02270 . 004Figure 1—figure supplement 1 . T cell lymphopenia in T-cell specific Pak2-deficient mice .", "( A ) Quantification of cell numbers of different lymphocyte subsets from Pak2F/F and Pak2F/F;Lck-Cre mice .", "Error bars: SD ( spleen [n = 5 mice per genotype] , pLn [n = 4 mice per genotype] , and mLn [n = 4 mice per genotype]; blood [n = 2 mice per genotype] ) .", "* , 0 . 01<p<0 . 05; ** , 0 . 001<p<0 . 01; *** , 0 . 0001<p<0 . 001; **** , p<0 . 0001 ( unpaired two-tailed Student's t test ) .", "( B ) Quantification of cell numbers of different subsets from Pak2F/F and Pak2F/F;Cd4-Cre mice .", "Error bars: SD ( spleen [n = 4 mice per genotype] , pLn [n = 4 mice per genotype] , mLn [n = 3 mice per genotype] , blood [n = 2 mice per genotype] ) .", "* , 0 . 01<p<0 . 05; ** , 0 . 001<p<0 . 01; *** , 0 . 0001<p<0 . 001; **** , p<0 . 0001 ( unpaired two-tailed Student's t test ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 02270 . 00410 . 7554/eLife . 02270 . 005Figure 1—figure supplement 2 . A T cell intrinsic role of Pak2 in T cell lymphopenia .", "( A ) Flow cytometry analysis of 1:1 mixed bone marrow chimeras .", "Chimeras were generated by transferring 1:1 mixed WT Pak2+/+ ( CD45 . 1+CD45 . 2+ ) and KO Pak2F/F;Cd4-Cre ( CD45 . 2+ ) donor bone marrows into lethally irradiated C57BL6 hosts that express CD45 . 1 .", "Data shown are representative of five bone marrow chimeras .", "CD4-CD8-double negative ( DN ) thymocytes generated from donor hematopoietic stem cells were gated using CD45 . 2 markers , and plotted with CD45 . 1 expression to distinguish T cells produced from WT ( CD45 . 1 positive ) and Pak2F/F;Cd4-Cre ( CD45 . 1 negative ) donors ( first panel ) .", "Reconstitution of CD19+ B cells from spleen or pLN by WT ( CD45 . 1 positive ) and Pak2F/F;Cd4-Cre ( CD45 . 1 negative ) donors showed similar contribution of donor BM cells from WT and Pak2F/F;Cd4-Cre mice ( second panel ) .", "In contrast , CD4 and CD8 T cells from Pak2F/F;Cd4-Cre ( CD45 . 1 negative ) donors were markedly underrepresented compared to those from WT ( CD45 . 1 positive ) donors ( third and fourth panels ) .", "( B ) Total lymphocytes generated either from Pak2+/+or Pak2F/F;Cd4-Cre donor bone marrow cells were gated and plotted as CD19 vs CD3 expression .", "( C ) Expression of CD4 and CD8 is shown in CD3 positive cells generated either from Pak2+/+ or Pak2F/F;Cd4-Cre donor bone marrow cells . DOI: http://dx . doi . org/10 . 7554/eLife . 02270 . 005 T-cell specific Pak2 deletion using Lck-Cre or Cd4-Cre transgenes resulted in severe T cell lymphopenia ( Figure 1D , F , G , J , Figure 1—figure supplement 1 ) .", "Both CD4 and CD8 peripheral T cells were reduced similarly .", "Naïve T cells ( CD62LhiCD44low ) were almost absent and the few remaining T cells were mostly CD44hi ( Figure 1E , H ) .", "We detected considerable expression of Pak2 in the CD44hi activated ( or memory ) cells , suggesting that residual CD44hi T cells had undergone lymphopenia-associated expansion from the few T cells that had escaped Cre-mediated deletion ( data not shown ) .", "Mixed bone marrow chimera ( 1:1 ratio ) experiments revealed that naive T cells generated from Pak2F/F;Cd4-Cre ( Figure 1I , Figure 1—figure supplement 2A–C ) or Pak2F/F;Lck-Cre ( data not shown ) hematopoietic stem cells had a cell-intrinsic disadvantage in reconstituting the secondary lymphoid organs .", "These results indicate that Pak2 is required for generation or homeostasis of peripheral T cells .", "To determine whether T cell lymphopenia in the absence of Pak2 is due to impaired T cell development , we analyzed thymocyte development .", "Onset of deletion of Pak2 at the DN2 stage in Pak2F/F;Lck-Cre mice inhibited the transition from the DN to DP stage and subsequent positive selection .", "Total , DP and CD4 and CD8 SP thymocyte numbers were markedly decreased ( Figure 2A , B ) .", "DN3 thymocytes in Pak2F/F;Lck-Cre mice were increased with impaired expression of intracellular TCR β-chain at DN3 but not DN4 stages , suggesting a relative block in development at the checkpoint mediated by the pre-TCR ( Figure 2C , D ) .", "The majority of TCRhi cells were CD4 or CD8 SP thymocytes in WT mice , but Pak2 deficiency substantially reduced TCRhi CD4 or CD8 SP thymocytes ( Figure 2E ) .", "To eliminate repertoire selection bias , we evaluated the influence of Pak2 deficiency in the context of the ovalbumin-peptide class II MHC specific OTII TCR transgene ( Barnden et al . , 1998 ) .", "The generation of OTII TCR transgene+ CD4SP thymocytes was greatly reduced , suggesting defective positive selection ( Figure 2F–H ) .", "In addition , the expression of CD5 was markedly lower in DP and CD4SP thymocytes ( Figure 2I ) with a reduced percentage of post-positive selection cells ( data not shown ) from OTII+;Pak2F/F;Lck-Cre mice , indicative of lower basal TCR signaling ( Azzam et al . , 1998 ) .", "Thus , deletion of Pak2 at the DN2 stage markedly impaired T cell development in the thymus by impairing both pre-TCR β-selection and positive selection . 10 . 7554/eLife . 02270 . 006Figure 2 . Pak2 is required for T cell development .", "( A ) Flow cytometry of lymphocytes from thymi of Pak2F/F or Pak2F/F;Lck-Cre mice ( n = 5 ) .", "( B ) Quantification of cell numbers of different thymic subsets from Pak2F/F and Pak2F/F;Lck-Cre mice .", "Error bars: SEM ( n = 5 mice per genotype ) .", "** , 0 . 001<p<0 . 01; *** , 0 . 0001<p<0 . 001; ( unpaired two-tailed Student's t test ) .", "( C ) Expression of CD44 and CD25 on DN thymocytes from Pak2F/F or Pak2F/F;Lck-Cre mice .", "( D ) Expression of intracellular TCRβ chain in DN3 or DN4 thymocytes .", "( E ) Expression of CD4 and CD8 on TCRhi ( or CD3hi ) thymocytes from Pak2F/F or Pak2F/F;Lck-Cre mice .", "( F ) Percentage of CD4SP thymocytes in OTII+;Pak2F/F;Lck-Cre mice ( n = 3 ) .", "( G ) Cell numbers of different thymic subsets from OTII+;Pak2F/F or OTII+;Pak2F/F;Lck-Cre mice .", "Graphs in this figure show mean ± SEM ( n = 3 ) .", "** , 0 . 001<p<0 . 01 ( H ) Expression of TCR transgene assessed by anti-Vα2 antibody in total thymocytes from OTII+;Pak2F/F or OTII+;Pak2F/F;Lck-Cre mice .", "( I ) Expression of CD5 was reduced on DP thymocytes or CD4SP thymocytes from OTII+;Pak2F/F;Lck-Cre mice .", "Shown are representative of three mice per genotype . DOI: http://dx . doi . org/10 . 7554/eLife . 02270 . 006 Although we found that Pak2 is required for pre-TCR β selection and positive selection in Pak2F/F;Lck-Cre mice , defects that occurred in DN and DP stages in these mice prevented us from studying the impact of Pak2 on a later developmental stage , namely , the SP stage .", "To define the effects of Pak2 deficiency at later stages of thymocyte development , we analyzed T cell development in thymi in Pak2F/F;Cd4-Cre mice , in which Pak2 is deleted at the DP stage using the Cd4 promoter-driven Cre transgene .", "Numbers of DP and CD4 and CD8 SP thymocytes were similar , suggesting generation of CD4 and CD8 SP thymocytes was normal in Pak2F/F;Cd4-Cre mice ( Figure 3A , B ) .", "Expression of CD5 , CD69 , and CD3 on DP thymocytes were similar between Pak2F/F and Pak2F/F;Cd4-Cre mice ( data not shown ) .", "The apparently normal development of DP and SP thymocytes in Pak2F/F;Cd4-Cre mice suggested that Pak2 may be dispensable for positive selection or a defect is masked by a compensatory mechanism in vivo .", "To eliminate compensatory changes in the TCR repertoire , we introduced the OTII transgene into Pak2F/F;Cd4-Cre mice .", "The generation of CD4SP thymocytes was markedly reduced in OTII+;Pak2F/F;Cd4-Cre mice ( Figure 3C–E ) , suggesting that Pak2 is required for positive selection . 10 . 7554/eLife . 02270 . 007Figure 3 . Defects in positive selection in OTII+;Pak2F/F;Cd4-Cre mice .", "( A ) Flow cytometry analyses of CD4 SP thymocytes of Pak2F/F ( WT ) , Pak2+/+;Cd4-Cre ( WT ) , Pak2F/+;Cd4-Cre ( Het ) , or Pak2F/F;Cd4-Cre ( KO ) mice .", "( B ) Quantification of cell numbers of different thymic subsets .", "Error bars: SEM ( n = 10 mice ) .", "( C ) CD4 and CD8 FACS analyses showing decreased percentage of CD4SP thymocytes in OTII+;Pak2F/F;Cd4-Cre mice .", "Shown are representative data from three mice from each genotype .", "( D ) Cell numbers of different thymic subsets from OTII+;Pak2F/F or OTII+;Pak2F/F;Cd4-Cre mice .", "Graphs in this figure show mean ± SEM ( n = 3 ) .", "* , p=0 . 014 .", "( E ) Expression of TCR transgene assessed by anti-Vα2 antibody in total thymocytes from OTII+;Pak2F/F or OTII+;Pak2F/F;Cd4-Cre mice .", "Shown are representative data from three mice from each genotype . DOI: http://dx . doi . org/10 . 7554/eLife . 02270 . 007 Severe T cell deficiency in the periphery of Pak2F/F;Cd4-Cre mice was at odds with the normal numbers of DP and SP thymocytes in Pak2F/F;Cd4-Cre mice .", "We hypothesized that Pak2 may play a key role in maturational events after positive selection .", "CD4SP thymocyte maturation can be further defined by transition from ‘semi-mature’ ( CD69hi CD62Llow ) to ‘mature’ ( CD69low CD62Lhi ) states ( Gabor et al . , 1997; Carlson et al . , 2006 ) .", "Remarkably , CD4SP thymocytes were blocked at the semi-mature stage in Pak2F/F;Cd4-Cre mice ( Figure 4A , B ) .", "Consistent with a defect at this developmental transition , expression of CD62L and integrin β7 were reduced , but CD69 expression remained high in CD4SP thymocytes ( Figure 4C , top panels ) .", "Expression of CD3 , TCRβ , and CD24 were similar between Pak2F/F and Pak2F/F;Cd4-Cre mice ( Figure 4C , bottom panels ) , but a subset of Pak2F/F;Cd4-Cre CD4SP thymocytes exhibited unusually high Qa2 expression ( Figure 4C , top right panel ) .", "However , these Qa2hi CD4SP thymocytes lacked other characteristics of maturation; for example , substantial numbers of thymocytes in this subset maintained high expression of CD69 and CD24 ( Figure 4E ) and they failed to increase CD62L expression ( Figure 4F ) . 10 . 7554/eLife . 02270 . 008Figure 4 . Inhibition of the semi-mature to mature transition of CD4SP thymocytes from Pak2F/F;Cd4-Cre mice .", "( A ) CD4SP TCRhi thymocytes were gated and analyzed by expression of CD69 and CD62L .", "Fraction A ( CD69hiCD62Llow ) , semi-mature stage; Fraction B ( CD69lowCD62Llow ) ; Fraction C ( CD69lowCD62Lhi ) , mature stage .", "Shown are representative data of ten mice per genotype .", "( B ) Quantification of cell numbers of A , B , and C fractions in CD4SP thymocytes .", "Error bars: SEM ( n = 10 ) .", "* , 0 . 01<p<0 . 05; *** , 0 . 0001<p<0 . 001 ( unpaired two-tailed Student's t test ) .", "( C ) Abnormal expression of maturation markers ( top panels ) in CD4 SP thymocytes .", "Pak2F/F ( WT , filled histogram ) ; Pak2F/F;Cd4-Cre ( KO , red ) .", "Expression of CD3 , TCRβ and CD24 in CD4 SP thymocytes from Pak2F/F and Pak2F/F;Cd4-Cre mice was similar ( bottom panels ) .", "Shown are representative data of three mice per genotype .", "( D ) Decreased expression of IL-7Rα on CD3hiDP , CD4SP and semi-mature ( CD69hiCD62Llow ) CD4SP thymocytes from Pak2F/F;Cd4-Cre mice .", "Shown are representative data of three mice per genotype .", "( E ) Abnormal expression of maturation markers in CD4SP thymocytes from Pak2F/F;Cd4-Cre mice .", "TCRhi CD4SP thymocytes were gated and analyzed by expression of CD69 vs Qa2 and CD24 vs Qa2 .", "Shown are representative of more than five mice .", "( F ) Abnormal expression of maturation markers in CD4SP thymocytes from Pak2F/F;Cd4-Cre mice .", "TCRhi CD4SP thymocytes were gated and analyzed by expression of Qa2 vs CD62L .", "Shown are representative of more than five mice . DOI: http://dx . doi . org/10 . 7554/eLife . 02270 . 00810 . 7554/eLife . 02270 . 009Figure 4—figure supplement 1 . Inhibition of the semi-mature to mature transition in CD4SP thymocytes from Pak2F/F;Lck-Cre or OTII+;Pak2F/F;Lck-Cre mice .", "( A ) CD4SP TCRhi thymocytes were gated and analyzed by expression of CD69 and CD62L .", "Semi-mature stage ( CD69hiCD62Llow ) and mature stage ( CD69lowCD62Lhi ) are shown .", "Note lack of the cells in the mature stage .", "( B ) Upper panels: expression of TCR transgene assessed by anti-Vα2 antibody in CD4SP thymocytes from OTII+;Pak2F/F or OTII+;Pak2F/F;Lck-Cre mice .", "Lower panels: Vα2hi thymocytes in CD4SP thymocytes were gated and analyzed by expression of CD69 and CD62L .", "Shown are representative data from three mice from each genotype . DOI: http://dx . doi . org/10 . 7554/eLife . 02270 . 009 Since we found that maturation of CD4SP thymocytes was impaired in Pak2F/F;Cd4-Cre mice , we analyzed the expression of CD69 and CD62L in Pak2F/F and Pak2F/F;Lck-Cre mice .", "We found that most Pak2F/F;Lck-Cre CD4SP thymocytes failed to increase CD62L expression and were blocked at the semi-mature stage ( Figure 4—figure supplement 1A ) .", "Moreover , Vα2 TCR transgene positive CD4SP thymocytes from OTII; Pak2F/F;Lck-Cre mice displayed marked inhibition at the transition from the semi-mature to the mature stage ( Figure 4—figure supplement 1B ) .", "Thus , decreased numbers of CD4SP thymocytes from Pak2F/F;Lck-Cre or OTII; Pak2F/F;Lck-Cre mice could be due to decreased positive selection or impaired maturation .", "While most DP thymocytes down-regulate expression of IL-7Rα , post-positive selection DP and SP thymocytes increase expression of IL-7Rα ( Van De Wiele et al . , 2004 ) .", "In the absence of Pak2 , IL-7Rα expression was partially decreased in some SP thymocytes , suggesting Pak2 deficiency could affect survival of CD4SP thymocytes ( Figure 4D ) .", "To determine reduced IL-7Rα expression could impact cell survival , we examined whether there was increased cell death or apoptosis by Pak2-deficient cells .", "The percentages of dead or apoptotic cells detected among resting DP and semi-mature and mature CD4SP thymocytes were comparable from WT and Pak2F/F;Cd4-Cre mice immediately after harvest ( Figure 5A ) or after 1–2 hr incubation in vitro ( Figure 5B ) .", "However , a twofold increase in cell death was observed in Pak2-deficient semi-mature CD4SP thymocytes when incubated in media for 24 hr , whereas mature CD4SP thymocytes from both WT and Pak2-deficient mice exhibited minimal cell death ( Figure 5C ) .", "Our results suggest that Pak2 plays a key role in the maturation and maintenance of semi-mature CD4SP thymocytes , but not in survival of mature CD4SP thymocytes . 10 . 7554/eLife . 02270 . 010Figure 5 . Apoptosis or cell death of semi-mature and mature CD4SP thymocytes in freshly isolated cells ex vivo or following in vitro culture .", "( A ) Apoptosis detected using 7AAD/Annexin V staining in freshly isolated Pak2F/F or Pak2F/F;Cd4-Cre CD4SP thymocytes .", "Semi-mature ( CD69hiCD62Llow , Fraction A in Figure 4A ) , mature ( CD69lowCD62Lhi , Fraction C in Figure 4A ) and CD69lowCD62Llow ( Fraction B in Figure 4A ) CD4SP thymocytes were gated and analyzed .", "Data are representative of two independent experiments .", "( B ) Intracellular staining of active caspase 3 following 0 , 1 , and 2 hr of incubation in 10% FBS serum containing media .", "Data are representative of two independent experiments .", "( C ) Apoptosis detected using 7AAD/Annexin V staining following 24 hr of incubation in 10% FBS serum containing media .", "Semi-mature and mature CD4SP thymocytes were gated and analyzed .", "Data are representative of two independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 02270 . 010 Abnormal expression of maturation markers such as CD62L , integrin β7 , Qa2 and IL-7Rα on CD4SP thymocytes from Pak2F/F;Cd4-Cre mice could be due to T cell-extrinsic factors .", "For example , the lymphopenic environment of T cell-specific Pak2-deficient mice might alter cytokine profiles in the thymus , which could impact expression of these molecules .", "To determine the effect of T cell-specific Pak2 deficiency on expression of these maturation markers in a non-lymphopenic environment , we analyzed thymic maturation using competitive bone marrow repopulation experiments .", "We identified thymocytes generated from WT or Pak2F/F;Cd4-Cre bone marrow cells using congenic CD45 markers , then compared expression of maturation markers on CD4SP thymocytes ( Figure 6A , B ) .", "We found that expression of CD62L and integrin β7 on CD4SP thymocytes from Pak2F/F;Cd4-Cre bone marrow donors was greatly decreased .", "Moreover , down-regulation of CD69 and CD24 was not efficient , suggesting Pak2 deficiency inhibits maturation of CD4SP thymocytes via a thymocyte intrinsic mechanism .", "Furthermore , CD4SP thymocytes generated from Pak2F/F;Cd4-Cre bone marrow donors displayed marked reduction in IL-7Rα expression , suggesting that regulation of IL-7Rα by Pak2 is cell autonomous ( Figure 6C ) . 10 . 7554/eLife . 02270 . 011Figure 6 . Defects in maturation of CD4SP thymocytes in the absence of Pak2 are T cell-intrinsic and Pak2 is required for maturation of fetal CD4SP thymocytes .", "( A ) Flow cytometry analysis of 1:1 mixed bone marrow chimeras generated by transferring WT ( Pak2+/+ , CD45 . 1+CD45 . 2+ ) and Pak2F/F;Cd4-Cre mice ( CD45 . 2+ ) donor bone marrow cells that contain hematopoietic stem cells ( HSCs ) into lethally irradiated C57BL6 hosts that express CD45 . 1+ .", "Thymocytes generated either from Pak2+/+ or Pak2F/F;Cd4-Cre donor bone marrow cells were identified using CD45 congenic markers and CD4 vs CD8 expression was shown .", "TCRhi CD4SP thymocytes were gated and expression of CD69 and CD62L was examined .", "Data shown are representative of five bone marrow chimeras .", "( B ) Aberrant expression of trafficking molecules and maturation markers in CD4 SP thymocytes generated from Pak2F/F;Cd4-Cre donor .", "( C ) Impaired expression of IL-7Rα in CD4SP T cells generated from Pak2F/F;Cd4-Cre donor bone marrow cells .", "( D ) Block in the semi-mature to mature transition of Pak2-deficient CD4 SP thymocytes in fetal thymic organ culture .", "CD4 vs CD8 ( top panels ) expression of total culture and CD69 vs CD62L expression within TCRhi CD4SP population ( bottom panels ) .", "Shown are data representative FTOC experiments of six embryos per each genotype .", "( E ) Altered expression of maturation markers in TCRhi CD4SP thymocytes from FTOC .", "( F ) IL-7Rα expression in DP , CD4SP , and semi-mature ( CD69hiCD62Llow ) CD4SP thymocytes from FTOC . DOI: http://dx . doi . org/10 . 7554/eLife . 02270 . 011 To further determine whether the generation of the mature CD4SP thymocytes was inhibited due to defective maturation , we performed fetal thymic organ culture ( FTOC ) ( Robinson and Owen , 1977 ) .", "Similar to what we observed in Pak2F/F;Cd4-Cre mice , CD4SP thymocytes from Pak2F/F;Cd4-Cre FTOC were blocked at the semi-mature stage ( Figure 6D , E ) .", "Interestingly , IL-7Rα expression was similar on DP and CD4SP thymocytes generated from WT and Pak2F/F;Cd4-Cre FTOCs ( Figure 6F ) , suggesting that the observed defects in IL-7Rα expression in adult CD4SP thymocytes from Pak2-deficient mice did not occur in fetal development .", "Importantly , these results indicate that fetal thymic maturation was still blocked at the semi-mature stage in the absence of Pak2 even when expression of IL-7Rα was not compromised .", "We conclude that Pak2 is required for transition from the semi-mature to the mature stage in fetal and adult CD4SP thymocytes .", "As SP thymocytes mature and acquire functional competency , they increase the expression of an egress receptor , S1P1 ( Carlson et al . , 2006; Weinreich et al . , 2009 ) .", "The transcription factor KLF2 plays a key role in controlling expression of S1P1 in SP thymocytes ( Carlson et al . , 2006 ) .", "Expression of KLF2 is controlled during thymocyte development .", "In DN and DP stages , expression of KLF2 is minimal to prevent premature egress of DN or DP thymocytes .", "SP thymocytes gradually increase expression of KLF2 , reaching highest expression amounts at the mature stage .", "KLF2 also controls expression of CD62L , which promotes T cell entry to the lymph nodes in the periphery .", "The signaling pathway that controls timed egress of thymocytes by increasing KLF2 has not been identified .", "Since CD62L expression was reduced in Pak2-deficient CD4SP thymocytes , we asked whether KLF2 expression is altered in the absence of Pak2 .", "Indeed , expression of KLF2 was greatly reduced in CD4SP thymocytes from Pak2F/F;Cd4-Cre mice ( Figure 7A ) .", "Moreover , we found that S1P1 expression was also markedly decreased ( Figure 7B ) , suggesting that Pak2-dependent signals regulate timed egress of SP thymocytes by controlling expression of KLF2 and S1P1 .", "Since Pak2 is required for the transition from the semi-mature to the mature stage , the decrease in KLF2 and S1P1 mRNA expression may reflect the lack of the mature CD4SP thymocytes in the absence of Pak2 .", "However , we observed a reproducible decrease in KLF2 expression in the semi-mature CD4SP thymocytes of Pak2F/F;Cd4-Cre mice ( Figure 7A , right panel ) , suggesting Pak2 contributes to expression of KLF2 when CD4SP thymocytes are just beginning to upregulate KLF2 . 10 . 7554/eLife . 02270 . 012Figure 7 . Pak2 plays a key role in functional maturation of CD4SP thymocytes .", "( A ) Defects in mRNA expression of KLF2 in mature CD4SP thymocytes from Pak2F/F;Cd4-Cre mice .", "KLF2 mRNA levels in semi-mature and mature CD4SP thymocytes relative to Pak2F/F DP thymocytes ( left panel , mean ± SD of triplicates , results are representative of three independent experiment ) ; KLF2 mRNA levels in mature Pak2F/F;Cd4-Cre CD4SP thymocytes relative to mature Pak2F/F CD4SP thymocytes ( middle panel , mean ± SEM; each dot represents one mouse , n = 3 ) ; KLF2 mRNA levels in semi-mature Pak2F/F;Cd4-Cre CD4SP thymocytes relative to semi-mature Pak2F/F CD4SP thymocytes ( right panel , mean ± SEM; each dot represents one mouse , n = 3 ) .", "*** , p=0 . 0001; ** , 0 . 001<p<0 . 01 .", "( B ) Defects in mRNA expression of S1P1 in mature CD4SP thymocytes from Pak2F/F;Cd4-Cre mice .", "S1P1 mRNA levels in semi-mature and mature CD4SP thymocytes relative to Pak2F/F DP thymocytes ( left panel , mean ± SD of triplicates , results are representative of four independent experiment ) ; S1P1 mRNA levels in mature Pak2F/F;Cd4-Cre CD4SP thymocytes relative to mature Pak2F/F CD4SP thymocytes ( middle panel , mean ± SEM; each dot represents one mouse , n = 4 ) ; S1P1 mRNA levels in semi-mature Pak2F/F;Cd4-Cre CD4SP thymocytes relative to semi-mature Pak2F/F CD4SP thymocytes ( right panel , mean ± SEM; each dot represents one mouse , n = 4 ) .", "**** , p<0 . 0001; ** , 0 . 001<p<0 . 01 .", "( C ) Proliferative defects of CD4 SP thymocytes from Pak2F/F;Cd4-Cre mice .", "Histograms shown are CFSE dilutions of CD24lowQa2hi CD4SP cells from Pak2F/F and Pak2F/F;Cd4-Cre mice following 72 hr of plate-bound anti-CD3 or anti-CD3/CD28 stimulation .", "Pak2F/F , resting ( grey ) ; Pak2F/F , plate-bound anti-CD3 or anti-CD3/CD28 stimulation ( blue histogram ) ; Pak2F/F;Cd4-Cre , plate-bound anti-CD3 or anti-CD3/ CD28 stimulation ( red histogram ) .", "Shown are data representative of two independent experiments .", "( D ) Phosphorylation of S6 in media or following plate-bound antibody stimulation for 24 hr ( top panels ) and 2 hr ( bottom panels ) in total CD4SP thymocytes .", "Shown are data representative of three ( 24 hr ) or two ( 2 hr ) independent experiments .", "Pak2F/F , blue; Pak2F/F;Cd4-Cre , red histogram .", "( E ) Immunoblotting analysis of phosphorylation status of p70S6K ( T389 ) in total thymocytes from Pak2F/F or Pak2F/F;Cd4-Cre mice in media or following plate-bound anti-CD3 stimulation for 2 , 5 , 30 and 60 min .", "Intensity of each band was measured , normalized by intensity of GAPDH ( as loading control ) , and shown in the graph as relative intensity .", "Shown are data representative of three independent experiments .", "( F ) Induction of Nur77 following plate-bound antibody stimulation for 24 hr .", "Shown are data representative of three independent experiments .", "Pak2F/F , blue; Pak2F/F;Cd4-Cre , red histogram .", "( G ) Phosphorylation of Erk1/2 in CD4SP cells in media or following plate-bound antibody stimulation for 10 min .", "Shown are data representative of three independent experiments .", "Pak2F/F , blue; Pak2F/F;Cd4-Cre , red histogram .", "( H ) Immunoblotting analysis of phosphorylation status of PLCγ1 ( Y783 ) and Erk1/2 ( T202/Y204 ) in total thymocytes from Pak2F/F or Pak2F/F;Cd4-Cre mice in media or following plate-bound anti-CD3 stimulation for 2 , 5 , 30 and 60 min .", "Intensity of each band was measured , normalized by intensity of GAPDH ( shown in Figure 7E ) , and shown in the graph as relative intensity .", "Shown are data representative of three independent experiments .", "See Figure 7—figure supplements 1 and 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 02270 . 01210 . 7554/eLife . 02270 . 013Figure 7—figure supplement 1 . Increased apoptosis or cell death in semi-mature CD4SP thymocytes from Pak2F/F;Cd4-Cre mice following 24 hr in vitro culture .", "( A ) Increased apoptosis in the Qa2hi CD4SP thymocyte subset from Pak2F/F;Cd4-Cre mice following 24 hr of incubation in media or in plate-bound CD3 or CD3/CD28 stimulation .", "Data are representative of two independent experiments .", "( B ) Increased apoptosis in CD4SP thymocytes from Pak2F/F;Cd4-Cre mice following 24 hr of incubation in media or in plate-bound CD3 or CD3/CD28 stimulation .", "Apoptosis detected using 7AAD/Annexin V staining .", "Data are representative of three independent experiments .", "( C ) Increased apoptosis in resting semi-mature ( CD62Llow ) CD4SP thymocytes from Pak2F/F;Cd4-Cre mice detected using Annexin V staining or active caspase 3 intracellular staining following 24 hr of incubation in 10% FBS serum containing media .", "Data are representative of three ( annexin V ) and two ( active caspase", "3 ) independent experiments .", "( D ) Increased apoptosis in semi-mature ( CD62Llow ) CD4SP thymocytes from Pak2F/F;Cd4-Cre mice detected using Annexin V staining following 24 hr of incubation in media or in plate-bound CD3 or CD3/CD28 stimulation .", "Data are representative of four independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 02270 . 01310 . 7554/eLife . 02270 . 014Figure 7—figure supplement 2 . mTOR-dependent phosphorylation of p70S6K and S6 . ( A ) Inhibition of MAPK pathway by UO126 , a MEK inhibitor .", "Effects of UO126 ( a MEK inhibitor ) and rapamycin ( an inhibitor of mTOR ) on phosphorylation of Erk1/2 and RSK were demonstrated by immunoblotting analysis of phosphorylation status of Erk1/2 ( T202/Y204 ) and RSK ( T359/S363 ) in total thymocytes from Pak2F/F or Pak2F/F;Cd4-Cre mice .", "Cells were preincubated in the presence of DMSO , rapamycin ( 25 nM ) , and UO126 ( 10 μM ) at the stated concentration for 30 min and treated with plate-bound anti-CD3 stimulation for 30 and 60 min .", "UO126 completely abrogated activation of MAPK and RSK , but rapamycin did not substantially inhibit activation of MAPK and RSK .", "( B ) Inhibition of mTORC1-mediated pathway by rapamycin .", "Effect of rapamycin was examined by immunoblotting analysis of phosphorylation status of p70S6K ( T389 ) .", "Phosphorylation of p70S6K at T389 was completely inhibited by rapamycin ( 25 nM ) .", "( C ) Phosphorylation of S6 at S235/S236 and p70S6K at T389 was completely dependent upon mTOR-mediated pathway .", "Cells were preincubated in the presence of DMSO , rapamycin ( 25 nM ) , and UO126 ( 10 μM ) at the stated concentration for 30 min .", "Cells were rested in media or treated with plate-bound anti-CD3 stimulation for 30 and 60 min .", "Immunoblotting analysis of phosphorylation status of S6 ( S235/S236 ) and p70S6K ( T389 ) in total thymocytes from Pak2F/F or Pak2F/F;Cd4-Cre mice in media ( resting ) or following plate-bound anti-CD3 stimulation for 30 and 60 min are shown .", "( D ) Intensity of each band shown in C was measured , normalized by intensity of GAPDH ( as loading control ) , and shown in the graph as fold increase .", "Shown are data representative of three independent experiments .", "( E ) Phosphorylation of S6 at S235/S236 and p70S6K at T389 were dependent upon activation of mTOR .", "Relative intensity of inhibitor-treated samples compared to DMSO-treated samples at 30 min or 60 min after stimulation are shown .", "Intensity of each band was normalized by DMSO-treated sample at each time point and displayed as relative intensity .", "Graphs in this figure show mean ± SEM ( n = 3 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 02270 . 014 Although CD4SP thymocytes from Pak2F/F;Cd4-Cre mice lack the CD69lowCD62Lhi mature subset and did not increase KLF2 or S1P1 expression , it is possible that they are functionally mature .", "Moreover , within CD4SP thymocytes there is a subset that increases Qa2 expression ( Figure 4E , F ) , which could represent a mature subset .", "Thus , we examined directly whether CD4SP thymocytes from Pak2F/F;Cd4-Cre mice contain functionally mature cells .", "One of the hallmarks of functional maturity of CD4SP thymocytes is their ability to proliferate in response to CD3 and CD28 stimulation , unlike semi-mature thymocytes that are more susceptible to cell death ( Hogquist et al . , 2005; Takada et al . , 2011 ) .", "We compared the proliferative responses of CD24lowQa2hi CD4SP cells from WT and Pak2F/F;Cd4-Cre mice following plate-bound TCR and CD28 stimulation .", "As expected , the CD24lowQa2hi CD4SP thymocytes from WT mice underwent 2–3 rounds of cell division following CD3 or CD3 and CD28 stimulation , suggesting these cells are functionally mature .", "In contrast , most CD4SP thymocytes from Pak2F/F;Cd4-Cre mice including the CD24lowQa2hi subset did not proliferate following TCR/CD28 stimulation ( Figure 7C ) .", "In addition , the Qa2hi CD4SP thymocytes from Pak2F/F;Cd4-Cre mice were more susceptible to cell death following stimulation ( Figure 7—figure supplement 1A ) , suggesting that these cells are not functionally mature .", "Of note , Pak2-deficient CD4SP thymocytes are more susceptible to cell death following 24 hr of incubation in resting or stimulated conditions because they contain more semi-mature thymocytes that undergo cell death ( Figure 7—figure supplement 1B–D ) .", "Together , these results indicate that functional maturity of CD4SP thymocytes depends on Pak2 .", "Since we found that Pak2 is required for proliferative response of CD4SP thymocytes following plate-bound anti-CD3 or anti-CD3/CD28 stimulation , we sought to determine the mechanism by which Pak2 affects signaling pathways activated by the TCR and CD28 stimulation .", "The PI3K/Akt/mTORC1 signaling pathway has been reported to be activated by the TCR and CD28 stimulation , reflected in the phosphorylation of S6 ribosomal protein ( S6 ) , a direct target of the mTORC1-activated p70S6 kinase ( p70S6K ) ( Hay and Sonenberg , 2004 ) .", "Activation of the mTORC1 pathway increases rates of mRNA translation and protein synthesis , which permits increased cell growth and proliferation ( Mills and Jameson , 2009 ) .", "Activation of mTORC1 pathway was absolutely required for phosphorylation of S6 at the S235/S236 sites and p70S6K at the T389 site , as phosphorylation of these sites were completely abrogated by rapamycin , an inhibitor of mTOR ( Figure 7—figure supplement 2B–E ) .", "Phosphorylation of S6 at S235/S236 in CD4SP thymocytes from WT mice was increased after 24 hr of stimulation ( Figure 7D , top panels ) .", "On the contrary , most of the CD4SP thymocytes from Pak2F/F;Cd4-Cre mice displayed minimal S6 phosphorylation .", "Since TCR-induced S6 phosphorylation reaches its optimal amounts after 1 or 2 hr of TCR stimulation and lasts up to 5 hr ( Salmond et al . , 2009; van den Brink et al . , 1999 ) , we examined S6 phosphorylation following 2 hr of plate-bound anti-CD3 stimulation .", "Phosphorylation of S6 at S235/S236 was substantially decreased in the absence of Pak2 ( Figure 7D , bottom panels ) .", "Activation of MAPK-RSK pathway contributes to phosphorylation of S6 ( Salmond et al . , 2009 ) .", "Indeed , treatment of thymocytes with a MEK inhibitor , UO126 , partially reduced phosphorylation of S6 at S235/S236 ( Figure 7—figure supplement 2C–E ) .", "However , since rapamycin treatment completely abrogated phosphorylation of S6 at these sites ( Figure 7—figure supplement 2C–E ) , the MAPK-dependent phosphorylation of S6 also seems to depend on activation of mTORC1-mediated pathway .", "To determine whether reduced phosphorylation of S6 in the absence of Pak2 is due to inhibition of mTORC1-mediated pathway , we examined phosphorylation of p70S6K at the T389 site because phosphorylation of this site was completely dependent on mTORC1-mediated pathway but independent of MAPK-mediated pathway ( Figure 7—figure supplement 2C–E ) .", "Phosphorylation of p70S6K at T389 was also decreased following 30 min and 1 hr of plate-bound CD3 stimulation in the absence of Pak2 ( Figure 7E ) .", "These findings demonstrate that Pak2 participates in mTORC1-mediated activation of p70S6K and phosphorylation of S6 following plate-bound TCR stimulation .", "To determine whether Pak2-deficient CD4SP thymocytes have a defect in optimal activation of integrated TCR-mediated signaling events , we examined expression of Nur77 , an orphan nuclear hormone receptor induced in response to TCR stimulation , as a reporter for TCR strength of signaling ( Moran et al . , 2011; Zikherman et al . , 2012 ) .", "Nur77 induction was greatly decreased in CD4SP thymocytes from Pak2F/F;Cd4-Cre mice following 24 hr of anti-CD3 or anti-CD3/CD28 stimulation ( Figure 7F ) .", "These results reveal that Pak2 is required for downstream events reflective of TCR signaling , including those inducing Nur77 induction and p70S6K activation .", "Decreased induction of Nur77 following TCR or TCR/CD28 stimulation suggests that Pak2 is required for TCR-dependent signaling events .", "Since Pak2 is required for multiple processes of development and maturation of thymocytes , it is possible that Pak2 regulates these processes by governing TCR-mediated signaling .", "Because activation of Erk1/2 following TCR stimulation plays a key role in thymic development as well as in induction of Nur77 ( van den Brink et al . , 1999 ) , we examined activation of Erk1/2 following TCR or TCR/CD28 stimulation .", "Phosphorylation of Erk1/2 was markedly reduced in the absence of Pak2 , when cells were stimulated by plate-bound anti-CD3 or anti-CD3/CD28 stimulation ( Figure 7G , H ) .", "In contrast , phosphorylation of Erk1/2 was not reduced when cells were stimulated by soluble anti-CD3 or anti-CD3/CD28 in the absence of Pak2 ( data not shown ) , suggesting that Pak2 is required for transducing signaling events that are specifically provoked by plate-bound TCR stimulation .", "TCR engagement by plating T cells on surfaces coated with antibodies to the TCR mimics T cell activation by antigen-presenting cells ( APCs ) and triggers dynamic changes in actin cytoskeletal architecture , such as formation and expansion of contacts bounded by continuously remodeled actin-rich rings ( Wear et al . , 2000; Bunnell et al . , 2001; Babich et al . , 2012 ) .", "These actin-rich rings are associated with extension of lamellipodia that requires assembly and disassembly of actin filament neworks ( Bunnell et al . , 2001 ) .", "Changes in actin cytoskeletal architecture induced by TCR engagement promote T cell signaling ( Babich et al . , 2012 ) .", "Interestingly , actin dynamics that mediate ongoing actin polymerization and retrograde flow promotes sustained PLCγ1 signaling during plate-bound TCR activation , while disruption of F-actin dynamics inhibits sustained PLCγ1 phosphorylation ( Babich et al . , 2012 ) .", "Since activation of PLCγ1 plays a critical role in activating Erk1/2 following TCR stimulation , we hypothesized that Pak2 participates in activation of Erk1/2 via promoting PLCγ1 activation by regulating actin cytoskeletal dynamics during T cell spreading triggered by TCR engagement .", "Indeed , we found that phosphorylation of PLCγ1 that is known to be associated with its activation status ( Kim et al . , 1991 ) is profoundly decreased in the absence of Pak2 , indicating that Pak2 is required for optimal PLCγ1 activation , possibly by regulating actin cytoskeletal reorganization ( Figure 7H ) .", "Our results suggest that Pak2 may participate in actin cytoskeletal dynamics during T cell spreading triggered by TCR stimulation .", "To determine whether the loss of Pak2 affects the actin cytoskeleton in CD4SP thymocytes , we first examined actin polymerization and Rac1 activation in resting conditions .", "To our surprise , the amount of polymerized filamentous actin ( F-actin ) was greatly increased in resting conditions ( Figure 8A ) , suggesting processes regulating actin assembly and disassembly were disrupted in the absence of Pak2 .", "Consistent with these data , Rac1 activation was consistently increased under resting conditions ( Figure 8B ) .", "However , despite increased F-actin and activated Rac levels , Pak2-deficient CD4SP thymocytes did not reorganize their actin cytoskeleton and spread when triggered by plate-bound TCR stimulation .", "WT CD4SP thymocytes exhibited intense spreading at 60 min of stimulation , when they were plated on the coverslips coated with antibodies to the TCR ( Figure 8D , upper left and middle panels ) , while polarizing F-actin of the cell body towards to the contact sites ( Figure 8D , upper right panel ) .", "In stark contrast , Pak2-deficient CD4SP thymocytes displayed poor spreading at the contact sites and actin polymerization at the contact sites was markedly impaired ( Figure 8D , lower left and middle panels ) , while F-actin in the cell was not polarized towards to the contact sites ( Figure 8D , lower right panel ) .", "As a result , areas where cells spread at the contact sites were significantly reduced in the absence of Pak2 ( Figure 8C ) .", "These findings revealed that Pak2 plays a vital role in regulating the actin cytoskeletal reorganization induced by TCR stimulation .", "Since the integrity of dynamic actin cytoskeletal structure and actin cytoskeletal reorganization triggered by TCR engagement are fundamental for the proper signal transmission in many biological processes of T cells , we propose that Pak2 controls multiple stages of T cell development in the thymus by governing dynamic rearrangement of the actin cytoskeleton . 10 . 7554/eLife . 02270 . 015Figure 8 . Pak2 regulates actin dynamics of CD4SP thymocytes .", "( A ) Increased actin polymerization in resting DP , CD4 and CD8 SP thymocytes from Pak2F/FCd4-Cre mice .", "The mean fluorescence intensity of phalloidin-Alexa488 in the DP , CD4SP and CD8SP thymocyte populations was normalized to the value of WT DP thymocytes at rest .", "( error bars; SEM , n = 3 ) .", "**** , p<0 . 0001 ( B ) Increased activation of Rac1 at resting from Pak2F/F;Cd4-Cre thymocytes using Rac1 pull-down assay .", "WCL; whole cell lysates .", "Shown are data representative of three independent experiments .", "( C ) Spreading areas of CD4SP thymocytes on the cover slips .", "Cells were allowed to spread for 60 min on coverslips coated with anti-CD3 .", "Z-stack images of F-actin staining of the cells were collected and spreading areas where cells contact the cover slips were analyzed .", "Shown are data representative of three independent experiments .", "( error bars; SEM , Pak2F/F [n = 21] , Pak2F/F;Cd4-Cre [n = 25] ) .", "**** , p<0 . 0001 .", "( D ) T cell spreading and actin polymerization triggered by plate-bound TCR stimulation .", "Shown are z-stack images of the cells from the contact sites where T cells touch the cover slides to the top of the cells .", "Left panels; maximal projection of z-stack images of F-actin staining ( F-actin staining at the contact site [green] and F-actin staining of the rest of the z-stacks [red] ) .", "Scale bar: 5 μm .", "Middle panels: F-actin staining on the contact site to visualize cell spreading .", "Right panels: z stack images of F-actin were complied and reconstructed as a 3D image .", "Shown is the orthogonal image viewed from the z-axis .", "Shown are data representative of three independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 02270 . 015" ], [ "We report that Pak2 , an effector for the small GTPase Rac and Cdc42 , is essential for multiple stages of thymocyte development and maturation .", "T cell-specific deletion of Pak2 at two different developmental stages using stage-specific Cre transgenic mice demonstrated that Pak2 is critical for T cell development , maturation and egress .", "We found that deletion of Pak2 at either the DN2 or DP stages using the Lck-Cre or Cd4-Cre transgenic mice resulted in severe T cell lymphopenia .", "Development of DP and SP thymocytes was inhibited in Pak2F/F;Lck-Cre mice due to impaired pre-TCR β-selection and positive selection , resulting in reduced numbers of DP and SP thymocytes .", "In contrast , generation of SP thymocytes was not affected , nor were numbers of DP and SP thymocytes reduced in Pak2F/F;Cd4-Cre mice .", "Apparent normal development was due to compensatory changes in the TCR repertoire because positive selection was impaired when the OTII transgene was introduced in Pak2F/F;Cd4-Cre mice .", "Pak2 deletion in Pak2F/F;Cd4-Cre mice presented a unique opportunity to decipher the role of Pak2 in maturation of CD4SP thymocytes after positive selection since generation of CD4SP thymocytes was not impaired .", "We found that Pak2 is required for the functional maturation and timed egress of CD4SP thymocytes in Pak2F/F;Cd4-Cre mice .", "To define the mechanisms by which Pak2 regulates thymocyte development and maturation , we sought to determine Pak2's function in TCR-induced signaling events .", "We found that Pak2 is required to remodel actin cytoskeletal architecture during T cell spreading induced by TCR stimulation .", "Since TCR engagement induces rapid changes in cytoskeletal reorganization in T cells , which is critical for transducing signals , it is possible that perturbation of the actin cytoskeletal reorganization processes in Pak2-deficient thymocytes prevents normal TCR-mediated signaling events .", "Indeed , inability to remodel the actin cytoskeleton during T cell spreading was accompanied by impaired activation of PLCγ1 and Erk1/2 , suggesting Pak2 is required to drive actin cytoskeleton-dependent signaling induced by the TCR .", "As a result , induction of Nur77 and phosphorylation S6 were inhibited , and proliferative responses were diminished in the absence of Pak2 .", "Activation of PLCγ1 ( Sommers et al . , 2005; Fu et al . , 2010 ) and Ras-mediated MAPK pathway leading to Erk1/2 activation are essential for positive selection ( Alberola-Ila et al . , 1995; Swan et al . , 1995; O'Shea et al . , 1996; Swat et al . , 1996; Pagès et al . , 1999; Fischer et al . , 2005; McGargill et al . , 2009 ) .", "Since positive selection is initiated by TCR/pMHC interaction between DP thymocytes and cTECs and controlled by avidity and affinity of this interaction ( Starr et al . , 2003 ) , inefficient signaling such as impaired activation of PLCγ1 and Erk1/2 could lead to defects in positive selection in the absence of Pak2 .", "We also confirmed that disruption of actin dynamics by administration of either the F-actin stabilizing agent jasplakinolide ( Bubb et al . , 1994; Murthy and Wadsworth , 2005 ) or the actin depolymerizing agent latrunculin ( Spector et al . , 1983; Coué et al . , 1987 ) completely abrogated signaling events such as induction of Nur77 and S6 phosphorylation following plate-bound TCR stimulation ( data not shown ) , suggesting actin cytoskeletal reorganization is critical for activation of TCR-induced signaling events .", "Together , these findings suggest that Pak2 is required to coordinate T cell signaling network by controlling actin cytoskeletal reorganization during thymocyte development .", "After completing positive selection , post-positive selection DP thymocytes migrate from the thymic cortex to the medulla and spend about 4–5 days there prior to exiting ( McCaughtry et al . , 2007 ) .", "Newly selected SP thymocytes acquire ‘mature’ features in the medulla before they egress ( McCaughtry et al . , 2007 ) .", "During this time , they decrease expression of CD69 and CD24 and increase expression of CD62L , integrin β7 , and Qa2 .", "In addition , SP thymocytes become functionally mature as they acquire competency to proliferate in response to TCR stimulation , rather than die like their immature precursors DP or semi-mature SP thymocytes .", "We found that CD4SP thymocytes from Pak2F/F;Cd4-Cre mice were arrested at the semi-mature stage , unable to increase expression of CD62L and integrin β7 .", "CD4SP thymocytes from Pak2F/F;Cd4-Cre mice did not contain a functionally mature subset because they failed to proliferate following CD3 or CD3/CD28 stimulation .", "Rather , CD4SP thymocytes from Pak2F/F;Cd4-Cre mice contain more semi-mature cells that undergo cell death when stimulated , confirming that Pak2 is required for functional maturation of CD4SP thymocytes .", "The effect of Pak2 deficiency in CD4-expressing SP thymocytes was specific to the transition from the semi-mature to the mature stage after positive selection , since the generation of semi-mature CD4SP thymocytes from DP thymocytes was normal in Pak2F/F;Cd4-Cre mice .", "The mechanism by which Pak2 mediates the transition from the semi-mature to the mature stage of CD4SP thymocytes is not clear .", "Since semi-mature CD4SP thymocytes from Pak2F/F;Cd4-Cre mice were more prone to cell death following 24 hr of in vitro culture , Pak2 may be required for maintenance of semi-mature CD4SP thymocytes .", "However , Pak2 deficiency in CD4-expressing SP thymocytes did not increase cell death or apoptosis in freshly isolated thymocytes ex vivo or after 1–2 hr of in vitro culture .", "Moreover , the number of semi-mature CD4SP thymocytes was not reduced in Pak2F/F;Cd4-Cre mice , suggesting that the effect of Pak2 deficiency may be specific to the semi-mature CD4SP thymocytes that received a maturation signal .", "For example , the maturation signal may activate Pak2 in semi-mature cells to provide protection from apoptosis and to make the transition to the mature stage .", "The nature of the upstream pathway that controls maturation and is also dependent upon Pak2 is currently unknown .", "Another key feature linked to SP thymocyte functional maturation is the increased expression of the egress receptor S1P1 as a result of expression of KLF2 , a crucial transcription factor that regulates expression of S1P1 and CD62L ( Carlson et al . , 2006; Weinreich and Hogquist , 2008 ) .", "Failure to generate mature CD4SP thymocytes in the absence of Pak2 greatly impeded both KLF2 and S1P1 expression .", "Although KLF2 plays a critical role in egress and trafficking , KLF2 does not contribute to functional maturation of thymocytes because KLF2-deficient SP thymocytes are not defective in proliferation following CD3/CD28 stimulation ( Takada et al . , 2011 ) .", "On the other hand , Pak2 plays essential roles in promoting functional maturation as well as KLF2 expression .", "These results indicate that Pak2-mediated functional maturation is required for the increased expression of KLF2 .", "We propose that timed egress of thymocytes by KLF2 is tightly coordinated by a Pak2-dependent maturation process as a quality checkpoint mechanism .", "As a consequence , Cd4-Cre mediated Pak2 deletion results in severe T cell lymphopenia due to Pak2's multiple roles in T cell maturation and timed egress of thymocytes .", "Since Pak2 is a serine/threonine kinase that phosphorylates multiple targets , it is of great interest to identify which substrates mediate Pak2's function in thymocyte development and maturation .", "Our results indicate that Pak2 facilitates actin cytoskeletal reorganization following TCR stimulation .", "Therefore , Pak2 may regulate thymic development by controlling TCR-mediated signaling strength by facilitating actin cytoskeletal reorganization .", "Among the known Pak2 substrates that are involved in cytoskeletal networks are: GEF-H1 ( Zenke et al . , 2004; Kosoff et al . , 2013 ) , ArhGAP15 ( Radu et al . , 2013 ) , filamin A ( Vadlamudi et al . , 2002 ) , Rho GDI ( DerMardirossian and Bokoch , 2006 ) , α and β PIX ( Koh et al . , 2001; Shin et al . , 2002; Rennefahrt et al . , 2007 ) , regulatory myosin light chain ( Ramos et al . , 1997; Chew et al . , 1998 ) , myosin heavy chain ( Yamashita and May 1998 ) , tubulin cofactor B ( Vadlamudi et al . , 2005 ) , LIMK ( Edwards et al . , 1999; Dan et al . , 2001 ) , and dynein light chain 1 ( Vadlamudi et al . , 2004 ) .", "Moreover , it is likely that Pak2 is required for transcriptional responses that mediate the transition from the semi-mature to the mature stage since loss of Pak2 severely impaired this transition .", "It is not clear whether Pak2 is directly involved in regulating transcription responses by phosphorylating components of transcriptional machinery which mediate this transition , or whether Pak2 phosphorylates a signaling component that ultimately leads to the transcription response facilitating this transition .", "Further studies to determine the mechanism of Pak2's function in thymic development and maturation are warranted .", "The Paks were the first Rho family GTPase-regulated kinases to be identified and are among the well-characterized effectors of Rac and Cdc42 ( Arias-Romero and Chernoff , 2008 ) .", "Here , we show that Pak2 is required for thymic development , including pre-TCR β selection and positive selection , and T cell activation , closely resembling the functions of Rac , Cdc42 or their activator Vav in T cell development and activation .", "Together , our findings suggest that Pak2 is the primary effector for mediating the functions of Rac and Cdc42 in T cell development and activation ." ], [ "To generate a conditional knock-out of Pak2 gene , a targeting vector was designed to flank exon 2 of Pak2 gene with loxP sites as previously described ( Kosoff et al . , 2013 ) .", "Mice with the Pak2 floxed allele ( Pak2F/+ ) were backcrossed at least nine times onto the C57BL/6 background .", "The Cd4-Cre ( Lee et al . , 2001 ) or Lck-Cre ( Hennet et al . , 1995 ) transgenic mice contain Cd4 or Lck promoter sequences , respectively , driving the expression of a Cre recombinase gene .", "The Cd4-Cre or Lck-Cre transgenes were introduced Pak2F/+ mice and backcrossed at least nine times onto the C57BL/6 backgrounds .", "Pak2F/+;Cd4-Cre or Pak2F/+;Lck-Cre mice in the C57BL/6 background were crossed to the OTII TCR transgenic mice ( Barnden et al . , 1998 ) .", "C57BL/6 , Cd4-Cre , Lck-Cre , OTII and BoyJ ( B6 mice with the CD45 . 1+ congenic marker B6 . SJL-Ptprca Pepcb/BoyJ ) used for breeding were originally obtained from the Jackson Laboratory .", "All animals were housed in a specific pathogen-free facility at Northwestern University and University of California , San Francisco according to university and National Institutes of Health guidelines .", "The floxed Pak2 mice were genotyped by genomic PCR isolated from tail clips .", "Amplification for the floxed Pak2 gene was carried out by standard PCR protocol .", "Primers used to screen the floxed Pak2 gene are; forward primer 5′-ATCTTCCCAGGCTCCTGACT , reverse primer 5′-TGAAGCTGCATCAATCTATTCTG .", "The Cre transgene in the Cd4-Cre or Lck-Cre mice was screened by standard PCR protocol using forward primer 5′-TGGGCGGCATGGTGCAAGTT and reverse primer 5′-CGGTGCTAACCA GCGTTTTC .", "Stimulatory armenian hamster anti-mouse CD3ε ( 2C11 ) antibody was purchased from BD Biosciences ( San Jose , CA ) .", "Goat anti-Armenian hamster IgG ( H and L chains ) Abs and goat anti-rabbit IgG Abs conjugated to either PE or allophycocyanin were obtained from Jackson ImmunoResearch Laboratories ( West Grove , PA ) .", "All antibodies for FACS analyses were obtained from BD Biosciences ( San Jose , CA ) .", "Phospho-S6 Ribosomal protein ( S235/236 ) ( 2F9 ) , phospho-p70S6K ( T389 ) ( 9234 ) , and phospho-Erk1/2 ( T202/Y204 ) ( 4377 ) antibodies were obtained from Cell Signaling Technologies ( Danvers , MA ) .", "Phospho-PLCγ1 ( Y783 ) ( 44-696G ) antibody was purchased from Life Technologies ( Grand Island , NY ) .", "Pak2 ( TA306346 ) antibody is from Origene ( Rockville , MD ) .", "Nur77 ( 12 . 14 ) antibody was purchased from eBiosciences ( San Diego , CA ) .", "For mixed bone marrow ( BM ) chimera experiments , BM chimeras were generated by transferring a 1:1 mixture of BM cells from WT ( CD45 . 1+CD45 . 2+ ) and Pak2F/F;Cd4-Cre mice ( CD45 . 2+ ) with different congenic markers into lethally irradiated BoyJ recipient mice ( B6 . SJL-Ptprca Pepcb/BoyJ , CD45 . 1+ ) .", "Mixed BM cells were resuspended in 1 . 0 ml ( 107 cells/ml ) PBS and 200 µl ( 2 × 106 mixed bone marrow cells ) were injected i . v . into lethally irradiated BoyJ recipients .", "BoyJ recipients were irradiated with two doses of 600 rads , 3–5 hr apart .", "After 6–8 weeks , thymi , spleen , lymph nodes , and blood of reconstituted chimeras were harvested and used for flow cytometry analysis .", "Single cell suspensions were prepared from thymi , lymph nodes ( LN ) , and spleens .", "Fc receptors were blocked with rat anti-CD16/32 ( 2 . 4G2; BD Biosciences ) .", "2 , 000 , 000 cells were stained with the indicated Abs and analyzed on a LSR II , FACSCanto or Fortessa ( BD Biosciences ) flow cytometry system .", "Forward and side scatter exclusion was used to identify live cells .", "Data analysis was performed using FlowJo ( version 9 . 6 . 2 ) software ( Tree Star ) .", "Statistical analysis and graphs were generated using Prism 6 ( GraphPad Software ) .", "Fetal thymi from embryos at embryonic day 16 were explanted and cultured on the top surface of a trans-well .", "The bottom surface of the trans-well was submerged in 10% FBS containing media in the bottom well , but the thymi were exposed to 5% CO2 during culture .", "The media in the bottom well was changed every day up to 7 days .", "In the end of 7 days of culture , single cell suspensions from the fetal thymi were prepared and used for flow cytometry .", "Plates ( 24 well ) were coated with anti-CD3 ( 1 μg/ml ) or anti-CD3 ( 1 μg/ml ) and anti-CD28 ( 10 μg/ml ) for overnight at 4°C .", "Single cell suspensions of thymocytes were washed with PBS without Ca2+ and Mg2+ twice , then resuspended at 2 × 107 cells/ml .", "A CFSE solution was added to the cells to a final concentration of 2 . 5 μM for 8 min at room temperature .", "After 8 min , fetal calf serum was directly added to the CFSE labeled cells and incubated for 1 min .", "Cells were washed twice with 5 min interval .", "2 , 000 , 000 of CFSE labeled cells were added to pre-coated plates .", "After 72 hr , cells were harvested and stained for surface markers as well as DAPI ( 4′ , 6-diamidino-2-phenylindole ) .", "Data were collected on the BD LSRII or FACSCanto flow cytometer and analyzed using FlowJo software .", "Thymocytes were harvested , washed in FACS buffer and stained for surface markers on ice .", "Following surface staining , cells were washed and fixed with Fixation/Permeabilization buffer ( eBiosciecne ) for 14 hr at 4°C following manufacturer's instruction .", "Cells were washed with 1X Permeabilization buffer ( eBioscience ) and stained with FITC-anti-phospho-S6 ( S235/236 ) ( Cell signaling technologies ) for 1 hr .", "Cells were washed twice in 1X permeabilization buffer ( eBioscience ) and resuspended in FACS buffer .", "Data were collected on the BD LSRII or FACSCanto flow cytometer and analyzed using FlowJo software .", "2 , 000 , 000 thymocytes were added to each well of 24-well plates that were coated with anti-CD3ε .", "After stimulation , cells were harvested using ice-cold PBS , transferred to FACS tubes , fixed for 10 min with equal volume of Cytofix ( BD Biosciences ) to terminate stimulation , and permeabilized using 95% ice-cold methanol ( EMS , Hatfield , PA ) for 30 min .", "Cells were washed twice , re-hydrated in staining buffer ( 2% BSA in PBS ) for 30 min , then stained with Nur77-PE ( eBioscience ) Ab in staining buffer for 1 hr .", "After Nur77 staining , cells were washed twice , stained with surface markers for 30 min .", "Data were collected on FACSCanto or LSRII flow cytometer ( BD Biosciences ) and analyzed using FlowJo software .", "Single cell suspensions from thymi were prepared and stained using CD4 , CD8 , CD69 , CD62L , CD24 and Qa2 surface markers as well as DAPI ( 4′ , 6-diamidino-2-phenylindole ) to identify DP , semi-mature ( CD69hiCD62Llow or CD24hiQa2low ) and mature ( CD69lowCD62Lhi or CD24lowQa2hi ) CD4SP thymocytes .", "Since the CD69lowCD62Lhi mature CD4SP thymocyte numbers were significantly reduced from Pak2F/F;Cd4-Cre mice , we sorted CD69lowCD62Lmed CD4SP thymocytes from Pak2F/F;Cd4-Cre mice .", "Alternatively , we used CD24 and Qa2 surface expression to sort the semi-mature CD24hiQa2low CD4SP thymocytes or the CD24lowQa2hi CD4SP thymocytes from Pak2F/F or Pak2F/F;Cd4-Cre mice in some experiments .", "Thymocytes were sorted using a MoFlo cell sorter .", "Cells were lysed and total RNA was prepared using RNeasy and QIAshredder kit ( Qiagen , Valencia , CA ) .", "First strand cDNA were synthesized using SuperScript III First-Strand Synthesis System ( Life Technologies ) .", "cDNA was analyzed in triplicate by QPCR amplification by incorporation of Platinum SYBR Green or Fast SYBR Green with a StepOnePlus Real-Time PCR System ( Applied Biosystems , Life technologies ) , and results are presented relative to the expression of Hprt ( encoding murine hypoxanthine phosphoribosyltransferase ) .", "Data were analyzed by comparative quantification .", "PCR primer pairs are as follows: HPRT forward , 5′-AGTCCCAGCGTCGTGATTAGC-3′; HPRT reverse , 5′-GAGCAAGTCTTTCAGTCCTGTCC-3′; KLF2 forward 5′-CTAAAGGCGCATCTGCGTA-3′; KLF2 reverse , 5′-TAGTGGCGGGTAAGCTCGT-3′; S1P1 forward 5′-GTGTAGACCCAGAGTCCTGCG-3′; S1P1 reverse , 5′-AGCTTTTCCTTGGCTGGAGAG-3′ .", "Amounts of polymerized actin in thymocytes were measured as previously described ( Phee et al . , 2010 ) .", "Specific isolation of Rac1-GTP was performed as described previously ( Phee et al . , 2010 ) .", "Untouched CD4SP thymocytes were enriched by removing DP and CD8 SP thymocytes using CD8 MACS beads ( Miltenyi Biotec , San Diego , CA ) and plated onto cover slips that are coated with anti-CD3 ( 10 μg/ml ) ( Bunnell et al . , 2001 ) .", "Following 60 min of stimulation , cells were fixed , permeabilized , stained with Alexa Flour488 Phalloidin , and mounted using ProLong Gold Antifade reagent ( Life Technologies ) as previous described ( Phee et al . , 2010 ) .", "Confocal images were collected using an A1R Resonant Scanning Multispectral Confocal Microscope ( Nikon Instruments Inc , Melville , NY ) .", "40X ( N . A . 1 . 3 ) Plan Fluor lens or 100X ( N . A . 1 . 45 ) PlanApo lens were used .", "Z-stack images were collected from the contact site where T cells touch the cover slides ( bottom ) to the top of the cells where no signals were detected ( top ) .", "In most cases , total distance of z stacks was 11–12 μm and Nyquist-compatible 0 . 25 μm step size was used to collect z-stack images .", "The contact areas where the cell spreads were measured by using the NIS-Elements Viewer ." ] ]

[ "The molecular mechanisms that govern thymocyte development and maturation are incompletely understood .", "The P21-activated kinase 2 ( Pak2 ) is an effector for the Rho family GTPases Rac and Cdc42 that regulate actin cytoskeletal remodeling , but its role in the immune system remains poorly understood .", "In this study , we show that T-cell specific deletion of Pak2 gene in mice resulted in severe T cell lymphopenia accompanied by marked defects in development , maturation , and egress of thymocytes .", "Pak2 was required for pre-TCR β-selection and positive selection .", "Surprisingly , Pak2 deficiency in CD4 single positive thymocytes prevented functional maturation and reduced expression of S1P1 and KLF2 .", "Mechanistically , Pak2 is required for actin cytoskeletal remodeling triggered by TCR .", "Failure to induce proper actin cytoskeletal remodeling impaired PLCγ1 and Erk1/2 signaling in the absence of Pak2 , uncovering the critical function of Pak2 as an essential regulator that governs the actin cytoskeleton-dependent signaling to ensure normal thymocyte development and maturation ." ]

[ "T cells are a key element of the immune system .", "There are many different types of T cells , and they all have their origins in hematopoietic stem cells that are found in the bone marrow .", "These stem cells leave the bone marrow and circulate in the body until they reach an organ called the thymus , where they become early thymic progenitor cells .", "These progenitor cells then undergo a process called differentiation to become specific types of T cells , which mature in the thymus before moving to the blood .", "Although various molecules and mechanisms are known to be involved in the development of T cells , many details of this process are not understood .", "One group of molecules that has been implicated in the differentiation of T cells is the p21-activated kinases .", "Kinases are proteins that activate or deactivate other proteins by adding phosphate groups to specific amino acids .", "Pak2 adds phosphorylate groups to various proteins that are involved in the reorganization of an important structure inside the cell called the cytoskeleton .", "A kinase called Pak2 has an important role in the reorganization of the cytoskeleton , and since this reorganization is involved in almost all aspects of T cell biology , it seems plausible that Pak2 is also involved in the development of T cells .", "However , it has not been possible to test this idea because deleting the gene for Pak2 in mice results in their death .", "Now , Phee et al . have overcome this problem by performing experiments in which the gene for Pak2 was only deleted in T cells .", "These mice had significantly fewer mature T cells than healthy mice .", "In particular , the absence of Pak2 in thymocytes ( the cells that become T cells ) prevented them from maturing into T cells , and also prevented them from producing a receptor protein that is needed for mature T cells to leave the thymus .", "This work implies that disruption of the Pak2-mediated signaling pathway that regulates the cytoskeleton may weaken the immune system in humans ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "neuroscience" ]

[ [ "The bilateral organization of the pathways mediating the orienting senses is optimized to detect stimuli occurring in the right and left halves of space relative to the body's midline .", "Generation of a unified representation of sensory space requires a functional interaction between the two sides of the pathway , which , in the case of vision and touch , occurs in the cerebral cortex predominantly via the corpus callosum .", "Hearing , however , is distinct amongst the senses in having extensive pre-thalamic processing in the brainstem .", "Here we report evidence demonstrating that the fundamental properties of sound frequency and level are influenced by extensive interactions between the left and right sides of the pathway at this more peripheral level .", "The sub-thalamic auditory system consists of chains of nuclei in the brainstem giving rise to multiple parallel pathways represented in mirror image about the midline .", "Processing in each side is dominated by the respective contralateral sound field .", "Each limb of the pathway culminates in the midbrain in the respective left or right inferior colliculi ( ICs ) whose outputs provide the primary ascending input to the auditory thalamus and cortex on each side .", "While the left and right pathways are distinct , they involve crossed connections at several levels ( Moore and Osen , 1979; Glendenning and Masterton , 1983; Glendenning et al . , 1985; Oliver , 2000; Cant and Benson , 2003 ) .", "Some of these crossed connections , such as those between the cochlear nuclei and the superior olivary complex , occur between different levels in the hierarchy and mediate interactions that extract the interaural disparities underlying sound localization .", "Such connections have been termed the acoustic chiasm ( Glendenning et al . , 1985 ) .", "In addition , there are commissural connections that interconnect mirror opposite nuclei .", "These are found as early in the pathway as the cochlear nuclei ( Adams and Warr , 1976; Cant and Gaston , 1982 ) , while , in the midbrain , the commissure of Probst connects the dorsal nuclei of the lateral lemniscus ( DNLLs ) ( Goldberg and Moore , 1967; Hutson et al . , 1991 ) .", "But the most prominent of these connections is the commissure of the inferior colliculus ( CoIC ) ( Beyerl , 1978; Adams , 1980; Aitkin and Phillips , 1984; Coleman and Clerici , 1987 ) .", "The CoIC interconnects the ICs in a systematic fashion that mirrors the spatial representation of frequency ( tonotopicity ) in these nuclei ( Adams , 1980; González-Hernández et al . , 1986; Saldaña and Merchán , 1992; Malmierca et al . , 1995 , 2009 ) .", "These fibers are predominantly excitatory although a significant minority ( ∼10–20% ) are GABAergic ( González-Hernández et al . , 1996; Hernández et al . , 2006; Nakamoto et al . , 2013 ) .", "This commissure is the final point of interaction between the two limbs of the ascending auditory pathway prior to the auditory cortex .", "The preponderance of these commissural inputs and the importance of interaural comparison in the analysis of sound suggest that the two ICs may operate in a more cooperative manner than has hitherto been considered .", "Recent in vivo studies in the IC in response to sound stimuli that were varied in azimuthal location have led to the speculation that the CoIC may mediate binaural processing between the ICs ( Malmierca et al . , 2005; Li and Pollak , 2013; Ono and Oliver , 2014 ) .", "Rather than each IC being an independent entity , we hypothesized that functionally the ICs should be considered as two halves of a whole .", "This commissural influence might manifest itself in one of two ways , either as a global regulation of firing , or as differential changes in the firing patterns of different unit types , indicating more functionally specific control .", "To test these hypotheses we have examined the extent to which two fundamental aspects of sound processing , neuronal responses to frequency and level , in one IC are governed by the commissural connections between them .", "We have recorded response properties of neurons in one IC of guinea pig while deactivating the other IC using two different methods , cooling using a cryoloop ( Lomber et al . , 1999; Orton et al . , 2012 ) or microdialysis of the short acting local anesthetic procaine ( Boehnke and Rasmusson , 2001 ) .", "These methods enabled us to address the causal impact of these connections on individual neurons .", "Our findings show that intercollicular commissural connections regulate firing and are a major determinant of the receptive fields of IC neurons .", "Intercollicular processing increases the heterogeneity of receptive field types with implications for enhancing the perception of frequency , and the ability of IC neurons to convey changes in sound level .", "Our results imply that the analysis of frequency and level , both fundamental components of hearing , and generally considered to be monaural processes , depend on bilateral interactions between the ICs .", "These findings demonstrate the two sides of the auditory midbrain operate collectively rather than independently of one another ." ], [ "We first verified the use of MDP in our preparation by recording multi-unit activity in the left IC adjacent to the microdialysis probe , before , during and after procaine dialysis .", "The schematic coronal section through the preparation in Figure 1A shows the placement of the microdialysis probe in the center of the exposed IC with a microelectrode inserted adjacent to the probe . 10 . 7554/eLife . 03764 . 003Figure 1 . Microdialysis of procaine into the IC produced a rapid , reversible deactivation of spiking .", "( A ) Schematic coronal image of the setup for recording the effects of procaine on neuronal activity in the left IC .", "( B ) Mean ( ±SD ) firing rate of multi-units recorded by an electrode adjacent to the probe , each point represents the average spikes per stimulus value in response to 100 repetitions of a CF tone at 20 dB above threshold .", "Procaine caused an immediate and persistent deactivation throughout infusion .", "After switching back to aCSF , firing recovered to near control levels after 20 min . ( C ) Multi-unit FRA recorded from an electrode adjacent to the microdialysis probe .", "( D ) Procaine infusion abolished firing at virtually all frequencies and levels .", "( E ) Response area shape and firing rate recovered similar to control . DOI: http://dx . doi . org/10 . 7554/eLife . 03764 . 003 Repeated PSTHs showed a stable firing rate with aCSF flowing through the microdialysis probe ( Figure 1B ) , and a rapid reduction in firing rate when procaine was dialyzed .", "Firing rate recovered over the course of 20 min following aCSF washout through the probe .", "Figure 1C shows the multi-unit FRA in the control condition .", "MDP abolished firing at almost all frequencies and levels of sound stimulation .", "The reduction in firing rate was similar across the entire response area .", "After 20 min of aCSF washout the response area returned to control .", "Insertion of the microdialysis probe into the left IC produced negligible trauma .", "The area of the IC and the area of the lesion caused by the probe were measured in Nissl stained sections .", "Lesions comprised just 1 . 49% ( ±0 . 31 ) of the area of the IC .", "While procaine has a high pKa , and consequently has low efficacy in vivo compared to other sodium channel blockers that are commonly used in deactivation studies ( Truant and Takman , 1959 ) , recovery from deactivation is faster than with amide local anesthetics such as lidocaine ( Sung and Truant , 1954 ) as ester local anesthetics are metabolized in plasma rather than liver ( Covino , 1981 ) .", "This avoided the spread of procaine deactivation to other areas of the brain as its metabolism begins in situ .", "Procaine has no effect of resting membrane potential ( Taylor , 1959 ) or spike amplitude ( Butterworth and Cole , 1990 ) .", "Microdialysis of procaine rapidly deactivated neurons without producing the mechanical disturbances associated with pressure injection ( Malmierca et al . , 2003 , 2005 ) .", "Frequency response areas in the IC are diverse; however , they can be grouped as being either V-shaped , with responses similar to those found in the in the auditory nerve , or into a more heterogeneous group collectively termed nonV .", "The latter are an emergent property of central processing .", "At least seven response area types can be defined within this group , but they represent points on a continuum rather than discrete classes ( Palmer et al . , 2013 ) .", "For convenience we will refer to these two classes as V and nonV FRAs , respectively .", "We measured the excitatory response area by finding frequency-level combinations within each FRA in which the firing rate exceeded the mean spontaneous firing rate by two standard deviations ( Ingham et al . , 2006 ) .", "We also measured the number of spikes elicited by the unit in each FRA .", "These two measures were compared between conditions .", "Similar effects were found in response to either cooling or MDP deactivation of the contralateral IC .", "Control units tested with cooling or MDP did not differ in characteristic frequency ( CF ) , FRA area or firing rate .", "For analysis we therefore pooled these responses .", "Figure 2 shows four examples from the sample of 49 V-shaped FRAs in our dataset .", "The area of V FRAs did not change in response to either cooling ( n = 37 ) or MDP ( n = 12 ) deactivation of the contralateral IC .", "The absence of changes in FRA area was apparent throughout recovery .", "Conversely , changes in firing rate within and across the existing response area occurred for most V FRAs in response to cooling and MDP deactivation .", "Both increases and decreases in firing rate were seen in the population and these recovered towards control levels after deactivation was stopped .", "With a 20% change in firing rate as a criterion of change ( LeBeau et al . , 2001 , Burger and Pollak , 2001; Park et al . , 2008 ) , 14% ( 7/49 ) V FRAs decreased in firing rate while 35% ( 17/49 ) increased . 10 . 7554/eLife . 03764 . 004Figure 2 . Deactivation of the contralateral IC influenced the firing rate of IC neurons with V responses but had little effect on their shape or area . Columns ( A , D , G , J ) show four examples of IC units in control ( top ) , deactivated ( middle ) and recovery ( bottom ) conditions with responses for each unit normalized to the maximum firing rate across conditions .", "While firing rate changes within V FRAs were common , the tuning and shape of these FRAs were unaffected by deactivation of the contralateral IC by either cooling or MDP .", "All changes in firing rate during deactivation ( B , E , H , K ) recovered following cessation of cooling or procaine infusion ( C , F , I , L ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 03764 . 004 We recorded examples of all previously reported nonV FRA response types , and in Figure 3 show four examples from the 50 in our dataset .", "In contrast to V FRAs , FRAs in the nonV group showed dramatic changes in area following deactivation of the other IC ( Figure 4C , D ) .", "For example , the neuron whose low-tilt FRA is shown in Figure 3A declined in both spontaneous and auditory driven firing rate and its FRA reduced in area by 52% ( Figure 3B ) .", "The narrow FRA type in Figure 3D reduced in area by 86% ( Figure 3E ) and the closed FRA in Figure 3G expanded in area by 234% ( Figure 3H ) and showed an elevation in firing rate for frequencies centered on its CF , the frequency to which it was most sensitive .", "The broad FRA in Figure 3J reduced in area by 40% ( Figure 3K ) .", "All of these changes reversed to near control levels on recovery ( Figure 3C , F , I , L ) .", "Quantitative analysis found that the firing rate of 34% ( 17/50 ) of nonV FRAs decreased in firing rate while 28% ( 14/50 ) increased . 10 . 7554/eLife . 03764 . 005Figure 3 . Deactivation of the contralateral IC influenced firing rate as well as the receptive field area and shape for nonV responses .", "( A ) Low tilt FRA with high spontaneous rate .", "( B ) Procaine in the contralateral IC caused a reduction in both driven and spontaneous firing and FRA area .", "( D ) Narrow FRA which ( E ) reduced in area on contralateral cooling but fired sporadically over a wider frequency range .", "( G ) Closed FRA which ( H ) expanded in area and increased in firing rate .", "( J ) Broad FRA which ( K ) reduced in rate and area on contralateral cooling .", "( C , F , I , L )", "All changes in firing rate and response area reversed on recovery . DOI: http://dx . doi . org/10 . 7554/eLife . 03764 . 00510 . 7554/eLife . 03764 . 006Figure 4 . Differential changes in firing rate and receptive field area by FRA class .", "( A ) Total spikes in FRA on deactivation of the contralateral IC as a function of control value .", "A wide range of changes in firing rate were noted for both V and nonV receptive field classes with deactivation .", "( B ) Cumulative distribution of change index ( see text ) for total spikes showing a similar range and distribution for V and nonV FRAs .", "( C ) Receptive field area on deactivation as a function of control .", "( D ) Cumulative distributions of change index of FRA area .", "The range of area change for nonV units was three times greater than for those with V response areas . DOI: http://dx . doi . org/10 . 7554/eLife . 03764 . 006 Plotting the spikes within each FRA in the deactivated condition as a function of FRA spikes in the control condition revealed changes in all FRA types ( Figure 4A ) .", "We quantified the change in spiking in both V and nonV FRAs by calculating a modulation index:MI= ( deactivated−control ) / ( deactivated+control ) .", "The distributions of change in firing rate for both V and nonV FRAs ( Figure 4B ) were similar ( two sample Kolmogorov-Smirnov test: D ( 97 ) = 0 . 20; p = 0 . 26 ) .", "There was a difference between the change in area for the two FRA classes .", "The distribution of V FRA areas was almost unchanged during deactivation ( Figure 4C ) , but the range of changes in nonV FRAs was more than double that of V FRAs ( Figure 4D ) .", "Comparison of FRA area change between V and nonV FRAs showed that nonV FRAs were modulated more than V ( D ( 97 ) = 0 . 32; p = 0 . 009 ) .", "Having revealed a distinction between the effect of commissural inputs on V and nonV frequency receptive fields , we next sought to investigate how changes in response type would influence the encoding of sound level .", "To this end , we generated RLFs for single units by recording responses to multiple presentations of pure tones at the unit CF at several sound levels .", "Effectively , this is a measure of a neuron's response to stimuli on a vertical line transecting the FRA at CF .", "Each RLF was classified into one of five previously reported types ( Rees and Palmer , 1988; Winter et al . , 1990 ) .", "Non-monotonic RLFs were defined using the criteria of Rees and Palmer ( 1988 ) , where a reduction in firing of 25% or more was required at a sound level above that which produced maximal firing .", "Non-monotonic RLFs comprised 12 of the 38 neurons recorded .", "A range of FRA types can give rise to non-monotonic RLFs .", "While examples of all RLF types showed firing rate changes during contralateral IC deactivation , only non-monotonic RLFs changed shape .", "Four of the 12 non-monotonic RLFs became monotonic ( Figure 5A , C ) , four became straight ( Figure 5B ) , while the remainder remained non-monotonic . 10 . 7554/eLife . 03764 . 007Figure 5 . Changes in the shape and half maximum firing rate of RLFs on deactivation of the contralateral IC .", "( A–C )", "Control RLFs ( black ) with non-monotonic response functions with increasing sound level .", "With deactivation ( red ) RLFs became more monotonic and in some ( B and C ) the curves shifted to higher sound levels .", "These changes reversed on recovery ( blue ) .", "( D–F )", "Normalizing the functions in the top row shows the sound level required to elicit half maximal firing increased during deactivation .", "( G ) The population of half maximum firing rates increased on deactivation of the contralateral IC .", "( H ) Changes in half maximum firing rates did not correlate with changes in threshold , indicating that the dominant effect of commissural input on firing rate was at supra-threshold levels . DOI: http://dx . doi . org/10 . 7554/eLife . 03764 . 007 To analyze these effects we constructed a 5 × 2 contingency table with each row comprising one of the five RLF types and each column the sum of a binary classifier applied to each RLF as to whether it changed in type ( 1 ) or maintained the same type ( 0 ) on contralateral deactivation .", "A Fisher's exact test found that the probability of the observed distribution occurring or one more extreme , was 0 . 0002 .", "Thus , non-monotonic RLFs in IC are a distinct type of RLF whose shape can be formed by CoIC processing .", "We assessed the impact commissural deactivation has on the supra-threshold encoding of changes in sound level by calculating the half maximum firing rate for RLFs in each condition ( Figure 5A–C ) .", "In the majority of units , deactivation shifted RLFs so that a higher sound level was required to evoke the half maximum firing rate than in the control condition ( Figure 5D–F ) .", "On recovery , the half maximum firing rate of these RLFs returned to values similar to the control condition .", "In the population of RLFs ( Figure 5G ) the median sound level required to elicit half maximal firing increased from 50 . 9 dB sound pressure level ( SPL ) ( 95% CI: 46 . 1 56 . 2 ) to 59 . 3 dB SPL ( 95% CI: 55 . 2 65 . 0 ) on deactivation .", "On recovery , the median half maximal level of the distribution returned to 51 . 3 dB SPL ( 95% CI: 47 . 5 61 . 1 ) .", "The increase in sound level required to reach half maximal firing rate during deactivation indicated that CoIC rescaling improved supra-threshold coding of sound level ( Friedman repeated measures ANOVA on ranks: χ2 ( 2 ) = 12 . 72; p = 0 . 001 ) .", "Post hoc analysis found a higher sound level was required to elicit half maximum firing during contralateral IC deactivation than during control recordings ( Z = −3 . 94; p = 0 . 00008 ) .", "There was no difference between the level required to evoke half maximal firing in the control and recovery conditions ( Z = −1 . 41; p = 0 . 15 ) .", "If this effect was due to a shift in the entire RLF to a higher sound level , changes in threshold would correlate with changes in the half maximum firing point on the RLF .", "To test this we performed a correlation analysis between the change in half maximum firing and the change in statistical threshold ( Ingham et al . , 2006 ) for neurons for which both FRAs and RLFs were collected ( Figure 5H ) .", "There was no correlation between the change in threshold and the change in half maximum firing ( rs ( 14 ) = 0 . 20; p = 0 . 45 ) .", "This demonstrates that the predominant effect of removing CoIC input was to rescale supra-threshold responses , consistent with the operation of a gain control of neuronal output .", "An important question is whether the changes in RLFs during deactivation altered the ability of units to signal changes in sound level .", "To investigate this we devised a measure to assess the discriminability of sound level changes for each RLF .", "We constructed an ROC curve from the responses to adjacent stimuli in each RLF in each condition ( Figure 6A ) and calculated the area under each curve for each pair ( Figure 6B ) before summing the absolute deviation of each receiver operating characteristic ( ROC ) curve from 0 . 5 .", "This produced a ‘Discriminability Index’ ( DI ) which quantified how well changes in sound level could be decoded by an ideal observer from changes in firing rate along each RLF . 10 . 7554/eLife . 03764 . 008Figure 6 . Deactivation of the contralateral IC reduced sound level discriminability in the IC .", "( A ) Control RLF of an IC neuron ( median ± IQR ) .", "( B ) ROC pairwise analysis of the responses in ( A ) with colors of ROC curves matching the corresponding paired values in ( A ) .", "( C ) An example RLF ( black ) which showed a reversible reduction in rate and slope on deactivation ( red ) that recovered to control values ( blue ) .", "( D ) A ‘discriminability index’ ( DI ) was calculated from the change in area under the ROC ( see text ) for each adjacent stimulus pair in each condition .", "The reduction in rate and slope in ( C ) on deactivation resulted in a reduction in DI from 1 . 5 to 1 . 2 with recovery to 1 . 4 .", "( E ) The median DI for the population of RLFs declined on deactivation of the contralateral IC , indicating a reduction in discriminability of sound level on in the absence of commissural input . DOI: http://dx . doi . org/10 . 7554/eLife . 03764 . 008 Figure 6C shows the RLFs from a neuron with a monotonic saturating response throughout the three conditions in the experimental paradigm .", "The DI of this unit in the control condition was 1 . 5 ( Figure 6D ) .", "The firing rate and slopes of the RLF were reduced on contralateral IC deactivation leading to a fall in DI to 1 . 2 .", "On recovery , the RLF returned to near control values and the DI increased to 1 . 4 .", "For the 38 units , the population DIs in the control condition ( median = 1 . 72; 95% CI: 1 . 52 1 . 99 ) were reduced during contralateral IC deactivation ( median = 1 . 54; 95% CI: 1 . 37 1 . 68 ) although some units did show an elevation ( Figure 6E ) .", "Deactivating the contralateral IC reduced the distribution of DIs ( Wilcoxon signed rank test: Z = −2 . 18; p = 0 . 028 ) .", "Such changes demonstrate that the supra-threshold gain control which the CoIC exerts over IC responses enhances the ability of IC neurons to discriminate changes in sound level ." ], [ "Our results show that neuronal responses and the formation of receptive fields in the auditory midbrain depend on interactions between the bilateral limbs of the auditory pathway .", "Specifically , activity mediated by commissural fibers connecting the ICs influences the responses of almost all the IC neurons we recorded in at least one of four ways .", "The majority of neurons in our sample showed changes in firing rate following deactivation of the other IC ( Figure 4A , B ) .", "Neurons with nonV frequency response areas also showed changes in receptive field shape , often ( but not exclusively ) becoming more V-like in the absence of commissural input ( Figure 4C , D ) .", "We observed that deactivating inputs from the opposite IC reduced supra-threshold firing rates to sounds of varying level ( Figure 5 ) .", "Finally , deactivation reduced the ability of IC neurons to signal changes in sound level by changes in firing rate ( Figure 6 ) .", "Similar changes were observed whether the IC was deactivated by cooling or MDP .", "Previous studies aimed at deactivating one colliculus were limited by their methodology so only a small number of units were recorded ( Malmierca et al . , 2003 , 2005 ) .", "Although the responses of individual units in those studies are consistent with our data , the population based patterns of change we reveal here were not apparent .", "The two neural deactivation methods we have applied in this study have the advantages of", "1 ) not needing direct interference with the preparation during deactivation , and", "2 ) rapid recovery from deactivation .", "We have shown that cooling as a method of deactivation is restricted to the IC when cooling cycle durations are less than 25 min ( Orton et al . , 2012 ) .", "Under these conditions , deeper structures , such as the DNLL , appear unaffected .", "We have also demonstrated reversible deactivation with a second method , MDP with effects consistent with cooling ( Figure 1 ) .", "The methods are complementary in that cooling effects are more extensive within the IC , and consequently produce more substantial changes in the other IC but MDP provides greater control over the extent of the deactivation , albeit with generally more modest effects .", "The similarity of the datasets using the two methods also gives confidence that they reflect the consequences of deactivating the contralateral IC .", "It is not impossible that some of the consequences of deactivation are mediated via descending pathways originating in the IC to lower brainstem structures , but this seems unlikely .", "Recordings of evoked potentials from the recorded IC while cooling the other show significant changes at latencies consistent with processing in the IC , but little or no effect on the incoming afferent volley to the IC ( Orton et al . , 2012 ) .", "The anatomical basis for these physiological observations is the CoIC , which tracer experiments demonstrate interconnect the frequency-band laminae in the IC ( González-Hernández et al . , 1986; Saldaña and Merchán , 1992; Malmierca et al . , 1995 , 2009 ) .", "Immunohistochemical studies suggest that the majority of commissural fibers are excitatory , with only 10–20% labelling for the inhibitory neurotransmitter GABA ( González-Hernández et al . , 1996; Hernández et al . , 2006; Nakamoto et al . , 2013 ) .", "The changes in response properties we observed are consistent with commissural input exerting both excitatory and inhibitory influences , with much greater inhibition than would be predicted from the ratio of excitatory to inhibitory fibers .", "This suggests that within the contralateral IC , excitatory commissural fibers can exert their effects di- or poly-synaptically via inhibitory neurons in the target IC .", "Evidence supporting this idea comes from intracellular recordings in IC slices in which electrical stimulation of the CoIC revealed inhibitory currents that were reduced by glutamatergic blockade consistent with di-synaptic inhibition ( Smith , 1992; Moore et al . , 1998 ) .", "Of particular interest is the effect of commissural inputs on the frequency receptive fields of IC neurons .", "Of the two major classes , V FRAs have many features in common with the responses of the primary fibers that emerge from the cochlea .", "However , unlike primary fibers , these neurons do receive inhibitory inputs , but this inhibition does not change receptive field shape ( Egorova et al . , 2001; Alkhatib et al . , 2006; Palmer et al . , 2013 ) .", "The increases in firing rate seen here within V response areas are similar to those obtained by iontophoretic blockade of GABA and glycine receptors in IC ( Vater et al . , 1992; Yang et al . , 1992; LeBeau et al . , 2001 ) .", "Commissural input serves to rescale the output of neurons with V-shaped responses areas by modifying the balance of local excitation and inhibition without changing area of the receptive field ( Figure 2 ) .", "In contrast , nonV receptive fields are more heterogeneous in appearance , and although different classes can be identified , many appear to be intermediate types supporting the existence of continua rather than discrete groups ( Palmer et al . , 2013 ) .", "Nevertheless all nonV responses are similar in appearing to be shaped from V responses by excitatory and/or inhibitory inputs .", "Whether this shaping occurs in the inferior colliculus or is inherited from earlier stages of the auditory pathway is still contentious .", "Both the firing rates and response areas of nonV units were modified by deactivation of the contralateral IC ( Figure 3 & Figure 4B , D ) .", "Often , but not always , nonV FRAs became more V-shaped ( Figure 3 ) .", "The changes in receptive field shape and firing rate we observed on deactivation of the other IC show that it is directly or indirectly a source of the inhibitory input that generates these receptive fields .", "Increasing diversity of spectral responses is a feature of the ascending auditory pathway that presumably reflects the increasing complexity of analyses performed at each level in the process of signal extraction .", "There is a greater diversity of FRA types in midbrain than in the brainstem ( Evans and Nelson , 1973; Hernández et al . , 2005; Ingham et al . , 2006; Palmer et al . , 2013 ) and other types not described in sub-thalamic nuclei are reported in auditory cortex ( Shamma et al . , 1993; Recanzone et al . , 2000; Bitterman et al . , 2008; Bartlett et al . , 2011; Atencio et al . , 2012; Grimsley et al . , 2012 ) .", "The contribution of commissural input to this process has not been previously considered .", "We have demonstrated that lateral connections between the two sides of the auditory pathway play an important role in shaping response areas in the IC , and thus increasing response-type diversity .", "It may not be the sole contributor to this process , and the extent of its contribution remains to be determined .", "The analysis of input-output functions for sound level shows that commissural input contributes to the non-monotonicity observed in many of these functions ( Figure 5A–C ) , as well as to the rescaling of responses that enhances the discriminability of sound level in the responses of IC neurons ( Figure 6E ) .", "The ubiquity of the commissural influence on firing rates supports the contention that the IC exerts an element of gain control on its contralateral counterpart ( Malmierca et al . , 2003 , 2005 ) .", "Given that a congruent representation of the auditory environment is dependent on combining the contralateral auditory hemifields represented in each IC , the commissure may operate to calibrate one with the other .", "Although the differential effects of commissural connections on V and nonV frequency receptive fields and level encoding are clear , how such changes enhance auditory analysis is less so .", "Stimuli that arrive at each of the two ears are commonly disparate in temporal , level or spectral characteristics .", "These disparities are fused by central processing into an auditory percept .", "The construction of left and right auditory hemi-fields is a consequence of monaural and binaural analysis in the brainstem leading to the encoding of sound source location in azimuth and elevation , but our behavioral sensitivity to changes in sound position is greatest around the midline where these representations meet ( Mills , 1958 ) .", "Furthermore , our ability to separate sound sources , for example to hear out an individual speaker in a noisy social situation , is enhanced by binaural hearing ( Litovsky and McAlpine , 2010 ) .", "Commissural modulation of frequency receptive fields in the ICs may assist sound source separation in acoustical cluttered situations .", "By modulating the spectral area of FRAs in each IC , this processing may increase FRA diversity to overcome correlated noise present in a neural population , and thus improve signal extraction and information content in a neuronal population ( Zohar et al . , 2013 ) .", "The IC seems the obvious site at which to interconnect the bilateral limbs of the auditory pathway .", "It is the first structure where the parallel pathways emerging from the cochlear nucleus ( Harrison and Warr , 1962; Osen , 1970; Cant and Benson , 2003 ) and further elaborated in the brainstem nuclei can be integrated .", "Thus , the different cues for determining sound location , ( interaural time and level disparities and pinna spectral cues ) are computed in separate nuclei and are first brought together in the ICs .", "Responses to changes in overall sound level at the periphery will always be congruent in the left and right ICs , while changes in azimuthal location will produce changes in firing rate in each IC antipodal to the other .", "Processing between the ICs likely takes advantage of the symmetrical organization of the CoIC , so that bilateral changes in firing rate in each IC can better represent changes in sound level and location ( Figure 6 ) .", "In conclusion , commissural processing between the ICs exerts control over the firing rate of most IC neurons , and contributes to the creation of receptive fields determining responses to two fundamental parameters of sound: frequency and level .", "This degree of interaction suggests that each IC should not be considered as independent of the other , but as two halves of a cooperative whole operating in concert to optimize the encoding of sounds ." ], [ "Data reported here are from experiments performed on 32 ( 19 male , 13 female ) outbred , pigmented guinea pigs ( Cavia porcellus ) .", "Animals were housed in pens in a breeding colony on a 12 hr light-dark cycle .", "The number of animals per pen varied from 1 to 6 depending on the numbers in the colony at any time .", "The walls of the pens were made from wire mesh so that all guinea pigs in the colony could see and hear other members of the colony .", "Experimental animals had a median age of 6 months ( Inter-quartile range ( IQR ) = 4 to 7; range = 2 to 13 ) .", "All animals showed a strong pinna reflex in response to a finger click above the head on the morning of each experiment .", "The median weight of animals was 796 g ( IQR = 678 to 894; range = 460 to 1093 ) .", "Anesthesia was induced with urethane ( 0 . 7–1 g/kg as 20% solution , intraperitoneal injection ( i . p . ) ) and supplemented by Hypnorm ( fentanyl citrate 0 . 315 mg/ml and fluanisone 10 mg/ml; 1 ml/kg , intramuscular injection ) .", "Atropine sulphate monohydrate ( BDH Chemicals; 0 . 05 mg/kg , subcutaneous injection ) was given to suppress bronchial secretions .", "Anesthesia was maintained with further doses of Hypnorm ( 1/3 original dose ) as required .", "A tracheotomy was performed and a cannula inserted into the stoma and secured with suture .", "The cannula provided an airway and a means through which the animal could be artificially ventilated if required .", "Animals were allowed to respire spontaneously .", "If breathing became labored , the animal was artificially respired with medical air via a modified small animal ventilator ( Harvard Apparatus , UK ) which maintained end-tidal CO2 at ∼5% .", "Core temperature was monitored with a rectal probe and maintained at 38 ± 1°C with a thermostatically controlled electric blanket ( Harvard Apparatus ) .", "Experiments were conducted inside a single walled , sound attenuating room ( IAC ) .", "Animals were placed in a stereotaxic frame ( Kopf , Tujunga , California ) in which the standard ear bars were replaced by hollow polymethyl methacrylate conical specula , the apices of which were placed in the auditory meatuses to permit sound delivery .", "A surgical microscope ( Zeiss , UK ) was used to visualize the tympanic membranes to check their condition and placement of the specula .", "A dorsal midsaggital incision was made along the scalp .", "The skin was reflected and the tissues overlying the skull were abraded .", "Two holes were trephined on either side of the midline to expose the occipital lobe covering the left and right ICs and each hole was extended with rongeurs .", "The dura mater was retracted bilaterally .", "The cortex overlying the left IC was aspirated with a glass Pasteur pipette attached to a vacuum pump until the dorsal surface of the left IC was visualized .", "The cryoloop and cooling system used in this study were as reported and validated previously ( Lomber et al . , 1999; Orton et al . , 2012 ) .", "A cryoloop was constructed from stainless steel tubing .", "A thermocouple ( Omega , UK ) was secured to the cryoloop tip to allow monitoring of the cryoloop temperature with a digital thermometer ( HH506RA , Omega ) .", "A peristaltic pump ( Gilson , UK ) pumped ethanol at −80°C around a hydraulic system .", "Regulating the pump speed controlled the flow rate through the system and thereby enabled control of cryoloop temperature .", "The cryoloop was curved to maximize contact with the dorso-lateral surface of the exposed IC .", "This cooled areas in which the density of neurons projecting to the contralateral IC is highest ( Saldaña and Merchán , 1992; Malmierca et al . , 2009 ) .", "Cooling cycles were kept as brief as possible to optimize the chance of holding the unit throughout recovery .", "We have verified this method as a consistent and reliable means to deactivate the left IC by making recordings and temperature measurements in the cooled IC ( Orton et al . , 2012 ) .", "Cooling reduced firing rates in response to sound stimuli in some cases almost completely and by at least half for units tuned to frequencies up to ∼8 kHz .", "Units tuned to higher frequencies were deactivated less or not at all .", "This established that cooling deactivated the dorsal half of the left IC .", "Cooling induced deactivation did not spread to the contralateral IC and did not modulate the afferent volley to the right IC indicating any changes imparted in the right IC were produced by removal of commissural input ( Orton et al . , 2012 ) .", "Concentric microdialysis probes were constructed as described previously ( Gartside et al . , 1996 ) .", "Stainless steel tubing ( AISI 302; Goodfellow , UK ) with an outside diameter 0 . 5 mm and an inside diameter 0 . 38 mm , was cut to three lengths: one at 15 mm to form the shaft and two 10 mm pieces to form the inlet and outlet tubes .", "The cut surfaces were filed with a watchmaker's broach to ensure all ends were smooth .", "Two 50 mm lengths of fused silica tubing ( SGE Analytical Science , UK ) with an outside diameter of 170 μm and an inside diameter of 110 μm were threaded inside the shaft with one threaded over the inlet and outlet tubes , respectively , to form a Y-shaped assembly .", "The end of the inlet silica tubing protruded from the end of the shaft by approximately 3 mm while the outlet tubing was drawn ∼5 mm up inside the shaft .", "Hollow fiber cellulosic membrane ( Filtral 12 , AN69 HF , Hospal , 300 μm diameter ) was cut to 10 mm and threaded over the silica tubing and inside the shaft .", "An epoxy adhesive ( Araldite ) was used to glue the microdialysis membrane to the steel with 3 . 5–4 . 0 mm protruding .", "A 0 . 5 mm epoxy plug was applied to seal the end of the membrane and epoxy was applied at the junction of the three steel tubes to hold the assembly together .", "The probe was implanted vertically into the center of the exposed IC and held in place by a stereotaxic manipulator .", "Using a syringe pump , the probe was continuously perfused at 2 μl/min with artificial cerebrospinal fluid ( aCSF: 140 mM NaCl; 3 mM KCl; 0 . 27 mM Na2H2PO4; 1 . 2 mM Na2HPO4; 2 . 4 mM CaCl2; 1 mM MgCl2; and 7 . 2 mM Glucose ) .", "The pH of the aCSF was adjusted to 7 . 4 .", "To deactivate neuronal activity , the sodium channel blocker procaine ( 0 . 1 M , Sigma , UK ) was dissolved in aCSF and infused through the probe .", "This concentration was selected as microdialysis probe efficacy is around 10% ( Boehnke and Rasmusson , 2001 ) .", "We estimate that this produced an infusion of ∼2 . 5% procaine into the IC .", "A switch between aCSF only and aCSF plus procaine was made at a junction 4 cm from the probe with the tubing clamped to prevent any mechanical disturbance .", "Stimuli were generated digitally by Tucker-Davis Technologies System 2 ( TDT2 , Alachua , FL ) hardware which was controlled by software that allowed the frequency and level of stimuli to be varied in real time .", "Pure tones were generated digitally and were cosine2 ramped at the onset and offset .", "Search stimuli were 50-ms tone bursts , roved manually between 0 . 1 to 20 kHz and between 0 to 99 dB attenuation , and presented at a repetition rate of 4 Hz .", "Stimuli were delivered through Sony ( UK ) MDR 464 earphones housed in an alloy enclosure and coupled to damped probe tubes ( 4 mm diameter ) that fitted into the Perspex specula ( Rees et al . , 1997 ) .", "The output of the system was calibrated using an eighth inch Bruel & Kjaer ( UK ) Type 4138 microphone , a Type 2639 preamplifier and Type 2610 measuring amplifier .", "The microphone was seated in a small tubing coupler that sealed the narrow end of the speculum holding each speaker .", "The maximum output of the system was approximately flat from 0 . 1 to 9 kHz ( 100 ± 8 dB SPL ) and then fell with a slope so that the maximum output of the system at 16 kHz was 78 dB from both the left and right speakers .", "Second and third harmonic components in the signal were approximately 60 dB of the fundamental at the highest output level .", "The majority of recordings were made in the flat region of the system below 10 kHz .", "All recordings were obtained using borosilicate glass-coated tungsten electrodes .", "The electrode position was controlled by a stepper-motor microdrive activated by a remote control outside the sound-attenuating room .", "Electrode penetrations were made through the cortex in a mirror opposite position to the center of the exposed IC .", "Extracellular action potentials were amplified ( ×10 , 000 ) and band-pass filtered ( 0 . 1–3 kHz ) by an amplifier ( Dam-80; World Precision Instruments , UK ) .", "Spikes were further high pass filtered ( 300 Hz ) via TDT2 hardware before being discriminated online , converted to logic pulses , and time stamped to an accuracy of 10 μs .", "Peri-stimulus time histograms ( PSTHs ) were generated to sound-driven multi-unit activity with an electrode inserted adjacent to the microdialysis probe .", "The stimuli to drive these responses were 100 repetitions of a CF tone at 20 dB above threshold .", "Multiple PSTHs and FRAs were recorded before , during and following infusion of procaine .", "In the IC contralateral to deactivation , only single units were recorded .", "On isolation of a single unit , the CF and minimum threshold to tones presented to the ear contralateral to the recorded IC were estimated to establish the settings for data collection .", "The parameters of the stimuli used to test each neuron were defined relative to this initial estimate .", "FRAs were generated from the pseudorandom presentation of pure tones to binaural diotic stimuli ( 75 ms duration , 5 Hz repetition rate , 5 ms cosine2 ramped rise/fall time ) from two octaves above to three octaves below the estimated CF in 1/10th octave intervals .", "The stimulus level could be varied from 10 to 90 dB attenuation from the maximum output of the system in 5 dB steps .", "The number of spikes produced in response to each stimulus was counted and displayed at the appropriate position in a plot of tone frequency vs attenuation , according to a color scale .", "RLFs were generated from responses to CF tones ( 75 ms duration , 4 Hz repetition rate ) presented in a pseudorandom manner from 10 to 90 dB attenuation at a resolution of 10 dB .", "The number of spikes fired to each sweep was stored .", "RLFs were constructed from either 20 or 50 repetitions of each stimulus .", "At the conclusion of an experiment the animal was deeply anesthetized by an i . p . injection of sodium pentobarbital ( Euthanal , Merial , UK , 200 mg/ml , 2 mls ) and the animal was perfused transcardially with 300 ml of a wash solution comprised of 0 . 1 M phosphate buffered saline ( PBS ) at a pH of 7 . 4 ( ±0 . 1 ) , followed by perfusion of 200 ml of a fixative solution ( 4% paraformaldehyde in PBS ) .", "After further fixation , the brain was cryoprotected in 30% sucrose in 4% paraformaldehyde in PBS solution until it sank before being embedded in Tissue-Tek ( Sakura , NL ) and frozen .", "The brain was sectioned at 60 μm in the coronal plane on a cryostat ( HM 560 , Microm , UK ) and stained for Nissl substance with cresyl violet .", "Sections through the IC were imaged with an Axio Imager microscope running AxioVision software with a motorized stage ( Zeiss ) .", "The MosaiX module was used to collect images from each section containing the entire IC .", "The perimeter of the IC and the lesion caused by the microdialysis probe were measured using the AxioVision outline module .", "Initial characterizations of each unit response to the FRA paradigm were designated as the control FRAs to which FRAs measured during and after contralateral IC deactivation were compared .", "In this regard , each neuron served as its own control .", "FRAs and RLFs were recorded throughout and following deactivations lasting less than 30 min .", "Units were allowed an hour to recover , although the majority of those that recovered did so within 30 min of terminating cooling or MDP .", "Owing to the need to minimize the period of deactivation to ensure recovery recordings could be made , multiple FRAs could not be collected in each condition .", "For this reason statistical comparisons for individual units between conditions were not possible .", "However , because each FRA is made up of the response to many hundreds of stimuli , there are strong grounds to support the contention that each FRA is a reliable and repeatable representation of that unit's frequency response ( Palmer et al . , 2013 ) .", "Furthermore recordings in control conditions show a high degree of repeatability for the analysis .", "The CF and threshold were acquired from each FRA ( Ingham et al . , 2006 ) and responses to the lowest level of stimulation across all frequencies within the FRA were used to define the mean spontaneous firing rate .", "Those bins within each FRA that contained values greater than two standard deviations above the mean spontaneous rate were identified .", "For each frequency the lowest stimulus level at which the spike rate exceeded the mean spontaneous rate plus two standard deviations was deemed to be the excitatory threshold for that frequency .", "The array of excitatory thresholds was fitted with a tenth order polynomial which produced the excitatory frequency tuning curve ( FTC ) of the FRA .", "The minimum point of the FTC was used to define the CF and threshold of the unit .", "The number of frequency-level response bins within each FRA contained within this classifier were counted and compared between conditions .", "For some FRAs ( n = 27 ) , a tenth order polynomial was not accurate in reflecting the FRA .", "For these FRAs the number of bins exceeding a given number ( 1 spike per bin for most of these neurons ) of spikes was counted .", "The number of spikes in each FRA was also counted and compared between conditions for each unit .", "The half maximal firing rate of RLFs was measured by normalizing to the maximum firing rate in each condition and calculating the point along the abscissa which formed a right angle to the half firing rate on the ordinate .", "ROC analyses for each unit involved the pairwise comparison of all adjacent stimulus values in the RLF .", "The area under ROC function for each comparison was used as a measure of discriminability between those two stimuli .", "The area under the curve for each pairwise comparison was summed to create the ‘discriminability index’ for each RLF .", "We calculated a modulation index for the changes between control and deactivation conditions for numerous measures .", "Each modulation index was constructed in the standard manner using the equation: MI = ( deactivated − control ) / ( deactivated + control ) .", "Non-parametric statistical tests have been used throughout .", "All reported p values are exact and two tailed .", "A two sample Kolmogorov-Smirnov test was employed to assess changes in FRA area and FRA spikes between the distributions of V-shaped and nonV FRAs .", "Friedman's repeated measures ANOVA on ranks was used in population analyses of changes in half maximal firing rate in RLFs and changes in ROC discriminability .", "For post hoc analyses , two Wilcoxon signed rank tests were performed: one between the control group and the deactivated group; the other between the control group and the recovery group .", "For these tests the α was Šidák corrected to 0 . 0253 because two post hoc tests were employed on each occasion .", "A Spearman rank correlation was used to investigate the relationship between changes in the half maximum firing level in the RLF and changes in threshold .", "Fisher's exact test , structured as a 5 × 2 ( rows x columns ) contingency table , was used to test changes in RLF type using a binary classification for each unit , with zero being no change and one being change .", "These two columns were summed for each of the five RLF types found in the dataset .", "We did not predetermine sample sizes as it was impossible to provide an accurate estimate of the effect of our experimental manipulation ." ] ]

[ "Connections unifying hemispheric sensory representations of vision and touch occur in cortex , but for hearing , commissural connections earlier in the pathway may be important .", "The brainstem auditory pathways course bilaterally to the inferior colliculi ( ICs ) .", "Each IC represents one side of auditory space but they are interconnected by a commissure .", "By deactivating one IC in guinea pig with cooling or microdialysis of procaine , and recording neural activity to sound in the other , we found that commissural input influences fundamental aspects of auditory processing .", "The areas of nonV frequency response areas ( FRAs ) were modulated , but the areas of almost all V-shaped FRAs were not .", "The supra-threshold sensitivity of rate level functions decreased during deactivation and the ability to signal changes in sound level was decremented .", "This commissural enhancement suggests the ICs should be viewed as a single entity in which the representation of sound in each is governed by the other ." ]

[ "The bilateral arrangement of our eyes and ears enables us to receive information from both sides of our body .", "This information is conveyed via various sensory pathways that take different routes through the brain to culminate in the cerebral hemispheres .", "The information is then processed in the brain's outer layer , which is called the cortex .", "In the visual system , information from both eyes is kept separate until it reaches the cortex .", "A similar arrangement exists for touch .", "However , hearing is unusual among our senses in that sounds undergo much more processing in the brainstem , which is located at the base of the brain , than other types of stimuli .", "Orton and Rees now show that , in contrast to vision and touch , information about sounds occurring to our left or right is refined by interactions between the two sides of the midbrain .", "To test for sideward interactions between the two limbs of the auditory pathway , electrodes were lowered into the brains of anesthetized guinea pigs so that neuronal responses to tones could be recorded .", "The electrodes were placed in the region of the midbrain that contains two structures called the inferior colliculi ( meaning ‘lower hills’ in Latin ) .", "Each inferior colliculus predominantly receives inputs from the opposite ear .", "However , recordings made in one colliculus when the other was deactivated revealed that one colliculus normally alters the response of the other .", "This shows that there is an important sideward interaction between the two halves of the auditory pathway in the midbrain that refines how fundamental aspects of sound , such as its frequency and intensity , are processed .", "This represents a marked departure from our previous understanding of auditory processing in the mammalian brain , and opens up new lines of investigation into the functioning of the auditory system in health and disease ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "immunology and inflammation" ]

[ [ "Innate immune system serves as the first line defense of invading microorganisms and endogenous danger signals .", "Host cells can recognize pathogen-associated molecular patterns ( PAMPs ) of invading pathogens via pattern recognition receptors ( PRRs ) , initiate signaling pathways to regulate the expression of proinflammatory cytokines and type I interferons ( IFN α/β ) ( Yin et al . , 2015 ) .", "Upon virus infection , host cells can also recognize viral components , especially nucleic acids derived from viral genome or produced during viral life cycle ( Gürtler and Bowie , 2013 ) .", "Viral RNAs are mainly detected by RIG-I-like receptors ( such as RIG-I and MDA5 ) that induce type I IFNs via MAVS ( mitochondrial antiviral-signaling protein , also known as IPS1 , Cardif and VISA ) –dependent activation of TANK-binding kinase 1 ( TBK1 ) –interferon regulatory factor 3 ( IRF3 ) signaling pathway ( Chan and Gack , 2015; Yoneyama et al . , 2015 ) .", "On the other hand , viral DNAs are detected by multiple DNA sensors that induce type I IFNs mainly via stimulator of interferon genes ( STING , also known as MITA , MPYS and ERIS ) –dependent TBK1–IRF3 signaling pathway ( Paludan and Bowie , 2013; Cai et al . , 2014 ) .", "Despite of these findings , the innate response to viruses has not been completely understood .", "Studies are still required to elucidate detailed signaling and regulatory mechanisms initiated by PRRs in antiviral innate response .", "Ubiquitin ( Ub ) modification is one type of the crucial posttranslational modification mechanisms , and has been implicated in regulation of both innate and adaptive immunity ( Jiang and Chen , 2011; Zinngrebe et al . , 2014 ) .", "As compared to the typical single subunit E3 ligases ( such as RING-finger type and HECT type E3 ligases ) , the Cullin-RING multisubunit ligase complexes are assembled on the cullin backbones and have been implicated in diverse processes ( Skaar et al . , 2013; 2014 ) .", "S phase kinase-associated protein 1 ( Skp1 ) –cullin 1 ( Cul1 ) –F-box protein ( SCF ) complex is example of the Cullin-RING multisubunit ligase complexes ( Skaar et al . , 2013; 2014 ) .", "In this complex , Skp1 binds to F-box domain of many F-box proteins .", "More than sixty F-box proteins have been identified up to date , and they have been classified into three categories: those with WD40 domain ( Fbxw ) , those with leucine-rich repeats ( Fbxl ) and those with other diverse domains ( Fbxo ) ( Jin et al . , 2004; Skaar et al . , 2013; 2014 ) .", "Despite great achievements have been made for elucidating the roles of SCF complexes in cancer ( Skaar et al . , 2014 ) , the function of SCF in immunity remains poorly understood .", "Possibly the best characterized SCF complexes associated with immunity are the F-box– and WD40 repeat–containing Fbxw1 ( also known as β-Trcp1 , Fbw1a and Fwd1 ) and Fbxw11 ( also known as β-TrCP2 , Fbw11 , Fbxw1b , Fbx1b and Hos ) complexes that have been implicated in regulating NF-κB signaling by degrading IκBα or processing of p100 ( also known as NFκB2 ) and p105 ( also known as NFκB1 ) ( Yaron et al . , 1998; Shirane et al . , 1999; Spencer et al . , 1999; Tan et al . , 1999; Wu and Ghosh , 1999; Orian et al . , 2000; Fong and Sun , 2002; Lang et al . , 2003; Amir et al . , 2004 ) .", "Since Fbxo proteins usually contain unidentified variable domains in addition to the F-box domain , the functions of Fbxo proteins remain largely unknown , especially in regulation of immunity .", "Apoptosis signal-regulating kinase 1 ( ASK1 , also known as Map3k5 ) is a mitogen-activated protein kinase ( MAPK ) kinase kinase that plays pivotal roles in stress and immune responses ( Ichijo et al . , 1997; Shiizaki et al . , 2013 ) .", "ASK1 can activate MKK4/6 and MKK3/7 , leading to activation of c-Jun N-terminal kinases ( JNK1/2 ) and p38 MAPKs ( Ichijo et al . , 1997; Shiizaki et al . , 2013 ) .", "ASK1 is crucial for apoptosis in response to multiple stresses and factors ( Ichijo et al . , 1997; Sagasti et al . , 2001; Tobiume et al . , 2001; Nishitoh et al . , 2002; Maruoka et al . , 2003; Takeda et al . , 2004; Matsuzawa et al . , 2005; Shiizaki et al . , 2013 ) .", "ASK1 has also been implicated in Toll-like receptor 4 ( TLR4 ) -triggered innate responses ( Matsuzawa et al . , 2005 ) .", "Evolutionarily , ASK1 is a conserved protein expressed by many species ranging from C . elegans and drosophila to mammals ( Ausubel , 2005; Irazoqui et al . , 2010 ) .", "ASK1 homologs in C . elegans ( NSY-1 ) and drosophila ( DASK1 ) are important in control of apoptosis or innate immunity ( Ausubel , 2005; Irazoqui et al . , 2010; Shiizaki et al . , 2013 ) .", "So ASK1-mediated signaling pathway may be conserved during evolution and be of great significance in control of innate response upon virus infection .", "Previously we have investigated the roles of polyubiquitination in the regulation of innate signaling and innate cytokine production ( Wang et al . , 2009; Yang et al . , 2011; Chen et al . , 2013; Xia et al . , 2013; Liu et al . , 2014 ) .", "To further explore roles of Ub modification in regulation of innate response , we screened Fbxo proteins in macrophages and identified Fbxo21 as an important regulator in antiviral innate immunity .", "Since roles of ASK1 in TLR response has been reported ( Matsuzawa et al . , 2005 ) while ASK1 is identified as the potential target of Fbxo21 in current study , we focused on the roles of Fbxo21 in antiviral response .", "We found that Fbxo21 , by joining with Skp1-Cul1-Rbx1 ( SCFFbxo21 ) , is required for the production of inflammatory cytokines and type I IFNs upon vesicular stomatitis virus ( VSV ) and herpes simplex virus 1 ( HSV-1 ) infection .", "Our study suggests that Fbxo21 facilitates non-proteolytic Ub-modification of ASK1 , adding insights into the mechanistic understanding of antiviral innate response ." ], [ "By using RAW264 . 7 cells treated with VSV and HSV-1 , we screened the expression of Fbxo proteins by using GeneChip Microarrays ( Figure 1A ) .", "We then searched Pubmed of NCBI for function-unknown Fbxo members .", "Taking into consideration of the BioGPS database for Fbxo molecules that demonstrated characteristics of high abundance and PAMP responsiveness , we selected Fbxo8 , Fbxo16 , Fbxo18 , Fbxo21 , Fbxo38 , Fbxo41 , Fbxo42 and Fbxo46 as potential Fbxo candidates involved in innate immunity ( Figure 1—figure supplement 1A ) .", "To confirm their functions in innate response , we performed Q-PCR assays of Ifnb expression in peritoneal macrophages ( PMs ) with knockdown of indicated Fbxo molecules ( Figure 1—figure supplement 1B ) and treated the cells with lipopolysaccharide ( LPS , TLR4 agonist ) and VSV .", "We found that Fbxo21 knockdown most significantly inhibited the mRNA levels of Ifnb upon LPS or VSV treatments ( Figure 1—figure supplement 1C , D ) .", "Moreover , we found that Fbxo21 is abundantly expressed by multiple tissues and macrophages ( Figure 1—figure supplement 1E ) .", "These data suggest that Fbxo21 is a potential candidate of Fbxo members involved in innate immunity . 10 . 7554/eLife . 14087 . 003Figure 1 . Knockdown of Fbxo21 inhibits innate antiviral response of macrophages .", "( A ) Heat map representation of F-box genes in RAW264 . 7 cells infected with VSV ( MOI = 1 ) or HSV-1 ( MOI = 5 ) .", "( B ) Efficiency of Fbxo21 knockdown in macrophages , as examined by immunoblot 48 hr after transient transfection of indicated control ( Ctrl ) or Fbxo21 siRNAs .", "One representative experiment of three was shown .", "PM , peritoneal macrophages; BMM , bone marrow-derived macrophages .", "( C–F )", "Cells ( 1 x 105 cells per 24-well ) in ( B ) were infected with or without ( Moc ) VSV ( C , D; MOI = 1 ) or HSV-1 ( E , F; MOI = 5 ) as indicated for 8 hr .", "IL-6 ( C , E ) and IFNβ ( D , F ) concentrations in supernatant were determined by ELISA .", "Error bars indicated for mean ± SD of triplicate samples .", "Data were analyzed by one-way ANOVA followed by Bonferroni multiple comparison using PRISM software ( ***p < 0 . 001; versus control siRNA group infected with VSV or HSV-1 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 14087 . 00310 . 7554/eLife . 14087 . 004Figure 1—figure supplement 1 . Identification of Fbxo21 as an important regulator of innate response .", "( A ) Selection of potential Fbxo molecules in innate immunity .", "'*' indicates that there are reports for indicated Fbxo proteins but no reports about their roles in innate immunity .", "'**' indicates for data of monocytes or RAW264 . 7 cells treated with LPS .", "'***' indicates that data are not available ( NA ) .", "( B ) Efficiency of indicated siRNAs was examined by Q-PCR .", "Error bars indicate for mean ± SD of triplicate samples .", "( C , D )", "Peritoneal macrophages were transiently transfected with control ( Ctrl ) siRNAs or indicated siRNAs for 48h , and then challenged with 100 ng/ml LPS ( C , 6h ) or VSV ( MOI = 1 , D; 8h ) .", "The mRNA levels of Ifnb were measured by Q-PCR .", "Error bars indicate for mean ± SD of triplicate samples .", "Data were analyzed by one-way ANOVA followed by Bonferroni multiple comparison using PRISM software ( *** , p < 0 . 001; versus control siRNA group treated with LPS or VSV ) .", "( E ) Expression profiles of Fbxo21 mRNA in tissues and cells were examined by Q-PCR .", "Error bars indicate for mean ± SD of triplicate samples .", "PM , peritoneal macrophages; BMM , bone marrow-derived macrophages; BMDC , bone marrow-derived dendritic cells .", "β-actin was used as internal control in the Q-PCR assays . DOI: http://dx . doi . org/10 . 7554/eLife . 14087 . 00410 . 7554/eLife . 14087 . 005Figure 1—figure supplement 2 . Knockdown of Fbxo21 impairs innate TLR4 response . 48 hr after transient transfection of indicated control ( Ctrl ) or Fbxo21 siRNAs , cells ( 1 × 105 cells per 24-well ) were treated with or without 100 ng/ml LPS ( A , B ) , 1 μM CpG-ODN ( C , D ) or 10 μg/ml poly ( I:C ) ( E , F ) as indicated for 6 hr .", "IL-6 ( A , C , E ) and IFNβ ( B , D , F ) concentrations in supernatant were determined by ELISA .", "Error bars indicated for mean ± SD of triplicate samples .", "Data were analyzed by one-way ANOVA followed by Bonferroni multiple comparison using PRISM software ( ns , not significant; ***p < 0 . 001; versus control siRNA group treated with TLR agonists ) .", "Med , medium; CpG , CpG-ODN; pIC , poly ( I:C ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 14087 . 005 We then analyzed the effects of Fbxo21 knockdown ( Figure 1B ) on innate immune response in detail .", "We found that upon LPS treatments , the production of IL-6 and IFNβ by PMs , bone marrow-derived macrophages ( BMMs ) and RAW264 . 7 cells was all significantly decreased; But Fbxo21 knockdown only minimally affected IL-6 and IFNβ production of macrophages treated with CpG-ODN ( TLR9 agonist ) and TLR3 agonist poly ( I:C ) ( Figure 1—figure supplement 2A–F ) .", "Upon infection with VSV and HSV-1 , the production of IL-6 and IFNβ was significantly impaired in macrophages after Fbxo21 knockdown ( Figure 1C–F ) .", "These data suggest that Fbxo21 is required for innate antiviral response in macrophages .", "We further validated the roles of Fbxo21 in antiviral response by depleting Fbxo21 expression using CRISPR-Cas9–mediated genome editing technique in RAW264 . 7 cells and L929 fibroblasts ( Figure 2A ) .", "Fbxo21-/- RAW264 . 7 cells demonstrated significantly impaired IL-6 and IFNβ production in response to LPS treatment , transfection of poly ( I:C ) , poly ( dA:dT ) and poly ( dG:dC ) , or infection with VSV and HSV-1 ( Figure 2B–E ) .", "More importantly , Fbxo21 deficiency significantly promoted the replication of VSV and HSV-1 in RAW264 . 7 cells and L929 fibroblasts ( Figure 2F , G ) .", "By using VSV expressing green fluorescent protein ( VSV-GFP ) , we confirmed that Fbxo21 deficiency promoted the replication of VSV ( Figure 2H ) .", "These data convincingly demonstrate that Fbxo21 is required for antiviral innate response . 10 . 7554/eLife . 14087 . 006Figure 2 . Deficiency of Fbxo21 impairs antiviral innate response .", "( A ) Confirmation of Fbxo21 depletion .", "Fbxo21 expression in single-clone-derived RAW264 . 7 cells and L929 cells established by CRISPR-Cas9 editing was examined by immunoblot .", "One representative experiment of three was shown .", "( B , C )", "Fbxo21+/+ and Fbxo21-/- RAW264 . 7 cells ( 1 × 105 cells per 24-well ) were treated with or without 100 ng/ml LPS ( for 6 hr ) , or transfected with 2 . 5 μg/ml of liposome-packaged poly ( I:C ) , poly ( dA:dT ) or poly ( dG:dC ) ( for 8 hr ) .", "IL-6 ( B ) and IFNβ ( C ) concentrations in supernatant were determined by ELISA .", "Med , medium; CpG , CpG-ODN; pIC , poly ( I:C ) .", "( D–G )", "Fbxo21+/+ and Fbxo21-/- RAW264 . 7 cells ( D–F ) or L929 cells ( G ) were infected with or without ( Mock ) VSV ( MOI = 1 ) or HSV-1 ( MOI = 5 ) as indicated for 8 hr ( D , E ) or 12 hr ( F , G ) .", "IL-6 ( D ) and IFNβ ( E ) concentrations in supernatant were determined by ELISA .", "The titers of viruses in cells were determined by plaque formation assay ( F , G ) .", "In ( B–G ) , error bars indicated for mean ± SD of triplicate samples .", "Data were analyzed by one-way ANOVA followed by Bonferroni multiple comparison using PRISM software ( ns , not significant; ***p < 0 . 001; versus corresponding Fbxo21+/+ group ) .", "( H ) Fbxo21+/+ and Fbxo21-/- RAW264 . 7 cells were infected with or without ( Mock ) VSV-GFP virus ( MOI = 1 ) for 8 hr .", "Then cells were viewed under immunofluorescence microscope .", "One representative experiment of three was shown .", "Scale bars: 200 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 14087 . 006 In Fbxo21-/- RAW264 . 7 cells , we transiently overexpressed Fbxo21 ( Figure 3A ) .", "We found that Fbxo21 dose-dependently increased the mRNA expression of Il6 and Ifnb induced by VSV and HSV-1 ( Figure 3B–E ) .", "Correspondingly , we found that Fbxo21 overexpression promoted the transactivation of AP-1 , IFNβ and IRF3 reporters , but not NF-κB reporter , upon VSV and HSV-1 infection ( Figure 3F–I ) .", "As further evidence , the nuclear levels of active DNA-bound c-fos and IRF3 , but not p65 , were significantly decreased in Fbxo21–/– cells ( Figure 3J–O ) .", "Therefore Fbxo21 may promote innate antiviral response by activating the AP-1 and IRF3 signaling pathways . 10 . 7554/eLife . 14087 . 007Figure 3 . Fbxo21 overexpression promotes innate antiviral response , and activates AP-1 and IRF3-IFNβ signaling pathway .", "( A ) Fbxo21-/- RAW264 . 7 cells were transfected with Fbxo21 vectors ( 0 , 0 . 1 , 0 . 2 , 0 . 5 and 1 μg respectively ) for 48 hr .", "Fbxo21 expression was evaluated by immunoblot .", "( B–E )", "Cells in ( A , transfected with 0 , 0 . 1 , 0 . 2 and 0 . 5 μg Fbxo21 vectors respectively ) were infected with VSV ( MOI = 1 ) or HSV-1 ( MOI = 5 ) for 8 hr .", "Then indicated cytokines were examined by Q-PCR .", "β-actin was used as internal control in the Q-PCR assays .", "( F–I )", "Cells in ( A , transfected with 0 , 0 . 1 , 0 . 2 and 0 . 5 μg Fbxo21 vectors respectively ) were also transfected with indicated reporters and infected with VSV or HSV-1 for 4 hr .", "Then reporter transactivation was measured by dual-luciferase activity assays .", "( J–O )", "Fbxo21+/+ and Fbxo21-/- RAW264 . 7 cells ( J–L ) or L929 cells ( M–O ) were infected with VSV ( MOI = 1 ) or HSV-1 ( MOI = 5 ) for 4 hr .", "Then nuclear extracts were examined for indicated transcription factors bound to specific DNA sequence by ELISA .", "Error bars indicated for mean ± SD of triplicate samples .", "Data were analyzed by one-way ANOVA followed by Bonferroni multiple comparison using PRISM software ( ns , not significant; ***p<0 . 001; versus corresponding Fbxo21+/+ group ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 14087 . 007 We then analyzed the signaling pathways in Fbxo21-/- RAW264 . 7 cells infected with VSV and HSV-1 .", "We found that Fbxo21 deficiency inhibited the phosphorylation of JNK1/2 and p38 but not ERK1/2 mitogen-activated protein kinases ( MAPKs ) , the type I IFNs-associated IRF3 , but not the NF-κB-associated IKKα/β-IκBα ( Figure 4A , B ) .", "In Fbxo21-/- L929 cells , the phosphorylation of JNK1/2 , p38 and IRF3 was also decreased ( Figure 4C ) .", "Moreover , the nuclear levels of c-fos and IRF3 , but not p65 , were lower in Fbxo21-/- RAW264 . 7 and L929 cells ( Figure 4D , E ) .", "These data together indicate that Fbxo21 deficiency impairs virus-induced activation of the AP-1 and IRF3 signaling pathways . 10 . 7554/eLife . 14087 . 008Figure 4 . Deficiency of Fbxo21 inhibits virus-induced activation of JNK/p38-AP-1 and IRF3 . ( A–C ) Fbxo21+/+ and Fbxo21-/- RAW264 . 7 cells ( A , B ) or L929 cells ( C ) were infected with VSV ( MOI = 1 ) or HSV-1 ( MOI = 5 ) as indicated .", "The phosphorylation of indicated molecules was examined by immunblot .", "( D , E )", "Fbxo21+/+ and Fbxo21-/- RAW264 . 7 cells ( D ) or L929 cells ( E ) were treated as in ( A–C ) , and the nuclear proteins were extracted and examined for indicated transcription factors by immunoblot .", "One representative experiment of three was shown . DOI: http://dx . doi . org/10 . 7554/eLife . 14087 . 008 We then went to analyze the potential target of Fbxo21 by using immunoprecipitations ( IPs ) of endogenous Fbxo21 in RAW264 . 7 cells plus liquid chromatography mass spectrometry ( LC-MS ) assays .", "The MS data indicated that Fbxo21 could interact with ASK1 ( Figure 5—figure supplement 1 ) .", "Using IP assays in RAW264 . 7 and L929 cells , we found that Fbxo21 did interact with ASK1 under resting state ( Figure 5A ) .", "After VSV and HSV-1 infection , we examined the dynamic changes of Fbxo21-ASK1 interactions in RAW264 . 7 cells .", "We found that the interaction of Fbxo21-ASK1 was increased 0 . 5–2 hr after virus infection , and was then decreased 3–4 hr after virus infection ( Figure 5B ) , indicating that the potential modulation of ASK1 by Fbxo21 mainly occurred within 0 . 5–2 hr after virus infection . 10 . 7554/eLife . 14087 . 009Figure 5 . Fbxo21 interacts with ASK1 . ( A , B ) Wild type RAW264 . 7 cells or L929 cells infected with ( B ) or without ( A ) VSV and HSV-1 were prepared for whole cell extracts ( WCE ) , which were immunoprecipitated ( IP ) with anti-Fbxo21 or IgG as indicated .", "The associated ASK1 was examined by immunoblot .", "( C ) Schema of ASK1 mutants .", "CC1 , coiled-coil domain 1; KD , kinase domain; CC2 , coiled-coil domain 2 .", "( D ) HEK293 cells were cotransfected with Fbxo21-Myc and indicated Flag-tagged ASK1 mutants ( 1–4 mutants as illustrated in ( C ) ) for 48 hr .", "Whole cell lysates immunoprecipitated ( IP ) with anti-Myc agaroses were examined for Flag-tagged ASK1 ( mutant ) by immunoblot .", "( E ) Schema of Fbxo21 mutants .", "F-box , F-box domain; CC , coiled-coil domain; YccV , YccV domain .", "( F ) HEK293 cells were cotransfected with ASK1-Flag and indicated Myc-tagged Fbxo21 mutants ( 1–4 mutants as illustrated in ( E ) ) for 48 hr .", "Whole cell lysates immunoprecipitated ( IP ) with anti-Flag agaroses were examined for Myc-tagged Fbxo21 ( mutant ) by immunoblot .", "( G ) GST pull-down assays of Fbxo21 interaction with recombinant ( Rec ) ASK1 .", "'0' indicates for GST alone , and 1–4 indicate for GST-fused Fbxo21 ( mutant ) as in ( E ) .", "One representative experiment of three was shown . DOI: http://dx . doi . org/10 . 7554/eLife . 14087 . 00910 . 7554/eLife . 14087 . 010Figure 5—figure supplement 1 . MS assays of Fbxo21-associated proteins . Wild type RAW264 . 7 cells were treated with or without 100 ng/ml LPS or VSV ( MOI = 1 ) as indicated .", "Whole cells lysates were immunoprecipitated ( IP ) with anti-Fbxo21 antibody plus protein A/G beads .", "The immune complexes were separated in a SDS-PAGE gel , and stained with silver buffer .", "The differential bands were subjected to MS assays .", "The identified unique peptides of ASK1 were shown at the right side . DOI: http://dx . doi . org/10 . 7554/eLife . 14087 . 01010 . 7554/eLife . 14087 . 011Figure 5—figure supplement 2 . Fbxo21 is a component of SCF complex .", "( A ) Wild type RAW264 . 7 cells were treated with or without VSV ( MOI = 1 ) or HSV-1 ( MOI = 5 ) as indicated for 2 hr .", "Whole cell lysates immunoprecipitated ( IP ) with anti-Fbxo21 antibody plus protein A/G agaroses were examined for indicated SCF components by immunoblot ( IB ) .", "( B–D )", "HEK293 cells were cotransfected with Myc-tagged Fbxo21 or Fbxo21 without F-box domain ( #2 ) and indicated Flag-tagged SCF components for 48 hr .", "Whole cell lysates immunoprecipitated ( IP ) with anti-Myc agaroses were examined for indicated Flag-tagged proteins by immunoblot ( IB ) .", "One representative experiment of three was shown . DOI: http://dx . doi . org/10 . 7554/eLife . 14087 . 011 We then mapped the domains in Fbxo21 and ASK1 mediating the interaction .", "After transfection of Fbxo21 with ASK1 ( mutants ) plasmids ( Figure 5C ) in HEK293 cells , we found that ASK1 or ASK1 with only the N-terminal segment could be coimmunoprecipitated with Fbxo21 ( Figure 5D ) .", "When ASK1 was cotransfected with Fbxo21 ( mutants ) ( Figure 5E ) in HEK293 cells , it was found that full-length Fbxo21 or C-terminal segment of Fbxo21 could be coimmunoprecipitated with ASK1 ( Figure 5F ) .", "As further evidence , we performed GST pull-down assays and found that GST fusion proteins of Fbxo21 and Fbxo21 ( 466–628 aa ) could pull down recombinant ASK1 ( Figure 5G ) .", "These data suggest that the C-terminal part of Fbxo21 that contains one coiled-coil domain and one YccV-like protein domain , may interact with the N-terminal part of ASK1 that contains one coiled-coil domain and one thioredoxin-binding domain .", "To validate that Fbxo21 is a component of the SCF complex , we examined the endogenous Skp1 , Cul1 and Rbx1 in Fbxo21 immune complexes , and found that Skp1 , Cul1 and Rbx1 could be coimmunoprecipitated with Fbxo21 ( Figure 5—figure supplement 2A ) .", "Then we cotransfected Fbxo21 with conserved SCF components ( Skp1 , Cul1 or Rbx1 ) in HEK293 cells .", "We found that Fbxo21 , but not Fbxo21 without F-box domain , could interact with Skp1 , Cul1 and Rbx1 ( Figure 5—figure supplement 2B–D ) .", "These data indicate that Fbxo21 could form a complex with Skp1 , Cul1 and Rbx1 ( SCFFbxo21 ) .", "We then asked how SCFFbxo21 affected the signaling pathways via ASK1 .", "Since SCF complexes usually mediate the Ub-modification and proteasome degradation of substrates ( Skaar et al . , 2013; 2014 ) , we evaluated the ubiquitination and degradation of ASK1 in Fbxo21-/- RAW264 . 7 cells .", "We found that ASK1 could be polyubiquitinated after VSV and HSV-1 infection , which was significantly impaired in Fbxo21-/- RAW264 . 7 cells ( Figure 6A ) .", "However , we found that the degradation of ASK1 in Fbxo21-/- RAW264 . 7 cells was not affected ( Figure 6B ) .", "These data indicate that SCFFbxo21 may mediate non-proteolytic polyubiquitination of ASK1 . 10 . 7554/eLife . 14087 . 012Figure 6 . SCFFbxo21 mediates virus-induced Lys29-linkage of ASK1 . ( A ) Fbxo21+/+ and Fbxo21-/- RAW264 . 7 cells were infected with VSV ( MOI=1 ) or HSV-1 ( MOI = 5 ) for 2 hr .", "Then polyubiquitination of ASK1 was examined by immunoblot ( IB ) after immunoprecipitations ( IP ) .", "( B ) Fbxo21+/+ and Fbxo21-/- RAW264 . 7 cells were infected with VSV ( MOI = 1 ) or HSV-1 ( MOI = 5 ) as indicated .", "ASK1 and Fbxo21 levels in whole cell lysates were examined by immunoblot .", "( C ) Myc-tagged Fbxo21 and Flag-tagged ASK1 were cotransfected with HA-tagged Ub ( mutant ) into HEK293 cells for 48 hr , and infected with VSV ( MOI = 1 ) for 2 hr .", "Then polyubiquitinated ASK1 was examined by immunoblot ( IB ) against HA after immuneprecipitations ( IP ) with anti-Flag agaroses .", "1 , wild type Ub; 2 , Ub with only the Lys6 residue unchanged ( K6O-Ub ) ; 3 , K11O-Ub; 4 , K27O-Ub; 5 , K29O-Ub; 6 , K33O-Ub; 7 , K48O-Ub; 8 , K63O-Ub .", "( D , E )", "Fbxo21-/- RAW264 . 7 cells were transfected with indicated amounts ( 0 , 1 or 5 μg ) Fbxo21-Myc and equal amounts of K29O-Ub-HA vectors for 48 hr , and then infected with VSV ( MOI = 1 ) or HSV-1 ( MOI = 5 ) for 2 hr in the presence or absence of MLN-4924 ( 10 nM ) .", "Then polyubiquitinated ASK1 was examined by immunoblot ( IB ) against HA after immuneprecipitations ( IP ) .", "( F ) After incubation for 30 min , the in vitro polyubiquitination system was boiled for 5 min , and then polyubiquitinated ASK1 was examined by immunoblot ( IB ) against Ub after immuneprecipitations ( IP ) .", "One representative experiment of three was shown . DOI: http://dx . doi . org/10 . 7554/eLife . 14087 . 01210 . 7554/eLife . 14087 . 013Figure 6—figure supplement 1 . Analysis of Fbxo21 expression and degradation . RAW264 . 7 cells were infected with VSV ( MOI = 1 ) or HSV-1 ( MOI = 5 ) as indicated in the presence ( B , C ) or absence ( A ) of actinomycin D ( Act D , 10 μM ) or MG132 ( 10 μM ) .", "Then the mRNA level of Fbxo21 ( A , error bars indicate for mean ± SD of triplicate samples ) and the protein levels of Fbxo21 ( B , C ) were examined by Q-PCR and Western blot , respectively .", "β-actin was used as internal control in the Q-PCR assays .", "One representative experiment of three was shown . DOI: http://dx . doi . org/10 . 7554/eLife . 14087 . 013 It was noted that Fbxo21 protein was also decreased after VSV and HSV-1 infection ( Figure 6B ) while the mRNA levels of Fbxo21 were differentially regulated by VSV and HSV-1 infection ( Figure 1A ) .", "By using Q-PCR assays , we confirmed that Fbxo21 mRNA was induced by HSV-1 infection but decreased by VSV infection ( Figure 6—figure supplement 1A ) .", "To reconcile this discrepancy , we examined the protein levels of Fbxo21 in RAW264 . 7 cells after VSV and HSV-1 infection in the presence of a transcription inhibitor ( actinomycin D , Act D ) or a proteasome inhibitor ( MG132 ) .", "In the presence of Act D , the amounts of Fbxo21 protein were decreased after VSV and HSV-1 infection ( Figure 6—figure supplement 1B ) , indicating that Fbxo21 itself may be regulated by proteasomal degradation .", "In the presence of MG132 that blocks the degradation of Fbxo21 , we found that VSV infection could not decrease Fbxo21 while HSV-1 infection could induce the expression of Fbxo21 protein 12 hr after infection ( Figure 6—figure supplement 1C ) , confirming that VSV and HSV-1 may differentially regulate the transcription of Fbxo21 .", "Given that Fbxo21 was abundantly expressed in many cell types , it may be inferred that Fbxo21 may act at an early stage of antiviral response while at a later stage the transcriptional regulation of Fbxo21 mRNA may contribute to the differences in innate response to RNA virus versus DNA virus .", "We then analyzed the Ub-modification type of ASK1 by SCFFbxo21 .", "We found that Ub with only the intact Lys29 residue ( K29O-Ub ) could be linked to ASK1 by Fbxo21 ( Figure 6C ) .", "In Fbxo21-/- RAW264 . 7 cells reconstituted with Fbxo21 , we found that Fbxo21 could promote the linkage of K29O-Ub to ASK1 after virus infection ( Figure 6D , E ) .", "When the cells were treated with MLN-4924 , an inhibitor of NEDD8-activating enzyme that has been implicated in the assembly of SCF complex ( Soucy et al . , 2009 ) , we found that Fbxo21-mediated Lys29-linkage of ASK1 was abrogated ( Figure 6D , E ) , indicating that Fbxo21-mediated ASK1 modification requires the assembly of SCFFbxo21 complex .", "These data suggest that Fbxo21 , by joining with Skp1-Cul1-Rbx1 ( SCFFbxo21 ) , is required for Lys29-linkage of ASK1 .", "As direct evidence , we performed in vitro polyubiquitination assays .", "Since the E2 enzyme ( s ) specific for Lys29-linkage of Ub chains have not been identified , we utilized conjugation fraction A that contains predominantly E1 and E2 enzymes instead of defined E2s in the in vitroin vitro polyubiquitination assays .", "In the presence of recombinant GST-Fbxo21 , ASK1 and Cul1-Rbx1-Skp1 , as well as the NEDD8 conjugation system ( neddylation ) , ASK1 could be efficiently polyubiquitinated; while in the presence of MLN-4924 , the polyubiquitination of ASK1 was significantly inhibited ( left panel , Figure 6F ) .", "To confirm the Lys29-linkage of Ub chains to ASK1 , we used 6xHis-Ub-K29O ( with only Lys29 in Ub ) and 6xHis-Ub-K29R ( with the Lys29 mutated into Arg in Ub ) in the in vitro assays , and found that the former , but not the later , could be linked to ASK1 ( right panel , Figure 6F ) .", "These data further validate that SCFFbxo21 could promote the Lys29-linkage of ASK1 .", "We then went to identify the receptor sites for Lys29-linked Ub chain in ASK1 .", "We predicted the ubiquitination sites in ASK1 by using the Bayesian discriminant method-prediction of ubiquitination sites algorithm ( Figure 7—figure supplement 1A ) .", "We also performed multiple alignment of ASK1 protein sequences derived from various species ( for evolution conservation; Figure 7—figure supplement 1B–-J ) .", "By these 2 rounds of selection , ASK1 was predicted to contain 29 ubiquitination sites .", "To verify these ubiquitination sites , we constructed 18 mutants for ASK1 ( K1-K18 , mutated indicated Lys residue ( s ) into Arg; Figure 7—figure supplement 2A ) , and examined the ubiquitin levels associated with ASK1 after VSV treatments in HEK293 cells .", "We found that the K13 mutant of ASK1 ( K13-ASK1; mutation of Lys946 , Lys950 , Lys951 , Lys952 , Lys953 and Lys957 into Arg ) could most significantly decrease the levels of polyubiquitinated ASK1 ( Figure 7—figure supplement 2B and Figure 7A ) .", "In Fbxo21-/- RAW264 . 7 cells reconstituted with Fbxo21 and infected with VSV or HSV-1 , we found that wild type ASK1 but not K13-ASK1 could be polyubiquitinated ( Figure 7B ) .", "Meanwhile , we found that K13-ASK1 demonstrated remarkably lower activity as compared to wild type ASK1 ( Figure 7C , D ) .", "These data suggest that the K13 mutant-covered region in ASK1 may be the major ubiquitination sites modified by Fbxo21 during virus infection , and Fbxo21-mediated Lys29-linkage of ASK1 may be required for ASK1 activation . 10 . 7554/eLife . 14087 . 014Figure 7 . Lys29-linkage of ASK1 is crucial for Fbxo21-mediated ASK1 activation .", "( A ) HEK293 cells transiently transfected with indicated vectors for 48 hr and infected with VSV ( MOI = 1 ) for 2 hr in the presence or absence of MLN-4924 ( 10 nM ) .", "Then ASK1 ( mutant ) was immunoprecipitated ( IP ) with anti-Flag agaroses , and polyubiquitination was evaluated by immunblot ( IB ) against HA .", "( B ) Fbxo21–/– RAW264 . 7 cells were transfected with indicated vectors for 48 hr and infected with VSV ( MOI = 1 ) or HSV-1 ( MOI = 5 ) for 2 hr .", "Then ASK1 ( mutant ) was immunoprecipitated ( IP ) with anti-Flag agaroses , and polyubiquitination was evaluated by immunblot ( IB ) against Ub .", "( C , D )", "Fbxo21-/–- RAW264 . 7 cells were transfected with indicated vectors for 48 hr and infected with VSV ( MOI = 1 , C ) or HSV-1 ( MOI = 5 , D ) for 2 hr .", "Then ASK1 ( mutant ) was immunoprecipitated ( IP ) with anti-Flag agaroses , and the phosphorylated ASK1 was examined by immunoblot .", "Otherwise , the ASK1 ( mutant ) after IP was incubated with recombinant MKK4 , and then immunoblotted for phosphorylated MKK4 .", "One representative experiment of three was shown . DOI: http://dx . doi . org/10 . 7554/eLife . 14087 . 01410 . 7554/eLife . 14087 . 015Figure 7—figure supplement 1 . Prediction of the Lys residue in ASK1 responsible for linkage with Ub chain .", "( A ) The predicted Ub modification sites in ASK1 by using the Bayesian discriminant method-prediction of ubiquitination sites algorithm .", "( B–J )", "Alignment of ASK1 protein sequences derived from multiple species .", "Black arrow heads indicate for conserved Lys residues , and the red arrow heads for not completely conserved Lys residues . DOI: http://dx . doi . org/10 . 7554/eLife . 14087 . 01510 . 7554/eLife . 14087 . 016Figure 7—figure supplement 2 . Confirmation of the Lys residue in ASK1 responsible for linkage with Ub chain .", "( A ) Summary of the mutated ASK1 with indicated Lys residue ( s ) mutated into Arg .", "( B ) HEK293 cells were transiently transfected with indicated vectors for 48 hr and infected with VSV ( MOI = 1 ) for 2 hr .", "Then Polyubiquitination of ASK1 ( mutant ) in immunoprecipitations ( IPs ) was evaluated by immunoblot ( IB ) .", "Arrow head indicates for the K13-ASK1 .", "1 to 18 mutants of ASK1 were corresponding to those in ( A , K1 to K18 ) .", "W , wild type ASK1 .", "Prediction of the Lys residue in ASK1 responsible for linkage with Ub chain .", "One representative experiment of three was shown . DOI: http://dx . doi . org/10 . 7554/eLife . 14087 . 01610 . 7554/eLife . 14087 . 017Figure 7—figure supplement 3 . Fbxo21 deficiency impairs poly-Ub modification of ASK1 and then ASK1 activation .", "( A ) Fbxo21+/+ or Fbxo21–-/- RAW264 . 7 cells were infected with VSV ( MOI = 1 ) or HSV-1 ( MOI = 5 ) for 2 hr in the presence or absence of MLN-4924 ( 10 nM ) .", "Then ASK1 was immunoprecipitated ( IP ) with anti-ASK1 antibody plus protein A/G agaroses , and the phosphorylated ASK1 was examined by immunoblot .", "Otherwise , the ASK1 after IP was incubated with recombinant MKK4 , and then immunoblotted for phosphorylated MKK4 .", "( B , C )", "Fbxo21+/+ or Fbxo21-/-RAW264 . 7 cells were infected with VSV ( MOI = 1 ) as indicated .", "Then the polyubiquitination of ASK1 ( B ) and activation of ASK1 ( C , examined as in A ) were analyzed after immunoprecipitations ( IP ) .", "One representative experiment of three was shown . DOI: http://dx . doi . org/10 . 7554/eLife . 14087 . 017 To further examine the contribution of SCFFbxo21 to ASK1 activation during antiviral innate response , we examined the phosphorylation and activity of ASK1 in Fbxo21-/- cells and in MLN-4924-treated cells .", "We found that the phosphorylation and activity of ASK1 were significantly impaired in both Fbxo21-/- cells and MLN-4924-treated cells after VSV or HSV-1 infection ( Figure 7—figure supplement 3A ) .", "These data suggest that SCFFbxo21 is crucial for the phosphorylation and kinase activity of ASK1 .", "However , the relationship of ASK1 polyubiquitination with ASK1 activation has not been clearly demonstrated .", "To determine the potential roles of ASK1 polyubiquitination in ASK1 activation , we examined the dynamics of ASK1 modifications after VSV infection in Fbxo21+/+ and Fbxo21-/- RAW264 . 7 cells .", "We found that Fbxo21 deficiency significantly decreased the poly-Ub levels of ASK1 ( Figure 7—figure supplement 3B ) .", "However , the K48- and K63-linked poly-Ub levels of ASK1 were not significantly affected .", "Moreover , the phosphorylation of ASK1 as well as the kinase activity of ASK1 both occurred later than poly-Ub modification of ASK1 ( Figure 7—figure supplement 3C ) .", "These data together indicate that SCFFbxo21-mediated Lys29-linkage of ASK1 may prejudge the activation of ASK1 .", "Previously ASK1 has been implicated in the regulation of TLR4-triggerred inflammatory cytokine production ( Matsuzawa et al . , 2005 ) .", "However , the roles of ASK1 in regulating innate antiviral response and type I IFN production have not been fully elucidated .", "So we established RAW264 . 7 cells deficient for ASK1 ( Map3k5−/− ) by CRISPR-Cas9 genome editing ( Figure 8A ) .", "In Map3k5−/− RAW264 . 7 cells , the production of IL-6 and IFNβ triggered by VSV and HSV-1 infection was significantly inhibited while the virus titers of VSV and HSV-1 were significantly increased ( Figure 8B–D ) .", "Meanwhile , we found that the activation of JNK1/2 , p38 and IRF3 was significantly impaired in Map3k5−/− RAW264 . 7 cells infected with VSV and HSV-1 ( Figure 8E ) .", "When the Map3k5−/− RAW264 . 7 cells were rescued by overexpression of ASK1 , K13-ASK1 or ASK1-K716A ( Lys716 mutated into Ala , kinase activity inactive ) , we found that ASK1 , but not K13-ASK1 or ASK1-K716A , could rescue the impaired IL-6 and IFNβ production and the inhibited antiviral response ( Figure 8A–D ) .", "Furthermore , we found that K13-ASK1 could not rescue the impairment in activation of JNK1/2 , p38 and IRF3 , phosphorylation on Thr845 of ASK1 , and the kinase activity of ASK1 ( Figure 8F , G ) .", "These data convincingly demonstrate that Lys29-linkage of ASK1 is required for ASK1 activation and the subsequent innate antiviral responses . 10 . 7554/eLife . 14087 . 018Figure 8 . Lys29-linkage of ASK1 is crucial for innate antiviral response .", "( A ) Map3k5+/+ RAW264 . 7 cells and Map3k5–/– RAW264 . 7 cells transfected with mock vector or indicated ASK1 vectors for 48 hr were examined for ASK1 expression by Western blot .", "( B–D )", "Map3k5+/+ and Map3k5–/– RAW264 . 7 cells ( as in A ) were infected with or without ( Mock ) VSV ( MOI = 1 ) or HSV-1 ( MOI = 5 ) as indicated for 8 hr ( B , C ) or 12 hr ( D ) .", "IL-6 ( B ) and IFNβ ( C ) concentrations in supernatant were determined by ELISA .", "The titers of viruses in cells were determined by plaque formation assay ( D ) .", "Error bars indicated for mean ± SD of triplicate samples .", "Data were analyzed by one-way ANOVA followed by Bonferroni multiple comparison using PRISM software ( ns , not significant; ***p < 0 . 001 ) .", "( EG )", "Cells in ( A ) were infected with or without ( Mock ) VSV ( MOI = 1 ) or HSV-1 ( MOI = 5 ) as indicated .", "Then whole cell lysate ( E , F ) or immunoprecipitates ( G ) were examined by Western blot .", "In ( G ) , the immunoprecipitated ASK1 was examined for the kinase activity in the presence of 10 ng recombinant MKK4 .", "One representative experiment of three was shown . DOI: http://dx . doi . org/10 . 7554/eLife . 14087 . 018 The above results have established an essential role of SCFFbxo21 in ASK1-dependent antiviral innate responses .", "However , the downstream signaling pathways responsible for ASK1-mediated effects during VSV or HSV-1 infection have not been elucidated .", "Since ASK1 is a kinase mainly involved in activation of JNK1/2 and p38 ( Ichijo et al . , 1997 ) , we tested the effects of JNK1/2 and p38 inhibitors in Fbxo21-mediated signaling pathway .", "We found that JNK1/2 inhibitor SP600125 significantly , and pan-p38 inhibitor SB203580 to a much lesser extent , reversed Fbxo21-mediated activation of IRF3 ( Figure 9A , B ) and the production of IL-6 and IFNβ ( Figure 9C , D ) after VSV or HSV-1 infection .", "These data suggest that Fbxo21-mediated antiviral innate response may mainly require ASK1-dependent activation of JNK1/2 and , to a lesser extent , p38 ( Figure 9—figure supplement 1 ) . 10 . 7554/eLife . 14087 . 019Figure 9 . JNK1/2 activation is crucial for Fbxo21-mediated innate response . Fbxo21–/– RAW264 . 7 cells transfected with mock or Fbxo21 vectors were infected with VSV ( MOI = 1 ) or HSV-1 ( MOI = 5 ) for 4 hr ( A , B ) or 8 hr ( C , D ) in the presence or absence of SP600125 ( 20 μM ) or SB203580 ( 20 μM ) .", "The indicated proteins were examined by immunoblot ( A , B ) , and the cytokine concentrations in the supernatant were measured by ELISA ( C , D ) .", "One representative experiment of three was shown ( A , B ) .", "Error bars indicated for mean ± SD of triplicate samples ( C , D ) .", "Data were analyzed by one-way ANOVA followed by Bonferroni multiple comparison using PRISM software ( ns , not significant; ***p < 0 . 001 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 14087 . 01910 . 7554/eLife . 14087 . 020Figure 9—figure supplement 1 . Proposed working model for Fbxo21-mediated innate antiviral response . Upon infection with RNA or DNA viruses , host cells can recognize nucleic acids of viruses and initiate MAVS- or STING-dependent signaling pathway to induce the production of inflammatory cytokines and type I interferons .", "When Fbxo21 is involved , it can mediate Lys29-linkage of ASK1 by joining with the SCF complex , and in turn , activate JNK1/2 ( inhibited by SP600125 ) and p38 ( inhibited by SB203580 ) , leading to elevated activation of AP-1 and IRF3 for induction of the cytokines . DOI: http://dx . doi . org/10 . 7554/eLife . 14087 . 020" ], [ "By initiating PRRs-triggered signaling pathway , host cells can mount the production of proinflammatory cytokines as well as type I IFNs , leading to inflammatory responses and the elimination of invading pathogens ( Gürtler and Bowie , 2013; Yin et al . , 2015 ) .", "Our study shows that ASK1 activation facilitated by SCFFbxo21 ubiquitin ligase complex plays an important role in promoting virus-triggered innate response , serving as a critical module in antiviral innate response .", "More importantly , our study reveals a new type of Ub-modification ( Lys29-linkage ) in regulating kinase activation , and suggests that SCF complexes may not only mediate degradation of protein substrates but non-proteolytic regulation of substrates .", "Considering that ASK1 is a remarkably conserved kinase crucial in defense of infection ( Ausubel , 2005; Irazoqui et al . , 2010 ) , our study may be of great significance for understanding of innate response in species other than mammals .", "However , in vivo roles of Fbxo21 may need to be confirmed by using Fbxo21 knockout mice .", "Polyubiquitination of protein substrates represents a key posttranslational modification .", "Many single subunit E3 ubiquitin ligases have been implicated in regulating innate immune response ( Jiang and Chen , 2011; Zinngrebe et al . , 2014 ) .", "However , little is known about the roles of the multisubunit ubiquitin ligases in innate immunity .", "In SCF complex , the recognition of substrate is determined by F-box proteins ( Skaar et al . , 2013; 2014 ) .", "Our study identifies Fbxo21 as a component of SCF complex and interacts with ASK1 to regulate the ubiquitination by SCF complex , thus expanding the family of SCF complex in immunity .", "Previously many SCF complexes have been reported whereas few of them are involved in innate immune response ( Skaar et al . , 2013; 2014 ) .", "The best examples may be SCFFbxw1 ( β-Trcp1 ) and SCFFbxw11 ( β-Trcp2 ) that mediate the processing of NF-κB components and degradation of IκB isoforms ( Yaron et al . , 1998; Shirane et al . , 1999; Spencer et al . , 1999; Tan et al . , 1999; Wu and Ghosh , 1999; Orian et al . , 2000; Fong and Sun , 2002; Lang et al . , 2003; Amir et al . , 2004 ) .", "The SCFFBXW7α has also been implicated in processing p100 subunit of NF-κB ( Busino et al . , 2012; Fukushima et al . , 2012 ) .", "Fbxo3 has been implicated in the degradation of Fbxl2 , leading to impaired degradation of TRAF proteins and exaggerated inflammatory response ( Chen et al . , 2013 ) .", "Interestingly , GogB from Salmonella can interact with human Fbxo22 and inhibit inflammatory response ( Pilar et al . , 2012 ) .", "Our study suggests that Fbxo21 is required for innate antiviral response by activating ASK1-JNK signaling pathway , thus revealing an important role of Fbxo21 in innate immunity .", "More importantly , we have identified a new modification of ASK1 , that is , Lys29-linkage of ASK1 .", "Ub-modification of ASK1 has been reported previously .", "E3 ligases , such as cIAP-1 , SOCS1 , zinc-finger protein A20 and Stub1 ( also known as CHIP ) , have been implicated in degradation of ASK1 and thus a negative regulator of JNK and/or p38 activation and apoptosis ( Zhao et al . , 2007; Yu et al . , 2009; Zhang et al . , 2009; Won et al . , 2010 ) .", "In all these studies , Ub-modifications lead to ASK1 degradation .", "Since most of SCF complexes mediate degradation of substrates ( Skaar et al . , 2013; 2014 ) , our finding of SCFFbxo21-mediated non-proteolytic polyubiquitination of ASK1 may indicate that SCF complexes may exert functions via non-proteolytic modification of substrates .", "Ubiquitin serves as a small molecular marker on proteins ( Jiang and Chen , 2011; Zinngrebe et al . , 2014 ) .", "Ideally , the seven lysine residues of Ub ( lys6 , lys11 , lys27 , lys29 , Lys33 , lys48 and Lys63 ) can all be transferred to protein substrates by the ubiquitin system .", "Lys48-linkage and Lys63-linkage of Ub have been most extensively investigated .", "Similar to the effects of Lys63-linkage , Lys33-linkage of CD3ζ promotes its phosphorylation and interaction with Zap70 and possibly the transport of CD3ζ to lysosomes for degradation ( Ouchida et al . , 2008; Huang et al . , 2010 ) .", "Recently our study revealed that K33-linkage of Zap70 by Nrdp1 promoted the dephosphorylation of Zap70 and thus terminated early TCR signaling in CD8+ T cells ( Yang et al . , 2015 ) .", "For the other types of Ub-modifications in innate response , little is known .", "TRAF6 has been implicated in Lys29-linkage but aggregation of huntingtin protein ( Zucchelli et al . , 2011 ) .", "The E3 ligase Itch may mediate Lys29-linked polyubiquitination and degradation of Notch ( Chastagner et al . , 2008 ) .", "Previously deubiquitinating enzyme USP9X is implicated in the removal of Lys29-linked ubiquitin from AMPK-related kinases ( Al-Hakim et al . , 2008 ) .", "Meanwhile , USP9X stabilizes ASK1 by removal of Ub chains from ASK1 and prevention of ASK1 degradation ( Nagai et al . , 2009 ) .", "Our study suggests that SCFFbxo21 may be the ligase responsible for Lys29-linkage of ASK1 by demonstrating that Fbxo21 is required for Lys29-linkage of ASK1 upon viral infection , leading to non-proteolytic modification of ASK1 and ASK1 phosphorylation .", "Our data also indicate that Lys29-linkage of ASK1 could facilitate its phosphorylation and activation , and a crosstalk may exist between phosphorylation and Lys29-linked polyubiquitination .", "ASK1 is an important serine and threonine kinase involved in stress and immune response ( Shiizaki et al . , 2013 ) .", "In Map3k5−/− MEFs , TNFα-induced apoptosis and JNK activation are attenuated ( Tobiume et al . , 2001 ) .", "In Map3k5−/− thymocytes , FasL-induced activation of JNK and p38 is decreased while the apoptosis of thymocytes is not significantly affected ( Tobiume et al . , 2001 ) .", "The primary neurons derived from Map3k5−/− mice are resistant to polyglutamine-induced cell death ( Nishitoh et al . , 2002 ) .", "More importantly , Map3k5−/− mice demonstrate increased resistance to TLR4 , but not TLR3 and TLR9 , engagement , accompanied by impaired p38 activation ( Matsuzawa et al . , 2005 ) .", "Upon infection with influenza virus , Map3k5−/− MEFs show decreased JNK and p38 activation and inhibited cell death ( Maruoka et al . , 2003 ) .", "Despite these convincing results , roles of ASK1 in innate antiviral response are not fully elucidated .", "Our study suggests that ASK1 activation by SCFFbxo21 is important in induction of type I IFN production , by using VSV and HSV-1 viruses as infection models .", "Our study also suggests that ASK1-mediated JNK activation , and to a lesser extent p38 , is indispensable for type I IFN production , which is similar to the previous reports investigating TNFα-death receptor interaction ( Tobiume et al . , 2001 ) .", "Activity of ASK1 is regulated by many molecules that complex with ASK1 ( ASK1 signalosome , about 1 , 500–2 , 000 kDa ) , such as ASK1 itself , ASK1 homologues , thioredoxin , TRAF2 , TRAF6 and Daxx etc . ( Shiizaki et al . , 2013 ) .", "More unidentified molecules in ASK1 signalosome may exist for modulation of ASK1 activation .", "It has been proposed that ASK1 is usually activated via autophosphorylation after TRAF2− and TRAF6−assisted homodimerization in TNFα response ( Tobiume et al . , 2002; Noguchi et al . , 2005 ) .", "In our study , we have found a complex containing Fbxo21 and SCF components , thus adding new components to ASK1 signalosome .", "Whether SCFFbxo21 associates with other components in ASK1 signalosome may need further investigation , and whether SCFFbxo21-mediated ASK1 activation needs TRAF2 and TRAF6 or upstream kinases may also be worthy of more studies .", "When we are preparing the manuscript , it is reported that ASK1 is required for type I IFN production during antiviral innate response ( Okazaki et al . , 2015 ) .", "In the current study , we also found that ASK1 deficiency led to impaired type I IFN production and increased virus replication .", "More importantly , we found that ASK1 deficient in Lys29-linkage could not rescue antiviral innate response in Map3k5−/− RAW264 . 7 cells .", "Therefore , Fbxo21-mediated Ub-modification and activation of ASK1 are essential for innate antiviral response .", "It is interesting to find that JNK inhibitor could decrease the activation of IRF3 and type I IFN production .", "Previously it has been reported that JNK1/2 may be involved in activating IRF3 by phosphorylating IRF3 on Ser173 ( Zhang et al . , 2009 ) or licensing TBK1-mediated IRF3 phosphorylation on Ser396 ( Nociari et al . , 2009 ) , which may explain the effects of JNK1/2 inhibition on decreased innate antiviral response mediated by Fbxo21 .", "Therefore , it can be inferred that Fbxo21-mediated ASK1 activation may be involved in regulation of antiviral innate response via JNK-dependent IRF3 activation .", "In sum , our study has shown that Fbxo21 plays an indispensable role in regulating innate antiviral response by promoting the Lys29-linkage and activation of ASK1 and subsequently ASK1-mediated JNK1/2 activation .", "Therefore Fbxo21-mediated regulation of ASK1 polyubiquitination and ASK1 activation may represent a new type of Ub modification by SCF complex and act as an intrinsic part of antiviral innate response ." ], [ "Wild type C57BL/6 mice ( 6–8 weeks old ) were purchased from Joint Ventures Sipper BK Experimental Animal ( Shanghai , China ) .", "All animal experiments were undertaken in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals , and with the approval of the Scientific Investigation Board of Second Military Medical University , Shanghai .", "HEK293 , RAW264 . 7 and L929 cells were obtained from ATCC ( Manassas , VA ) and cultured as recommended .", "Peritoneal macrophages and bone marrow-derived macrophages were obtained , prepared and cultured as described ( Tang et al . , 2014 ) .", "Recombinant M-CSF , GM-CSF and IL-4 were from R&D Systems ( Minneapolis , MN ) .", "Antibodies specific to Flag-tag ( M2 , ab49763 ) , HA-tag ( HA . C5 , ab18181 ) , His-tag ( HIS . H , ab18184 ) , Myc-tag ( Myc . A7 , ab18185 ) , β-actin ( AC-15 , ab6276 ) , c-fos ( EPR883 ( 2 ) , ab134122 ) , Cullin 1 ( EPR3103Y , ab75817 ) , Fbxo21 ( EPR13163 , ab179818 ) , IKKβ ( Y466 , ab32135 ) , and ubiquitin ( Ubi-1 , ab7254 ) , and the agaroses used in immunoprecipitations were from Abcam Inc . ( Cambridge , MA ) .", "Abs specific for ASK1 ( D11C9 , #8662 ) , ERK1/2 ( 137F5 , #4695 ) , GSK3β ( D5C5Z , #12456 ) , IkBa ( 44D4 , #4812 ) , IRF3 ( #4302 ) , JNK1/2 ( 56G8 , #9258 ) , K48-linkage specific polyubiquitin ( D9D5 , #8081 ) , K63-linkage specific polyubiquitin ( D7A11 , #5621 ) , p38 ( D13E1 , #8690 ) , p65 ( L8F6 , #6956 ) , phospho-ASK1 ( Thr845 ) ( #3765 ) , phospho-ERK1/2 ( Thr202/Tyr204 ) ( 197G2 , #4377 ) , phospho-GSK3α/β ( Ser21/9 ) ( D17D2 , #8566 ) , phospho-IkBa ( Ser32/Ser36 ) ( 5A5 , #9246 ) , phospho-IKKα/β ( Ser176/Ser180 ) ( C84E11 , #2078 ) , phospho-IRF3 ( Ser396 ) ( 4D4G , #4947 ) , phospho-JNK1/2 ( Thr183/Tyr185 ) ( 81E11 , #4668 ) , phospho-p38 ( Thr180/Tyr182 ) ( 12F8 , #4631 ) , Rbx1 ( D3J5I , #11922 ) and Skp1 ( D3J4N , #12248 ) were from Cell Signaling Technology ( Beverly , MA ) .", "The inhibitors , including MLN-4924 , SP600125 and SB203580 , were obtained from MedChemExpress ( Monmouth , NJ ) .", "C-class CpG ODN ( ODN 2395 ) , poly ( dA:dT ) /LyoVec and poly ( dG:dC ) /LyoVec were obtained from Invivogen ( San Diego , California ) .", "Poly ( I:C ) and LPS ( 0111:B4 ) were purchased from Sigma ( St . Louis , MO ) .", "The other non-specified reagents were form Sigma .", "The recombinant vectors encoding mouse ASK1 ( GenBank No . NM_008580 . 4 ) , Fbxo21 ( GenBank No . NM_145564 . 3 ) and the indicated mutations were constructed as described ( Wang et al . , 2009; Yang et al . , 2011 ) .", "Flag-tagged vectors for Skp1 , Cullin-1 and Rbx1 were from Origene ( Beijing , China ) and subcloned into pcDNA3 . 1 vector as described ( Wang et al . , 2009 ) .", "For transient transfection of plasmids in RAW264 . 7 and L929 cells , the X-tremeGENE HP reagents were used according to manufacturer’s instructions ( Roche , Welwyn Garden City , UK ) .", "For transient knockdown of Map3k5 or Fbxo21 , the siRNA duplexes specific for Map3k5 ( sc-29749; Santa Cruz Biotechnology , Dallas , TX ) or Fbxo21 ( 5’ GCAGAAAGCTGGGTTAGAA 3’ ) were transfected using the INTERFERin-HTS according to the standard protocol ( Polyplus-transfection Company , Illkirch , France ) .", "The nonsense sequence 5’-TTCTCCGAACGTGTCACG-3’ was used as control siRNA .", "For the depletion of Fbxo21 or ASK1 , pc3-U6-guide RNA-CMV-RED ( encoding guiding RNA and red fluorescent protein ) and Cas9-IRES-EGFP ( encoding Cas9 and green fluorescent protein ) plasmids ( kind gifts from Shanghai Biomodel Organism Science & Technology Development Co . , Shanghai , China ) were cotransfected into RAW264 . 7 or L929 cells ( Tang et al . , 2014 ) .", "Four target sequences for each guiding RNA synthesis against Fbxo21 and Map3k5 were tested , and the target sequences ( 5’ GGTAGCGGGGGACAGCGCTA 3’ for Fbxo21 , and 5’ TTTCGTTCAGCTTTTGAAGG 3’ for Map3k5 ) were mostly efficient in depletion of Fbxo21 and ASK1 .", "Cells with both red and green fluorescence were then sorted by using Gallios Flow Cytometer ( Beckman Coulter , Brea , CA ) .", "Sorted cells were cultured for 3–5 days , and clones propagated from single cell were picked out .", "The depletion of Fbxo21 or ASK1 was confirmed by both Western blot and DNA sequencing .", "Total cellular RNA was extracted using Trizol reagent ( Invitrogen Corporation , CA , USA ) , and cDNA was synthesized by using the AMV Reverse Transcriptase kit ( Promega , Madison , WI ) .", "Specific primers used for Q-PCR assays and the mRNA quantification methods were as described ( Tang et al . , 2014 ) .", "β-actin was used as the internal quantitative control .", "Microarray expression analysis was performed using a high-density oligonucleotide array ( Affymetrix GeneChip array , Affymetrix , Santa Clara , CA , USA ) as described ( Yang et al . , 2015 ) , and microarray expression analysis was done according to the instruction manual .", "The extraction of total mRNA was done using Trizol ( Invitrogen Corporation ) .", "The obtained mRNA was hybridized to each array .", "Hybridization of biotin-labeled cRNA fragment to Mouse Genome 430 2 . 0 array , washing , staining with streptavidin-phycoerythrin ( Molecular Probes ) , and signal-amplification were performed according to the manufacturer's instructions .", "The changes of the interested genes were presented in heat map generated from the microarray results by using R Statistics .", "ELISA kits for mouse IFNβ and IL-6 were from R&D Systems ( Minneapolis , MN ) .", "The concentrations of cytokines in the culture supernatants were determined as described ( Wang et al . , 2009; Tang et al . , 2014 ) .", "The AP-1 , NF-κB , IRF3 and IFNβ luciferase reporter plasmids were obtained from Panomics of Affymetrix ( Santa Clara , CA ) or as described ( Wang et al . , 2009; Yang et al . , 2011 ) .", "Luciferase activities were measured with Dual-Luciferase Reporter Assay System ( Promega , Madison , WI ) .", "The determination of reporter transactivation was performed as described previously ( Wang et al . , 2009; Yang et al . , 2011 ) .", "For propagation of VSV and HSV-1 , Vero cells were infected with virus at a MOI of 2 , and the viruses produced by one replication cycle were harvested .", "For purification of virus , the cell cultures were first freeze-thawed twice , and the supernatants were clarified by centrifugation at 3000 ×g for 1 hr , followed by pelleting of the virus by ultracentrifugation at 45 , 000 ×g for 1 hr .", "For quantification of virus in infected cells , cells were homogenized in MEM supplemented with 2% FCS just before use .", "The homogenates were pelleted by centrifugation at 1600 ×g for 30 min , and the supernatants ( 100 μl ) were used for plaque assays on monolayers of Vero cells .", "The cells were left overnight to settle and were infected by incubation for 1 hr at 37°C with serial dilutions .", "After 1 hr , 1 ml of medium was added , and the plates were incubated for 2 days .", "After incubation , the plates were stained with 0 . 03% methylene blue to allow quantification of plaques .", "The numbers of plaque formation unit ( PFU ) were counted , and the results were expressed as PFU/ml of homogenates .", "Immunoprecipitated Fbxo21 immune complexes were separated on SDS-PAGE gel as described previously ( Yang et al . , 2015 ) .", "After silver staining , each differential gel-band was excised and then analyzed by nano-ultra-performance liquid chromatography–electrospray ionization tandem mass spectrometry .", "The immunoprecipitations using anti-Fbxo21 , anti-Flag or anti-Myc , and the immunoblot assays were performed as described previously ( Wang et al . , 2009; Yang et al . , 2015 ) .", "Nuclear proteins were extracted by NE-PER Protein Extraction Reagent ( Pierce , Rockford , IL ) according to the manufacturer’s instructions .", "Protein concentration was determined by the BCA protein assay ( Pierce ) .", "For the analysis of nuclear transcription factors that could bind with specific DNA sequences , nuclear extracts were examined for c-fos , IRF3 and p65 by using the TransAM Transcription Factor ELISA Kits from Active Motif Inc . ( Carlsbad , CA ) as recommended .", "Mouse wild-type or truncation coding sequences for Fbxo21 were inserted 3’ and in frame to glutathione S-transferase ( GST ) coding sequence in pGEX-2T vector ( GE Health , Piscataway , NJ ) .", "Escherichia coli BL21 ( DE3 ) cells ( 50 ml at an optical density 600 nm of 0 . 6 ) harboring the recombinant expression vectors were incubated with 1 mM isopropyl β-D-thiogalactopyranoside ( IPTG; Sigma ) at 37°C for 3 hr .", "For GST fusion protein purification , cells were harvested by centrifugation and suspended in 10 ml PBST containing 2 mM EDTA , 0 . 1% β-mercaptoethanol , 0 . 2 mM PMSF , and 5 mM benzamidine .", "Cells were lysed by passing through a French press twice at 1200 lb/in2 .", "1 ml of bacterial supernatant was mixed with 100 μl 50% ( v/v ) glutathione-agarose beads ( Pierce of Fisher , Rockford , IL ) and incubated for 30 min at 4°C with gentle rotating .", "The agarose beads were washed four times with 10 ml ice-cold PBST or lysis buffer .", "The fusion proteins were eluted by 100 μl of 10 mM glutathione in 50 mM Tris ( pH 8 . 0 ) at 4°C .", "The purity and quantity of fusion proteins were examined by SDS-PAGE followed by Coomassie blue staining .", "The GST pull-down assays were conducted as described previously ( Wang et al . , 2009; Yang et al . , 2015 ) .", "For in vitro kinase assays , 100 μg proteins contained in total cell extracts or nuclear extracts were immunoprecipitated with indicated antibodies plus protein A/G beads by gently rocking at 4°C for 2 hr followed by centrifugation at 4°C for 5 min .", "The pellets were washed with the lysis buffer ( 20 mM Tris–HCl , pH 7 . 4 , 150 mM NaCl , 1 mM EDTA , 1% Triton X-100 , 2 . 5 mM sodium pyrophosphate , 1 mM β-glycerolphosphate , 1 mM Na3VO4 and 1 mM PMSF ) .", "Each washed pellet was resuspended in the kinase assay buffer ( 25 mM Tris-HCl , pH 7 . 5 , 5 mM β-glycerolphosphate , 2 mM DTT , 0 . 1 mM Na3VO4 , and 10 mM MgCl2 ) and supplemented with 10 μM ATP and 0 . 5 μg of recombinant MKK4 ( Abcam ) in a total volume of 30 μl at 30°C for 30 min .", "Then phosphorylated MKK4 was examined by Western blot .", "For analysis of ubiquitination of ASK1 in vivo , whole-cell extracts prepared with radioimmunoprecipitation assay buffer ( 50 mM Tris , pH 8 . 0 , 150 mM NaCl , 1% ( vol/vol ) Nonidet-P40 , 0 . 5% ( wt/vol ) sodium deoxycholate , 1% ( wt/vol ) SDS and proteinase inhibitors ) supplemented with 10 mM N-ethylmaleimide , were immunoprecipitated with indicated antibodies and analyzed by immunoblot as described ( Wang et al . , 2009; Yang et al . , 2015 ) .", "The in vitro analysis of ubiquitination of ASK1 was performed as described ( Chen et al . , 2013 ) with modifications .", "The ubiquitination of ASK1 was performed in 50 μl buffer containing 40 mM Tris-HCl , pH 7 . 1 , 40 mM NaCl , 1 mM β-ME , 5 mM MgCl2 supplemented with 2 mM ATP , 1 μM ubiquitin aldehyde , 1 μg Conjugation Fraction A ( F-340 , BostonBiochem , Cambridge , MA ) , 1 μg/μl ubiquitin and derivatives ( BostonBiochem ) , 10 ng/μl recombinant ASK1 ( MAP3K5-9515M ) and Cul1-Rbx1-Skp1 ( CUL1-147H ) ( Creative Biomart , Shirley , NY ) and 10 ng/μl GST-Fbxo21 in the presence or absence of Nedd8 Conjugation System ( #J3150 , UBPBio , Aurora , CO ) and 5 μM MLN-4924 for 30 min at 37°C .", "Reaction products were finally processed for Ub immunoblotting .", "All the experiments were independently repeated at least three times .", "Dr . X . Z . who is blinded to group design performed the analysis .", "Results are given as mean ± SE or mean ± SD .", "Multiple comparisons were done with one-way ANOVA followed by Bonferonni’s multiple comparison test .", "Statistical significance was determined as p<0 . 05 .", "Complete microarray data set , GSE72077 ." ] ]

[ "Protein ubiquitination regulated by ubiquitin ligases plays important roles in innate immunity .", "However , key regulators of ubiquitination during innate response and roles of new types of ubiquitination ( apart from Lys48- and Lys63-linkage ) in control of innate signaling have not been clearly understood .", "Here we report that F-box only protein Fbxo21 , a functionally unknown component of SCF ( Skp1–Cul1–F-box protein ) complex , facilitates Lys29-linkage and activation of ASK1 ( apoptosis signal-regulating kinase 1 ) , and promotes type I interferon production upon viral infection .", "Fbxo21 deficiency in mice cells impairs virus-induced Lys29-linkage and activation of ASK1 , attenuates c-Jun N-terminal kinase ( JNK ) and p38 signaling pathway , and decreases the production of proinflammatory cytokines and type I interferon , resulting in reduced antiviral innate response and enhanced virus replication .", "Therefore Fbxo21 is required for ASK1 activation via Lys29-linkage of ASK1 during antiviral innate response , providing mechanistic insights into non-proteolytic roles of SCF complex in innate immune response ." ]

[ "The innate immune system is the body’s first line of defense against being infected by viruses and other microbes .", "Upon recognizing a virus , host cells trigger the innate immune response in an effort to eliminate the threat .", "However , innate immune responses must be carefully controlled because an excessive response can cause inflammation that harms the body .", "The innate immune response involves a variety of cells and processes that are each activated through a series of communication systems called signaling pathways .", "While much has been learned about which parts of a virus trigger the innate immune response , it is not clear how the immune response to the virus is controlled .", "It has been suggested that a process known as ubiquitination could be involved in regulating the activity of signaling pathways that activate the innate immune response .", "During ubiquitination , enzymes attach a small molecule called ubiquitin to a specific target protein .", "Ubiquitin often acts as a label that targets a particular protein for destruction .", "Enzymes called E3 ubiquitin ligases play central roles in identifying specific target proteins for ubiquitination .", "Some of these enzymes consist of a single protein unit that acts alone , but other E3 ubiquitin ligases are formed by groups ( or “complexes” ) of several proteins working together .", "Members of the F-box only protein family are components of some ubiquitin ligase complexes .", "Here , Yu et al . used a “microarray” technique to assess which F-box only proteins in mice are produced during an immune response to two viruses .", "The experiments identified an F-box protein called Fbxo21 as a potential candidate for a role in regulating the innate immune response .", "Additional experiments revealed that Fbxo21 is involved in adding ubiquitin to a specific location on a signaling protein called ASK1 , which is known to be crucial for innate immune responses .", "Instead of targeting ASK1 for destruction , this ubiquitination activates ASK1 .", "Therefore , Yu et al . ’s findings demonstrate that Fbxo21 plays an important role in regulating innate immune responses .", "A future challenge is to investigate exactly how ASK1 is activated by the ubiquitin ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "computational and systems biology", "genetics and genomics" ]

[ [ "Amyloid plaques consisting of the amyloid beta ( Aß ) peptide are a pathological hallmark of Alzheimer’s disease ( AD ) , the most common cause of dementia and a leading global cause of morbidity with very high societal and economic impact ( Ballard et al . , 2011; World Health Organization , 2012 ) .", "Although most cases of AD are sporadic and of uncertain cause , rare familial forms of the disease also exist ( Campion et al . , 1999 ) .", "These inherited forms of dementia typically have earlier onset and are caused by high penetrance mutations in multiple loci , including in the amyloid precursor protein ( APP ) gene , which encodes the protein from which Aß is derived by proteolytic cleavage ( O'Brien and Wong , 2011 ) .", "Several mutations in PSEN1 and PSEN2 , the genes coding for the secretases performing sequential cleavage of APP , also lead to autosomal dominant forms of AD .", "The two most abundant isoforms of Aß generated upon cleavage are 42 and 40 amino acids ( aa ) in length , with the longer Aß peptide associated with increased aggregation in vitro and cellular toxicity ( Meisl et al . , 2014; Sandberg et al . , 2010 ) .", "The amyloid state is a thermodynamically low energy state but , both in vitro and in vivo , the spontaneous formation of amyloids is normally very slow because of the high kinetic barrier of fibril nucleation ( Knowles et al . , 2014 ) .", "The process of nucleation generates oligomeric Aß species that have been hypothesized to be particularly toxic to cells and that then grow into fibrils ( Michaels et al . , 2020; Bolognesi et al . , 2010; Cleary et al . , 2005 ) .", "Fourteen different mutations in the Aß peptide have been reported to cause familial Alzheimer’s disease ( fAD ) , with all but two having a dominant pattern of inheritance ( Weggen and Beher , 2012; Van Cauwenberghe et al . , 2016 ) .", "However , it is not clear why these particular mutations cause fAD ( Weggen and Beher , 2012; Van Cauwenberghe et al . , 2016 ) , and these 14 mutations represent only 3 . 7% of the possible 378 single nucleotide changes that can occur in Aß .", "As for nearly all disease genes , therefore , the molecular mechanism by which mutations cause the disease remains unclear and the vast majority of possible mutations in Aß are variants of uncertain significance ( VUS ) .", "This makes the clinical interpretation of genetic variation in this locus a difficult challenge ( Starita et al . , 2017; Gelman et al . , 2019 ) .", "Moreover , given the human mutation rate and population size , it is likely that nearly all of these possible variants in Aß actually exist in at least one individual currently alive on the planet ( Conrad et al . , 2011 ) .", "A comprehensive map of how all possible mutations affect the formation of Aß amyloids and how these changes relate to the human disease is therefore urgently needed .", "More generally , amyloid fibrils are associated with many different human diseases ( Knowles et al . , 2014 ) , but how mutations alter the propensity of proteins to aggregate into amyloid fibrils is not well understood and there has been no large-scale analysis of the effects of mutations on the formation of any amyloid fibril .", "Here , we address this shortcoming by quantifying the rate of fibril formation for >14 , 000 variants of Aß .", "This provides the first comprehensive map of how mutations alter the propensity of any protein to form amyloid fibrils .", "The resulting data provide numerous mechanistic insights into the process of Aß fibril nucleation .", "Moreover , they also accurately classify all the known dominant fAD mutations , validating the clinical relevance of a simple cell-based model and providing a comprehensive resource for the interpretation of clinical genetic data ." ], [ "To globally quantify the impact of mutations on the nucleation of Aß fibrils , we used an in vivo selection assay in which the nucleation of Aß is rate-limiting for the aggregation of a second amyloid , the yeast prion [PSI+] encoded by the sup35 gene ( Chandramowlishwaran et al . , 2018 ) .", "Aggregation of Sup35p causes read-through of UGA stop codons , allowing growth-based selection using an auxotrophic marker containing a premature termination codon ( Figure 1A and Figure 1—figure supplement 1A ) .", "We generated a library containing all possible single nucleotide variants of Aß42 fused to the nucleation ( N ) domain of Sup35p and quantified the effect of mutations on the rate of nucleation in triplicate by selection and deep sequencing ( Faure et al . , 2020; see Materials and methods ) .", "The selection assay was highly reproducible , with enrichment scores for aa substitutions strongly correlated between replicates ( Figure 1B and Figure 1—figure supplement 1B ) .", "Comparing our in vivo enrichment scores to the qualitative effects of 16 mutations analysed in vitro across 10 previous publications validated the assay , with mutational effects matching the effects on in vitro nucleation previously reported for 14 Aß variants out of 16 .", "( Supplementary file 1 ) .", "Moreover , the in vivo scores correlate extremely well with the rate of nucleation of Aß variants in positions 21 , 22 , 23 ( Yang et al . , 2018; Törnquist et al . , 2018; Figure 1C and Figure 1—figure supplement 1C ) .", "We henceforth refer to the in vivo enrichment scores as ‘nucleation scores’ ( NS ) .", "A prior deep mutational scan quantified the effects of mutations on the abundance of Aß fused to an enzymatic reporter ( Gray et al . , 2019 ) .", "These ‘solubility scores’ do not predict the effects of mutations on Aß nucleation ( Figure 1—figure supplement 1D ) .", "Previously we identified a principal component of aa properties ( principal component 1 [PC1] , related to changes in hydrophobicity ) that predicts the aggregation and toxicity of the amyotrophic lateral sclerosis ( ALS ) protein TDP-43 when it is expressed in yeast ( Bolognesi et al . , 2019 ) .", "PC1 is also not a good predictor of Aß nucleation ( Figure 1D ) but it does predict the previously reported changes in Aß solubility ( Figure 1E ) , suggesting that Aß is aggregating by a similar process to TDP-43 in this alternative selection assay ( Gray et al . , 2019 ) but by a different mechanism in the nucleation selection .", "The distribution of mutational effects for Aß nucleation has a strong bias towards reduced nucleation , with 56% of single aa substitutions reducing nucleation but only 16% increasing it ( Z-test , false discovery rate [FDR] = 0 . 1 , Figure 2A ) .", "Moreover , mutations that decrease nucleation in our dataset typically have a larger effect than those that increase it , with many mutations reducing nucleation to the background rate observed for Aß variants containing premature termination codons ( Figure 2A ) .", "In addition to covering all aa changes obtainable through single nt mutations , our mutagenesis library was designed to contain a substantial fraction of double mutants .", "In total , we quantified the impact of 14 , 015 double aa variants of Aß .", "Double mutants were even more likely to reduce nucleation , with 63% decreasing and only 5 . 5% increasing nucleation ( Z-test , FDR = 0 . 1; Figure 2B ) .", "Therefore , mutations more frequently decrease rather than increase Aß nucleation .", "Inspecting the heatmap of mutational effects for aa changes at all positions in Aß reveals strong biases in the locations of mutations that increase and decrease nucleation ( Figure 2C and D , and Figure 2—figure supplement 1A ) .", "Mutations that decrease nucleation are highly enriched in the C-terminus of Aß , whereas mutations that increase nucleation are enriched in the N-terminus ( Figure 2E ) .", "Indeed , >84% of mutations in the C-terminus ( residues 27-42 ) reduce nucleation and only 9 . 6% increase it ( FDR = 0 . 1 ) .", "In contrast , the effects of mutations are smaller ( Figure 2F ) and also more balanced in the first 26 aa of the peptide , with 38 . 6% decreasing and 20% increasing nucleation ( FDR = 0 . 1 ) .", "These differences in the direction and strength of mutational effects between the N- and C-terminal regions of Aß suggest a modular organization of the peptide .", "This modularity is also reflected in the primary sequence of Aß , which has a hydrophobic C-terminus and a more polar and charged N-terminus ( eight out of nine charged residues in Aß are found before residue 24 and the peptide consists entirely of hydrophobic residues from position 29 ) ( Figures 2C and 3A ) .", "Consistent with this modular organization , mutations in the few hydrophobic residues in the N-terminus have effects that are more similar to mutations in polar residues in the N-terminus rather than in hydrophobic residues in the C-terminus .", "Similarly , mutations in the most C-terminal charged residue ( K28 ) frequently strongly reduce nucleation , just as they do in the adjacent hydrophobic positions ( Figure 3A ) .", "Considering the entire Aß peptide , there are only seven positions in which mutations are not more likely to decrease rather than increase nucleation ( FDR = 0 . 1; Figure 2D ) .", "Strikingly , these positions , which we refer to as ‘gatekeepers’ of nucleation ( Rousseau et al . , 2006; Pedersen et al . , 2004 ) , include five of the six negatively charged residues in Aß .", "The sixth gatekeeper is an unusual hydrophobic residue in the N-terminus , L17 , where seven mutations increase nucleation and only one decreases it ( FDR = 0 . 1; Figure 2D ) .", "The final aa of the peptide , A42 , also has an unusual distribution of mutational effects that is different to the rest of the C-terminus , with four mutations increasing and three mutations decreasing nucleation ( FDR = 0 . 1; Figure 2D ) .", "Taken together , on the basis of mutational effects , we therefore distinguish the following mutually exclusive positions in Aß: the C-terminus ( aa 27-41 ) where the majority of mutations strongly decrease nucleation , the N-terminus ( aa 2-26 ) where mutations have smaller and more balanced effects , and seven gatekeeper residues ( D1 , E3 , D7 , E11 , D22 , L17 , A42 ) where mutations frequently increase nucleation .", "We consider each of these classes below .", "Mutations in the C-terminus nearly all decrease nucleation ( Figure 3A ) .", "This is consistent with the C-terminus forming part of the tightly packed amyloid core of all known structural polymorphs of both Aß42 ( Colvin et al . , 2016; Meier et al . , 2017; Wälti et al . , 2016; Xiao et al . , 2015; Gremer et al . , 2017; Lührs et al . , 2005; Schmidt et al . , 2015 ) and Aß40 ( Kollmer et al . , 2019; Lu et al . , 2013; Qiang et al . , 2012; Sgourakis et al . , 2015; Paravastu et al . , 2008; Schütz et al . , 2015 ) .", "Consistent with this , we quantified the nucleation of three C-terminal fragments of the peptide ( aa 22-42 , 24-42 , 27-42 ) and found that they nucleate similarly or better than full length Aß ( Figure 3—figure supplement 1C ) .", "Mutations to polar and charged residues in this region nearly all decrease nucleation , but so too do most changes to other hydrophobic residues ( Figure 3B ) , suggesting specific side chain packing in this region is important for nucleation .", "The relative effects of different mutations are only partially captured by changes in hydrophobicity ( Figure 3F; Pearson correlation coefficient , R = 0 . 45 ) and by predictors of aggregation potential ( Figure 3—figure supplement 1A ) .", "Only a few mutations in this region increase nucleation: substitutions to isoleucine at positions 30 , 34 , and 39; mutations to valine at positions 29 , 30 , and 34; a change to threonine at position 30; changes to leucine and methionine at 36; and a mutation to phenylalanine at position 41 ( FDR = 0 . 1 ) .", "Mutations in the N-terminus of Aß have a more balanced effect on nucleation , and these effects are not well predicted by either hydrophobicity or predictors of aggregation potential ( Figure 3—figure supplement 1B , D and E ) .", "The effects of introducing particular aa are , however , biased , with the introduction of asparagine , isoleucine , and valine most likely to increase nucleation ( Figure 3C and Figure 3—figure supplement 2 ) .", "As at the C-terminus , the introduction of negative charged residues typically strongly reduces nucleation ( Figure 3B and C ) .", "However , in contrast to what is observed in the C-terminus ( Figure 3B ) , the effects of introducing positive charge are less severe ( Figure 3C ) .", "Interestingly , the effects of mutations to proline , isoleucine , valine , and threonine in the N-terminus depend on the position in which they are made: mutations in the first 12 residues typically decrease nucleation , whereas mutations in the next four to nine residues increase nucleation ( Figure 3E ) .", "The conformational rigidity of proline and the beta-branched side chains of isoleucine , valine , and threonine that disfavour helix formation suggest that disruption of a secondary structure in this region may favour nucleation .", "Interestingly , this same region was highlighted as the part of the peptide remaining most disordered across different states of the solution ensemble of Aß in molecular dynamics simulations , with the same region also making extensive long-range contacts in different states of the kinetic ensemble ( Löhr et al . , 2021 ) .", "At five of six negatively charged positions in Aß , mutations frequently increase nucleation ( Figures 2D and 3A ) .", "Moreover , the introduction of negative charge at other positions strongly decreases nucleation ( Figure 3A ) , suggesting that negatively charged residues act as gatekeepers ( Pedersen et al . , 2004; Rousseau et al . , 2006 ) to limit nucleation ( Figure 3D and Figure 3—figure supplement 1D ) .", "In contrast , mutations in the three positively charged residues ( R5 , K16 , K28 ) mostly decrease nucleation ( Figure 2D ) .", "Mutating the negatively charged gatekeepers to the polar aa glutamine and asparagine , to positively charged residues ( arginine and lysine ) , or to small side chains ( glycine and alanine ) increases nucleation ( Figure 3D ) .", "Mutating the same positions to hydrophobic residues typically reduces nucleation ( Figure 3D ) .", "This is consistent with a model in which the negative charge at these positions acts to limit nucleation , but that the overall polar and unstructured nature of the N-terminus must be maintained for effective nucleation .", "To further investigate the role of charge in controlling Aß nucleation , we extended our analyses to the double mutants .", "Including double mutants allows the net charge of Aß to vary over a wider range and it also allows comparison of the nucleation of peptides with the same net charge but a different total number of charged residues ( e . g . , a net charge of −3 can result from a negative/positive aa composition of 6/3 , as in wild-type Aß , or compositions of 7/4 , 5/2 , etc . ) .", "Considering all mutations between charged and polar residues or glycine reveals that , although reducing the net charge of the peptide from −3 progressively increases nucleation ( Figure 3G ) , the total number of charged residues is also important: for a given net charge , nucleation is increased in peptides containing fewer charged residues of any sign ( Figure 3G and Figure 3—figure supplement 1F and G ) .", "Thus , both the overall charge and the number of charged residues control the rate of Aß nucleation .", "In addition to the five negatively charged gatekeeper residues , mutations most frequently increase nucleation of Aß in two specific hydrophobic residues: L17 and A42 ( Figure 2C and D ) .", "At position 17 , changes to polar , aromatic , and aliphatic aa all increase nucleation , as does the introduction of a positive charge and mutation to proline .", "Only a mutation to cysteine reduces nucleation ( Figure 2C ) .", "This suggests a specific role for leucine at position 17 in limiting nucleation , perhaps as part of a nucleation-limiting secondary structure suggested by the mutational effects of proline , isoleucine , valine , and threonine in this region ( Figure 3E ) .", "Finally , the distribution of mutational effects at position 42 differs from that in the rest of the hydrophobic C-terminus of Aß , with mutations most often increasing nucleation ( Figure 2D; FDR = 0 . 1 ) .", "The mutations that increase nucleation are all to other aliphatic residues ( Figures 2C and 3A ) .", "The distinction of position 42 is interesting because of the increased toxicity and aggregation propensity of Aß42 compared to the shorter Aß40 APP cleavage product ( Meisl et al . , 2014; Sandberg et al . , 2010 ) .", "To investigate how nucleation in the cell-based assay relates to the human disease , we considered all the mutations in Aß known to cause fAD .", "In total , there are 11 mutations in Aß reported to cause dominantly inherited fAD and one additional variant of unclear pathogenicity ( H6R ) ( Janssen et al . , 2003 ) .", "These 12 known disease mutations are not well discriminated by commonly used computational variant effect predictors ( Figure 4 and Figure 4—figure supplement 1A ) or by computational predictors of protein aggregation and solubility ( Figure 4 and Figure 4—figure supplement 1B ) .", "They are also poorly predicted by the previous deep mutational scan of Aß designed to quantify changes in protein solubility , suggesting the disease is unrelated to the biophysical process quantified in this assay ( Gray et al . , 2019; Figure 4—figure supplement 1C ) .", "In contrast , the scores from our in vivo nucleation assay accurately classify the known dominant fAD mutations , with all 12 mutations increasing nucleation ( Figure 4 , area under the receiver operating characteristic curve , ROC−AUC = 0 . 9 , two-tailed Z-test , p<2 . 2e-16 ) .", "This suggests the biophysical events occurring in this simple cell-based assay are highly relevant to the development of the human disease .", "Consistent with the overall mutational landscape , the known fAD mutations are also enriched in the N-terminus of Aß ( Figure 2C ) .", "In some positions the known fAD mutations are the only mutation or one of only a few mutations that can increase nucleation .", "For example , based on our data , K16N is likely to be one of only two fAD mutations in position 16 .", "However , in other positions , there are several additional variants that increase nucleation as much as the known fAD mutation .", "At position 11 , for example , there are five mutations with a NS higher than the known E11K disease mutation ( Figure 2C and D ) .", "Overall , our data suggest there are likely to be many additional dominant fAD mutations beyond the 12 that have been reported to date ( Supplementary file 2 ) .", "In addition to the 12 known dominant fAD mutations , two additional variants in Aß have been suggested to act recessively to cause fAD ( Di Fede et al . , 2009; Tomiyama et al . , 2008 ) .", "One of these variants is a codon deletion ( E22Δ ) and is not present in our library .", "The other variant , A2V , does not have a dominant effect on nucleation in our assay ( Figure 2C ) , consistent with a recessive pattern of inheritance and a different mechanism of action , such as reduced ß-cleavage and increased Aß42 generation , as previously proposed ( Benilova et al . , 2014 ) .", "More generally , of the hundreds of aa changes possible in the peptide , our data prioritize 63 as candidate fAD variants ( Supplementary file 2 ) ; 131 variants are likely to be benign , and 262 reduce Aß nucleation and so may even be protective .", "These include variants already reported in the gnomAD database of human genetic variation ( Figure 4—figure supplement 1D ) .", "With the currently available data for patients carrying fAD mutations , we could not observe a correlation between NS and disease age-of-onset ( Ryman et al . , 2014; Figure 4—figure supplement 1E ) ." ], [ "Taken together , the data presented here provides the first large-scale analysis of how mutations promote and prevent the aggregation of an amyloid .", "The results reveal a modular organization for the impact of mutations on the nucleation of Aß .", "Moreover , they show that the rate of nucleation in a cell-based assay identifies all of the mutations in Aß that cause dominant fAD .", "The dataset therefore provides a useful resource for the future clinical interpretation of genetic variation in Aß .", "A majority of mutations in the C-terminal core of Aß disrupt nucleation , consistent with specific hydrophobic contacts in this region being required for nucleation .", "In contrast , mutations that increase nucleation are enriched in the polar N-terminus with mutations in negatively charged gatekeeper residues and the L17 gatekeeper being particularly likely to accelerate aggregation .", "Indeed , decreasing both the net charge of the peptide and the total number of charged residues increases nucleation .", "Little is known about the structure of Aß during fibril nucleation , but the results presented here are in general consistent with the nucleation transition state resembling the known mature fibril structures of Aß where the C-terminal region of the peptide is located in the amyloid core and the N-terminus is disordered and solvent exposed ( Figure 5 and Figure 5—figure supplements 1 and 2 ) .", "Although the N-terminus is not required for nucleation , it does affect the process when present and most mutations that accelerate nucleation are located in the N-terminus .", "Interestingly , the effects of mutations in residues immediately before position 17 suggest that the formation of a structural element in this region may interfere with nucleation .", "That accelerated nucleation is a common cause of fAD is also supported by the effects of mutations in APP outside of Aß and by the effects of mutations in PSEN1 and PSEN2 .", "These mutations destabilise enzyme-substrate complexes , increasing the production of the longer Aß peptides that more effectively nucleates amyloid formation ( Szaruga et al . , 2017; Veugelen et al . , 2016 ) .", "In addition , Aß42 oligomers are hypothesised to be more toxic ( Michaels et al . , 2020; Bolognesi et al . , 2010 ) .", "It is possible that the effects of some of the mutations reported here on nucleation are also mediated by a change in the concentration of Aß rather than by an increase in a kinetic rate parameter .", "Some of the variants evaluated here may have additional effects , for example , altering cleavage of APP .", "Future work will be needed to test these hypotheses .", "Comparing our results to the effects of mutations on Aß solubility quantified in a previous high-throughput analysis ( Gray et al . , 2019 ) provides evidence that , in the same type of cell ( yeast ) , Aß can aggregate in at least two different ways .", "Moreover , the different performance of the two sets of scores from these datasets in classifying fAD mutations suggests that one of these aggregation processes ( quantified by the nucleation assay employed here ) is likely to be very similar to the aggregation that occurs in the human brain in fAD .", "The other pathway of aggregation ( quantified by the solubility assay; Gray et al . , 2019 ) , however , is less obviously related to the human disease , because mutations that cause fAD do not consistently affect it .", "This second aggregation pathway is , at least to a large extent , driven by changes in hydrophobicity , similar to what we previously reported for the aggregation in yeast of the ALS protein , TDP-43 ( Bolognesi et al . , 2019 ) .", "More generally , our results highlight how the combination of deep mutational scanning and human genetics can be a general ‘genetic’ strategy to quantify the disease relevance of biological assays .", "Many in vitro and in vivo assays are proposed as ‘disease models’ in biomedical research with their relevance often justified by how ‘physiological’ the assays seem or how well phenotypes observed in the model match those observed in the human disease .", "The range of phenotypes that can be assessed and their similarity to the pathology of AD human brains are appealing features of many animal models of AD and many important insights have been derived – and will continue to be derived – from animal models ( Sasaguri et al . , 2017 ) .", "However , there are applications where animal models cannot be realistically used , for example , for high-throughput compound screening for drug discovery and for testing hundreds or thousands of genetic variants of unknown significance .", "For these applications , in vitro or cell-based ( Pimenova and Goate , 2020; Veugelen et al . , 2016 ) assays are required and an important challenge is to evaluate the ‘disease relevance’ of different assays .", "Our study highlights an approach to achieve this , which is to use the complete set of known disease-causing mutations to quantify the ‘genetic agreement’ between an assay and a disease .", "Thus , although the yeast-based assay that we employed here might typically be dismissed as ‘non-physiological , ’ ‘artificial , ’ or ‘lacking many features important for a neurological disease , ' unbiased massively parallel genetic analysis provides very strong evidence that it is reporting on biopysicall events that are extremely similar to – or the same as – those that cause the human disease . Indeed , one could argue that this simple system is now better validated as a model of fAD than many others , including animal models where the effects of only one or a few mutations ( including control mutations ) have ever been tested . Similarly strong agreement between mutational effects in a cellular assay and the set of mutations already known to cause a disease is observed for other diseases ( Starita et al . , 2017; Gelman et al . , 2019 ) , suggesting the generality of this approach . We suggest therefore that the combination of deep mutational scanning and human genetics provides a general strategy to quantify the disease relevance of in vitro and cell-based assays . We encourage that deep mutagenesis should be employed early in discovery programmes to ‘genetically validate’ ( or invalidate ) the relevance of assays for particular diseases .", "The concordance between mutational effects in an assay and a disease is an unbiased metric that can be used to prioritize between different assays .", "Quantifying the ‘genetic agreement’ between an assay and a disease will help prevent time and resources being wasted on research that actually has little relevance to a disease .", "Finally , the strikingly consistent effects of the dominant fAD mutations in our assay further strengthen the evidence that fAD is a ‘nucleation disease’ ultimately caused by an increased rate of amyloid nucleation ( Aprile et al . , 2017; Cohen et al . , 2018; Knowles et al . , 2009 ) .", "This accelerated nucleation can be caused by the direct effects of mutations in Aß — such as those quantified here — or by changes in upstream factors ( Szaruga et al . , 2017 ) .", "If this hypothesis is correct , then nucleation is the key bioph step to target to prevent or treat AD .", "We suggest that the ‘genetic validation’ of assays by mutational scanning and comparison to sets of known disease-causing mutations will be increasingly important in assay development and drug discovery pipelines ." ], [ "The plasmid PCUP1-Sup35N-Aβ42 used in this study was a kind gift from the Chernoff lab ( Chandramowlishwaran et al . , 2018 ) .", "The Aβ coding sequence and two flanking regions of 52 bp and 72 bp , respectively , upstream and downstream of Aβ were amplified ( primers MS_01 and MS_02 , Supplementary file 3 ) by error-prone PCR ( Mutazyme II DNA polymerase , Agilent ) .", "Thirty cycles of amplification and 0 . 01 ng of initial template were used to obtain a mutagenesis rate of 16 mutations/kb , according to the manufacturer’s protocol .", "The product was treated with DpnI ( FastDigest , Thermo Scientific ) for 2 hr and purified by column purification ( MinElute PCR Purification Kit , Qiagen ) .", "The fragment was digested with EcoRI and XbaI restriction enzymes ( FastDigest , Thermo Scientific ) for 1 hr at 37°C and purified from a 2% agarose gel ( QIAquick Gel Extraction Kit , Qiagen ) .", "In parallel , the PCUP1-Sup35N-Aβ42 plasmid was digested with the same restriction enzymes to remove the WT Aβ sequence , treated with alkaline phosphatase ( FastAP , Thermo Scientific ) for 1 hr at 37°C to dephosphorylate the 5’ ends , and purified from a 1% agarose gel ( QIAquick Gel Extraction Kit , Qiagen ) .", "Mutagenised Aβ was then ligated into the linearised plasmid in a 5:1 ratio ( insert:vector ) using a ligase treatment ( T4 , Thermo Scientific ) overnight .", "The reaction was dialysed with a membrane filter ( Merck Millipore ) for 1 hr , concentrated 4x , and transformed in electrocompetent Escherichia coli cells ( 10-beta Electrocompetent , NEB ) .", "Cells were recovered in SOC medium and plated on LB with ampicillin .", "A total of 4 . 1 million transformants were estimated , ensuring that each variant of the library was represented more than 10 times; 50 ml of overnight E . coli culture was harvested to purify the Aβ plasmid library with a midi prep ( Plasmid Midi Kit , Qiagen ) .", "The resulting library contained 29 . 9% of WT Aβ , 23 . 8% of sequences with 1 nt change , and 21 . 8% of sequences with 2 nt changes .", "Saccharomyces cerevisiae [psi-pin-] ( MATa ade1-14 his3 leu2-3 , 112 lys2 trp1 ura3-52 ) strain ( also provided by the Chernoff lab ) was used in all experiments in this study ( Chandramowlishwaran et al . , 2018 ) .", "Yeast cells were transformed with the Aβ plasmid library starting from an individual colony for each transformation tube .", "After an overnight pre-growth culture in YPDA medium at 30°C , cells were diluted to OD600 = 0 . 3 in 175 ml YPDA and incubated at 30°C 200 rpm for ~5 hr .", "When cells reached the exponential phase , they were harvested , washed with milliQ , and resuspended in sorbitol mixture ( 100 mM LiOAc , 10 mM Tris pH 8 , 1 mM EDTA , 1M sorbitol ) .", "After a 30 min incubation at room temperature ( RT ) , 5 µg of plasmid library and 175 μl of ssDNA ( UltraPure , Thermo Scientific ) were added to the cells .", "PEG mixture ( 100 mM LiOAc , 10 mM Tris pH 8 , 1 mM EDTA pH 8 , 40% PEG3350 ) was also added and cells were incubated for 30 min at RT and heat-shocked for 15 min at 42°C in a water bath .", "Cells were harvested , washed , resuspended in 350 ml recovery medium ( YPD , sorbitol 0 . 5M , 70 mg/L adenine ) and incubated for 1 . 5 hr at 30°C 200 rpm .", "After recovery , cells were resuspended in 350 ml -URA plasmid selection medium and allowed to grow for 50 hr .", "Transformation efficiency was calculated for each tube of transformation by plating an aliquote of cells in -URA plates .", "Between 1 and 2 . 5 million transformants per tube were obtained .", "Two days after transformation , the culture was diluted to OD600 = 0 . 02 in 1 l -URA medium and grown until the exponential phase .", "At this stage , cells were harvested and stored at −80°C in 25% glycerol .", "Three independent replicate selection experiments were performed .", "Tubes were thawed from the −80°C glycerol stocks and mixed proportionally to the number of transformants in a 1 l total -URA medium at OD600 = 0 . 05 .", "A minimum of 3 . 7 million yeast transformants were used for each replicate to ensure the coverage of the full library and reaching therefore a 10x coverage of each variant .", "Once the culture reached the exponential phase , cells were resuspended in 1 l protein inducing medium ( -URA , 20% glucose , 100 µM Cu2SO4 ) at OD600 = 0 . 05 .", "As a result , each variant was represented at least 100 times at this stage .", "After 24 hr the input pellets were collected by centrifuging 220 ml of cells and stored at −20°C for later DNA extraction ( input pellets ) .", "In parallel , 18 . 5 million cells of the same culture underwent selection , with a starting coverage of at least 50 copies of each variant in the library .", "For selection , cells were plated on -ADE-URA selective medium in 145 cm2 plates ( Nunc , Thermo Scientific ) and let grow for 7 days at 30°C .", "Colonies were then scraped off the plates and recovered with PBS 1x to be centrifuged and stored at −20°C for later DNA extraction ( output pellets ) .", "For individual testing of specific variants , cells were plated on -URA ( control ) and -ADE-URA ( selection ) plates in three independent replicates .", "Individual growth was calculated as the percentage of colonies growing -ADE-URA relative to colonies growing in -URA .", "The input and output pellets ( three replicates , six tubes in total ) were thawed and resuspended in 2 ml extraction buffer ( 2% Triton-X , 1% SDS , 100 mM NaCl , 10 mM Tris pH 8 , 1 mM EDTA pH 8 ) , and underwent two cycles of freezing and thawing in an ethanol-dry ice bath ( 10 min ) and at 62°C ( 10 min ) .", "Samples were then vortexed together with 1 . 5 ml of phenol:chloroform:isoamyl 25:24:1 and 1 . 5 g of glass beads ( Sigma ) .", "The aqueous phase was recovered by centrifugation and mixed again with 1 . 5 ml phenol:chloroform:isoamyl 25:24:1 .", "DNA precipitation was performed by adding 1:10 V of 3M NaOAc and 2 . 2 V of 100% cold ethanol to the aqueous phase and incubating the samples at −20°C for 1 hr .", "After a centrifugation step , pellets were dried overnight at RT .", "Pellets were resuspended in 1 ml resuspension buffer ( 10 mM Tris pH 8 , 1 mM EDTA pH 8 ) and treated with 7 . 5 μl RNase A ( Thermo Scientific ) for 30 min at 37°C .", "The DNA was finally purified using 75 μl of silica beads ( QIAEX II Gel Extraction Kit , Qiagen ) , washed and eluted in 375 μl elution buffer .", "DNA concentration in each sample was measured by quantitative PCR , using primers ( MS_03 and MS_04 , Supplementary file 3 ) that anneal to the origin of replication site of the plasmid at 58°C .", "The library was prepared for high-throughput sequencing in two rounds of PCR ( Q5 High-Fidelity DNA Polymerase , NEB ) .", "In PCR1 , the Aβ region was amplified for 15 cycles at 68°C with frame-shifted primers ( MS_05 to MS_18 , Supplementary file 3 ) with homology to Illumina sequencing primers; 300 million of molecules were used for each input or output sample .", "The products of PCR1 were purified with an ExoSAP-IT treatment ( Affymetrix ) and a column purification step ( QIAquick PCR Purification Kit ) and then used as the template of PCR2 .", "This PCR was run for 10 cycles at 62°C with Illumina indexed primers ( MS_19 to MS_25 , Supplementary file 2 ) specific for each sample ( three inputs and three outputs ) .", "The six samples were then pooled together equimolarly .", "The final library sample was purified from a 2% agarose gel with silica beads ( QIAEX II Gel Extraction Kit , Qiagen ) ; 125 bp paired-end sequencing was run on an Illumina HiSeq2500 sequencer at the CRG Genomics Core Facility .", "FastQ files from paired-end sequencing of the Aß library before ( ‘input’ ) and after selection ( ‘output’ ) were processed using a custom pipeline ( https://github . com/lehner-lab/DiMSum ) .", "DiMSum ( Faure et al . , 2020 ) is an R package that uses different sequencing processing tools such as FastQC ( http://www . bioinformatics . babraham . ac . uk/projects/fastqc/ ) ( for quality assessment ) , Cutadapt ( Martin , 2011 ) ( for constant region trimming ) , and USEARCH ( Edgar , 2010 ) ( for paired-end read alignment ) .", "Sequences were trimmed at 5′ and 3′ , allowing an error rate of 0 . 2 ( i . e . , read pairs were discarded if the constant regions contained more than 20% mismatches relative to the reference sequence ) .", "Sequences differing in length from the expected 126 bp or with a Phred base quality score below 30 were discarded .", "As a result of this processing , around 150 million total reads passed the filtering criteria .", "At this stage , unique variants were aggregated and counted using Starcode ( https://github . com/gui11aume/starcode ) .", "Variants containing indels and nonsynonymous variants with synonymous substitutions in other codons were excluded .", "The result is a table of variant counts which can be used for further analysis .", "For downstream analysis , variants with less than 50 input reads in any of the replicates were excluded and only variants with a maximum of two aa mutations were used .", "On the basis of variant counts , the DiMSum pipeline ( Faure et al . , 2020; https://github . com/lehner-lab/DiMSum ) was used to calculate nucleation scores ( NS ) and their error estimates .", "For each variant in each replicate NS was calculated as:Nucleationscore=ESi−ESwtwhere ESi =log ( Fi OUTPUT ) −log ( Fi INPUT ) for a specific variant and ESwt =log ( Fwt OUTPUT ) −log ( Fwt INPUT ) for Aß WT .", "DiMSum models measurement error of NS by assuming that variants with similar counts in input and output samples have similar errors .", "Based on errors expected from Poisson-distributed count data , replicate-specific additive and multiplicative ( one each for input and output samples ) modifier terms are fit to best describe the observed variance of NS across all variants simultaneously .", "After error calculation , NS were merged by using the error-weighted mean of each variant across replicates and centered using the error-weighted means frequency of synonymous substitutions arising from single nt changes .", "Merged NS and NS for each independent replicate , as well as their associated error estimates , are available in Supplementary file 4 .", "Nonsense ( stop ) mutants were excluded for the analysis except when indicated ( Figure 2A and C and Figure 2—figure supplement 1A ) .", "We used K-medoids , or the partitioning around medoids algorithm , to cluster the matrix of single aa variant NS estimates by residue position with the number of clusters estimated by optimum average silhouette width , for values of K in [1 , 10] .", "The silhouette width is a measure of how similar each object ( in this case residue position ) is to its own cluster .", "In order to take into account uncertainty in NS estimates in the determination of the optimum number of clusters , we repeated this analysis after random resampling from the NS ( error ) distributions of each single aa variant ( n = 100 ) .", "Based on this clustering , we defined the N-terminus as aa 2-26 and the C-terminus as aa 27-41 ( Figure 2—figure supplement 1B ) .", "Seven positions where as many ( or more ) single mutations increase as decrease nucleation were defined as ‘gatekeepers’ ( D1 , E3 , D7 , E11 , L17 , E22 , A42 ) and excluded from the N- and C-terminus classes .", "Only those positions where most mutations are significantly different from WT ( FDR = 0 . 1 ) were considered for the definition of gatekeepers .", "Nucleation scores were correlated with aa properties and scores from aggregation , solubility , and variant effect prediction algorithms .", "Pearson correlations were weighted based on the error terms associated with the NS of each variant using the R package ‘weights . ' The aa property features were retrieved from a curated collection of numerical indices representing various aa physicochemical and biochemical properties ( http://www . genome . jp/aaindex/ ) . We also used a principal component of these aa properties from a previous work ( PC1; Bolognesi et al . , 2019 ) that relates strongly to changes in hydrophobicity . For each variant ( single and double aa mutants ) , the values of a specific aa property represent the difference between the mutant and the WT scores . For the aggregation and solubility algorithms ( Tango [Fernandez-Escamilla et al . , 2004] , Zyggregator [Tartaglia and Vendruscolo , 2008] , CamSol [Sormanni et al . , 2015] , and Waltz [Oliveberg , 2010] ) , individual residue-level scores were summed to obtain a score per aa sequence . We then calculated the log value for each variant relative to the WT score ( single and double aa mutants for Tango , Zyggregator , CamSol and single aa mutants for Waltz ) . For the variant effect predictors ( Polyphen [Adzhubei et al . , 2013] and CADD [Rentzsch et al . , 2019] ) , we also calculated the log value for each variant ( only single aa mutants ) but in this case values were scaled relative to the lowest predicted score . The table of fAD mutations used in this study was taken from https://www . alzforum . org/mutations/app . Allele frequencies of APP variants were retrieved from gnomAD ( Karczewski , 2020 ) ( https://gnomad . broadinstitute . org/ ) and the clinical significance of variants was taken from their Clinvar ( Landrum et al . , 2014 ) classification ( https://www . ncbi . nlm . nih . gov/clinvar ) . ROC curves were built and AUC values were obtained using the ‘pROC’ R package .", "The coordinates of the following PDB structures were used for Figure 5 , Figure 5—figure supplements 1 and 2: 5OQV , 2NAO , 5KK3 , 2BEG , 2MXU , 5AEF , 6SHS , 2LMN , 2LMP , 2LNQ , 2MVX , 2M4J , 2MPZ ( Gremer et al . , 2017; Colvin et al . , 2016; Wälti et al . , 2016; Lührs et al . , 2005; Xiao et al . , 2015; Schmidt et al . , 2015; Kollmer et al . , 2019; Lu et al . , 2013; Qiang et al . , 2012; Sgourakis et al . , 2015; Schütz et al . , 2015 ) ." ] ]

[ "Plaques of the amyloid beta ( Aß ) peptide are a pathological hallmark of Alzheimer’s disease ( AD ) , the most common form of dementia .", "Mutations in Aß also cause familial forms of AD ( fAD ) .", "Here , we use deep mutational scanning to quantify the effects of >14 , 000 mutations on the aggregation of Aß .", "The resulting genetic landscape reveals mechanistic insights into fibril nucleation , including the importance of charge and gatekeeper residues in the disordered region outside of the amyloid core in preventing nucleation .", "Strikingly , unlike computational predictors and previous measurements , the empirical nucleation scores accurately identify all known dominant fAD mutations in Aß , genetically validating that the mechanism of nucleation in a cell-based assay is likely to be very similar to the mechanism that causes the human disease .", "These results provide the first comprehensive atlas of how mutations alter the formation of any amyloid fibril and a resource for the interpretation of genetic variation in Aß ." ]

[ "Alzheimer’s disease is the most common form of dementia , affecting more than 50 million people worldwide .", "Despite more than 400 clinical trials , there are still no effective drugs that can prevent or treat the disease .", "A common target in Alzheimer’s disease trials is a small protein called amyloid beta .", "Amyloid beta proteins are ‘sticky’ molecules .", "In the brains of people with Alzheimer’s disease , they join to form first small aggregates and then long chains called fibrils , a process which is toxic to neurons .", "Specific mutations in the gene for amyloid beta are known to cause rare , aggressive forms of Alzheimer’s disease that typically affect people in their fifties or sixties .", "But these are not the only mutations that can occur in amyloid beta .", "In principle , any part of the protein could undergo mutation .", "And given the size of the human population , it is likely that each of these mutations exists in someone alive today .", "Seuma et al . reasoned that studying these mutations could help us understand the process by which amyloid beta forms new aggregates .", "Using an approach called deep mutational scanning , Seuma et al . mutated each point in the protein , one at a time .", "This produced more than 14 , 000 different versions of amyloid beta .", "Seuma et al . then measured how quickly these mutants were able to form aggregates by introducing them into yeast cells .", "All the mutations known to cause early-onset Alzheimer’s disease accelerated amyloid beta aggregation in the yeast .", "But the results also revealed previously unknown properties that control how fast aggregation occurs .", "In addition , they highlighted a number of positions in the amyloid beta sequence that act as ‘gatekeepers’ .", "In healthy brains , these gatekeepers prevent amyloid beta proteins from sticking together .", "When mutated , they drive the protein to form aggregates .", "This comprehensive dataset will help researchers understand how proteins form toxic aggregates , which could in turn help them find ways to prevent this from happening .", "By providing an ‘atlas’ of all possible amyloid beta mutations , the dataset will also help clinicians interpret any new mutations they encounter in patients .", "By showing whether or not a mutation speeds up aggregation , the atlas will help clinicians predict whether that mutation increases the risk of Alzheimer’s disease ." ]

[ "Results", "Discussion", "Materials and methods" ]

[ "structural biology and molecular biophysics", "tools and resources" ]

[ [ "We validate DISC using simulated single-molecule trajectories using kinetic parameters obtained from our recent studies exploring the regulatory mechanisms of cyclic nucleotide binding domains ( CNBDs ) from hyperpolarization-activated cyclic nucleotide gated ion channels ( HCN ) which regulate pacemaking in heart and brain cells ( Materials and methods ) ( Goldschen-Ohm et al . , 2017; Goldschen-Ohm et al . , 2016 ) .", "In these experiments , isolated CNBDs are tethered into ZMWs whereupon we monitor the binding and unbinding dynamics of fluorescent cyclic nucleotides ( e . g . fcAMP ) at physiological concentrations to uncover the elementary dynamics associated with channel gating .", "While ligand binding has been observed at the single-molecule level via both FRET and CoSMoS ( co-localization ) , we adapt our simulations to the latter case so our dynamics are not limited in time by acceptor photobleaching .", "Notably , trajectories obtained with CoSMoS exhibit heterogeneous bound intensity values which vary with each binding event ( Figure 3—figure supplement 1 ) .", "While we are uncertain as to the exact source of this fluctuation , it is likely caused by shifts of the molecule in the heterogeneous excitation field of the ZMW or dye photodynamics ( Levene et al . , 2003; Dempsey et al . , 2009 ) .", "While the excitation field changes particularly sharply in ZMWs , TIRF and confocal microscopy also contain a heterogenous excitation field ( Moerner and Fromm , 2003 ) .", "Minor changes in apparent dye brightness due to dye conformational or photodynamics ( such as in Protein-induced fluorescence enhancement , PIFE ) , shifts of dye orientation , or partial quenching via electron transfer are all commonly observed ( Stennett et al . , 2015 ) .", "Thus , heterogeneous intensity values are a common and inconvenient feature in real life single-molecule fluorescence data .", "Including this additional noise source in our simulations yields a closer representation of experimentally obtained data .", "In total , we simulated 4000 trajectories composed of 2000 data points each , totaling 8 × 106 data points .", "Each trajectory is 200 s in duration collected at frame rate of 10 Hz .", "We varied the complexity of the trajectory by simulating one to four independent CNBDs inside a given ZMW ( two to five intensity states ) and vary the signal to noise ratio ( SNR ) according to typical values from a ZMW experiment using Gaussian noise ( Materials and methods ) ( Goldschen-Ohm et al . , 2017; Goldschen-Ohm et al . , 2016 ) .", "We include the observed heterogenous bound intensities by randomly modulating each binding event according to our fit of the experimentally observed data ( Figure 3—figure supplement 2 ) .", "Given that each simulation features a different number of possible states , the total time spent within each state changes; therefore , these simulations also address the ability to capture states with unequal and even rare observation probabilities ( see Figure 3—source data 1 ) .", "We benchmark the results of DISC against commonly used HMM and CP-HAC methods: vbFRET and STaSI ( Bronson et al . , 2009; Shuang et al . , 2014 ) .", "These algorithms were chosen following the results of a recent comparative study that determined these to be the best performers among their class of analysis methods ( Hadzic et al . , 2018 ) .", "In addition , DISC , STaSI and vbFRET all perform trajectory-by-trajectory idealization and are written entirely in MATLAB ( MathWorks ) which standardizes computational performance ( Materials and methods ) .", "Across all the simulations , DISC provides the highest average accuracy , precision and recall ( Figure 3a , terms defined in Materials and methods ) .", "While no algorithm can idealize a trajectory in the presence of SNR = 1 , DISC returns the lowest accuracy at SNR = 2 .", "We suspect this result is due to the use of robust BIC for state detection; accuracy would likely be improved with less penalizing objective functions , such as AIC .", "While vbFRET performs the best at SNR = 2 , the overall accuracy is still quite low: an average accuracy value for each number of simulated states is near chance .", "This low value demonstrates the inability of many algorithms to analyze data in presence of high noise and reinforces the common practice of discarding noisy data to create a more reliable dataset .", "For SNR > 3 , which accounts for most of our experimentally obtained data ( Figure 3—figure supplement 1c ) , DISC performs exceptionally well with highest average accuracy ( 0 . 91 ± 0 . 05 ) and is robust against false positives ( precision = 0 . 96 ± 0 . 04 ) and false negatives ( recall = 0 . 93 ± 0 . 03 ) across all simulated conditions ( Figure 3a ) .", "While vbFRET matches the recall of DISC in this SNR range ( 0 . 94 ± 0 . 05 ) , the tendency to overfit the number of states at higher SNR lowers precision ( 0 . 80 ± 0 . 18 ) and overall accuracy ( 0 . 76 ± 0 . 19 ) .", "We find STaSI returns the lowest overall accuracy ( 0 . 47 ± 0 . 17 ) likely do to an overfitting the number of states ( precision = 0 . 57 ± 0 . 2 ) and a tendency to miss transitions ( recall = 0 . 75 ± 0 . 10 ) .", "Notably , DISC is the only method unaffected by inclusion of heterogeneous state intensities of fcAMP likely due to the use of a Gaussian derived BIC for state selection ( Figure 3—figure supplement 3 ) .", "Critically , DISC not only returned high accuracy results , DISC was also much faster than the other methods .", "Idealization of all 4000 trajectories by DISC was completed in just over two minutes , whereas STaSI took over fifteen minutes and vbFRET took over twelve hours .", "To thoroughly explore the computational efficiency of DISC , we simulated data with increasing durations per trajectory at a constant SNR and number of states .", "Remarkably , we find DISC is 400-fold to 1 , 200-fold faster than vbFRET and 2-fold to 8 , 700-fold faster than STaSI due to STaSI’s quadratic time dependence ( Figure 4a; Shuang et al . , 2014 ) .", "For example , a trajectory of 106 data points can be analyzed by DISC in 10 s compared to 3 hr for vbFRET and 27 hr for STaSI .", "Thus , DISC can handle the analysis of long trajectories , unlike CP-HAC methods .", "While fluorescence measurements from a single fluorophore at room-temperature rarely contain this many data points , large trajectory lengths are common in non-fluorescence experiments or fluorescence experiments with replenishing fluorescent labels such as in single-molecule genome sequencing and studies of catalysts via fluorogenic reactions ( Eid et al . , 2009; English et al . , 2006; Sambur et al . , 2016 ) .", "To evaluate performance on more typical data , we compared the results of each algorithm on simulated smFRET trajectories that are limited in duration by acceptor photobleaching ( Figure 4—figure supplement 1 , Materials and methods ) .", "For simulations featuring two or three states FRET , we find DISC 5 . 5-fold faster than STaSI and 235-fold faster than vbFRET while maintaining the highest accuracy .", "This result validates the use of DISC for the analysis of large volumes of shorter fluorescence trajectories , especially compared to HMM approaches .", "This feature is particularly important as advances in hardware such as CMOS cameras and lab-on-chip methods generate larger smFRET data sets ( Juette et al . , 2016 ) .", "Finally , we evaluate the effect of trajectory duration on DISC accuracy .", "As expected , we find that the accuracy increases with increasing number of data points per trajectory .", "This result also indicates the minimum number of data points needed for an accurate idealization for a given SNR ( Figure 4b ) .", "Overall , the results of our simulations suggest DISC is more or comparably accurate and critically , is substantially faster than standard idealization approaches , making it an enabling technology for analysis of high-throughput single-molecule experiments .", "To verify performance of DISC in an experimental configuration with high volumes of experimental data , we analyzed a large single-molecule data set obtained from ZMWs that explore HCN dynamics ( Figure 5a ) .", "Previous macroscopic studies of HCN channel gating have revealed that ligand binding to CNBDs exhibits both positive and negative cooperativity depending on the ligation state and the membrane potential ( Kusch et al . , 2012; Thon et al . , 2015 ) .", "Cyclic AMP regulates cardiac pacemaking via HCN channels and , therefore , this unusual allostery has significant physiological implications .", "However , as this allosteric analysis was based on global fits of ensemble binding data , the reliability of model parameters remains an open question ( Hines et al . , 2015 ) .", "Evaluating cooperativity is an experiment well-suited for single-molecule investigation given the ability to observe the total time a molecule spends in each liganded state and directly extract state transition probabilities .", "Therefore , to directly assess the cooperativity between HCN2 CNBDs upon ligand binding , we use single-molecule fluorescence and monitor the binding of individual fcAMP molecules to our previously described tetramerized CNBDs inside ZMWs .", "( Figure 5a; Goldschen-Ohm et al . , 2016 ) .", "Our initial dataset included 13 , 670 ZMWs each monitored for 800 s at a sampling rate of 10 Hz ( Materials and methods ) .", "All trajectories were obtained in the presence of 1 µM fcAMP which is near the ligand dissociation constant for individual CNBDs ( Goldschen-Ohm et al . , 2016 ) .", "As shown with other high-throughput collection platforms , an essential part of analysis at this scale is the application of stringent criteria to select traces that yield meaningful information about the system ( Chen et al . , 2014; Juette et al . , 2016 ) .", "Therefore , we first analyzed all trajectories with DISC to find reliable data prior to trace selection .", "DISC successfully processed this entire data set within 20 min using a standard MacBook Air ( 1 . 6 GHz Intel Core i5 ) .", "The same analysis completed with STaSI yielded unphysical results in 4 hr ( Figure 5—figure supplement 1 ) .", "We estimated analysis with vbFRET would take weeks to complete and was therefore not performed .", "While correcting for non-specific binding is often a necessity in CoSMoS experiments , we find the passivated surfaces within the ZMWs greatly reduce non-specific absorption of fcAMP to either the metallic or glass surfaces , thus minimizing this concern ( Figure 5—figure supplement 2; Smith et al . , 2019; Eid et al . , 2009; Foquet et al . , 2008 ) .", "Using the idealized fits obtained from DISC , we screened our data to select reliable trajectories for our analysis .", "Standard cut-offs in state separation , the total number of observed states , and a filter for kinetic activity ensured that each trajectory arose from a ZMW featuring a singly occupied and functional tetrameric CNBD .", "Notably , we noticed an asynchronous decay of protein activity over excitation time that may be caused by singlet-oxygen formation and subsequent inhibition of cyclic nucleotide binding to CNBDs ( Figure 5—figure supplement 3; Idikuda et al . , 2018 ) .", "In conclusion , we retained 293 molecules totaling 1 . 2 × 105 s of combined protein activity across 53 , 474 events .", "To determine if the binding of cAMP to CNBDs is cooperative , we first calculated the total time each molecule spends in each of the liganded states ( 0 to 4 fcAMPs ) using the state assignments from the idealized fits ( Figure 5b , Figure 5—figure supplement 4 ) .", "The resulting distribution of state occupancies is fit with a binomial distribution to evaluate the independence of each CNBD ( Figure 5c , Materials and methods ) .", "Our binomial fit matches the distribution well and returns the probability of occupancy at 1 µM fcAMP for a single CNBD in the tetramer as 31% which is similar to our previous monomeric CNBDs studies ( Goldschen-Ohm et al . , 2016 ) , suggesting a lack of cooperativity between the CNBDs .", "Notably , our measured state-occupancy distribution is strikingly different that than the unusual cooperativity modeled from either activated or non-activated channels ( Figure 5—figure supplement 5; Kusch et al . , 2012; Thon et al . , 2015 ) .", "We further explored the underlying dynamics of our data using the idealized single-molecule transitions obtained from DISC .", "Using QuB , we built a simple HMM of sequential ligand binding across four binding sites that was globally optimized across each molecule’s idealized state trajecotry ( Nicolai and Sachs , 2013; Qin et al . , 2000; Figure 5d , Materials and methods ) .", "As expected for non-cooperative processes , the optimized kon and koff rates for each state transition exhibit a strong linear relationship ( Figure 5e ) .", "Combined , these results strongly suggest that CNBD units act independently during ligand binding .", "We postulate that the macroscopically observed cooperativity is either an artifact of model fitting or that it requires the presence of the transmembrane domains of the HCN channel and is not an intrinsic property of the CNBDs ." ], [ "We developed a new algorithm for rapid and accurate unsupervised idealization of single-molecule trajectories .", "Our approach combines unsupervised statistical learning of discrete states with the event detection power of the Viterbi algorithm to quickly identify both significant states and transitions in a model-independent manner .", "Software implementing the DISC algorithm that includes a graphical user interface is available at https://github . com/ChandaLab/DISC ( Chanda et al . , 2019; copy archived at https://github . com/elifesciences-publications/DISC ) .", "Like CP-HAC methods , DISC is not a fully probabilistic approach .", "While fully probabilistic HMM training approaches are beneficial for providing unbiased estimates of the parameter distributions , their high accuracy comes at the cost of significantly increased computational time .", "This cost is especially apparent for the recently developed infinite HMM approaches that aim to learn the true number of states from a potentially infinite number of possibilities .", "However , accomplishing this task costs hundreds of iterations per trace to provide a reproducible fit with some approaches taking days to analyze single trajectories ( Hines et al . , 2015; Sgouralis and Pressé , 2017; Sgouralis et al . , 2018 ) .", "Thus , while exhaustive search algorithms may be desirable in other contexts , they are clearly not suited for large datasets associated with high-throughput experiments .", "In contrast , by simulating data closely resembling the binding of fcAMP to pacemaker channels , we find that DISC surpasses the accuracy of common CP-HAC and HMM algorithms with a dramatic improvement in computational speed .", "Therefore , DISC satisfies the need for accuracy and speed in high-throughput analysis .", "In this regard , DISC is like the SKM algorithm for estimating the parameters of an HMM without direct HMM training .", "However , unlike SKM which relies on user-specified states , DISC uses unsupervised statistics to learn the states .", "Therefore , DISC offers the idealization power of SKM with the state-learning capabilities of CP-HAC .", "We used DISC to analyze a large dataset obtained from ZMWs to evaluate cooperativity between CNBDs from pacemaker ion channels upon ligand binding .", "The rapid and robust idealization provided by DISC enabled stringent trace selection to ensure only reliable trajectories were analyzed .", "These data show that , in contrast to model inferences from ensemble measurements of HCN2 channels , CNBD tetramers do not exhibit cooperative ligand binding .", "This result suggests that allosteric interactions between binding sites may be coordinated by the channel’s transmembrane domains .", "Although we have demonstrated the use of DISC on single-molecule fluorescence data , the framework can be easily extended to other data paradigms due to its modular nature .", "For example , the use of BIC for state determination or the Student’s t-test for change-point analysis could be interchanged with other information theoretic approaches or merit functions where appropriate .", "To allow for easy comparison of a given data set , the provided software and graphical user interface ( GUI ) allows the user to select from several options the desired parameters such as choice of information criteria .", "This flexibility makes DISC suitable for a wide array of experimental data provided it can be described as a series of transitions between discrete states , including , for example , single-channel current recordings , force spectroscopy and smFRET .", "However , there is no inherent knowledge within DISC to consider various sources of experimental noise , such as photo-blinking or baseline drift; therefore , correcting for these noise sources prior to DISC analysis will likely improve idealization accuracy .", "Finally , our results show that DISC provides a dramatic improvement in computational speed over current state-of-the-art approaches while either improving or maintaining high accuracy for both state determination and event detection .", "This increase in speed is directly applicable to analyzing the growing datasets obtained in single-molecule fluorescence paradigms to adequately sample population dynamics .", "For example , the use of sCMOS camera enables smFRET measurements of tRNA conformational changes during protein translations across thousands of molecules simultaneously with millisecond resolution ( Juette et al . , 2016 ) .", "Additionally , magnetic tweezers have enabled week-long mechanical measurements of single-protein folding and unfolding , shifting observable dynamics to pathological time-scales and allowing the detection of rare events ( Popa et al . , 2016 ) .", "Thus , highly computationally efficient and robust algorithms such as DISC may be well suited for analysis of a wide variety of single molecule datasets beyond the standard smFRET data ." ], [ "Single-molecule trajectories were simulated as a Markov process of transitions between discrete states .", "All simulations were performed with a frame rate of 10 Hz and featured variable total durations , SNR , and number of states .", "The primary kinetic scheme used was adapted from our recent studies of fcAMP binding to isolated monomeric CNBDs ( Goldschen-Ohm et al . , 2016 ) .", "This model is a four-state scheme where both the unbound ( U ) and bound states ( B ) exhibit conformational changes ( U’ ⇔ U ⇔ B ⇔ B’ ) , yet exhibit only two different observable states ( ie , U’/U are indistinguishable via fluorescence intensity , as are B/B’ ) .", "fcAMP binding occurs between U and B . The rate constants ( s−1 or M−1 s−1 ) are: kU’U = 0 . 15; kU , U’=0 . 04; kU , B = 2 . 3x10−6 * [fcAMP]; kB , U = 0 . 95; kB , B’=0 . 51; kB’ , B = 0 . 31 at 1 µM fcAMP .", "To mimic the tetrameric nature of HCN channels with no cooperativity , we extrapolated up to four bound states by summing independent CNBD trajectories prior to the addition of noise .", "To include realistic SNR , state-intensities , and heterogeneity distribution of bound intensities , we analyzed the direct fcAMP excitation and emission trajectories following acceptor photobleaching from the monomeric CNBD dataset used in our previous work ( Figure 3—figure supplement 1; Goldschen-Ohm et al . , 2016 ) .", "This dataset consisted of 861 single molecules for a combined acquisition time of 44 , 090 s ( 4775 total binding events ) .", "All trajectories had a SNR >2 and all events persisted for longer than two frames , which resulted in an imbalance in the bound and unbound events .", "For each simulated trajectory , state intensities were each drawn from log normal distributions fit to monomeric CNBD single-molecule data , with average intensities between subsequent states being uniform .", "Gaussian noise was applied to trajectories at specified SNR .", "To quantitate the heterogeneous intensities from fcAMP binding , the mean of individual bound event intensities were taken for each identified event , so long as the event was >2 frames in duration .", "Heterogeneity was computed as the absolute percent difference for each event vs the mean bond intensity for the given trajectory by: ( 14 ) Percent Heterogenity =| <I>event− <I>bound <I>bound |× 100% The heterogeneity of unbound events was minimal and was therefore not included in the simulations .", "For each simulated event , heterogenous bound intensity emissions were each drawn from an exponential fit monomeric CNBD single-molecule data .", "Gaussian noise was added to trajectories as specified .", "Simulated smFRET data were downloaded from the kinSoftChallenge on June 11th , 2019 ( https://sites . google . com/view/kinsoftchallenge/home ) .", "Data used came from the provided training data sets titled: ‘Level 1’ and ‘Level 2’ with folder names ‘sim_190212_194543_level1’ and ‘sim_190212_202530_level2’ .", "DISC , STaSI , and vbFRET are all written entirely in MATLAB ( MathWorks ) .", "Each algorithm was used outside of their graphical user interfaces ( GUIs ) to more accurately compare the computational time of native functions within each algorithm .", "User parameters in DISC include: the confidence interval of CP detection and the objective function for clustering .", "Unless otherwise stated , a 95% confidence interval was applied for CP detection and BIC was used for all clustering .", "For analysis with STaSI and vbFRET , we used the recommended default values set by their authors ( Bronson et al . , 2009; Shuang et al . , 2014 ) .", "For STaSI , this means a 99 . 8% confidence interval of CP detection .", "In vbFRET , users must provide the number of states and fitting attempts per trace ( left at the default value of 10 ) .", "To circumvent providing the number of states , we modified the provided vbFRET_no_gui . m script to perform analysis outside of the vbFRET GUI .", "The modified script begins by fitting the trace to one state and increases the number of states until two more beyond the number of states with the maximum evidence to ensure the maximum fit has been obtained .", "As no changes were made to native vbFRET functions , implementing this script has no effect on vbFRET’s accuracy .", "We expect changing parameters in both STaSI and vbFRET may lead to different results; however , it was not our goal to optimize the use of these algorithms .", "Also , as a thorough investigation into the performance of STaSI and vbFRET has been conducted elsewhere , we did not investigate why these algorithms presented lower performance than DISC ( Hadzic et al . , 2018 ) .", "All quantifications of computational time were performed using the tic and toc functions in MATLAB .", "For idealization accuracy , each event returned by a given algorithm is classified as a True Positive ( TP ) , False positive ( FP ) , or False Negative ( FN ) .", "We define a TP as being in the correct state ( ±10% the correct intensity level’s standard deviation ) and correct event duration ( ±1 frame ) for a given simulated event .", "FPs are either added events or correct events in the wrong state .", "FNs are missed events .", "For each trajectory , we computed accuracy , precision , and recall as: ( 15 ) Accuracy = TP ( TP+FP+FN ) ( 16 ) Precision= TP ( TP+FP ) ( 17 ) Recall= TP ( TP+FN ) Accuracy represents the overall performance , whereas precision and recall highlight the false positive error rate ( overfitting the data ) and false negative rate ( underfitting the data ) , respectively .", "The expression , purification , biotinylation , and fluorescence labeling of tetrameric CNBDs were performed as previously described ( Goldschen-Ohm et al . , 2016 ) .", "Non-commercial arrays of ZMWs were purchased from Pacific Biosciences .", "These waveguides featured a polyphosphonate passivation layer on the aluminum walls and a biotinylated polyethylene glycol ( PEG ) layer on the glass surface to reduce non-specific binding ( Foquet et al . , 2008; Eid et al . , 2009 ) .", "The PEG-Biotin surface was incubated with 0 . 05 mg/mL streptavidin ( Prospec , cat # PRO-791 ) for 5 min in a buffer containing: 40 mM HEPES , 600 mM NaCl , 20% glycerol , 2 mM TCEP , 0 . 1% LDAO ( Sigma , cat no . 40236 ) , 2 mg/mL bovine serum albumin ( BSA ) , 1 mM Trolox ( Sigma , cas no . 53188-07-1 ) , 2 . 5 mM protocatechuic acid ( Sigma , cas no . 99-50-3 ) ( PCA ) , pH 7 . 5 ( Buffer A ) .", "After incubation , the ZMW chip was thoroughly rinsed with Buffer A to remove unbound streptavidin .", "Next , biotinylated tetrameric-CNBDs were diluted in Buffer A with the addition of the PCA/PCD oxygen scavenging system by adding 250 nM of protocatechuate 3 , 4-dioxygenase ( PCD ) from Pseudomonas sp .", "( Sigma , cas no . 9029-47-4 ) to between 100 pM and 2 nM for surface immobilization in ZMWs ( Buffer B ) ( Aitken et al . , 2008 ) .", "This resulted in ≈100 occupied ZMWs out of the total ≈1000 ZMWs per field of view identified by fluorescence bleach steps of DY-650 that labels each of the four CNBDs .", "Fluorescently labeled cAMP ( fcAMP; 8- ( 2-DY-547]-aminoethylthio ) adenosine-3’ , 5’-cylic monophosphate ) ( BioLog , cat # D 109 ) was added at 1 µM for all single-molecule experiments in Buffer B . ZMW chips were placed on top of an inverted microscope ( Olympus IX-71 , 100X , NA 1 . 49 ) and imaged under 532 nm ( 60 W/ cm2 ) or 640 nm ( 25 W/ cm2 ) ( Coherent ) as described previously ( Goldschen-Ohm et al . , 2017; Goldschen-Ohm et al . , 2016 ) .", "The only notable difference is that unlike previous experiments we did not use FRET to monitor binding in order to obtain data for extended periods ( Goldschen-Ohm et al . , 2016 ) .", "We excited DY-650 with 640 nm to identify ZMWs featuring DY-650-labeled tetrameric-CNBDs .", "Next , fcAMP was continuously imaged with 532 nm for 8000 frames at 10 Hz to monitor binding activity .", "All emission spectra were split with a 650 nm long pass dichroic ( Semrock Brightline FF650 ) and bandpass filtered using pairs of edge filters ( 532–623 . 8 nm , 632 . 9–945 nm; Semrock Cy3/Cy5-A-OMF ) and imaged onto two separate EMCCDs ( Andor iXon Ultra X-9899 ) using Metamorph software ( Molecular Devices ) .", "All data were collected using ZMWs of 150–200 nm diameter which are large enough to accommodate tetrameric CNBD complex ( each monomeric CNBD is 4 × 6×20 nm ) ( Goldschen-Ohm et al . , 2016 ) .", "All analysis was performed using custom software written in MATLAB ( Mathworks ) or ImageJ .", "Single-molecule trajectories of each ZMW were extracted from tiff stacks saved by Metamorph software using MATLAB .", "Locations of ZMWs were obtained using a threshold mask of the brightfield image of the whole ZMW array .", "ZMW locations were refined with a 2D Gaussian fit to the local intensity height map .", "The time-dependent fluorescence at each ZMW was obtained by projecting the average image intensity in a 5-pixel diameter circle onto the ZMW location throughout each image in the stack .", "A total of 13 , 670 individual ZMWs ( 1 . 1 × 108 data points ) were processed from the single-molecule tetrameric CNBD experiments .", "Each trajectory was idealized with DISC using a 95% confidence interval for CP detection and BIC for state selection ( Figure 5—figure supplement 1 ) .", "To reflect the ability to cleanly resolve the individual occupation states , we computed the separation of each sequential state vs the noise within a state by: ( 18 ) State Separation = 1K−1∑i=2K ( μi− μi−1 ) σi−1where K is the total number of states , µ is the mean intensity value of a state , and σ is the standard deviation of the data points belonging to a state .", "This ensures that states are separated well enough to resolve , as would be expected for sequential ligand binding .", "Traces featuring 4 to 6 identified states with state separation ≥ 3 were retained for further analysis .", "To ensure a given trajectory contained a functional tetrameric CNBD , we kept traces that spent less than 50% of the time in the unbound state , resulting in a total of 480 trajectories for visual inspection .", "The observed asynchronous decay of protein activity was corrected using the CP detection method to identify the most likely point in a given trajectory where protein behavior dramatically changed .", "This was accomplished using MATLAB's findchangepoint function using the change in standard deviation as the statistic .", "Data points following the identified CP location were discarded from the analysis to include only the frames of consistent fcAMP binding to presumably functional proteins ( Figure 5—figure supplement 3 ) .", "In conclusion , a total of 293 molecules totaling 1 . 2 × 105 s ( ≈34 . 5 hr ) of combined protein activity across 53 , 474 events was included for the final analysis .", "Each trajectory exhibited four or five conformational states ( 3 to 4 fcAMPs bound ) .", "Binomial fitting of the total time spent in each state was performed using MATLAB’s mle function .", "HMM modeling of single-molecule binding events was performed with QuB ( Nicolai and Sachs , 2013; Qin et al . , 2000 ) .", "Idealized trajectories from DISC were exported to QuB with the first and last events removed .", "A sequential model of 0 to 4 ligand binding sites was globally optimized to simultaneously describe the idealized binding trajectories for all molecules ." ] ]

[ "Single-molecule approaches provide enormous insight into the dynamics of biomolecules , but adequately sampling distributions of states and events often requires extensive sampling .", "Although emerging experimental techniques can generate such large datasets , existing analysis tools are not suitable to process the large volume of data obtained in high-throughput paradigms .", "Here , we present a new analysis platform ( DISC ) that accelerates unsupervised analysis of single-molecule trajectories .", "By merging model-free statistical learning with the Viterbi algorithm , DISC idealizes single-molecule trajectories up to three orders of magnitude faster with improved accuracy compared to other commonly used algorithms .", "Further , we demonstrate the utility of DISC algorithm to probe cooperativity between multiple binding events in the cyclic nucleotide binding domains of HCN pacemaker channel .", "Given the flexible and efficient nature of DISC , we anticipate it will be a powerful tool for unsupervised processing of high-throughput data across a range of single-molecule experiments ." ]

[ "During a chemical or biological process , a molecule may transition through a series of states , many of which are rare or short-lived .", "Advances in technology have made it easier to detect these states by gathering large amounts of data on individual molecules .", "However , the increasing size of these datasets has put a strain on the algorithms and software used to identify different molecular states .", "Now , White et al . have developed a new algorithm called DISC which overcomes this technical limitation .", "Unlike most other algorithms , DISC requires minimal input from the user and uses a new method to group the data into categories that represent distinct molecular states .", "Although this new approach produces a similar end-result , it reaches this conclusion much faster than more commonly used algorithms .", "To test the effectiveness of the algorithm , White et al . studied how individual molecules of a chemical known as cAMP bind to parts of proteins called cyclic nucleotide binding domains ( or CNDBs for short ) .", "A fluorescent tag was attached to single molecules of cAMP and data were collected on the behavior of each molecule .", "Previous evidence suggested that when four CNDBs join together to form a so-called tetramer complex , this affects the binding of cAMP .", "Using the DISC system , White et al . showed that individual cAMP molecules interact with all four domains in a similar way , suggesting that the binding of cAMP is not impacted by the formation of a tetramer complex .", "Analyzing this data took DISC less than 20 minutes compared to existing algorithms which took anywhere between four hours and two weeks to complete .", "The enhanced speed of the DISC algorithm could make it easier to analyze much larger datasets from other techniques in addition to fluorescence .", "This means that a greater number of states can be sampled , providing a deeper insight into the inner workings of biological and chemical processes ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "structural biology and molecular biophysics" ]

[ [ "Seminal plasma ( SP ) is a unique biological fluid , harboring unusually high concentrations of proteases and protease inhibitors ( Laflamme and Wolfner , 2013 ) , immunomodulatory cytokines such as TGF-β ( Robertson et al . , 2002 ) , and metals such as zinc , all of which serve important functions in promoting reproductive success .", "Thus far , SP is also the only human biological fluid known to contain endogenous amyloid fibrils in a non-disease state ( Usmani et al . , 2014 ) .", "Two classes of semen amyloids have been identified: those derived from proteolytic fragments of prostatic acid phosphatase ( PAP ) which polymerize to form amyloids named semen-derived enhancer of viral infection ( SEVI ) , and those derived from PSA-generated fragments of semenogelins which polymerize to form amyloids named SEM fibrils .", "Both sets of amyloids markedly enhance HIV infection by electrostatically binding HIV virions and increasing their propensity to bind to and infect cellular targets ( Münch et al . , 2007; Kim et al . , 2010; Roan et al . , 2011; Arnold et al . , 2012; Roan et al . , 2014 ) .", "Because the ability of semen and SP to enhance HIV infection directly correlates with endogenous levels of these fibrils ( Kim et al . , 2010; Roan et al . , 2014 ) , inhibiting the activity of semen amyloids may decrease HIV transmission rates .", "Indeed , semen fibrils are currently being pursued as targets for HIV microbicide development ( Olsen et al . , 2010; Roan et al . , 2010; Hartjen et al . , 2012; Lump et al . , 2015 ) .", "While the effects of SP amyloids on HIV infection have been extensively studied , the normal physiological function of these fibrils is unclear .", "SEM proteins have undergone extensive positive selection over evolutionary time ( Hurle et al . , 2007; Ferreira et al . , 2013 ) , suggesting an important role for these proteins in evolutionary fitness .", "Studies analyzing orthologs of the human amyloidogenic SEM peptide from 12 non-human primate species revealed that the amyloidogenic potential of these orthologous peptides and their virus-enhancing properties are conserved amongst great apes ( Roan et al . , 2014 ) .", "Whether this selective pressure occurs at the level of the fibrils or the parent protein is not known , but the existence of amyloidogenic semen peptides from multiple primate species suggest that these structures may serve a physiological function .", "Furthermore , the observation that SP amyloids promote infection by multiple sexually transmitted viruses ( Tang et al . , 2013; Torres et al . , 2015 ) suggests that they should be selected against during primate evolution unless they serve a significant physiological purpose .", "Here , we examined the effects of semen amyloid fibrils on sperm function , and show that they participate in sperm selection and disposal ." ], [ "Because parallels exist between the fusion of HIV to cells and the fusion of sperm to oocyte ( Doncel , 2006 ) , we first examined whether semen fibrils promote fertilization .", "Because endogenous semen fibrils behave similarly to synthetic versions of the fibrils and are difficult to isolate as purified materials ( Roan et al . , 2014; Usmani et al . , 2014 ) , we used fibrils derived from synthetic peptides for the majority of our studies .", "Synthetic SEVI and SEM peptides were confirmed to form fibrils by thioflavin T ( ThT ) staining and electron microscopy ( Figure 1—figure supplement 1 ) .", "These synthetic fibrils , like their endogenous counterparts , include both fibrils and fibrillar oligomers as well as prefibrillar oligomers , as determined by their reactivity with amyloid conformer-specific antibodies OC and A11 ( Figure 1—figure supplement 2 ) .", "In the remainder of this manuscript , we use the term ‘fibrils’ to refer to the synthetic form of the amyloids , and ‘endogenous amyloids’ when fibrils were purified from semen .", "For ethical reasons , we conducted in vitro fertilization ( IVF ) using mouse instead of human gametes .", "Given that the infection-promoting effects of the fibrils are driven by electrostatic forces and is not receptor-specific ( Roan et al . , 2009 , 2011 ) , if fertilization is enhanced by the fibrils , then the effect should be species-independent .", "Contrary to our hypothesis , both SEVI and SEM fibrils decreased IVF rates in a dose-dependent manner ( Figure 1 ) .", "Significant inhibition of IVF was observed at fibril concentrations of 50 µg/ml , and near complete inhibition was achieved with 250 µg/ml .", "Because concentrations of amyloidogenic peptides in semen range from 28 to 267 µg/ml ( Münch et al . , 2007; Roan et al . , 2011 , 2014 ) , these results suggest that physiologically relevant concentrations of semen fibrils suppress IVF .", "This inhibition was not due to fibril-induced cytotoxicity to the spermatozoa , oocytes , or embryos , as evidenced by propidium iodide ( PI ) staining experiments ( data not shown ) . 10 . 7554/eLife . 24888 . 003Figure 1 . SEVI and SEM fibrils inhibit IVF in a dose-dependent manner . The indicated concentrations of SEVI and SEM fibrils were added to mouse spermatozoa and oocytes , and monitored for IVF rates as detailed in the Materials and methods section .", "*p<0 . 05 ( two-tailed Student’s t test ) .", "n . s . = non-significant .", "Error bars reflect variation between different experiments conducted using gametes from different mice , and correspond to data averaged from 3 to 5 experiments .", "In experiments with SEVI , the number of oocytes fertilized were 197/272 ( 0 µg/ml SEVI ) , 112/179 ( 10 µg/ml SEVI ) , 67/219 ( 50 µg/ml SEVI ) , and 0/77 ( 250 µg/ml SEVI ) .", "In experiments with SEM fibrils , the number of oocytes fertilized were 78/116 ( 0 µg/ml SEM ) , 91/162 ( 10 µg/ml SEM ) , 39/128 ( 50 µg/ml SEM ) , and 6/35 ( 250 µg/ml SEM ) .", "The 250 µg/ml condition lacks error bars as it was only tested in two experiments due to limited cell numbers; in both of these experiments treatment with 250 µg/ml SEVI led to complete abrogation of IVF ( 0% fertilized oocytes ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 24888 . 00310 . 7554/eLife . 24888 . 004Figure 1—figure supplement 1 . Confirmation of fibril formation by SEVI and SEM peptides .", "( A ) SEVI and SEM fibrils were mixed with 5 µM thioflavin T , and emission at 482 nm was recorded as a measure of fibril formation .", "( B ) Electron micrograph of SEVI ( left ) or SEM ( right ) fibrils .", "The scale bar on the left image corresponds to 500 nm , and the scale bar on the right image corresponds to 200 nm . DOI: http://dx . doi . org/10 . 7554/eLife . 24888 . 00410 . 7554/eLife . 24888 . 005Figure 1—figure supplement 2 . Amyloid conformer analysis of seminal plasma and synthetic semen fibrils . Seminal plasma ( SP ) , blood plasma , SEVI fibrils , SEM fibrils , or the corresponding native peptides were spotted onto nitrocellulose and then blotted with either OC antibody ( which recognizes fibrils and fibrillar oligomers ) or A11 antibody ( which recognizes prefibrillar oligomers ) .", "Following incubation with secondary antibodies , dot blots were developed by chemiluminescence .", "OC and A11 reacted with seminal plasma and the fibrils , and not with blood plasma or the native peptide precursors of SEVI and SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 24888 . 00510 . 7554/eLife . 24888 . 006Figure 1—figure supplement 3 . Semen fibrils trap mouse spermatozoa and inhibit their progressive motility . Spermatozoa were allowed to swim out of epididymis of euthanized C57Bl/6N mice into buffer and allowed to capacitate at 37°C for 1 hr .", "Spermatozoa were stained with nuclear stain Hoechst 33342 ( green ) and amyloid fibrils were stained with Proteostat amyloid staining dye ( red ) .", "Spermatozoa ( 107/ml ) were mixed with the fibrils and images were acquired for 20 s with an interval of 1 s on a laser scanning confocal microscope using a 20X air objective .", "Shown are still images from a representative time-lapse experiment with mock ( A ) or 50 µg/ml SEVI-treated ( B ) mouse spermatozoa .", "Scale bar corresponds to 50 µm .", "Results are representative of two independent experiments .", "See also Video 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 24888 . 006 To clarify the mechanism underlying reduced IVF rates in the presence of the fibrils , we performed live cell imaging .", "We found that the fibrils seem to inhibit fusion of sperm to oocyte by entrapping mouse sperm cells ( Figure 1—figure supplement 3; Video 1 ) .", "Fibrils similarly entrapped human spermatozoa in a dose-dependent manner , as assessed by both manual quantitation as well as computer-assisted sperm analysis ( CASA ) ( Figure 2A , Figure 2—figure supplement 1 ) .", "Close examination of fibril-exposed human spermatozoa by cryosection electron microscopy revealed that the fibrils directly interacted with the plasma membranes of both sperm heads and tails , and that points of contact tended to extend the membrane away from the base of the sperm head and tail ( Figure 2B ) .", "To confirm that endogenous amyloids also associate with sperm , we fractionated human SP pooled from 20 donors and obtained a fraction containing endogenous amyloids as demonstrated by ThT binding ( Figure 2—figure supplement 2 ) .", "Microscopic analysis showed that these purified endogenous amyloids associated with spermatozoa , as did endogenous amyloids present in fresh liquefied ejaculates ( Figure 2—figure supplement 3 ) .", "Furthermore , purified endogenous amyloids , like synthetic fibrils , efficiently entrapped spermatozoa ( Figure 2C ) . 10 . 7554/eLife . 24888 . 007Figure 2 . Semen fibrils directly bind and immobilize human spermatozoa .", "( A ) Human spermatozoa incubated with semen fibrils were imaged at 37°C for 5–10 min and then assessed for % entrapped spermatozoa as described in the Materials and methods section .", "Native peptide corresponds to monomeric , non-fibrillized peptide .", "( B ) Spermatozoa were incubated in the absence ( i ,", "ii ) or presence ( iii ,", "iv ) of SEM fibrils and then imaged by sectioning electron microscopy .", "Image in panel", "( iii ) shows two sperm heads and image in panel", "( iv ) shows two sperm tails , with arrows highlighting examples of interactions between the fibrils and the tail .", "( C ) Spermatozoa treated with fractions containing ( Positive Fraction ) or lacking ( Negative Fraction ) endogenous semen amyloids were imaged for 5–10 min at 37°C and then assessed for % entrapped spermatozoa as described in the Materials and methods section .", "Treatment of spermatozoa with synthetic SEVI fibrils was used as a positive control for entrapment .", "The buffer only and negative fraction controls exhibited 0% entrapment .", "( D ) Sperm motility was assessed before", "( i ) or after ( ii–vii ) perfusion with 50 µg/ml of SEM fibrils", "( ii ) , SEM1 ( 68–85 )", "( iii ) , SEM1 ( 108–159 )", "( iv ) , SEVI", "( v ) , the O . garnettii ( Galago ) SEM2 repeat amyloid fibrils", "( vi ) , or Aβ ( 1–42 )", "( vii ) .", "Within each pair of images , the first corresponds to t = 0 , whereas the second corresponds to t = 0 . 6 s .", "Numbers correspond to the number of beats that occurred within the length of each movie ( total time = 2 s ) .", "In instances where two numbers are shown , the first corresponds to the spermatozoon on the left and the second to the spermatozoon on the right .", "Red text highlights samples where spermatozoa were immobilized .", "Yellow arrows highlight tail regions within the second frame that moved relative to the first frame .", "Scale bars = 5 µm .", "Data for each treatment are representative of at least two independent experiments examining >5 individual spermatozoa per treatment . DOI: http://dx . doi . org/10 . 7554/eLife . 24888 . 00710 . 7554/eLife . 24888 . 008Figure 2—figure supplement 1 . Semen fibrils immobilize spermatozoa in CASA . Spermatozoa , at a concentration of 5 , 000/µl ( ‘High Sperm’ ) or 500/µl ( ‘Low Sperm’ ) , were treated with 50 µg/ml of SEM or SEVI fibrils , or scrambled SEM peptide control , and assessed for the % of motile spermatozoa following normalization to a PBS control .", "SEM1 protein , also known to inhibit sperm motility ( Silva et al . , 2013 ) , served as a positive control for motility inhibition ( sperm concentration = 5000/µl ) .", "The data demonstrate that semen fibrils immobilize spermatozoa , and higher ratios of sperm:fibril lower the proportion of immobilized spermatozoa .", "Shown are cumulative results from eight experiments .", "Bonferroni’s multiple comparisons test was used for statistical analysis . DOI: http://dx . doi . org/10 . 7554/eLife . 24888 . 00810 . 7554/eLife . 24888 . 009Figure 2—figure supplement 2 . Confirmation of fibrillar nature of purified endogenous amyloids by thioflavin T . SP was fractionated and the amyloid-containing fraction ( positive fraction ) and an amyloid-deficient fraction ( negative fraction ) were stained with thioflavin T ( ThT ) , and emission was measured at the indicated wavelengths .", "Synthetic SEVI fibrils were used as a positive control .", "Emission at 482 nm is reflective of the presence of amyloids . DOI: http://dx . doi . org/10 . 7554/eLife . 24888 . 00910 . 7554/eLife . 24888 . 010Figure 2—figure supplement 3 . Spermatozoa interact with endogenous amyloids in human semen .", "( A ) Endogenous amyloids purified from SP associate with spermatozoa .", "Purified human spermatozoa were either imaged alone ( top panels ) , in the presence of purified endogenous amyloids ( middle panels ) , or in the presence of synthetic fibrils ( bottom panels ) .", "The panels on the left show differential interference contrast ( DIC ) images .", "Amyloids were detected by the amyloid-binding dyes Proteostat ( red ) and pFTAA ( blue ) .", "Sperm cells were identified by Hoechst 33342 staining ( green ) .", "In the overlay images , overlaps of Proteostat and pFTAA stains are shown in pink .", "Scale bar = 20 µm .", "( B ) Endogenous amyloids present in freshly liquefied semen associate with spermatozoa .", "Liquefied ejaculates were stained with the amyloid binding dye pFTAA and imaged by confocal microscopy .", "The image on the left shows a DIC image where individual sperm cells can be detected .", "Scale bar = 10 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 24888 . 01010 . 7554/eLife . 24888 . 011Figure 2—figure supplement 4 . Fibril-immobilized spermatozoa are metabolically active and viable .", "( A ) Spermatozoa were stained with the mitochondrial activity indicator Mitotracker , attached onto coverslips , and examined by live microscopy .", "A motile sperm cell with fluorescence localized towards the midpiece region ( where most mitochondria are localized ) was imaged before perfusion of SEM fibrils ( top images ) .", "Following perfusion of the fibrils , the sperm cell was immobilized without a loss of fluorescence ( middle images ) .", "Triton X-100-treated spermatozoa served as a positive control for a loss in sperm viability ( bottom images ) .", "The channel detecting the Green FM signal in Triton X-100-treated spermatozoa did not detect any fluorescence signal indicating loss of cellular viability .", "( B ) Human spermatozoa were incubated in the absence or presence of semen fibrils for 0 . 5 hr , stained with propidium iodide ( PI ) , and then assessed for % viable cells by flow cytometry .", "( C , D )", "Spermatozoa were treated with control scrambled peptide or SEM fibrils , and then capacitated in vitro .", "Capacitation was assessed by enumerating acrosome-reacted spermatozoa identified by the Pisum sativum agglutinin staining assay ( examples of acrosome-intact sperm ( labeled ‘1’ ) and acrosome-reacted sperm ( labeled ‘2’ ) are indicated by arrowheads ) ( top panel ) , or quantitating the levels of phosphotyrosine relative to acetylated tubulin ( bottom panel ) .", "Scale bars = 5 µm .", "n . s . = non-significant ( two-tailed Student’s t test ) .", "Data are representative of at least two independent donors . DOI: http://dx . doi . org/10 . 7554/eLife . 24888 . 01110 . 7554/eLife . 24888 . 012Video 1 . Semen fibrils entrap mouse spermatozoa . 105 mouse spermatozoa stained with Hoechst 33342 ( green ) were incubated in the absence ( A ) or presence ( B ) of 50 µg/ml of semen fibrils at 37°C in a final volume of 100 µl for 15–20 min .", "Images were acquired for 20 s with an interval of 1 s on an LSM710 confocal microscope ( Zeiss ) using a 20X air objective . DOI: http://dx . doi . org/10 . 7554/eLife . 24888 . 012 To examine the effects of the fibrils on sperm motility at the single cell level , we assayed the motility of spermatozoa from freshly ejaculated human semen by video microscopy .", "Under HEPES-buffered conditions , spermatozoa attached by their heads to coverslips exhibited continuous beating motions of the tail ( Video 2A ) .", "Remarkably , within 10 min after perfusion of the SEM fibrils , some spermatozoa became completely immotile , while others exhibited twitching movements ( Video 2B , Figure 2D ) .", "In the presence of the fibrils , 89 . 8 ± 9 . 3% of spermatozoa ( average data from three donors ) were fully or partly immobilized as defined by at least 50% of the tail being surface-immobilized .", "Of note , this immobilization is distinct from the previously described ability of the SEM1 holoprotein to limit sperm motility by binding to the sperm-associated EPPIN complex ( Mitra et al . , 2010 ) , since that activity , mapped to cysteine 239 of SEM1 , is not part of the amyloidogenic SEM1 fragment ( Roan et al . , 2014 ) .", "SEM fibril-induced immobilization did not cause changes in PI uptake , mitochondrial activity , or ability to capacitate relative to control peptide ( Figure 2—figure supplement 4 ) , showing that immobilized spermatozoa were not compromised in viability due to exposure to amyloid structures .", "In addition , perfusion of seminal proteases onto fibril-immobilized spermatozoa partially restored sperm motility ( Video 3 ) further confirming lack of cytotoxicity .", "Because proteolytic degradation of amyloidogenic peptides in semen occurs gradually over the course of liquefaction ( Roan et al . , 2014 ) , immobilization of sperm is likely most efficient during the earliest stages post-ejaculation . 10 . 7554/eLife . 24888 . 013Video 2 . Spermatozoa from fresh ejaculates are immobilized by SEM fibrils .", "( A ) Spermatozoa were isolated as detailed in the supplemental experimental procedures , attached onto coverslips , and examined for motility by live microscopy .", "( B ) Cells were then perfused with 50 µg/ml SEM1 ( 86–107 ) fibrils and motility was assessed by video microscopy after 10 min .", "Of note , the same four spermatozoa are shown in the two panels . DOI: http://dx . doi . org/10 . 7554/eLife . 24888 . 01310 . 7554/eLife . 24888 . 014Video 3 . Immobilization of surface-associated sperm cells by SEM1 ( 86–107 ) fibrils is reversible . Motile spermatozoa were examined before ( A ) and 10 min after ( B ) perfusion with SEM1 ( 86–107 ) amyloid fibrils .", "0 . 45 µm-filtered seminal plasma was then perfused in at a concentration of 20% as a source of seminal proteases , and 20 min later sperm cells were assessed for motility ( C ) .", "Data are representative of n = 6 experiments from two sperm donors . DOI: http://dx . doi . org/10 . 7554/eLife . 24888 . 014 We further demonstrated that the ability of SEM fibrils to immobilize spermatozoa was linked to its fibrillar structure by showing that SEM1 ( 68–85 ) , a naturally-occurring SEM-derived peptide in semen that does not form fibrils ( Roan et al . , 2011 ) , did not inhibit motility ( Video 4A , Figure 2D ) .", "Motility was also not inhibited by SEM1 ( 108–159 ) , a SEM-derived peptide that is non-fibrillar but highly cationic ( pI = 10 . 12 ) ( Roan et al . , 2011 ) ( Video 4B , Figure 2D ) .", "In contrast , human SEVI ( Münch et al . , 2007 ) and the previously described SEM-derived fibril from the non-human primate Otolemur garnettii ( referred to as Galago ) ( Roan et al . , 2014 ) both immobilized spermatozoa ( Video 5A , B , Figure 2D ) , while Aβ ( 1–42 ) fibrils , not naturally present in semen , did not cause immobilization ( Video 5C , Figure 2D ) .", "All together , these data suggest that semen amyloid fibrils , and not native semen peptides or pathological amyloids , are distinct in their ability to immobilize sperm cells .", "The differential effects of semen fibrils vs . Aβ ( 1–42 ) fibrils on sperm motility could be due to differences in fibril charge and/or distribution of amyloid conformers . 10 . 7554/eLife . 24888 . 015Video 4 . PSA-generated non-fibrillar fragments SEM1 ( 68–85 ) and SEM1 ( 108–159 ) do not inhibit sperm motility . Motile spermatozoa were examined for motility before and 10 min after perfusion with 50 µg/ml SEM1 ( 68–85 ) ( A ) or SEM1 ( 108–159 ) ( B ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 24888 . 01510 . 7554/eLife . 24888 . 016Video 5 . SEVI and O . garnettii SEM2 repeat fibrils inhibit sperm motility , whereas Aβ ( 1–42 ) fibrils do not . Motile spermatozoa were examined for motility before and 10 min after perfusion with 50 µg/ml SEVI fibrils ( A ) , O . garnettii ( Galago ) SEM2 repeat fibrils ( B ) , or Aβ ( 1–42 ) fibrils ( C ) .", "An Aβ ( 1–42 ) fibril concentration of 100 µg/ml , corresponding to an equimolar amount of 50 µg/ml SEM1 ( 86–107 ) , was also tested and did not inhibit sperm motility ( data not shown ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 24888 . 016 Having established that semen fibrils do not promote fusion of sperm to egg , but that they do uniquely immobilize sperm cells , we next sought to address what effect this may have on reproduction .", "Sexual intercourse elicits a massive infiltration of neutrophils and macrophages into the female reproductive tract ( FRT ) ( Pandya and Cohen , 1985; Sharkey et al . , 2012 ) , presumably to mediate clearance of microorganisms and remnant sperm cells , and perhaps to filter out morphologically abnormal and/or non-functional sperm cells ( Tomlinson et al . , 1992; Oren-Benaroya et al . , 2007 ) .", "Thus , we next tested whether spermatozoa entrapped by the fibrils are preferentially phagocytosed .", "Macrophages were differentiated from human monocytes obtained from female donors , confirmed for phagocytic activity ( Figure 3—figure supplement 1A ) , and then imaged by confocal microscopy following incubation with fluorescently-labeled spermatozoa .", "Macrophages that had taken up multiple sperm cells could be readily observed ( Figure 3—figure supplement 1B , Videos 6 and 7 ) .", "Higher throughput imaging with the Amnis Imagestream revealed that some macrophages had taken up a single spermatozoon while others had taken up multiple ones ( Figure 3—figure supplement 1C ) .", "Having demonstrated the ability to assess phagocytosis of spermatozoa in vitro , we next used a biochemical method to assess whether fibrils increase this process .", "Spermatozoa were added to cultured macrophages , and at various timepoints the macrophages were washed extensively to remove surface-associated spermatozoa and cell lysates were prepared for Western blotting .", "Acetylated tubulin , a ciliary protein expressed at high levels in sperm but not in somatic cells , was used as a marker for phagocytosed sperm cells .", "As shown in Figure 3A , phagocytosis of spermatozoa was apparent by 3 hr , and markedly increased by semen fibrils .", "Acetylated tubulin was not detected when the assay was performed at 4°C to prevent phagocytic activity ( Figure 3A ) , verifying detection of actual phagocytosis as opposed to surface binding of sperm cells . 10 . 7554/eLife . 24888 . 017Figure 3 . Semen fibrils promote phagocytosis of sperm cells .", "( A ) Spermatozoa were added to monocyte-derived macrophages for the indicated number of hours in the presence or absence of 100 µg/ml SEM fibrils , washed , and then blotted for acetylated tubulin ( to detect spermatozoa ) or β-actin ( to detect macrophages ) .", "Negative controls include macrophages in the absence of spermatozoa , and incubation of macrophages with spermatozoa at 4°C to prevent phagocytosis .", "( B ) Motile sperm cells purified by the swim-up method were labeled with eFluor 670 and then left at room temperature or damaged by five sequential rounds of freeze-thaw with liquid nitrogen .", "Spermatozoa were then added to monocyte-derived macrophages for 0 . 5 hr at 37°C in the presence or absence of 100 µg/ml SEM fibrils , washed , and then assessed by flow cytometry .", "Macrophages were identified by gating on CD14+CD33+ cells , and phagocytosis was assessed by determining the percentages of macrophages that were eFluor 670+ .", "Results are representative of data from five different donors .", "( C ) Comparison of eFluor 670+ macrophages after incubation with labeled spermatozoa at 37°C vs . 4°C ( temperature at which phagocytosis is inhibited ) .", "These data suggest that in the presence of SEM fibrils , a small number of macrophages have surface-associated spermatozoa .", "( D ) Macrophage-mediated phagocytosis of healthy vs . damaged spermatozoa in the presence of SEM fibrils was compared in triplicates .", "Shown values are those where the levels of eFluor 670+ macrophages in the presence of the SEM fibrils under 4°C conditions were subtracted out .", "This normalization was performed to discount surface-associated spermatozoa ( which is present to some extent as demonstrated in panel C ) from the analysis .", "*p<0 . 05 ( by 2-tailed t test ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 24888 . 01710 . 7554/eLife . 24888 . 018Figure 3—figure supplement 1 . Internalization of spermatozoa by macrophages .", "( A ) Confirmation of phagocytic activity of monocyte-derived macrophages .", "The Vybrant kit from Molecular Probes was used to confirm the phagocytic activity of monocyte-derived macrophages .", "Phagocytosis was assessed by incubating macrophages with killed , fluorescently-labeled bacteria for 2 hr , followed by quenching fluorescence of the extracellular bacteria .", "Fluorescence ( excitation: 480 nm , emission: 520 nm ) , reflecting the amount of endocytosed bacteria protected from quenching , was then measured .", "Fluorescence of bacteria in the absence of macrophages corresponds to background signal from incomplete fluorescence quenching .", "*p<0 . 05 ( two-tailed Student’s t test ) .", "( B ) Internalization of multiple spermatozoa by a macrophage .", "Monocyte-derived macrophages labeled with the membrane dye Vybrant DiO ( green ) were incubated with eFluor 670-stained sperm cells ( red ) for 3 hr and then imaged on a Nikon Eclipse Ti-E inverted microscope .", "( C ) ImageStream images of primary macrophages incubated with human spermatozoa .", "Macrophages are shown in green ( FITC-conjugated anti-CD14 ) while spermatozoa are shown in red .", "Phase contrast images are shown in the first column , followed by each individual channel , followed by merged images .", "Shown at the bottom is a control where the assay was conducted at 4°C instead of 37°C .", "Under these conditions , spermatozoa remain on the outside of macrophages .", "Data are representative of at least two independent donors . DOI: http://dx . doi . org/10 . 7554/eLife . 24888 . 01810 . 7554/eLife . 24888 . 019Figure 3—figure supplement 2 . Macrophage-mediated phagocytosis of sperm cells can be detected by flow cytometry .", "( A ) Monocyte-derived macrophages were assessed for phagocytosis of spermatozoa after 0 . 5 hr co-incubation at 37°C .", "A CD14+CD33+ gate was to identify macrophages ( top row ) , which were assessed for phagocytosis of eFluor 670-labeled spermatozoa ( bottom row ) .", "Cytochalasin D treatment , and 4°C instead of 37°C treatment , were used as conditions where phagocytosis is inhibited .", "( B ) Phagocytic macrophages exhibit high viability .", "Viability of the monocyte-derived macrophages used in phagocytosis assays was assessed by use of an amine-reactive fluorescent dye .", "Greater than 99% of the macrophages used in phagocytosis assays were viable , in contrast to the positive control of staurosporine-treated macrophages of which 82% of the cells were viable .", "Data are representative of at least two independent donors . DOI: http://dx . doi . org/10 . 7554/eLife . 24888 . 01910 . 7554/eLife . 24888 . 020Figure 3—figure supplement 3 . Effect of damage induction and fibrils on sperm phagocytosis .", "( A ) Sperm damage induced by liquid nitrogen and electromagnetic radiation both increase susceptibility of spermatozoa to macrophage-mediated phagocytosis .", "Monocyte-derived macrophages were incubated for the indicated number of hours at 37°C with swim-up sperm cells that were either mock-treated , or damaged by five successive rounds of freeze-thaw in liquid nitrogen , or by radiofrequency electromagnetic radiation by microwave .", "Results are gated on CD14+CD33+ cells , and the percentages of macrophages with phagocytosed spermatozoa are shown within the gates .", "( B ) SEM fibrils increase the kinetics of sperm phagocytosis .", "Monocyte-derived macrophages were incubated for the indicated number of hours at 37°C with eFluor 670-labeled swim-up sperm cells that were either mock-treated , or damaged with five successive rounds of freeze-thaw in liquid nitrogen .", "Results are gated on CD14+CD33+ cells , and the percentages of macrophages with phagocytosed spermatozoa are shown within the gates .", "After 6 hr , the percentages of macrophages that have phagocytosed healthy vs . damaged spermatozoa in the presence of SEM fibrils are similar , suggesting that the fibrils increase the kinetics of damaged sperm phagocytosis .", "The bottom row presents results obtained at 4°C to show percentages of eFluor 670+ macrophages that are surface-associated after 6 hr .", "Data are representative of at least two independent donors . DOI: http://dx . doi . org/10 . 7554/eLife . 24888 . 02010 . 7554/eLife . 24888 . 021Figure 3—figure supplement 4 . High number of spermatozoa internalized by a single macrophage in the presence of semen fibrils . Monocyte-derived macrophages labeled with the membrane dye Vybrant DiO ( green ) were incubated with eFluor 670-stained sperm cells ( red ) in the presence of 100 µg/ml SEM fibrils for 3 hr and then imaged on a Nikon Eclipse Ti-E inverted microscope .", "More than a dozen sperm heads can be detected in the macrophage shown . DOI: http://dx . doi . org/10 . 7554/eLife . 24888 . 02110 . 7554/eLife . 24888 . 022Figure 3—figure supplement 5 . Semen fibrils increase the percentage of macrophages that have phagocytosed both healthy and damaged spermatozoa . Monocyte-derived macrophages were incubated for 0 . 5 hr at 37°C with a 1:1 ratio of eFluor 670-labeled healthy spermatozoa and Celltracker Blue CMAC-labeled liquid nitrogen-damaged spermatozoa in the absence or presence of SEM fibrils .", "CD14+ macrophages were then analyzed for the presence of phagocytosed healthy ( eFluor 670+ ) or damaged ( CMAC+ ) spermatozoa .", "The data reveal that SEM fibrils increase the percentage of macrophages that have taken up both healthy and damaged spermatozoa ( upper right hand quadrant ) , resulting in an overall increase in the proportions of macrophages that have taken up damaged spermatozoa .", "Data are representative of at least two independent donors . DOI: http://dx . doi . org/10 . 7554/eLife . 24888 . 02210 . 7554/eLife . 24888 . 023Video 6 . Internalization of spermatozoa by macrophage ( rotational view ) .", "Monocyte-derived macrophages labeled with a membrane dye ( green ) were incubated with sperm cells ( red ) for 3 hr and then imaged by confocal microscopy . DOI: http://dx . doi . org/10 . 7554/eLife . 24888 . 02310 . 7554/eLife . 24888 . 024Video 7 . Internalization of spermatozoa by macrophage ( z-stacks view ) .", "Monocyte-derived macrophages labeled with a membrane dye ( green ) were incubated with sperm cells ( red ) for 3 hr and then imaged by confocal microscopy . DOI: http://dx . doi . org/10 . 7554/eLife . 24888 . 024 To quantify spermatozoa uptake by macrophages at the single-cell level , we developed a FACS-based phagocytosis assay .", "Spermatozoa were fluorescently labeled with eFluor 670 and then co-cultured with macrophages .", "Cell surface expression of CD14 and CD33 was used to differentiate the macrophages from the sperm cells .", "Macrophages that had taken up spermatozoa were identified by eFluor 670 fluorescence .", "As demonstrated in Figure 3—figure supplement 2A , a distinct population of macrophages that had phagocytosed spermatozoa was readily apparent after 0 . 5 hr of co-culture .", "This population was abrogated when the assay was conducted in the presence of the phagocytosis inhibitor cytochalasin D or when the assay was conducted at 4°C instead of 37°C ( Figure 3—figure supplement 2A ) .", "Thus , detection of sperm-harboring macrophages was not due to leakage of dye from spermatozoa , or from cell-surface binding of the spermatozoa to the macrophages .", "Furthermore , >99% of the macrophages at the time of harvest were viable ( Figure 3—figure supplement 2B ) , excluding non-specific effects due to autofluorescence of dying cells .", "To assess whether semen fibrils promote phagocytosis of spermatozoa , we conducted the FACS-based phagocytosis assay in the absence and presence of semen fibrils .", "Consistent with the Western blot data , addition of fibrils increased phagocytosis of spermatozoa , from 4 . 61% to 22 . 6% ( Figure 3B ) .", "Because removal of damaged or defective sperm cells from the reproductive tract may be important to rapidly clear the lower FRT of potentially immunogenic male antigens , we also assessed phagocytosis of damaged spermatozoa .", "Sperm cells damaged by multiple freeze/thaw cycles in liquid nitrogen or electromagnetic radiation were taken up at higher rates than healthy sperm cells ( Figure 3—figure supplement 3A ) .", "Because liquid nitrogen exerted a more potent effect ( up to 3 . 4-fold increased uptake , as opposed to up to 2 . 3-fold increased uptake for radiation-damaged spermatozoa ) , we selected this method of damage induction for subsequent experiments .", "In the absence of the fibrils , the rate of phagocytosis of liquid nitrogen-damaged spermatozoa was higher than that of fresh spermatozoa ( 31 . 3% versus 4 . 61% , respectively ) ; this was also observed in the presence of the fibrils but at higher percentages ( 49 . 9% versus 22 . 6% , respectively ) ( Figure 3B ) .", "Importantly , the absolute numbers of spermatozoa added to each co-culture condition were the same to allow direct comparisons between conditions .", "Interestingly , with longer incubation times high levels of phagocytosis of both healthy and damaged sperm cells were achieved in the presence of fibrils , suggesting that the fibrils promote the kinetics of sperm phagocytosis ( Figure 3—figure supplement 3B ) .", "Confocal microscopy revealed that fibrils also increased the number of spermatozoa engulfed by individual macrophages , frequently resulting in a single macrophage endocytosing more than a dozen sperm heads ( Figure 3—figure supplement 4 , Videos 8 and 9 ) . 10 . 7554/eLife . 24888 . 025Video 8 . High number of spermatozoa internalized by a single macrophage in the presence of semen fibrils ( rotational view ) .", "Monocyte-derived macrophages labeled with a membrane dye ( green ) were incubated with sperm cells ( red ) for 3 hr in the presence of 100 µg/ml SEM fibrils and then imaged by confocal microscopy . DOI: http://dx . doi . org/10 . 7554/eLife . 24888 . 02510 . 7554/eLife . 24888 . 026Video 9 . High number of spermatozoa internalized by a single macrophage in the presence of semen fibrils ( z-stacks view ) .", "Monocyte-derived macrophages labeled with a membrane dye ( green ) were incubated with sperm cells ( red ) for 3 hr in the presence of 100 µg/ml SEM fibrils and then imaged by confocal microscopy . DOI: http://dx . doi . org/10 . 7554/eLife . 24888 . 026 We consistently observed in multiple donors that in the presence of the fibrils damaged spermatozoa are preferentially phagocytosed over healthy ones ( Figure 3B , Figure 3—figure supplement 3A ) .", "To confirm this phenomenon , healthy and damaged spermatozoa were co-cultured with macrophages in the absence or presence of SEM fibrils , both at 37°C as well as 4°C to block phagocytic activity .", "As shown in Figure 3C , the highest level of phagocytosis was observed with damaged spermatozoa in the presence of the fibrils , in line with our prior experiments .", "However , macrophage/sperm samples incubated at 4°C with SEM fibrils also exhibited positive events within the phagocytosis gate , suggesting that SEM caused some level of sperm sticking to the macrophage surface ( Figure 3C ) .", "To normalize for the contribution of cell-surface sticking , we subtracted out the contribution of the positive events at 4°C for each sample .", "Levels of phagocytosis of damaged spermatozoa in the presence of fibrils remained significantly higher than that of healthy spermatozoa under these conditions ( Figure 3D ) .", "All together , these results suggest that: ( 1 ) macrophages preferentially phagocytose damaged spermatozoa both in the absence and presence of semen fibrils , consistent with prior reports that increased levels of macrophages are associated with decreased levels of abnormal spermatozoa in semen ( Tomlinson et al . , 1992 ) , and ( 2 ) within the first hour , the highest levels of damaged sperm phagocytosis are observed in the presence of semen fibrils .", "To directly assess the preference for phagocytosis of damaged spermatozoa , we established a competition assay enabling visualization of normal and damaged spermatozoa within the same well by labeling the two populations with different fluorescent dyes .", "Notably , macrophages that had taken up only damaged spermatozoa or healthy spermatozoa as well as combinations of both could be detected ( Figure 3—figure supplement 5 ) .", "We found that although damaged spermatozoa were preferentially phagocytosed both in the absence and presence of the fibrils , the highest rate of damaged sperm phagocytosis occurred in the presence of fibrils , when 36 . 8% of macrophages contained damaged spermatozoa ( Figure 3—figure supplement 5 ) .", "Of these , 26 . 3% had only damaged spermatozoa , while 10 . 5% had both healthy and damaged spermatozoa .", "These results confirm that damaged sperm cells are efficiently phagocytosed in the presence of semen fibrils .", "Spermatozoa are highly prone to undergo apoptosis , and this process is accelerated by reactive oxygen species produced by surrounding leukocytes as well as the sperm cells themselves ( Aitken et al . , 2012 ) .", "One of the hallmarks of apoptotic sperm cells is externalization of phosphatidylserine , a negatively charged phospholipid membrane component .", "Because all known semen fibrils are highly cationic and bind to anionic membrane components ( Roan et al . , 2009 , 2011 ) , we reasoned they may also entrap and promote phagocytosis of apoptotic sperm cells .", "To test this , we incubated swim-up sperm cells for 24 hr at 25°C .", "Because swim-up sperm cells are no longer in the antioxidant environment of seminal plasma , they are prone to undergo their default cascade of apoptotic cell death ( Aitken et al . , 2012 ) .", "Annexin V staining confirmed that compared to fresh sperm cells , a higher proportion of the 24 hour-treated sperm cells were apoptotic ( Figure 4A , B ) .", "The fresh vs . treated sperm cells were then added to macrophages in the absence or presence of fibrils , and phagocytosis was monitored .", "Like damaged spermatozoa , the highest levels of phagocytosis of apoptotic sperm cells was observed under conditions where semen fibrils were present ( Figure 4C ) . 10 . 7554/eLife . 24888 . 027Figure 4 . Apoptotic sperm cells are efficiently phagocytosed in the presence of fibrils .", "( A ) Incubation of spermatozoa for 24 hr at 25°C increases the proportion of apoptotic sperm cells .", "Spermatozoa from fresh ejaculates were purified by the swim-up technique and then assessed immediately for cell surface expression of the apoptotic marker Annexin V by flow cytometry , or incubated for 24 hr at 25°C prior to staining and analysis .", "Results are representative of 3 independent donors .", "Presented are flow cytometric plots showing the percentages of Annexin-negative ( non-apoptotic ) and Annexin-positive ( apoptotic ) spermatozoa as indicated .", "( B ) Results from experimental triplicates of each condition described in panel A . *p<0 . 05 ( two-tailed Student’s t test ) .", "Results are representative of 3 independent donors .", "( C ) Phagocytosis of fresh spermatozoa or spermatozoa incubated for 24 hr at 25°C .", "Motile spermatozoa purified by the swim-up method were labeled with eFluor 670 and then fed immediately to macrophages or incubated for 24 hr at 25°C to induce apoptosis prior to incubation with macrophages .", "Assays were conducted in the presence or absence of 100 µg/ml SEM fibrils , and in all cases phagocytosis was allowed to proceed for 0 . 5 hr prior to flow cytometric analysis .", "Macrophages were identified by gating on CD14+CD33+ cells , and phagocytosis was assessed by determining the percentages of macrophages that were eFluor 670+ .", "Results are representative of data from three different donors . DOI: http://dx . doi . org/10 . 7554/eLife . 24888 . 027" ], [ "In conclusion , we demonstrate that semen fibrils promote phagocytosis of sperm cells by macrophages .", "Because the highest rates of damaged sperm phagocytosis occurred in the presence of semen fibrils , the fibrils may participate in quality control by promoting the efficiency of sperm selection by macrophages .", "Moreover , by also promoting phagocytosis of non-damaged sperm , the fibrils appear to additionally participate in rapidly clearing the lower reproductive tract of remaining sperm cells ( whether damaged or not ) , which may help avoid the development of an inappropriate cell-mediated immune response against sperm antigens .", "The rapid removal of sperm cells is consistent with the observation that the lower reproductive tract largely returns to its pre-mating state by 24 hr post-coitus ( Pandya and Cohen , 1985 ) , and may help explain why only a minute fraction of deposited spermatozoa actually reach the oviduct isthmus ( Hunter , 1993; Eisenbach and Giojalas , 2006 ) .", "This in turn may be important for fertilization and blastocyst development since high concentrations of spermatozoa can result in increased polyploidy rates and be detrimental for human embryonic development ( Mahadevan and Trounson , 1984; Diamond et al . , 1985; Englert et al . , 1986; Dumoulin et al . , 1992 ) .", "Future studies should assess the molecular basis by which semen fibrils entrap sperm and promote their elimination , including the nature of the interaction between the fibrils and the sperm membrane .", "In addition , because semen amyloids are structurally diverse ( Usmani et al . , 2014 ) and include both fibrillar and pre-fibrillar oligomers ( Figure 1—figure supplement 2 ) , further studies to determine whether specific conformers are responsible for sperm binding and entrapment are warranted .", "Of note , semen fibrils may not be the only structures in semen that can participate in sperm disposal; neutrophil extracellular traps ( NETs ) – chromatin- and protein-containing extracellular fibers extruded by neutrophils to trap and kill bacteria ( Brinkmann et al . , 2004 ) – have been shown to entrap spermatozoa ( Alghamdi and Foster , 2005 ) , which could conceivably promote their subsequent phagocytic uptake .", "Importantly , however , NETs are different from the semen fibrils because they inhibit rather than enhance HIV infection ( Saitoh et al . , 2012 ) .", "As such , while semen fibrils have been proposed as targets for HIV microbicide development ( Olsen et al . , 2010; Roan et al . , 2010; Hartjen et al . , 2012; Roan and Münch , 2015 ) , this would not apply to NETs .", "Should semen fibril antagonists advance to clinical stages , one should be cautious as to potential effects on sperm cells and fertilization , given the data presented herein .", "Amyloid fibrils have long been considered a result of aberrant protein folding that is almost exclusively associated with systemic or localized amyloidosis , or various chronic degenerative diseases ( Chiti and Dobson , 2006 ) .", "However , it is becoming increasingly clear that fibrils may also form for beneficial reasons ( Kelly and Balch , 2003; Hou et al . , 2011; Bergman et al . , 2016 ) .", "Two prominent examples of functional amyloid in humans are HD6 ( human α-defensin 6 ) forming amyloid nets to protect host cells from invasion by enteric bacterial pathogens ( Chu et al . , 2012 ) , and Pmel17 fibrils facilitating the formation of melanosomes with covalently linked melanin ( Fowler et al . , 2006 ) .", "Prior studies have also suggested that amyloids generated by the male reproductive tract may play beneficial roles in bacterial clearance and sperm maturation ( Whelly et al . , 2012; Easterhoff et al . , 2013 ) .", "The present findings add SEVI and SEM fibrils to the growing list of functional amyloids in humans by demonstrating that these fibrils can play an active role in defective sperm disposal ." ], [ "Sequences of the semen-derived peptides to generate the SEVI and SEM fibrils have been previously described ( Münch et al . , 2007; Roan et al . , 2011 , 2014 ) .", "These peptides were chemically synthesized by Celtek Peptides ( Nashville , TN ) , CPC Scientific ( Sunnyvale , CA ) , or U-PEP ( Ulm , Germany ) , and dissolved in PBS ( pH 7 . 0 ) at a concentration of 2 . 5 mg/ml .", "To accelerate nucleation of fibril formation ( Giehm and Otzen , 2010 ) , all peptide samples were agitated overnight in PBS at 37°C at 1400 rpm in an Eppendorf Thermomixer ( Hauppauge , NY ) .", "Synthetic Aβ ( 1–42 ) peptides lyophilized from hexafluoroisopropanol ( HFIP ) solution were purchased from rPeptide ( Bogart , GA ) , diluted to 100 µg/ml , and amyloid formation was promoted by agitating peptides at 1400 rpm for 2 days at 37°C .", "Amyloid formation was confirmed by both electron microscopy and Thioflavin-T ( ThT ) analysis as described ( Roan et al . , 2014 ) .", "Nitrocellulose ( Hybond ECL , GE Healthcare , Chicago , IL ) was pre-wetted with PBS-T ( PBS containing 0 . 1% Tween 20 ) and then spotted under vacuum with 50 µl 10% seminal plasma , 10% blood plasma , or 20 µg/ml semen amyloids ( SEVI or SEM1 ( 86–107 ) fibrils ) or the corresponding monomeric peptides .", "Wells were then washed once with PBS-T under vacuum .", "The nitrocellulose membrane was then immediately transferred to a blocking solution ( PBS-T containing 5% non-fat dry milk and 1% BSA ) and incubated at 4°C with gentle shaking for 2 hr .", "The membrane was then washed three times for 5 min before incubation with anti-Amyloid Fibrils OC Antibody ( EMD Millipore , Billerica , MA ) or Anti-Amyloid Oligomers A11 antibody ( Abcam , Cambridge , United Kingdom ) ( both used at 1:2500 in PBS-T with 1% BSA ) .", "Primary antibody incubation was allowed to proceed overnight at 4°C with gentle shaking .", "The membrane was then washed three times for 5 min each before addition of HRP-conjugated rabbit IgG ( GE Healthcare ) ( used at 1:5000 in PBS-T with 1% BSA ) .", "Secondary antibody incubation was allowed to proceed for 2 hr at 4°C with gentle shaking .", "The membrane was then washed three times for 10 min each , and then developed by 1 min incubation with the Western Lightning ECL Pro ( Perkin Elmer , Waltham , MA ) , and exposed using chemiluminescence film ( Amersham Hyperfilm ECL , GE Healthcare ) .", "C57Bl/6N or NMRI mice were purchased from JANVIER SAS ( Le Genest Saint Isle , France ) and bred in-house at the TVZ University of Ulm .", "All animals were allowed to adjust to the facility for at least one week before they were used in experiments .", "The animals were maintained in separate microventilated cages ( Sealsafe Next IVC Blue Line , Tecniplast , Buguggiate , Italy ) , with a 12 hr light-dark cycle and food and water ad libitum .", "The use of animals was approved by the Regierungspräsidium Tübingen , registration number 0 . 185 , and was in accordance with existing regulations of the German Federal Law on Care and Use of Laboratory Animals .", "Mouse spermatozoa were collected directly from the vas deferens and cauda epididymis of euthanized C57Bl/6N mice .", "For the entrapment assay , spermatozoa were allowed to swim out of the epididymis , squeezed out of the vas deferens , and then incubated for 1 hr at 37°C to allow for capacitation .", "Capacitation media consisted of Human Tubal Fluid ( HTF ) medium ( Irvine Scientific , Newtownmountkennedy , Ireland ) supplemented with 15 mg/ml BSA ( Fraction V , Carl Roth GmbH , Karlsruhe , Germany ) .", "For cryopreservation of spermatozoa for subsequent IVF assay , spermatozoa of NMRI mice with proven fertility were equilibrated for 10 min in cryoprotectant media ( 18% raffinose and 3% skim milk ) , aliquoted in cryovials , and placed in the vapor phase of liquid nitrogen for 10 min .", "The cryovials were then flash frozen with liquid nitrogen and stored in the liquid phase until further use .", "Female 5–7 week old C57Bl/6N mice were administered intraperitoneally with 0 . 1 ml containing 5 IU of Pregnant Mare Serum Gonadotropin ( PMSG , Intervet , Germany ) , followed by 0 . 1 ml containing 5 IU of human chorionic gonadotropin ( hCG , Intervet ) 47–48 hr later .", "Cryopreserved mouse NMRI spermatozoa were rapidly thawed 13 hr post-hCG and added to tissue culture dishes at a concentration of 4 . 8 × 104 cells/ml in the presence of the indicated concentrations of fibrils .", "Females were euthanized by cervical dislocation and cumulus-oocyte complexes ( COCs ) were isolated by tearing up the swollen ampulla in pre-warmed M2 medium ( Sigma-Aldrich , St . Louis , MO ) .", "Following a 6 hr incubation , COCs were washed in HTF medium and added to the fertilization dishes in a final volume of 0 . 5 ml .", "All dishes were covered with mineral oil and allowed to equilibrate for at least 6 hr at 37°C in a humidified atmosphere and 5% CO2 in air .", "Zygotes were extensively washed in M16 media ( Sigma-Aldrich ) and further cultured until fertilization assessment the next day .", "Fertilization rates were determined by counting the total number of 2 cell embryos 22–24 hr post-IVF .", "Freshly isolated mouse spermatozoa ( 107/ml in 100 μl ) were stained with the nuclear stain Hoechst 33342 ( shown in green ) and incubated with SEVI amyloid fibrils stained with the Proteostat Amyloid Plaque Detection Kit ( Enzo Life Sciences , Farmindale , NY ) ( shown in red ) in HTF medium for 15–20 min at 37°C .", "Proteostat has been used for detection of a diverse array of amyloid fibrils ( Wang et al . , 2012; Navarro and Ventura , 2014; Navarro et al . , 2016; Taglialegna et al . , 2016 ) including those from human semen ( Arnold et al . , 2012; Usmani et al . , 2014; Lump et al . , 2015 ) .", "Images were acquired on an LSM710 confocal microscope for 20 s with an interval of 1 s using a 20X air objective .", "Results are representative of a total of 3–5 experiments performed for each animal ( n = 2 ) .", "De-identified fresh human semen samples were obtained the from University of California , San Francisco ( UCSF ) / San Francisco General Hospital ( SFGH ) Positive Health Program Research Group ( The Options Project , IRB # 10–00301 ) and the Kinderwunsch-Zentrum ( Ulm , Germany , Approval # 351/10 and 156/13 ) after giving informed consent .", "For single-cell motility assays , confocal microscopy , Western blotting , and flow cytometry , sperm cells were isolated via the sperm swim-up assay similar to methods previously described ( Lishko et al . , 2011 ) .", "Briefly , 2 ml of semen liquefied at room temperature was carefully underlaid into a 50 ml conical tube containing 8 ml of in-house generated HTF ( consisting of 97 . 8 mM NaCl , 5 mM KCl , 0 . 2 mM MgSO4 , 0 . 37 mM KH2PO4 , 2 mM CaCl2 , 20 mM HEPES , 20 mM lactic acid , 0 . 4 mM Na-Pyruvate , and 3 mM glucose ) and incubated for 1 hr at 37°C .", "The HTF buffer containing motile spermatozoa was then collected without disturbing the solution of semen underneath .", "For population analyses ( entrapment assays ) , spermatozoa were instead purified by density gradient centrifugation using the PureCeption kit ( KB Biosystem Deutschland , ART-2004 , Germany ) .", "To confirm fibrillation of synthetic amyloids , samples ( 500 µg/ml ) were prepared with parlodion-filmed carbon-coated grids and 2% potassium phosphotungstate , pH6 . 5 , and analyzed and photographed as previously described ( Roan et al . , 2009 ) .", "To visualize the physical interaction between sperm cells and the fibrils , swim-up spermatozoa isolated as described above were incubated in the absence or presence of 100 µg/ml SEM fibrils for 1 hr , and then fixed in a 0 . 1 M sodium cacodylate buffer solution ( pH 7 . 4 ) containing 2% glutaraldehyde .", "The samples were then loaded into 200 µm diameter cellulose capillary tubes ( Leica Microsystems Inc . , Buffalo Grove , IL ) , post-fixed in 2% osmium tetroxide in the same buffer , stained en block with 2% aqueous uranyl acetate , dehydrated in acetone , infiltrated , and embedded in LX-112 resin ( Ladd Research Industries , Burlington , VT ) .", "Samples were ultrathin-sectioned on a Reichert Ultracut S ultramicrotome and counter-stained with 0 . 8% lead citrate .", "Grids were examined on a JEOL JEM-1230 transmission electron microscope ( JEOL USA , Inc . , Peabody , MA ) and photographed with the Gatan Ultrascan 1000 digital camera ( Gatan Inc . , Warrendale , PA ) .", "Swim-up sperm cells were stained with the nuclear dye Hoechst 33342 while amyloid fibrils were stained with the amyloid-binding dye Proteostat or pFTAA ( a luminescent-conjugated oligothiophene dye ) as previously described ( Usmani et al . , 2014 ) .", "A total of 105 spermatozoa ( final concentration 107/ml ) were mixed with 0 , 2 , 10 , 50 and 250 µg/ml of the indicated synthetic amyloid fibril or purified endogenous amyloids ( from the equivalent of 0 . 34 , 0 . 17 , or 0 . 8 ml SP , see details below ) in a final volume of 50–100 µl , and then incubated at 37°C for 15–20 min .", "Images were acquired for 20 s with an interval of 1 s using a Plan-Apochromat 63X/1 . 40 oil objective lens on a LSM710 confocal microscope ( Zeiss , Germany ) equipped with Zen-Software ( Zeiss ) .", "All experiments were performed at 37°C .", "A total of 3–5 experiments was performed for each donor to calculate the number of entrapped spermatozoa .", "Only live and motile spermatozoa were considered for analysis .", "The total number of free spermatozoa and spermatozoa trapped within the amyloid network was determined from all frames over a 20 s period and then the percentage of entrapped cells was calculated .", "Fresh semen samples from 20 donors were allowed to liquefy and then immediately frozen at −20°C .", "To isolate fractions enriched for amyloids , all samples were thawed simultaneously , pooled ( constituting ~26 ml ) , and processed as described ( Münch et al . , 2007 ) .", "Briefly , samples were centrifuged ( 17 , 000 x g ) to separate spermatozoa and SP .", "The SP was then diluted with 25 ml extraction buffer ( 1 M acetic acid , 20 mM ascorbic acid , 1 mM EDTA , 2 M sodium chloride , pH 2 . 0 ) and stirred for 10 min at 4°C .", "The extract was then diluted to a volume of 1 . 5 L with ultrafiltration buffer ( 0 . 1 M acetic acid , 20 mM ascorbic acid , 1 mM EDTA , pH 3 . 0 ) , and then ultrafiltered using a 30 kDa polysulfon membrane ( Pall , Quattro Flow Fluid Systems , Pall GmbH , Dreiich , Germany ) .", "The filtrate was diluted with water to a volume of 2 L and then applied to a conditioned cation exchange column ( Fractogel TSK SP ( S ) Merck , Darmstadt , Germany , column size: 2 × 12 . 5 cm ) .", "After loading , the column was washed with two column volumes of water at pH 2 . 5 , and eluted with the following buffers: ( A ) 0 . 1 M Na2HPO4 , pH 7 . 4 , volume 150 ml , ( B ) 1 M ammonium acetate , pH 7 . 0 , volume 180 ml , ( C ) water pH 7 . 0 , volume 180 ml , ( D ) 0 . 1 M NaOH , pH 13 , volume 200 ml .", "The eluates B-D were pooled , the pH was adjusted to 3 . 0 and the pooled eluates were applied to a reverse phase column ( Source RPC polystyrol , particle size15 µm , column size 1 × 12 . 5 cm , Pharmacia , Freiburg , Germany ) .", "Bound peptides were eluted using a linear gradient from 95% A ( water and 0 . 1% [v/v] TFA ) to 60% B ( 80% [v/v] acetonitrile and 0 . 1% [v/v] TFA ) in 55 min , from 60% B to 100% B in 10 min using a flow rate of 1 . 5 ml/min .", "Protein elution was monitored with an absorbance detector at 214 nm .", "A total of 40 fractions was collected , freeze-dried and used for ThT analysis to identify a fraction containing amyloids .", "The presence of amyloid was further verified by microscopy , and staining with the amyloid-binding dyes Proteostat and pFTAA .", "After all the purification steps , ~60% of the total material was recovered , accounting for ~15 ml of semen .", "Because the entrapment assays used 2 . 3% of the starting material and 3-fold dilutions thereof , this corresponded to semen volume equivalents of 0 . 34 ml , 0 . 17 ml , and 0 . 08 ml .", "To demonstrate the presence of amyloids by thioflavin T , 10 µl synthetic fibrils or fractions from pooled SP ( each corresponding to 5% of total of fractionated material , or 0 . 75 ml SP ) were diluted in 80 µl of PBS and then stained with ThT ( final concentration 20 µM , from Sigma-Aldrich ) .", "Samples were incubated in the dark at room temperature for 15 min with constant shaking ( 350 rpm ) before being measured in an Infinite M1000 Pro microplate reader ( Tecan Group Ltd . , Switzerland ) .", "Samples were excited at 435 nm and emission spectra were measured between 470 and 650 nm , with a 5 nm bandwidth and a manual gain set to 150 .", "Endogenous amyloids from fractionated or unfractionated SP were visualized by staining samples with Proteostat and/or pFTAA , using approaches previously described ( Usmani et al . , 2014 ) .", "Briefly , fractionated or unfractionated SP were incubated with Proteostat and/or pFTAA for 15 min at room temperature , and then transferred into an Ibidi Chamber Slide ( #80826 from GmbH ) .", "Stained samples were imaged on a Zeiss LSM710 AxioObserver confocal microscope equipped with a Plan-Apochromat 63/1 . 40 oil objective lens and Zen-Software v2010 ( Zeiss , Germany ) .", "Spermatozoa were simultaneously imaged by DIC using transmitted light detector ( T-PMT ) and appropriate condenser settings .", "Where indicated , spermatozoa were additionally visualized by staining with Hoechst 33342 .", "Swim-up human spermatozoa were plated onto 5 mm coverslips ( WPI , Sarasota , FL ) in HEPES buffer solution ( HS ) containing 130 mM NaCl , 5 mM KCl , 1 mM MgSO4 , 2 mM CaCl2 , 5 mM glucose , 1 mM sodium pyruvate , 10 mM lactic acid , and 20 mM HEPES ( pH 7 . 4 ) .", "Coverslips with sperm cells were then placed in a recording chamber ( Warner instruments , Hamden , CT ) containing HS solution .", "Sperm movement was recorded at room temperature with a high-speed GX-1 Memrecam camera ( NAC , Simi Valley , CA ) attached to an Olympus IX71 microscope .", "Sperm motility was always confirmed before any peptide/fibril treatment and recorded .", "Following confirmation of sperm motility , HS containing 50 µg/ml of the appropriate peptide or amyloid fibril was perfused into the chamber , and then incubated with spermatozoa for 10 min .", "The speed of recording was 1000 frames per second ( fps ) , but all supplemental videos were slowed down five-fold for clarity .", "Viability of immobilized spermatozoa was confirmed by monitoring mitochondrial activity with Mitotracker ( Invitrogen , Grand Island , NY ) .", "Motility at the population level was confirmed by CASA using methods previously described ( Mitra et al . , 2010 ) .", "Peptides , fibrils , and SEM1 protein ( G26-R281 , from [Silva et al . , 2012] ) were all used at a final concentration of 50 µg/ml , while PBS alone served as the negative control .", "Sperm concentrations were diluted to 5 , 000/µl or 500/µl as indicated .", "The % motility inhibition mediated by SEM1 ( 86–107 ) between the concentration range of 50–800 µg/ml was not statistically different , as high concentrations of the fibrils just caused larger aggregates to form , leading to immobilization of the same % of spermatozoa .", "Because each sperm sample had different % motility , each experiment was normalized by dividing each treatment by the % motility of the untreated sample and multiplying by 100 .", "This set the % motility of the untreated sample to 100% and allowed comparison between different experiments using different sperm samples .", "Human spermatozoa ( 107/ml ) were treated with PBS or 50 µg/ml SEVI fibrils at 37°C for 30 min , and then incubated with 50 µg/ml of PI .", "Toxicity was assessed by monitoring uptake of PI by flow cytometry using a BD FACSCanto II ( BD Biosciences , San Jose , CA ) .", "Spermatozoa were assessed for the ability to capacitate by monitoring acrosome reaction and capacitation-mediated induction of tyrosine phosphorylation ( Arcelay et al . , 2008 ) .", "Spermatozoa from fresh ejaculates were prepared by the swim-up method and then added to 6-well dishes containing 22 mm coverslips ( WPI , Sarasota , FL ) in HS buffer and allowed to settle for 30 min .", "Wells were then incubated with PBS , or 50 µg/ml SEM1 ( 86–107 ) amyloid fibrils or the scrambled control , for an additional 15 min .", "Spermatozoa were washed with HS , and then capacitated for 4 hr at 37°C with HS in the presence of 25 mM NaHCO3 and 20% fetal bovine serum ( FBS ) .", "Cells were then either fixed with ice cold 95% ethanol for 30 min at 4°C for acrosomal staining , or lysed in 2X Laemmli sample buffer for Western blot analysis .", "For acrosomal staining , spermatozoa fixed in 95% ethanol were allowed to air dry .", "Coverslips were then incubated with fluorescein isothiocyanate ( FITC ) -conjugated Pisum sativum agglutinin ( Sigma-Aldrich ) for 10 min , and then washed with ultra-pure water .", "Cells were then analyzed for acrosome reaction as described ( Lishko et al . , 2010 ) .", "For phosphotyrosine analysis , 4% β-mercaptoethanol ( β-ME ) was added to cells lysed in 2X Laemmli sample buffer .", "Lysates were boiled for 5 min at 100°C and then adjusted to a final β-ME concentration of 8% .", "Samples were transferred to 4–12% polyacrylamide gels and blotted onto PVDF membranes .", "Membranes were blocked with 3% γ-globulin-free BSA in PBS containing 0 . 1% Tween ( PBS-T ) for 30 min at room temperature .", "Subsequently , membranes were incubated for 1 hr at room temperature with mAB 4G10 Platinum , anti-phosphotyrosine ( Millipore , Billerica , MA ) or anti-acetylated alpha-tubulin ( Thermo Scientific , Waltham , MA ) diluted 1:5000 in PBS-T containing 1% γ-globulin-free BSA .", "After washing three times with PBS-T , the membranes were incubated for 1 hr at room temperature with goat anti-mouse HRP-conjugated IgG ( Millipore ) diluted 1:20 , 000 in PBS-T containing 1% γ-globulin-free BSA .", "Protein bands were detected by SuperSignal West Pico Chemiluminescent Substrate ( Thermo Scientific ) on a FluorChem M imaging system ( Protein Simple , Santa Clara , CA ) .", "Phagocytic activity of macrophages was assessed using the Vybrant Phagocytosis Assay Kit ( Thermo Fisher ) .", "Peripheral blood mononuclear cells ( PBMCs ) were isolated from fresh TrimaLeuko reduction chambers from female donors ( obtained through the Blood Centers of the Pacific Blood Systems ) using Ficoll-Hypaque density gradients .", "CD14+ monocytes were then purified using CD14+ microbeads ( Miltenyi Biotec , Bergisch Gladbach , Germany ) .", "Monocytes were differentiated into macrophages by culturing 106 cells/well in clear flat-bottom 6-well plates ( Corning Primaria , Corning , NY ) in RPMI supplemented with 10% FBS , 2 mM L-glutamine , 50 U/ml penicillin , and 50 µg/ml streptomycin , in the absence or presence of 100 ng/ml human recombinant MCSF ( R&D Systems , Minneapolis , MN ) .", "Five days later , cells were fed with fresh media , and 48 hr later all cultures were replaced with fresh media lacking MCSF .", "The cells cultured in the absence or presence of MCSF were assessed for phagocytic activity on dead fluorescent bacteria following guidelines provided by the manufacturer .", "For analysis by ImageStream , swim-up cells were centrifuged at 526 x g and then the pellet containing the sperm cells was resuspended in 0 . 05 µM Cell Proliferation Dye eFluor 670 ( eBioscience , San Diego , CA ) .", "For flow cytometry experiments , a concentration of 2 . 5 µM eFluor 670 was instead used .", "Sperm cells were labeled for 10 min at room temperature and washed three times with PBS prior to use in phagocytosis assays .", "To compare healthy ( fresh and motile ) vs . damaged spermatozoa , the sperm cells were evenly divided following fluorescence labeling with eFluor 670 .", "One half , containing the healthy and motile spermatozoa , was incubated at room temperature while the other half was damaged by five rounds of freezing/thawing using liquid nitrogen .", "A complete loss of motility in the freeze/thawed but not the mock-treated sample was confirmed by visual inspection by light microscopy .", "Alternatively , to induce apoptosis , swim-up spermatozoa were incubated for 24 hr at 25°C after eFluor 670 labeling .", "To simultaneously monitor two populations of spermatozoa within the same sample , two different dyes were used .", "Unstained sperm cells were isolated using approaches described above and divided into two equal parts .", "The first population was labeled with eFluor 670 as described above , while the second population was labeled with a 160 µM Celltracker Blue CMAC ( Thermo Fisher ) for 1 hr at 37°C .", "The CMAC labeled cells were then washed twice with PBS and were then damaged by five rounds of freeze/thaw using liquid nitrogen .", "Macrophages were differentiated from monocytes ( 106 cells / condition ) in the presence of MCSF as described above .", "A total of 1 . 5–2 × 106 spermatozoa for each treatment condition were incubated in the absence or presence of 100 µg/ml SEM1 ( 86–107 ) fibrils for 15 min at 37°C , and then added to the macrophages .", "Unlabeled spermatozoa were used for Western blot experiments , while spermatozoa labeled using methods described above were used for flow cytometry and ImageStream experiments .", "Phagocytosis was allowed to proceed for the designated amount of time ( 0 . 5 , 1 . 5 , 3 , or 6 hr ) at 37°C or 4°C as appropriate .", "At each timepoint , macrophages were washed three times with RPMI and treated with 2 . 5 μg/ml of Heparin ( Sigma-Aldrich ) in RPMI for 0 . 5 hr at 37°C to help remove surface-bound fibrils .", "Following three additional washes with PBS , cells were either lysed for Western blot analysis , or stained for phenotyping by flow cytometry or Imagestream .", "For comparing phagocytosis of spermatozoa with different proportions of apoptotic spermatozoa , eFluor 670 stained spermatozoa were either added to the macrophages immediately after being stained and washed , or they were incubated at room temperature for 24 hr to induce apoptosis prior to being added to the macrophages .", "Phagocytosis of these spermatozoa was allowed to proceed for 0 . 5 hr and then analyzed by flow cytometry .", "At the designated timepoint , macrophages were lysed in 25–35 µl 2X Laemmli’s buffer ( 263 mM Tris pH 6 . 8 , 80% glycerol , 142 mM of SDS , 29 mM of bromophenol blue ) , containing protease inhibitor cocktail ( Roche , Basel , Switzerland ) and 0 . 1 mM PMSF .", "Lysates were heated for 5 min at 97°C immediately before loading 20 µl on a 7 . 5% Criterion Tris-HCl polyacrylamide gel ( Bio-Rad , Hercules , CA ) .", "Proteins were transferred for 2 hr at 65 V onto nitrocellulose Immuno-Blot PVDF membranes ( Bio-Rad ) .", "Membranes were then blocked for 0 . 5 hr with 3% BSA in PBS-Tween ( PBS-T ) , incubated at a 1:2000 dilution with a primary antibody against acetylated tubulin ( Sigma-Aldrich ) which exhibits specificity for sperm cells ( Piperno and Fuller , 1985 ) , followed by washing and a secondary anti-mouse sheep IgG HRP antibody at a 1:5000 dilution ( GE Healthcare , Chicago , IL ) .", "Immunoblots were developed using Western Lightning ECL ( Perkin Elmer , Waltham , MA ) .", "At the appropriate timepoint , macrophages were trypsinized using 1 ml of 0 . 05% Trypsin containing 0 . 53 mM EDTA , washed , and then incubated for 0 . 5 hr at 4°C with anti-CD14 ( Clone M5E2 , conjugated to FITC ) and anti-CD33 , ( Clone WM53 , conjugated to PE ) .", "The samples were then washed , fixed with 1% PFA , and run on an LSRII flow cytometer ( Becton Dickinson , Franklin Lakes , NJ ) .", "Macrophages were identified on the LSRII by gating on CD14+CD33+ cells .", "For Imagestream analysis , cells were identified by CD14 expression .", "Negative controls for fluorescence labeling and autofluorescence included the following: unlabeled sperm cells , labeled sperm cells , SEM1 ( 86–107 ) fibrils , labeled sperm cells + SEM1 ( 86–107 ) fibrils , macrophages , and macrophages + SEM1 ( 86–107 ) fibrils .", "Monocyte-derived macrophages were first stained with 2 . 5 µg/ml of Vybrant DiO Cell-Labeling Solution ( Thermo Scientific ) diluted in PBS .", "The cells were then washed three times with PBS and incubated with eFluor 670 spermatozoa in the absence or presence of SEM fibrils using conditions described above .", "After 3 hr , macrophages were washed 3x with PBS , trypsinized , and stored in 1% PFA at 4°C in the dark until imaging .", "Confocal imaging was carried out using a Nikon Eclipse Ti-E inverted microscope equipped with a Yokogawa CSU22 spinning disk confocal scanner , a Sutter emission Lambda filter wheel adapter with ET460/50m , ET525/50m , ET645/65m and ET700/75m filters , a Prior motorized stage with Piezo Z-drive , and a Photometrics Evolve 512 Delta EMCCD Camera .", "Images were acquired with Micro-Manager software version 1 . 4 . 21 .", "Image scanning was executed employing a Plan Apo VC 100x/1 . 4 Oil ( DIC N2/100X I ) objective using 488 nm and 640 nm , 100 mW Coherent OBIS lasers .", "The Piezo Z-drive was set for fast acquisition and z-steps at 0 . 40 µm .", "Image analysis was performed using ImageJ software version 1 . 48 with the Deconvolution lab ( EPFL ) and 3D viewer ( B . Schmid ) plugins installed .", "All acquisition and analysis parameters were maintained constant for all samples .", "Fresh spermatozoa and spermatozoa incubated for 24 hr at 25°C were stained for the apoptotic marker phosphatidylserine by use of FITC-conjugated Annexin V ( eBioscience ) followed by flow cytometric analysis on an LSRII ( Becton Dickinson ) , following manufacturer’s protocol ." ] ]

[ "Unlike other human biological fluids , semen contains multiple types of amyloid fibrils in the absence of disease .", "These fibrils enhance HIV infection by promoting viral fusion to cellular targets , but their natural function remained unknown .", "The similarities shared between HIV fusion to host cell and sperm fusion to oocyte led us to examine whether these fibrils promote fertilization .", "Surprisingly , the fibrils inhibited fertilization by immobilizing sperm .", "Interestingly , however , this immobilization facilitated uptake and clearance of sperm by macrophages , which are known to infiltrate the female reproductive tract ( FRT ) following semen exposure .", "In the presence of semen fibrils , damaged and apoptotic sperm were more rapidly phagocytosed than healthy ones , suggesting that deposition of semen fibrils in the lower FRT facilitates clearance of poor-quality sperm .", "Our findings suggest that amyloid fibrils in semen may play a role in reproduction by participating in sperm selection and facilitating the rapid removal of sperm antigens ." ]

[ "Seminal plasma , the fluid portion of semen , helps to transport sperm cells to the egg during sexual reproduction .", "Seminal plasma contains numerous proteins that help the sperm to survive and , in recent years , researchers discovered that it also harbours protein deposits known as amyloid fibrils .", "Such protein deposits are generally associated with neurodegenerative diseases such as Alzheimer's and Parkinson’s disease , where a build-up of fibrils can damage the nervous system .", "Semen amyloids , however , are present in the absence of disease , but can boost infection by HIV and other sexually transmitted viruses , by shuttling virus particles to their target cells .", "Despite these damaging effects , some researchers had suggested that amyloids in semen could be beneficial for humans , though it was unclear what these benefits might be .", "Roan et al . now set out to assess how semen amyloids affect human sperm activity .", "The results show that semen amyloids bind to damaged sperm cells and immobilize them , which are then quickly cleared away by immune cells .", "This could ensure that only the fittest sperm cells reach the egg .", "These findings suggest that amyloids can potentially serve beneficial roles for reproduction .", "A next step will be to investigate how semen amyloids trap unwanted sperm and how immune cells know when to remove it .", "More research is needed to investigate if problems in these processes could lead to infertility in men ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "developmental biology", "evolutionary biology" ]

[ [ "Mechanical forces produced at the cellular level are known to shape tissues during morphogenesis ( see Lecuit et al . , 2011 , for a recent review ) .", "Molecular motors and cytoskeletal elements generate these mechanical forces , which cause tissues to deform and change shape ( Mammoto and Ingber , 2010 ) .", "Until recently , such tissue-level aspects of morphogenesis have received relatively little attention in the field of evolutionary developmental biology .", "The evolution of developmental processes is generally attributed to changes in genetic regulation ( see for example , Carroll et al . , 2009; Davidson and Erwin , 2006; Peter and Davidson , 2015; Wilkins , 2002 ) .", "To date , it is not fully understood how a developing organism integrates the mechanical and genetic factors necessary to shape a tissue , or how this interplay between tissue mechanics and genetics is contributing to the evolution of development .", "We focus on this latter aspect by studying how a continuous epidermal layer is formed by epithelial fusion during dorsal closure in a non-model organism , the scuttle fly Megaselia abdita ( Diptera: Phoridae ) .", "Epithelial fusion is a fundamental morphogenetic mechanism in animal development where two opposing epithelial sheets are brought together to subsequently seam and result in a single continuous epithelial layer ( Jacinto et al . , 2001 ) .", "Dorsal closure in Drosophila melanogaster ( Diptera: Drosophilidae ) is a classical model system to study epithelial fusion ( Jacinto et al . , 2000 ) .", "This process is promoted by the mechanical action of different players: a contractile actomyosin cable forming at the leading edge of the epidermal flanks , the extraembryonic amnioserosa which covers the dorsal opening and generates contractile forces during epidermal flank advancement , and the eventual seaming of the epidermis through a mechanism involving microtubule-based cellular protrusions ( Eltsov et al . , 2015; Hutson et al . , 2003; Kiehart et al . , 2000; Saias et al . , 2015 ) .", "Genetically , the c-Jun N-terminal kinase ( JNK ) pathway and the transforming growth factor beta ( TGF-β ) family gene decapentaplegic ( dpp ) play an essential regulatory role in the process ( Fernández et al . , 2007; Glise and Noselli , 1997; Jacinto et al . , 2002; Knust , 1997 ) .", "The expression of dpp localizes to the leading edge of the epidermal flanks and depends on the activity of the D . melanogaster JNK gene ( basket , bsk ) .", "Embryos lacking bsk activity show downregulation of dpp at the epidermal leading edge , failure of dorsal closure progression , and a dorsal-open phenotype in the larval cuticle ( Glise and Noselli , 1997; Sluss et al . , 1996 ) .", "At the molecular level , activation of the JNK/Dpp signaling pathways promotes the formation and maintenance of the actomyosin cable at the epidermal leading edge ( Ducuing et al . , 2015 ) and , thus , progression of the opposing epidermal flanks toward the dorsal midline where they meet .", "At the final stage of dorsal closure , the opposing epidermal flanks ‘zipper’ or ‘seam’ through the action of microtubules that align toward the dorsal opening and promote the formation of filopodial protrusions at both epidermal leading edges ( Jacinto et al . , 2002; Jankovics and Brunner , 2006; Millard and Martin , 2008 ) .", "Dorsal closure is a conserved morphogenetic process that occurs in all insects ( Chapman , 1998 ) .", "Although in D . melanogaster it involves two tissues , the embryonic epidermis and the extraembryonic amnioserosa , in most insects it involves three: the embryonic epidermis , an extraembryonic amnion , and a separate extraembryonic serosa ( Panfilio , 2008; Schmidt-Ott and Kwan , 2016 ) .", "These complex anatomical differences raise the question whether the mechanisms responsible for epithelial fusion in a simple two-tissue system are conserved in a three-tissue system .", "The phorid scuttle fly M . abdita ( placed in an early branching cyclorraphan lineage ) presents a three-tissue system of dorsal closure and has been established as a model to study the evolution of developmental processes ( Bullock et al . , 2004; Rafiqi et al . , 2008; Schmidt-Ott et al . , 1994; Stauber et al . , 2000; Wotton et al . , 2015 ) .", "Thus , M . adbita offers the opportunity to compare the three-tissue system of dorsal closure to the two-tissue system present in D . melanogaster .", "Here , we perform a quantitative characterization of dorsal closure in M . abdita .", "Combining molecular tools with live imaging , we show that dorsal closure in M . abdita embryos occurs in three distinct phases:", "( i ) serosa rupture and retraction ,", "( ii ) serosa contraction and progression of opposing epidermal flanks , and", "( iii ) a dual seaming process to eventually form a fused continuous epidermis .", "Despite the significant morphological differences with D . melanogaster , the regulation of dorsal closure in M . abdita involves a conserved role for the JNK/Dpp signaling pathway to form and maintain an epidermal actomyosin cable surrounding the dorsal opening .", "More specifically , we find that following an actomyosin-dependent contraction of the serosa , two consecutive microtubule-dependent seaming events take place in the amnion as well as in the epidermis .", "In both cases , apical microtubule bundles align and extend toward the site of closure suggesting a general epithelial fusion mechanism .", "Altogether , our results provide a dynamic and quantitative description of epithelial fusion in a complex three-tissue system .", "They indicate that the evolutionary transition from a three-tissue to a two-tissue system of dorsal closure involves changes in the number and sequence of morphogenetic events , rather than changes in the spatio-temporal activity of the main signaling pathways that control closure progression ." ], [ "In order to map the spatial arrangement of tissues involved in dorsal closure of M . abdita embryos , we obtained confocal projections of fixed non-devitellinized embryos with stained nuclei .", "Nuclear anatomy and staining have been used previously to identify extraembryonic tissues in the flour beetle Tribolium castaneum ( Panfilio et al . , 2013 ) .", "In M . abdita , staining fixed embryos with the nuclear dye DAPI allowed us to distinguish three types of tissues: ( 1 ) The extraembryonic serosa , which constitutes the outermost extraembryonic layer and envelops the entire M . abdita embryo before the onset of dorsal closure ( magenta in Figure 1A , A’ and and B , B’ ) .", "Its cells have very large nuclei ( average size 125± 21 µm2 , SD , n = 150 cells ) and show discontinuous or ‘punctuated’ DAPI staining ( magenta in Figure 1A and A’ and Figure 1—figure supplement 1A–A’ and B–B’’ ) .", "( 2 ) The extraembryonic amnion , which is one to two cells wide , localizes in between the serosal and epidermal tissues ( blue in Figure 1A , A’ and and B , B’ ) .", "Its cells also have large nuclei ( average size 77 ± 16 µm2 , SD , n = 150 cells ) and show a more continuous , ‘compact’ DAPI staining ( blue in Figure 1A and A’ and Figure 1—figure supplement 1A–A’ and B–B’’ ) .", "( 3 ) The embryonic epidermis , which contains numerous small nuclei ( average size 14 ± 3 µm2 , SD , n = 150 cells ) that are tightly packed ( gray in Figure 1A and A’ ) .", "In order to obtain a closer view of the spatial arrangement of tissues in live M . abdita embryos , we injected DAPI at the embryo poles during dorsal closure stage and obtained confocal projections .", "This staining showed that amnion cells sit on top of yolk granules and are positioned adjacent to the embryonic epidermis ( blue arrowheads in Figure 1—figure supplement 1C–C” ) .", "When fixing M . abdita embryos at dorsal closure stage , devitellinization also removes the serosa together with the vitelline membrane ( Figure 1—figure supplement 2A ) .", "Devitellinization and serosa cells removal resulted in a gap on the dorsal side of the embryo , seen as lack of phalloidin staining ( Figure 1—figure supplement 2B ) .", "Amnion cells ( 1–2 cell rows adjacent to the epidermis , blue arrowheads in Figure 1—figure supplement 2B ) remained apposed to an intact epidermis .", "In a few cases , devitellinization left some intact serosa cells on top of M . abdita embryos ( very large cells highlighted by phalloidin and DAPI counterstains , white arrowhead in Figure 1—figure supplement 2C and C’ ) .", "An optical re-slice of confocal projections of the intact serosa and amnion cells showed that these two cells types are apposed ( yellow arrowhead in Figure 1—figure supplement 2C’’ ) .", "In summary , the anatomy of M . abdita embryos at dorsal closure reveals a three-tissue system , where large serosa cells surround the embryo and are apposed to the amnion cells at the dorsal-most end of the embryo .", "Amnion cells form a row , in turn apposed to the adjacent epidermis ( Figure 1B and B’ ) .", "This three-tissue geometry poses an interesting challenge for the process of dorsal closure , since the apposed serosa and amnion need to undergo dramatic rearrangements to achieve epidermal fusion at the end of dorsal closure .", "How does M . abdita solve this problem ?", "At early stages of dorsal closure , the serosa surrounding the embryo undergoes an abrupt rupture ( Wotton et al . , 2014 ) .", "Serosa rupture initiates close to the posterior pole of the embryo .", "Spread of the rupture occurs anteriorly through the ventral side of the embryo and results in a retraction of the serosa toward the dorsal end of the embryo ( Figure 1C ) .", "During this process , serosa cells accumulate at the dorsal opening ( magenta arrowheads in Figure 1D , and magenta cells in 1 G-G’’ ) .", "In the meantime , the amnion remains in place , apposed to the serosa ( blue arrowheads in Figure 1D , and blue cells in 1 G-G’’ ) , and adjacent to the embryonic epidermis ( white arrowheads in Figure 1D , and green line in 1 G-G’’ ) .", "To gain quantitative evidence on the dynamics of dorsal closure in M . abdita , we labeled live embryos with the fluorescent lipophylic dye FM 4–64 and followed the process using confocal imaging ( Video 1 ) .", "We used the fusion of the dorsal ridge ( merging of the ridge primordia at the dorsal midline , magenta bar in Figure 1—figure supplement 3A and A’ ) as a developmental landmark for the initiation of dorsal closure ( T = 0 min ) ( Campos-Ortega and Hartenstein , 1997; VanHook and Letsou , 2008 ) .", "Under this time frame , the serosa ruptures at T = 25 min ( ±8 min , SD; n = 15 embryos ) and dorsal closure ( seaming of the embryonic epidermal flanks at the dorsal midline , see yellow line in Figure 1—figure supplement 3A’’ ) concludes at T = 74 min ( ±10 min , SD , n = 15 embryos ) .", "To relate serosa retraction to the kinetics of dorsal closure , we measured the relative changes in area of the serosa covering the embryo and the relative changes in height ( h ) of the dorsal opening over time ( see red and blue curves in Figure 1F , yellow dashed line in Figure 1E , blue line in Figure 1—figure supplement 3A- and Materials and methods ) .", "At early stages of dorsal closure , the leading edge of the epidermis straightens ( yellow line in Figure 1—figure supplement 3A and A'' ) and the dorsal opening increases progressively in height ( h ) ( blue curve in Figure 1F ) .", "After dorsal ridge fusion ( pink area in Figure 1F ) , the serosa ruptures , retracts and accumulates onto the dorsal opening ( Figure 1C , magenta arrowheads in 1D , yellow dashed line in 1E , magenta cells in 1G’-G’’ ) .", "This is concurrent with a fast reduction in dorsal opening height ( red and blue curves in Figure 1F , yellow line in Figure 1—figure supplement 3A and A'' ) .", "Visualizing the process from an orthogonal view , we observe that following serosa accumulation on top of the dorsal opening , this extraembryonic tissue bends inwards and serosa cells undergo an apicobasal elongation resulting in the internalization of a large part of the serosa cells into the yolk prior to epidermal fusion ( Video 2 and Figure 1—figure supplement 3B and C–C’ ) .", "Optical re-slices of confocal projections along the anterio-posterior axis of fixed embryos show that extraembryonic tissues internalizes during dorsal closure and do not accumulate underneath the fused epidermis after completion of the process ( Figure 1—figure supplement 4 ) .", "Thus , at late dorsal closure stages , the serosa is fully internalized ( magenta cells in Figure 1G’’ ) and the two epidermal flanks continue progressing toward the dorsal midline , covering the dorsal opening and eventually fusing completely ( Figure 1—figure supplement 3A'' ) .", "Since the JNK and Dpp signaling pathways are known to regulate dorsal closure in D . melanogaster embryos , and their impairment results in the failure of dorsal closure and in a dorsal-open phenotype ( Glise and Noselli , 1997; Sluss et al . , 1996 ) , we wondered whether JNK and Dpp signaling would also be important regulators of dorsal closure and serosa retraction in M . abdita embryos .", "Using in situ hybridization and cuticle preparations , we observed that in wild-type embryos , M . abdita dpp ( Mab_dpp ) is expressed along the leading edge of the epidermis ( black arrowheads in Figure 2A ) and progression of dorsal closure results in the deposition of a continuous larval cuticle ( Figure 2A’ ) , very similar to D . melanogaster .", "Next , we perturbed the M . abdita JNK pathway , using gene knockdown by RNA interference ( RNAi , see Materials and methods ) against M . abdita bsk ( Mab_bsk ) in pre-gastrulating embryos .", "Around 90% of Mab_bsk dsRNA-injected embryos developed to at least germband retraction stage ( 821 out of 922 embryos ) .", "Mab_bsk RNAi knock-down resulted in both a disrupted pattern of Mab_dpp expression ( including a complete absence of expression at the leading edge of the epidermis , magenta arrowheads in Figure 2B ) and a dorsal-open phenotype in the larval cuticle ( magenta arrowheads in Figure 2B’ ) of ~64% of RNAi-injected and developed embryos ( 528 out of 821 embryos ) .", "Both phenotypes are similar to the ones that occur in D . melanogaster embryos after JNK/Dpp signaling perturbation .", "Interestingly , live imaging of RNAi-injected embryos reveals that serosa rupture and retraction still occur ( yellow dashed line in Figure 2C–C’’ and Video 3 ) , despite a failure of progression of the epidermal flanks ( yellow arrowheads in Figure 2—figure supplement 1B; note that the embryo from Video 3 and Figure 2C corresponds to the same embryo in Figure 2—figure supplements 1B , 24 hr after RNAi injection ) .", "In summary , these results indicate that JNK and Dpp signaling regulate progression of dorsal closure in M . abdita as in D . melanogaster embryos but are not required for the regulation of serosa rupture and retraction .", "In D . melanogaster embryos , both a JNK/Dpp-dependent contractile epidermal cable and the contraction of the amnioserosa tissue ( through actomyosin contractility and volume decrease ) power the progression of dorsal closure ( Hutson et al . , 2003; Kiehart et al . , 2000; Saias et al . , 2015 ) .", "The actomyosin cytoskeleton is therefore an essential component in force generation during this process .", "To gain insights into the structures generating forces during dorsal closure in M . abdita , we stained fixed embryos with phalloidin ( to reveal F-actin ) or a phosphoMyosin antibody .", "Both stains showed accumulation at the leading edge of the epidermis ( green arrowheads in Figure 3A and B ) and at the surface of serosa cells during internalization ( red arrowheads in Figure 3A and B ) .", "Measurements from time-lapse sequences showed that serosa cell area reduces over time during their accumulation at the dorsal opening , from an average of 212 ± 52 µm2 ( SD; n = 20 cells ) to 76 ± 18 µm2 ( SD; n = 20 cells ) to 33 ± µm2 ( SD; n = 20 cells ) at 20 , 30 and 40 min after serosa rupture , respectively ( Figure 3—figure supplement 1B ) .", "This cell area reduction correlates with an apical accumulation of actin at early stages of serosa internalization ( yellow arrowheads in Figure 1—figure supplement 4a’’ , b’’ and c’’ ) , suggesting an apical constriction mechanism through actomyosin contraction during serosa internalization .", "In contrast , amnion cells show low levels of actin and myosin , even during late stages of dorsal closure after full serosa internalization ( blue arrowhead in Figure 3A and B , Figure 3C’’ and Figure 3—figure supplement 1A ) .", "Closer observation also reveals the presence of actin-enriched filopodia-like extensions protruding from the actomyosin cable ( yellow arrowheads in Figure 3—figure supplement 1C and C’ ) .", "RNAi knock-down of Mab_bsk strongly reduces the level of actin accumulation at the epidermal leading edge compared to wild-type embryos ( white arrows in Figure 3—figure supplement 2A and B ) .", "This suggests that the embryonic epidermal cable is similar in structure and regulated by the JNK/Dpp signaling pathway in both M . abdita and D . melanogaster embryos .", "A timed sequence of phalloidin-stained M . abdita embryos ( Figure 3C–C’’’ ) reveals further details concerning the dynamics of dorsal closure: upon serosa internalization , the amniotic flanks ( devoid of actomyosin ) move to the dorsal midline where the two flanks merge ( white arrowhead in Figure 3C’’ ) .", "Upon merging of amnion flanks , the actomyosin cable propels the epidermal flanks to the dorsal midline where epidermal seaming takes place ( yellow arrowheads in Figure 3C’ , C’’ and C’’’ ) .", "To affect actomyosin-based tissue contractility , we injected M . abdita embryos with the Rho kinase ( ROCK ) inhibitor Y-27632 .", "This drug has been extensively used in D . melanogaster to inhibit actomyosin contractility by blocking ROCK activity and , consequently , downstream targets including myosin phosphorylation ( Czerniak et al . , 2016; Monier et al . , 2010; Sommi et al . , 2011 ) .", "Injection of Y-27632 at early stages of M . abdita dorsal closure reduces actomyosin accumulation at the epidermal leading edge compared to wild-type embryos ( white arrowheads in Figure 3—figure supplement 2A and C ) .", "The progression of the epidermal leading edge is arrested upon treatment ( Figure 3—figure supplement 2D–D’’ and Video 4 ) .", "The internalization of serosa cells is also abolished as observed by the lack of inward bending and apicobasal elongation of this tissue in orthogonal view ( Figure 3—figure supplement 2E–E’’ and Video 5 ) .", "These observations indicate that actomyosin contractility contributes to both the internalization of the serosa and progression of the epidermal leading edge .", "Interestingly , the kinetics of serosa retraction in Y-27632-injected embryos seemed less affected during the first half of the process than the second half ( purple curve in Figure 3D ) , suggesting that actomyosin-based cell contraction is taking place mainly at the final stage of retraction and during serosa internalization .", "Taken together , these results indicate that actomyosin-based contractility within the serosa and the cable surrounding the dorsal opening are required for the progression of dorsal closure in M . abdita embryos .", "In addition to an actomyosin cable , the microtubule cytoskeleton is known to be essential during the last step of dorsal closure in D . melanogaster .", "In epidermal cells at the leading edge , microtubules align in apical bundles parallel to the dorsoventral axis and protrude into stable filopodia .", "These aligned epidermal microtubules are required for proper epidermal seaming and their depolymerization leads to defects in seaming and incomplete closure in D . melanogaster ( Jankovics and Brunner , 2006 ) .", "We investigated whether such a microtubule configuration is observable during dorsal closure in M . abdita as well .", "Staining fixed M . abdita embryos at different stages of late dorsal closure with a β-tubulin antibody , we observed that microtubules in the epidermis also orient toward the dorsal midline ( Figure 4—figure supplement 1A and B ) and protrude from the epidermal leading edge ( white arrowheads in Figure 4—figure supplement 1B ) forming apical bundles ( Figure 4—figure supplement 1B’ ) .", "This epidermal microtubule alignment is maintained throughout epidermal flank advancement and during epidermal seaming ( Figure 4—figure supplement 1C–C’’’ ) .", "Interestingly , we find a similar alignment of microtubules in the extraembryonic amnion , where microtubule bundles localize apically ( Figure 4B’ ) and orient toward the internalizing serosa ( blue arrowheads in Figure 4A and B and Figure 4—figure supplement 1A ) .", "This apical microtubule alignment in the amnion seems to follow cell elongation .", "To support this observation , we estimated apical cell surface area by performing a Voronoi tessellation around the nuclei of extraembryonic cells .", "We could observe that amniotic cells present an elongated apical cell surface area ( 285 ± 81 µm2 , SD; n = 12 cells; blue arrowheads in Figure 4—figure supplement 1D ) compared to serosa cells ( 57 ± 23 µm2 , SD; n = 108 cells; red arrowheads in Figure 4—figure supplement 1D ) .", "Both apical microtubule alignment and elongated apical cell surface area in the amnion are maintained during serosa internalization and merging of amnion cells from opposite flanks ( blue arrowheads in Figure 4B and Figure 4—figure supplement 1C–C’ ) .", "Amnion microtubule alignment is subsequently lost after the opposite amnion flanks meet at the dorsal midline ( blue arrowhead in Figure 4—figure supplement 1C’’ ) .", "Since microtubule alignment is necessary for epidermal seaming during dorsal closure in D . melanogaster , we reasoned that a similar microtubule alignment in the amnion of M . abdita embryos could indicate epithelial amniotic fusion through seaming in this species .", "To follow the last steps of M . abdita dorsal closure more closely , we imaged FM 4–64-labeled embryos during the late stage of dorsal closure ( Video 6 ) .", "We observed that upon serosa internalization , the opposing amniotic flanks of M . abdita embryos meet and fuse at the dorsal midline , suggesting an amniotic seaming process ( blue-shaded area in Figure 4C , C’ and C’’ ) .", "Immediately after amniotic seaming occurred , the epidermal flanks progressed dorsally and also seamed on top of the continuous amnion ( green dashed line in Figure 4C , C’ and C’’ ) .", "An estimation of seaming velocities in the amnion and epidermis ( 8 . 1 ± 2 µm/min and 3 . 8 ± 1 . 7 µm/min , SD , n = 5 embryos , respectively ) shows a variation in speed between the two processes that could result from a difference in the mechanical properties of the fusing epithelia or the seaming angle at each canthi .", "To investigate the functional role of microtubules in amnion cell elongation and seaming , we injected embryos with the microtubule depolymerizing drug colcemid ( Jankovics and Brunner , 2006 ) .", "Injection of colcemid does not perturb the kinetics of serosa retraction ( Figure 4—figure supplement 2A ) , actin accumulation in serosa cells ( red arrowheads in Figure 4—figure supplement 2B and B’ ) , straightening of the epidermal leading edge ( white arrowheads in Figure 4D ) , or actin cable formation ( white arrowheads in Figure 4—figure supplement 2B and B’ ) .", "An orthogonal view from a time-lapse sequence also shows that the initial inward bending and apicobasal elongation of the serosa cells during internalization occurs in both wild type and colcemid injected embryos ( Video 7 and magenta dashed lines in Figure 1—figure supplement 3C–C’ and Figure 4—figure supplement 2C–C’ ) .", "In contrast , dorsal closure arrests during the internalization of the serosa ( red arrowhead in Figure 4D’’ ) and does not progress toward epithelial seaming ( Video 8 and red curve in Figure 4E ) .", "We observe that microtubule polymerization does not occur in colcemid-injected embryos ( blue and white arrowheads for amnion and epidermal cells , respectively , in Figure 4—figure supplement 2D compared to 2D’ ) and that amnion cells initially elongate toward the dorsal midline as in wild-type conditions , although they relax and retract from the amnion merging site ( Figure 4—figure supplement 4A and Video 9 ) .", "Thus , microtubule depolymerization does not affect amnion cell elongation and dorsal convergence but rather a subsequent amniotic seaming .", "In order to test the role of the microtubule assembly , specifically in the amnion , we deactivated the depolymerizing effect of colcemid treatment on dorsal closure progression with UV light .", "UV-irradiation was performed in a region between the epidermis and the internalizing serosa , corresponding to the amnion ( magenta area in Figure 4—figure supplement 3A’’ and Video 10 ) .", "In UV-irradiated embryos , dorsal closure progressed further , reducing the height ( h ) of the dorsal opening , compared to colcemid-treated embryos , although it did not progress to the extent of wild-type control embryos ( Figure 4—figure supplement 3B ) .", "Taken together , these observations indicate that the microtubule cytoskeleton in the amniotic tissue is required for seaming of the amnion and the completion of dorsal closure .", "In summary , the results presented in this section indicate the presence of subsequent microtubule-based seaming processes in both the amnion and the epidermis during dorsal closure in M . abdita .", "First , opposing amniotic flanks fuse at the dorsal midline upon serosa ingression , followed by epidermal seaming ( see schematics in Figure 4F and F’ and lower panels of Figure 4—figure supplement 4b–b’’ ) .", "Both processes are necessary for the completion of epidermal seaming and dorsal closure , to result in a continuous embryonic epidermal sheet .", "Our results indicate that serosa contraction contributes to bringing amniotic flanks into close proximity and that amniotic seaming is a microtubule-dependent event ." ], [ "In this study , we provide a detailed characterization of epithelial fusion during dorsal closure in the non-drosophilid scuttle fly M . abdita .", "In this species , dorsal closure involves three different tissues: the embryonic epidermis , as well as the extraembryonic amnion and serosa .", "Dorsal closure in M . abdita occurs in three distinct phases:", "( i ) rupture and retraction of the extraembryonic serosa surrounding the embryo ,", "( ii ) concurrent contraction of both an epidermal actomyosin cable and the serosal tissue in the dorsal region of the embryo leading to internalization of the serosa into the dorsal opening , and", "( iii ) successive seaming processes fusing first the amnion and then the epidermis .", "Even though genetic regulation of dorsal closure by the JNK and Dpp signaling pathways appears to be conserved between M . abdita and D . melanogaster , the sequence of morphogenetic rearrangements is very different between the two species .", "These differences , however , result in the same output: a continuous epidermal layer covering the dorsal region of the embryo .", "In M . abdita , the serosa encloses the whole embryo .", "It is apposed to the amnion , which in turn is apposed to the epidermis at the edge of the dorsal opening ( schematics in Figure 1B and B’ ) ( see also Rafiqi et al . , 2008 ) .", "The rupture of the serosa is the first step of a series of complex morphogenetic events ( see Figure 4—figure supplement 5 ) .", "Although the initiation signal for serosal rupture is not yet known , we can discard a purely mechanical trigger since injection of the embryo prior to dorsal closure did not induce serosal rupture and global retraction , despite resulting in a small wound and a slight retraction of the tissue around the injection site .", "In addition , rupture still occurs in embryos injected with Rho-kinase ( ROCK ) inhibitor , which reduces actomyosin contractility .", "Lastly , rupture always initiates at a very specific ventral-posterior site .", "Taken together , these observations indicate that rupture is not triggered exclusively by global straining and non-autonomous forces applied to the serosa tissue .", "Instead , rupture seems to be triggered by a specific localized cue .", "Upon rupture , the remaining serosal tissue retracts and constricts dorsally through an actomyosin-dependent mechanism , in a way similar to serosa rupture and retraction in the beetle T . castaneum ( Hilbrant et al . , 2016; Panfilio et al . , 2013 ) .", "The retracting serosa then internalizes into the dorsal opening of M . abdita .", "Concomitant with serosal internalization , a JNK/Dpp-dependent actomyosin cable forms at the epidermal leading edge of M . abdita embryos .", "It promotes the advancement of the opposing epidermal flanks toward the dorsal midline , and the eventual seaming of the two flanks .", "This stage of dorsal closure occurs in a similar fashion to D . melanogaster , but differs in comparison with T . castaneum , where no actomyosin epidermal cable appears to be involved in epidermal flank advancement ( Panfilio et al . , 2013 ) .", "In contrast to D . melanogaster , dorsal closure in M . abdita involves an additional amniotic seaming process .", "Our experimental data indicate that amniotic seaming is microtubule-dependent and essential for dorsal closure to occur .", "Thus , similar to epidermal seaming in D . melanogaster embryos , where microtubules align dorsoventrally prior to tissue fusion ( Jankovics and Brunner , 2006 ) , the two sequential amniotic and epidermal seaming processes in M . abdita also involve a dorsoventral alignment of microtubules .", "Why microtubules align in this way remains unclear .", "One possible scenario is that shape elongation of amniotic cells toward the retracting and internalizing serosa could promote microtubule reorientation in the direction of contractile cells .", "Interestingly , cellular fusion in the developing trachea of D . melanogaster involves cell elongation and microtubules orientation toward the site of fusion ( Kato et al . , 2016 ) .", "Elongation of cells toward a contractile tissue also occurs during gastrulation in D . melanogaster ( Rauzi et al . , 2015 ) and neural tube closure in the chordate Ciona intestinalis ( Hashimoto et al . , 2015 ) .", "It is not known whether microtubule alignment also occurs in the latter processes to promote epithelial fusion .", "If this is the case , the microtubule-dependent seaming that we describe might reflect a common mechanism for epithelial tissues to fuse .", "It remains unclear whether microtubule-dependent epithelial seaming is a process that can generate forces contributing to dorsal closure .", "In the case of D . melanogaster , laser-ablation of epidermal canthi ( i . e . the epidermal corners where opposing epidermal flanks meet ) slows down the last stages of dorsal closure ( Wells et al . , 2014 ) .", "However , F-actin-enriched epidermal seaming still occurs between the opposing leading edges of the epidermis despite the removal of the canthi .", "Dorsal closure in embryos of M . abdita presents two sequential seaming events that share a common feature: transient microtubule reorganization .", "It would be interesting to explore if the cytoskeletal basis ( i . e . microtubule reorganization ) of epithelial seaming events is conserved in other insect species with a three-tissue system of dorsal closure , for example T . castaneum .", "Our work suggests that the evolutionary transition from a three-tissue to a two-tissue system of dorsal closure not only involves the reduction of extraembryonic tissue , for example from distinct amnion and serosa to a fused amnioserosa ( see Horn et al . , 2015; Rafiqi et al . , 2008; Rafiqi et al . , 2010; Schmidt-Ott and Kwan , 2016 ) .", "In addition , it requires changes in epidermal progression and seaming events .", "Further development of imaging and molecular tools in M . abdita will help us better understand the subcellular , cellular , and tissue dynamics that led to this evolutionary transition .", "In the case of dipteran dorsal closure , it appears that the evolutionary modulation of tissue remodeling is mainly driven by morphological rearrangements rather than large changes in gene expression .", "This study provides the first detailed analysis of tissue anatomy and dynamics complemented with gene expression assays to understand the evolution of a morphogenetic process .", "In this respect , dipterans provide a powerful model to understand the interplay between tissue rearrangement and gene expression during the evolution of development ." ], [ "Our M . abdita fly culture was maintained as previously described ( Rafiqi et al . , 2011a ) .", "Embryos were collected at 25°C for 4 hr , and then incubated at 19°C until they reached stages 13–15 , corresponding to dorsal closure as described in Wotton et al . ( 2014 ) .", "Mab_bsk was cloned using sequence data from a published early embryonic transcriptome ( http://diptex . crg . es; gene ID: Mab_bsk: MK10 ) ( Jiménez-Guri et al . , 2013 ) .", "Briefly , open-reading frames ( ORFs ) were PCR-amplified based on cDNA from 0 to 5 hr-old M . abdita embryos .", "Amplified fragments were cloned into PCRII-TOPO ( Invitrogen , Carlsbad , CA ) or pGEM-T ( Promega , Madison , WI ) vectors using the following specific primers ( 5’/3’ ) : Mab_bsk , TGCCCGTCATCAGTTTTACA and GACGACGCGGGACTACTTTA .", "dsRNA was performed using the Ambion MEGAscript kit ( Life Technologies , Carlsbad , CA ) .", "The following specific primers ( 5’/3’ ) containing a T7 promoter sequence at their 5’ end were used: Mab_bsk , GGTGGGCGACACAAGATT and AAACAGGCATCGGGGAAT .", "RNAi injection was performed using previously published protocols ( Rafiqi et al . , 2008; Rafiqi et al . , 2010; Rafiqi et al . , 2011d; Wotton et al . , 2015 ) .", "Dechorionated embryos were injected prior to gastrulation at a concentration of 5 µM for Mab_bsk , then incubated at 25°C .", "The injected dsRNA construct comprised 798 nucleotides ( base pairs 369–1166 of the ORF ) for Mab_bsk .", "In situ hybridization in heat-fixed M . abdita embryos was performed according to a previously published protocol from D . melanogaster ( Crombach et al . , 2012 ) .", "Digoxigenin-labeled Mab_dpp probe is from Jiménez-Guri et al . ( 2013 ) .", "Fixation , devitellinization and immunostaining of M . abdita embryos were performed as previously described ( Rafiqi et al . , 2011c; Rafiqi et al . , 2012 ) with slight modifications .", "Briefly , embryos undergoing dorsal closure were dechorionated and fixed for 25 min in heptane and PEMS ( 100 mM PIPES , 2 mM EGTA and 1 mM MgSO4 , pH 6 . 9 ) , in a 3:1 PEMS:methanol solution , and a final concentration of 6 . 5% formaldehyde .", "Embryos were postfixed and hand devitellinized as described ( Rafiqi et al . , 2012 ) .", "Microtubules were stained using a monoclonal primary antibody ( mouse ) against β-tubulin ( E7 , Developmental Studies Hybridoma Bank ) at a dilution 1:100 , and a secondary antibody conjugated to Alexa 488 dye ( Invitrogen , Carlsbad , CA ) at a dilution of 1:1000 .", "For phalloidin staining , embryos were fixed for 1 hr using PEMS and a final concentration of 8% formaldehyde , hand-devitellinized as described for D . melanogaster embryos ( Fernández et al . , 2007; Kaltschmidt et al . , 2002; Rothwell and Sullivan , 2000 ) , and incubated with phallodin-Alexa488 or phalloidin-Alexa563 ( Invitrogen , Carlsbad , CA ) at a dilution of 1:200 for 1 hr .", "When double-staining against phalloidin and microtubules , embryos were fixed , hand-devitellinized , and stained for phalloidin first , followed by incubation with the β-tubulin primary and secondary antibodies as above .", "Nuclei were counterstained using DAPI ( 1:1000 ) .", "Embryos were washed in PBT ( PBS , with 0 . 1% Triton X-100 ) , and mounted using ProLong Gold Antifade ( Invitrogen , Carlsbad , CA ) .", "Cuticle preparations of M . abdita embryos were performed as previously described ( Rafiqi et al . , 2011b ) with slight modifications .", "Briefly , embryos were fixed and hand-devitellinized before preparing and mounting the cuticles .", "Images of cuticle preparations were taken using a phase-contrast microscope .", "Time-lapse imaging was performed with dechorionated M . abdita embryos .", "RNAi-injected embryos were imaged using a Zeiss Cell Observer with a controlled temperature chamber at 25°C and phase contrast settings .", "For fluorescence imaging , wild type embryos at dorsal closure stage were desiccated for 5 min , aligned , oriented , and immobilized on a coverslip with heptane glue , covered with halocarbon oil and injected at the embryo poles with 1 mM ( needle concentration ) of the lipophilic dye FM 4–64 ( Molecular Probes ) .", "Embryos were imaged at room temperature using an Andor Revolution XD spinning-disk confocal microscope .", "ROCK inhibitor Y-27632 ( Sigma-Aldrich , St . Louis , MO ) and colcemid ( Santa Cruz Biotechnology , Santa Cruz , CA ) were prepared to 10 mM and 500 µg/ml ( needle concentration ) , and also injected at the embryo poles during dorsal closure stage .", "Control embryos were injected with water or DMSO , respectively .", "Final needle concentrations of dye and/or drugs were prepared in injection buffer ( 10 mM HEPES , 180 mM NaCl , 5 mM KCl and 1 mM MgCl2 , pH 7 . 2 ) , and delivered to the interstitial space formed between the serosa and the embryo .", "Confocal projections of ~10 z-stack images ( 1 µm spacing ) were used to generate time-lapse sequences in dorsal view .", "Orthogonal views from time-lapse live imaging were obtained by reslicing confocal z-stacks of 0 . 25 µm spacing .", "The height ( h ) of the dorsal opening is the maximum perpendicular distance from the dorsal midline to the epidermal leading edge ( Hutson et al . , 2003 ) .", "The changes in area of the serosa covering the embryo over time were determined by approximating the embryonic shape using an ellipse and resizing manually to follow the serosa edge on one lateral side during retraction , assuming that serosa retraction occurs symmetrically on both sides of the embryo after rupture along the ventral midline .", "Detection of serosal edge morphology from bright-field time-lapse sequences was performed by subtracting images at time t + 1 from images at time t .", "This operation rendered the contour of the retracting serosa visible .", "The identification of extraembryonic tissues was performed using nuclear anatomy , staining profiles and z position of the nuclei in confocal stack images obtained from fixed embryos labeled with DAPI .", "Staining profiles , nuclear areas and z position in the embryo were measured in 150 cells for each cell type ( serosa and amnion ) from 15 different embryos .", "UV irradiation experiments to deactivate colcemid were performed as follows: dechorionated , desiccated , FM 4–64-labeled and colcemid-injected M . abdita embryos were immobilized in heptane glue , mounted in halocarbon oil , and imaged dorsally in an inverted Leica TCS SP5 laser-scanning confocal microscope in resonant scanner mode .", "Colcemid injection was performed during dorsal ridge fusion and prior to serosa rupture .", "Imaging acquisition started ~30 min after injection .", "A region of interest ( ROI ) was selected comprising an area between the epidermal flanks and the internalizing serosa , corresponding to the extraembryonic amnion .", "The ROI was scanned for at least 30 s using a 405 nm UV laser and imaging was resumed after irradiation .", "Fixed , immunostained embryos were imaged as follows: images were acquired using an inverted Leica TCS SP5 laser-scanning confocal microscope .", "All post-acquisition image processing and analysis was done using ImageJ software ( NIH ) .", "For Voronoi analysis , the center of mass of cell nuclei was detected manually with Fiji and used as seed for Voronoi tessellation with Matlab ." ] ]

[ "Evolution of morphogenesis is generally associated with changes in genetic regulation .", "Here , we report evidence indicating that dorsal closure , a conserved morphogenetic process in dipterans , evolved as the consequence of rearrangements in epithelial organization rather than signaling regulation .", "In Drosophila melanogaster , dorsal closure consists of a two-tissue system where the contraction of extraembryonic amnioserosa and a JNK/Dpp-dependent epidermal actomyosin cable result in microtubule-dependent seaming of the epidermis .", "We find that dorsal closure in Megaselia abdita , a three-tissue system comprising serosa , amnion and epidermis , differs in morphogenetic rearrangements despite conservation of JNK/Dpp signaling .", "In addition to an actomyosin cable , M . abdita dorsal closure is driven by the rupture and contraction of the serosa and the consecutive microtubule-dependent seaming of amnion and epidermis .", "Our study indicates that the evolutionary transition to a reduced system of dorsal closure involves simplification of the seaming process without changing the signaling pathways of closure progression ." ]

[ "For a single fertilized egg to become an animal with many millions of cells , complex networks of genes must control the different stages of development .", "These gene networks create all the patterns needed to form different parts of the body .", "Changes to these patterns can create new species , with different sizes , body shapes , colors and lifestyles .", "Researchers often examine how evolution can create new species by altering gene networks to change patterns of development .", "Yet , some differences in development may not directly result from changes to gene networks .", "Other causes could include how the tissues are organized to begin with .", "One way to better understand this kind of difference is to compare developmental processes between two or more related species .", "Dorsal closure , for example , is a stage in a fly’s development when one layer of cells – the epidermis – closes over the back of the developing embryo .", "Dorsal closure in the scuttle fly ( Megaselia abdita ) involves the epidermis and two other layers of cells , yet it only involves one other cell layer in the vinegar fly ( Drosophila melanogaster ) .", "The different number of layers means that dorsal closure must happen differently in the two species .", "Yet , Fraire-Zamora et al . now report that the genetic control behind the process is very similar in both species .", "Instead , it is differences in the arrangement and shape of cell layers that lead to the changes in dorsal closure .", "Dorsal closure involves physical changes to several cell layers , and is driven by protein structures that give shape and strength to cells .", "These findings highlight how living systems adapt to evolutionary changes .", "Fraire-Zamora et al . suggest that the same concepts could be adapted further , helping to design and build complex organs in the laboratory .", "This in turn could help scientist develop new approaches to repairing tissues and healing wounds ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "immunology and inflammation" ]

[ [ "Engagement of the BCR results in proliferation of B cells and their differentiation into either antibody-secreting plasma cells or memory B cells ( Rajewsky , 1996 ) .", "In its monomeric form , the BCR is a 1:1 complex between the membrane-bound immunoglobulin ( mIg ) molecule and the Igα/Igβ-heterodimer ( Schamel and Reth , 2000; Tolar et al . , 2005 ) .", "The cytoplasmic tails of both Igα and Igβ carry an immunoreceptor tyrosine-based activation motif ( ITAM ) , the tyrosines of which are phosphorylated by Src family protein tyrosine kinases such as Lyn and the spleen tyrosine kinase ( Syk ) ( Schmitz et al . , 1996; Pao et al . , 1998 ) .", "However , the two kinases interact with the BCR in different ways .", "In contrast to Lyn , which predominantly phosphorylates only the first ITAM tyrosine , Syk phosphorylates both tyrosines and subsequently binds the double-phosphorylated ITAM ( ppITAM ) via its tandem SH2 domains ( Futterer et al . , 1998 ) .", "The binding of Syk to a ppITAM sequence results in the rapid phosphorylation of ITAM tyrosines of neighboring receptors .", "The resulting increased Syk recruitment generates a positive feedback that amplifies signal transduction from the BCR ( Rolli et al . , 2002; Mukherjee et al . , 2013 ) .", "A PLA study showed that Syk interacts only with the activated but not with the resting BCR ( Infantino et al . , 2010 ) .", "How the ITAM sequence is shielded from the action of this kinase in resting B cells is not known but may involve cytoskeletal elements .", "Indeed , B cells can be activated not only by exposure to antigen but also to the F-actin inhibitor Latrunculin A ( Lat-A ) , suggesting that the actin cytoskeleton protects the resting BCR ( Treanor et al . , 2010 ) .", "The cytoskeleton is also limiting the free diffusion of receptors ( Kusumi et al . , 2005 ) .", "In line with this , B cell activation is accompanied by cytoskeleton remodeling and increased BCR mobility .", "Furthermore , it has been shown that BCR mobility is reduced in membrane areas enriched in ezrin-radixin-moesin ( ERM ) proteins ( Harwood and Batista , 2011 ) .", "However , and complicating matters further , the cytoskeleton not only plays an inhibitory , but also an activating role during B cell activation , as it is required for microcluster formation and receptor internalization ( Brown and Song , 2001; Cheng et al . , 2001 ) .", "Mature B lymphocytes coexpress an IgM-BCR and an IgD-BCR on their surface , the latter being the dominant receptors on these cells .", "These two receptors have distinct signaling functions and diffusion behaviors on the B cell surface but the molecular basis for these differences is poorly understood ( Kim and Reth , 1995; Treanor et al . , 2010 ) .", "The cross-linking model ( CLM ) assumes that most of the 100 , 000 BCR complexes on the surface of a B cell are monomeric and that cross-linking of two BCR monomers by a polyvalent antigen initiates the signaling process ( Metzger , 1992 ) .", "Similarly , the conformation-induced oligomerization model ( CIOM ) proposes that antigen binding induces a conformational change of the monomeric BCR , resulting in the dimerization of the membrane proximal CH domain of the BCR followed by receptor signaling ( Tolar and Pierce , 2010 ) .", "However , recent studies show that antigen receptors on resting T and B cells are not uniformly distributed but are rather organized in nanoclusters inside protein islands ( Lillemeier et al . , 2006 , 2010; Mattila et al . , 2013 ) .", "In earlier biochemical studies employing the blue-native polyacrylamide gel electrophoresis ( BN-PAGE ) technique , we provided the first evidence for the oligomeric organization of the BCR ( Schamel and Reth , 2000 ) .", "Furthermore , with a quantitative bifluorescence complementation ( BiFC ) assay , we found that the resting BCR forms stable dimers on the cell surface while a BCR mutant that cannot form dimers is hyperactive and cannot be stably expressed on the B cell surface ( Yang and Reth , 2010b ) .", "We thus have proposed the dissociation-activation model ( DAM ) whereby BCR activation is initiated by the dissociation of signaling-inert BCR oligomers ( Yang and Reth , 2010a ) .", "We here explore the nanoscale organization of the BCR at a 10–20 nm resolution with a modified version of PLA .", "With this technique , we have found direct evidence for the opening of BCR dimers during B cell activation and show that the kinase Syk plays an essential role in this process ." ], [ "BCR monomers or dimers have sizes in the 10–20 nm range and thus , these forms cannot be distinguished by live cell imaging techniques with a diffraction barrier of 250 nm ( Huang et al . , 2010 ) .", "To analyze the BCR conformation in the nanoscale range , we employ different versions of the PLA method using DNA-oligo-coupled antibodies , a rolling circle amplification and detection by fluorescence-coupled oligonucleotides ( Soderberg et al . , 2008 ) .", "The classical PLA method ( 2-PLA ) involves secondary antibodies and detects the proximity of two molecules in the 10–80 nm range .", "By coupling the DNA oligos directly to primary antibodies ( 1-PLA ) , or to Fab fragments ( Fab-PLA ) , we narrowed the detection range of this assay down to 10–40 nm and 10–20 nm , respectively ( Figure 1A–C ) .", "We then used these three PLA assays , and a monoclonal antibody directed against the constant part of IgM , to analyze the BCR organization on resting or activated TKO-MD B cells stimulated for 5 min with antigen ( Figure 1D–F ) .", "TKO-MD is a pro-B cell line derived from RAG , Lambda5 , SLP-65 triple-deficient mouse bone marrow which is transfected to express an IgM- and IgD-BCR specific for the hapten 4-hydroxy-5-iodo-3-nitrophenyl acetyl ( NIP ) ( Meixlsperger et al . , 2007 ) .", "As indicated by the numbers of fluorescent dots , the 2-PLA and 1-PLA are more sensitive than the Fab-PLA , a phenomenon that is explained by the avidity associated with divalent antibody binding .", "However , only the Fab-PLA showed a clear difference in the relative IgM:IgM conformation or distance between resting and activated B cells ( Figure 1F ) .", "Importantly , we are using non-permeabilized , fixed B cells in these assays .", "This allows us to analyze the organization of molecules specifically on the cell surface without detecting intracellular complexes of these molecules . 10 . 7554/eLife . 02069 . 003Figure 1 . Nanoscale analysis of the IgM:IgM BCR proximity on the surface of TKO-MD B cells by PLA of different settings .", "( A–C )", "Scheme of the three PLA settings .", "Plus and minus oligos for annealing and rolling circle amplification were coupled to either ( A ) secondary antibodies ( 2-PLA ) , ( B ) primary antibodies ( 1-PLA ) or ( C ) Fab fragments ( Fab-PLA ) .", "The theoretical detection ranges are noted under the double-heads-arrows for each setting .", "( D–F )", "Representative confocal microscopic images of IgM:IgM PLA of resting ( left ) and antigen ( NIP-BSA ) activated ( right ) TKO-MD cells by ( D ) 2-PLA , ( E ) 1-PLA or ( F ) Fab-PLA .", "Nuclei were stained with DAPI and presented as blue signals while the PLA signals are presented as red color dots .", "Scale bar: 5 μm .", "( G ) Quantification of the results of IgM:IgM BCR proximity for resting ( R ) and NIP-BSA-stimulated ( S ) TKO-MD cells analyzed by 2-PLA , 1-PLA and Fab-PLA .", "For each experiment , the PLA signals ( counts per cell ) of each sample were counted from a minimum of 50 cells and then normalized to the PLA signal of the resting cells with Fab-PLA ( set to 100% ) .", "Data represent the mean and SEM of five independent experiments .", "Significant difference between samples is highlighted by stars .", "( H and I )", "Representative confocal microscopic images ( H ) and quantified results ( I ) of IgM:IgM Fab-PLA of resting and NIP-BSA stimulated TKO-MD cells using different concentrations of Fab reagents .", "Data represent the mean and SEM of a minimum of three independent experiments .", "For each experiment , the PLA signals ( counts per cell ) of each sample were counted from a minimum of 1000 cells and were then normalized to the PLA signal of the resting B cells probed with the 1/100 Fab dilution .", "( J ) Schematic drawing showing the spatial organization of the BCR as monitored by Fab-PLA on resting and stimulated B cells ( left and middle panel ) or 1-PLA on stimulated B cells ( right panel ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 02069 . 003 The PLA data were quantified by the BlobFinder software ( Allalou and Wahlby , 2009 ) and showed that the Fab-PLA differences between resting and activated B cells are statistically significant ( Figure 1G ) .", "PLA is a sampling method that , by measuring a few interactions per cell , allows conclusions to be drawn about the behavior of a larger pool of molecules .", "If we increase the Fab concentration , we can measure many more IgM:IgM interactions on resting but not on activated B cells ( Figure 1H , I ) .", "However , at these concentrations , the signals ( red blobs ) are often overlapping , making it more difficult to quantify .", "We therefore use a concentration of the oligo-labeled Fab fragment that detects 5–10 interactions on each cell and collect data from hundreds of B cells in each experiment .", "All experiments are then repeated 3–5 times .", "The IgM–IgM PLA studies suggests that , upon B cell activation , mIgM molecules move further than 20 nm apart from each other so that their proximity can be still detected by 1-PLA but no longer by Fab-PLA ( Figure 1J ) .", "To provide further proof that the Fab-PLA method is able to distinguish oligomeric from monomeric IgM , we studied different forms of soluble IgM bound to NIP-coupled latex beads ( Figure 2A ) .", "For this we purified monomeric and pentameric IgM from the supernatant of the anti-NIP specific hybridoma cell line B1-8 ( Figure 2B ) .", "NIP-coupled latex beads were exposed to low amounts of either monomeric or pentameric IgM and equal IgM loading was determined by FACScan using anti-µ fluorescent antibodies ( Figure 2C , lower left panel ) .", "The beads were then subjected to Fab-PLA and 1-PLA .", "The FACScan analysis of these beads shows that Fab-PLA detects the pentameric IgM but not the loosely spaced monomeric IgM whereas 1-PLA detects both forms of IgM equally well ( Figure 2C , compare upper and lower panels ) .", "This result clearly shows that the Fab-PLA specifically detects oligomeric forms of IgM be it the soluble IgM pentamer or the oligomeric IgM-BCR on the B cell surface .", "In a recent structural study of pentameric IgM , it was shown that the constant domains of IgM are no further apart from each other than 5–6 nm ( Czajkowsky and Shao , 2009 ) , a distance that can be easily bridged by the anti-IgM Fabs used for the Fab-PLA method . 10 . 7554/eLife . 02069 . 004Figure 2 . Fab-PLA but not 1-PLA is able to distinguish the oligomeric and monomeric BCR .", "( A ) Scheme of the experiment setting .", "Latex beads coupled with low density of NIP-BSA were loaded with either the monomer or pentamer form of NIP-specific IgM antibodies .", "Due to the theoretical difference in their detection range ( Figure 1J ) , 1-PLA is expected to obtain positive signals for beads loaded with either the monomeric or the pentameric IgM , while Fab-PLA is expected to show positive only for beads bound with pentameric IgM .", "( B ) The size and purity of monomeric and pentameric IgM preparations were verified by size exclusion chromatography .", "( C ) Comparison of beads loaded with monomeric ( left ) or pentameric ( right ) IgM measured by FACS after Fab-PLA ( up ) or 1-PLA ( low ) assay .", "Results were gated for the beads based on SSC and FSC .", "MFI of the PLA signals for the beads are marked on the upper right corner of each plot .", "The amount of IgM bound to the beads were monitored by anti-µ staining and shown at the lower right corner .", "Data are representative of three independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 02069 . 00410 . 7554/eLife . 02069 . 005Figure 2—figure supplement 1 . Similar binding efficiency of Fab-PLA probes on resting and stimulated B cells .", "( A ) Schematic drawing of Fab-PLA probes and control probes used for the binding assay .", "( B ) FACScan analysis of anti-IgM Fab-PLA plus ( upper left ) and minus ( lower left ) , anti-IgD Fab-PLA plus ( upper middle ) and minus ( lower middle ) , anti-CD19 Fab-PLA plus ( upper right ) and anti-CD20 Fab-PLA minus ( lower right ) probes binding on resting or differently ( NIP-BSA , Pervanadate , Latrunculin-A ) , stimulated B1-8 B cells .", "( C–F )", "Nanoscale proximity of heavy chain ( HC ) and light chain ( LC ) examined by Fab-PLA remained unaltered in TKO ( C and D ) and B1-8 B cells ( E and F ) upon activation with different stimuli .", "Representative microscopic images ( left ) and quantified results ( right ) show the proximity of ( C and E ) μm HC or ( D and F ) δm HC to λ LC of resting or activated B cells stimulated for 5 min with the antigen NIP-BSA , the oxidant pervanadate ( Perv ) and the F-actin inhibitor Lat-A .", "Scale bar: 5 μm .", "Data represent the mean and SEM of four independent experiments .", "For each experiment , PLA signals ( counts per cell ) of each sample were counted from a minimum of 1000 cells and were then normalized to the PLA signal of the resting B cells . DOI: http://dx . doi . org/10 . 7554/eLife . 02069 . 005 To exclude that the loss of the PLA signal in activated B cells is due to a loss of binding of the Fab fragment rather than the oligomer to monomer transition we measured the binding of all our Fab reagents by flow cytometry and found no alteration of binding between resting and activated B cells ( Figure 2—figure supplement 1 ) .", "In addition we used the same anti-Ig Fabs to monitore the heavy ( H ) and light ( L ) chain interaction by H:L Fab-PLA and found , as expected , no alteration upon B cell activation .", "In contrast to the H:L , the IgM:IgM and IgD:IgD ( see below ) Fab-PLA signal is consistently lost in activated B cells suggesting that the later assays detect dynamics states of a protein complex and we think that this is the oligomer to monomer transition of the BCR .", "In case of the IgD-BCR , we found direct evidence for this notion , because the IgD:IgD Fab-PLA detects the wild-type receptor but barely detects a mutant IgD-BCR that is defective in oligomer formation ( Figure 3 ) .", "For the latter assay , we used transfected S2 cells because unlike B cells they allow the equal expression of wild-type and monomeric mutant IgD-BCR ( Yang and Reth , 2010b ) . 10 . 7554/eLife . 02069 . 006Figure 3 . Fab-PLA detects IgD-BCR oligomers on the S2 cell surface .", "( A ) Schematic drawing of wild type and double-mutant IgD-BCRs ( δm transmembrane mutations and removal of the S–S bridge of the Igα/Igβ heterodimer ) and their expression on transfected S2 cells analyzed by FACScan with a fluorescent 1NIP-peptide .", "Positively transfected S2 cells are indicated by the expression of a cotransfected GFP vector .", "The double-positive ( GFP+ , BCR+ ) S2 cell population , indicated by red square , were sorted and used for Fab-PLA .", "( B ) Confocal microscopy analysis of the IgD:IgD Fab-PLA reaction of GFP+ S2 cells expressing wild type ( left panel ) or double-mutant ( right panel ) IgD-BCR .", "PLA signals are shown as red dots and nuclei were visualized by DAPI staining ( blue ) .", "Scale bar: 5 μm .", "( C ) Quantification of IgD:IgD Fab-PLA ( each dot represents the amounts of Fab-PLA signals per S2 cell ) .", "The data were analyzed by the mann-whitney test and the median values are shown as red line . DOI: http://dx . doi . org/10 . 7554/eLife . 02069 . 006 We next used the Fab-PLA method to further analyze the IgM:IgM , and the IgD:IgD proximity on the surface of resting or activated spleen cells from the B1-8 mouse , whose B cells express a NIP-specific BCR ( Sonoda et al . , 1997 ) .", "For this , we stimulated the B cells for 5 min with the antigen NIP-BSA ( Figure 4A ) .", "The quantification of the Fab-PLA analysis of B1-8 ( Figure 4A , right panel ) and TKO-MD ( Figure 4B ) B cells shows that in each case , the IgM:IgM and IgD:IgD Fab-PLA signal is lost upon exposure of B cells to antigen .", "The Fab-PLA signal is also lost when we stimulated B1-8 B cells apart from antigen with BCR-activating drugs such as pervanadate ( Perv ) and Lat-A ( Figure 4C ) , which alter the phosphorylation/dephosphorylation equilibrium and inhibit actin polymerization , respectively ( Secrist et al . , 1993; Treanor et al . , 2010 ) .", "The same results were also obtained by an analysis of human peripheral blood B cells ( Figure 4D ) .", "Together , these studies suggest that B cell activation is accompanied by the opening of tightly packed BCR oligomers as predicted by the DAM hypothesis ( Yang and Reth , 2010a , 2010b ) . 10 . 7554/eLife . 02069 . 007Figure 4 . Fab-PLA study of the nanoscale organization of the IgM-BCR and the IgD-BCR on resting and activated B cells .", "( A ) The IgM:IgM ( upper ) and IgD:IgD ( lower ) proximity of antigen receptors on resting or activated B1-8 splenic B cells were examined by Fab-PLA and shown as representative microscopic images ( left ) and quantified results ( right ) .", "( B–D )", "Quantified Fab-PLA results indicate the IgM:IgM ( upper ) and IgD:IgD ( lower ) proximity on resting or activated TKO-MD cells ( B ) , B1-8 splenic B cells ( C ) and human IgM+IgD+ naïve B cells isolated from peripheral blood ( D ) .", "Scale bar: 5 μm .", "Quantified data represent the mean and SEM of a minimum of four independent experiments .", "For each experiment , PLA signals ( counts per cell ) of each sample were counted from a minimum of 100 cells and were then normalized to the PLA signals of the resting cells . DOI: http://dx . doi . org/10 . 7554/eLife . 02069 . 007 The above analysis shows that BCR opening does not always require the direct engagement of the receptor but can also be mediated by drugs such as Lat-A and pervanadate which alter the cytoskeleton or kinase/phosphatase equilibrium , respectively .", "These treatments also result in strong ITAM phosphorylation .", "We thus wondered whether the detected BCR opening is associated with the Syk/ITAM signal amplification process we have described previously ( Rolli et al . , 2002 ) and that was recently confirmed by another study ( Mukherjee et al . , 2013 ) .", "To test this , we pretreated splenic B cells with the Src-family kinase inhibitor PP2 , or the Syk inhibitor R406 , prior to their activation with Lat-A ( Braselmann et al . , 2006 ) .", "While the PP2-treatment only delays the opening of the IgM-BCR oligomer , this process no longer occurs in the presence of the Syk inhibitor R406 ( Figure 5A , B ) .", "The dependence on Syk activity was also found for B cells stimulated with the antigen NIP-BSA ( Figure 5—figure supplement 1 ) .", "To exclude that the block in BCR opening by PP2 or R406 is not due to off-target effects of these inhibitors , we analyzed the BCR conformation in Lyn- or Syk-deficient splenic B cells before and after their activation with Lat-A ( Figure 5C ) .", "The IgM:IgM Fab-PLA results of these genetically altered B cells were similar to those previously observed using kinase inhibitors and suggests that Syk not only signals downstream but also alters the BCR conformation via an inside-out signaling process . 10 . 7554/eLife . 02069 . 008Figure 5 . Syk activity is required for an efficient IgM-BCR dissociation after B cell activation . Representative microscopic images of the IgM:IgM ( A ) and the Igα:Syk ( D ) proximity detected by Fab-PLA .", "B1-8 B cells were analyzed at different time points ( 0 , 1 , 5 , 10 min ) after their activation with Lat-A in the absence ( upper panels ) or presence of the src-family kinase inhibitor PP2 ( middle panels ) or the Syk inhibitor R406 ( bottom panels ) .", "Scale bar: 5 μm .", "Quantification of the IgM:IgM ( B ) and the Igα:Syk ( E ) Fab-PLA plotted as mean and SEM of a minimum of three independent experiments .", "For each experiment , PLA signals ( counts per cell ) of each sample were counted from a minimum of 500 cells and were then normalized to the PLA signals of resting untreated B1-8 B cells .", "Quantification of the IgM:IgM ( C ) and the Igα:Syk ( F ) proximity detected by Fab-PLA .", "B cells isolated from spleens of wild type , Lyn-KO and inducible , B cell-specific Syk-KO mice were analyzed at different time points ( 0 , 1 , 5 , 10 min ) after their activation with Lat-A .", "For each experiment , PLA signals ( counts per cell ) of each sample were counted from a minimum of 500 cells and were then normalized to the PLA signals of resting untreated wild type B cells .", "Data represent the mean and SEM of a minimum of three independent experiments .", "( G ) , Quantification of the IgM:IgM Fab-PLA of TKO-M cells stimulated for 5 min with the indicated amounts ( 2 , 10 , 50 , 100 , 250 , 10000 ng/ml ) of the antigen NIP-BSA at different times ( 0 , 10 , 30 , 60 min ) after the pretreatment of the B cells with the Syk inhibitor R406 .", "PLA signals ( signal counts ) of each sample were counted from a minimum of 500 cells .", "Data are representative of three independent experiments .", "( H ) Schematic drawing showing the spreading and amplification of the BCR/Syk signal by an outside-in and inside-out signaling mechanism .", "( 1 ) Formation of the BCR/Syk seed complex by antigen-engaged BCRs; ( 2 ) ITAM phosphorylation of neighboring ( unengaged ) BCR complexes; ( 3 ) Binding of Syk to the phosphorylated ITAM opens the receptor from the inside ( inside-out signaling ) and results in further signal spreading . DOI: http://dx . doi . org/10 . 7554/eLife . 02069 . 00810 . 7554/eLife . 02069 . 009Figure 5—figure supplement 1 . Syk activity is required for an efficient IgM-BCR dissociation on antigen stimulated B cells . Representative microscopic images ( A ) and quantification ( B ) of the IgM:IgM proximity detected by Fab-PLA .", "B1-8 B cells were analyzed at different time points ( 0 , 1 , 5 , 10 min ) after their stimulation with the antigen NIP-BSA in the absence ( upper panels , black line ) or presence of the Syk inhibitor R406 ( bottom panels , red line ) .", "Scale bar: 5 μm .", "The Fab-PLA was quantified and plotted as mean and SEM of a minimum of three independent experiments .", "For each experiment , PLA signals ( counts per cell ) of each sample were counted from a minimum of 500 cells and were then normalized to the PLA signals of resting untreated B1-8 B cells . DOI: http://dx . doi . org/10 . 7554/eLife . 02069 . 009 An inside-out signaling mechanism has previously been described for integrins such as LFA-1 , where the binding of talin to the intracellular tails changes the outside conformation and binding behavior of this receptor ( Wang , 2012 ) .", "Accordingly , it may be that Syk binding to the phosphorylated ITAM tyrosines induces receptor opening .", "We therefore used Igα:Syk Fab-PLA to analyze whether or not the kinase inhibitors PP2 and R406 influence the binding of Syk to the BCR in Lat-A-stimulated B cells .", "For this study , the B cells were fixed and permeabilized to allow probing of the inner surface of the plasma membrane .", "This analysis shows that the BCR/Syk interaction is induced within 1 min of Lat-A treatment ( Figure 5D , E ) .", "This interaction is delayed by PP2 whereas the Syk inhibitor R406 completely blocks the recruitment of Syk to the BCR .", "The inside-out signaling function of Syk is thus likely to be mediated not only by its kinase activity , but also by the binding of Syk to the ITAM sequences of the BCR .", "To again exclude off-target effects of inhibitors , we studied the ITAM/Syk association also in Lyn- and Syk-deficient splenic B cells before or after their activation with Lat-A ( Figure 5F ) .", "Note that the ITAM/Syk interaction is delayed but not prevented in Lyn-deficient B cells , whereas the Igα:Syk Fab-PLA in Syk-deficient B cells gave no signal due to the absence of Syk .", "The Syk/BCR inside-out signaling process , described here for the first time , may allow B cells to become fully active when exposed to low amounts of antigens .", "If this were true , one would predict that under conditions of Syk inhibition , B cells would require more antigen for the opening of the many IgM-BCR oligomer complexes on their cell surface .", "To test for this , we exposed TKO-M cells for different times to the Syk inhibitor R406 ( 5 μM ) and then stimulated these B cells for 5 min with increasing doses of antigen ( Figure 5G ) .", "Note that in the cytosol there exists an equilibrium between a closed autoinhibited and an open active form of Syk .", "As the inhibitor is interacting only with the latter form , it takes some time for the majority of Syk molecules in the cell to be blocked by the inhibitor .", "Syk-sufficient cells require 10 ng/ml of antigen to lose the IgM:IgM Fab-PLA signal whereas B cell exposed for 60 min to the Syk inhibitor lose the IgM:IgM Fab-PLA signal only at 10–50 times higher ( 250–500 ng/ml ) antigen doses .", "This titration experiment supports the notion that the Syk/BCR inside-out signaling increases the sensitivity of B cells to low doses of antigen .", "B cell activation may thus involve two distinct phases ( Figure 5H ) .", "First an outside-in signal where limited amounts of antigens open a few BCR dimers , allowing them to form active BCR/Syk seed complexes that then diffuse in the membrane and phosphorylate neighboring BCR oligomers .", "This process then results in more Syk recruitment , BCR opening and the amplification of the BCR signal via an inside-out signaling mechanism .", "To show the influence of Syk on the BCR conformation more directly , we performed a gain-of-function experiment in the S2 Schneider cell system , which allowed us to express the BCR , either alone or together with its signaling components ( Yang and Reth , 2012 ) .", "On IgM-BCR-expressing S2 cells , we detect receptor dimers by IgM:IgM Fab-PLA ( Figure 6A , B ) .", "The IgM:IgM Fab-PLA signal is drastically reduced on S2 cells co-expressing the IgM-BCR together with a GFP-Syk fusion protein ( Figure 6A , second panel and Figure 6B , second bar ) .", "The opening of the BCR by Syk could be either due to the phosphorylation of the ITAM tyrosines or to the binding of the tandem SH2 domains to the ppITAM sequences .", "To distinguish between these possibilities , we separated the enzymatic and binding domain of Syk from each other .", "For this , we expressed in S2 cells the chimeric Syk protein nLyn-Syk-KD consisting of the N-terminal myristoylation anchor of Lyn and the isolated Syk kinase domain either alone or in combination with a GFP- ( SH2 ) 2 construct containing the tandem SH2 domains of Syk .", "S2 cells expressing the IgM-BCR only together with nLyn-Syk-KD show tyrosine phosphorylation of Igα ( data not shown ) but no loss of the IgM:IgM Fab-PLA signal ( Figure 6A , third panel and Figure 6B , third bar ) .", "However , the oligomeric IgM-BCR opened when we co-expressed nLyn-Syk-KD together with either the GFP- ( SH2 ) 2 or the kinase negative GFP-Syk-K- constructs that alone did not alter the IgM-BCR conformation ( compare relevant panels and bars in Figure 6A , B ) .", "Together these data indicate that it is not the phosphorylation of the ITAM tyrosines but rather the binding of the Syk to the receptor that results in the opening of BCR oligomers ( Figure 6C ) . 10 . 7554/eLife . 02069 . 010Figure 6 . SH2 domains of Syk bind to phosphorylated ITAM and open the BCR . Representative microscopic images ( A ) and quantified results ( B ) show the IgM:IgM proximity of S2 cells expressing IgM-BCR together with the indicated constructs .", "GFP is shown as green , nuclei are stained with DAPI and shown as blue signals .", "The PLA signal is indicated by the red color .", "Scale bar: 5 µm .", "For the quantification , PLA signals ( signal counts ) of each sample were counted from a minimum of 500 cells .", "( C ) Schematic drawing showing that not the ITAM phosphorylation tyrosines but the binding of the two tandem SH2 domains of Syk to the phosphorylated ITAM tyrosines is opening the BCR . DOI: http://dx . doi . org/10 . 7554/eLife . 02069 . 010 Signaling from the BCR is controlled and further amplified by BCR co-receptors , most prominently by the CD19 molecule ( Sato et al . , 1995; Fujimoto et al . , 2000 ) .", "CD19-deficient B cells have a compromised immune response to antigen and are defective in BCR microcluster formation ( Rickert et al . , 1995; Depoil et al . , 2008 ) .", "We therefore studied the IgM:CD19 and IgD:CD19 organization on resting and activated TKO-MD B cells by Fab-PLA ( Figure 7A , B ) .", "This analysis shows that CD19 is only found in close association with the activated but not with the resting IgM-BCR ( Figure 7A ) .", "Interestingly , in this assay , the results for the IgD-BCR were opposite to those observed for IgM .", "CD19 is found together with the resting IgD-BCR and dissociates from this receptor upon BCR activation ( Figure 7B ) .", "The quantification of these data shows , for the first time , that the CD19:BCR interaction is Ig class specific and remodeled upon B cell activation ( Figure 7A , B , right panel ) .", "Compared to CD19 , less is known about the function of CD20 on the B cell surface but this molecule plays an important role as the target of the therapeutic antibody rituximab ( Korhonen and Moilanen , 2010 ) .", "By IgM:CD20 and IgD:CD20 Fab-PLA , we found that CD20 displays the same class-specific BCR interaction as CD19 ( Figure 7A , B ) .", "The BCR:co-receptor analysis described above for TKO-MD cells was repeated with human blood ( Figure 7C , D ) and murine splenic B1-8 ( Figure 7E , F ) B cells and showed the same class-specific co-receptor association . 10 . 7554/eLife . 02069 . 011Figure 7 . Fab-PLA of the nanoscale proximities of the IgM-BCR and IgD-BCR to the coreceptor molecules CD19/CD20 and the GM-1 Ganglioside . Representative microscopic images ( left ) and quantified results ( right ) of the proximity of CD19 or CD20 to the IgM-BCR ( A , C , E ) or the IgD-BCR ( B , D , F ) on resting or activated ( A and B ) TKO-MD , ( C and D ) human B cells and ( E and F ) B1-8 B cells stimulated for 5 min with NIP-BSA .", "( G and H )", "The proximity of the GM-1 Ganglioside ( detected by CTB ) to ( G ) the IgM-BCR or ( H ) the IgD-BCR on resting or activated B1-8 B cells stimulated for 5 min with the antigen NIP-BSA , the oxidant pervanadate and the F-actin inhibitor Lat-A , are shown as representative microscopic images ( left ) and quantified results ( right ) .", "Scale bar: 5 μm .", "For the quantified results , data represent the mean and SEM of a minimum of three independent experiments .", "For each experiment , PLA signals ( counts per cell ) of each sample were counted from a minimum of 250 cells and were then normalized to the PLA signals of either ( in A , C , E ) the activated , or ( in B , D , F , H ) the resting B cells , or in ( G ) the Lat-A activated cells . DOI: http://dx . doi . org/10 . 7554/eLife . 02069 . 01110 . 7554/eLife . 02069 . 012Figure 7—figure supplement 1 . Nanoscale IgM-BCR and IgD-BCR dissociation and coreceptor reorganization on B cells expressing only one BCR isotype .", "( A–C )", "Representative microscopic images ( left ) and quantified results ( right ) of the IgM:IgM ( A ) , the IgM:CD19 ( B ) , and the IgM:CD20 ( C ) Fab-PLA of resting or activated TKO-M cells stimulated for 5 min with the antigen NIP-BSA , the oxidant pervanadate and the F-actin inhibitor Lat-A .", "( D–F )", "Representative microscopic images ( left ) and quantified results ( right ) of the IgD:IgD ( D ) , the IgD:CD19 ( E ) , and the IgD:CD20 ( F ) Fab-PLA of resting or activated TKO-D cells stimulated for 5 min with the antigen NIP-BSA , the oxidant pervanadate and the F-actin inhibitor Lat-A .", "Scale bar: 5 μm .", "The quantified data represent the mean and SEM of a minimum of three independent experiments .", "For each experiment , PLA signals ( counts per cell ) of each sample were counted from a minimum of 1000 cells and were then normalized to the PLA signal of the resting ( A , D , E , F ) or NIP-BSA stimulated ( B and C ) cells . DOI: http://dx . doi . org/10 . 7554/eLife . 02069 . 01210 . 7554/eLife . 02069 . 013Figure 7—figure supplement 2 . Fab-PLA of the nanoscale proximities of the IgM-BCR and IgD-BCR to the lipid raft marker CD52 . ( A ) Representative microscopic images of the IgM:CD52 Fab-PLA on resting ( upper ) or NIP-BSA stimulated ( lower ) TKO-M cells ( left ) or the IgD:CD52 Fab-PLA on resting ( upper ) or NIP-BSA stimulated ( lower ) TKO-D cells ( right ) .", "Scale bar: 5 μm .", "( B ) Quantified results of IgM:CD52 ( left ) and IgD:CD52 ( right ) Fab-PLA of resting and stimulated TKO-M or TKO-D cells .", "Data represent the mean and SEM of a minimum of three independent experiments .", "For each experiment , PLA signals ( counts per cell ) of each sample were counted from a minimum of 1000 cells and were then normalized to the PLA signal of the resting ( IgD:CD52 ) or NIP-BSA stimulated ( IgM:CD52 ) cells . DOI: http://dx . doi . org/10 . 7554/eLife . 02069 . 013 BCR opening , as well as the reorganization of co-receptors , does not require the co-expression of the IgM and IgD on the cell surface .", "TKO-M and TKO-D cells that only express one class of BCR behave similarly to the TKO-MD cells ( Figure 7—figure supplement 1 ) .", "Resting TKO-D cells display a close proximity between the IgD-BCR and the CD19/CD20 module but lose this interaction upon BCR stimulation , while the IgM-BCR gains contact to the CD19/CD20 module only upon B cell activation .", "These results suggest that on resting B cells , the CD19/CD20 module resides in a membrane compartment that is also targeted by the IgD-BCR but that is not dependent on IgD expression .", "To learn more about the lipid composition of this compartment , we labeled the cholera toxin B-subunit ( CTB ) with one of the PLA oligos .", "CTB binds with high affinity to GM1 gangliosides , glycolipids that are highly expressed in lipid ordered domains ( Kenworthy et al . , 2000 ) .", "The IgD:CTB and IgM:CTB PLA showed that on resting B cells , GM1 gangliosides are found in close proximity to the IgD-BCR , whereas the IgM-BCR gains access to large amounts of these lipids only upon B cell activation ( Figure 7H , G ) .", "The GPI-linked protein CD52 ( Treumann et al . , 1995 ) showed a distribution by Fab-PLA similar to that seen for the GM1 gangliosides ( Figure 7—figure supplement 2 ) .", "Together , these studies suggest that the IgM-BCR and the IgD-BCR are localized on the B cell surface in different membrane areas that are reorganized upon B cell activation ." ], [ "The Fab-PLA method we have developed , makes it possible , for the first time , to explore the organization of receptors on the surface of normal B cells in the 10–20 nm range .", "Our analysis suggest that antigen-dependent B cell activation is accompanied by a transition from a tightly packed BCR oligomer to a more loosely spaced BCR-antigen or BCR-Syk clusters that can no longer be detected by Fab-PLA ( Figure 1J , Figure 5H ) .", "Our findings support the DAM hypothesis ( Yang and Reth , 2010a , 2010b ) , and in addition show for the first time that the kinase Syk is necessary and sufficient for the opening of the oligomeric BCR .", "Importantly , all these observations could only be made with Fab-PLA but not with 1-PLA or the classical 2-PLA techniques , indicating that these receptor reorganization processes occur at 10–20 nm distances .", "The reason why only Fab-PLA can reliably monitor the alterations of the BCR conformation may be related to the structure of this receptor .", "Like an antibody , the BCR contains flexible Fab arms and it maybe the extension of these arms that moves the BCR monomers apart from each other so that Fab-PLA can no longer detect their proximity ( Figure 1J ) .", "Another important advantage of Fab-PLA is that it does not require the overexpression of wild type or altered ( tagged ) proteins in cell lines but allows studying the nanoscale organization of unchanged proteins on primary cells .", "The Fab-PLA is a surprisingly robust technique that detects the same BCR alterations in B cell lines or normal B cells of either mouse or human origin , using three different stimulation protocols and different sets of Fab fragments ( Figure 4 ) .", "Previous live cell imaging studies of the behavior of the BCR on activated B lymphocytes have detected the formation of higher organized structures such as receptor microclusters and immunological synapses ( Treanor and Batista , 2007; Tolar et al . , 2008; Harwood and Batista , 2011 ) .", "These studies have suggested that receptor aggregation rather than dissociation is the earliest event in B cell activation .", "However , due to the diffraction barrier of visible light of 250 nm ( Huang et al . , 2010 ) , these live cell imaging studies could never directly monitor the conformation and nanoscale organization of the BCR on resting or activated B cells .", "In contrast to the expectations , several studies have now shown that antigen receptors on resting lymphocytes are not uniformly distributed monomers but rather pre-organized in nanoclusters or protein islands .", "The existence of these structures was first indicated by electron microscopy studies ( Wilson et al . , 2000 ) and recently confirmed by photo-activated localization microscopy ( PALM ) ( Lillemeier et al . , 2006 , 2010; Sherman et al . , 2011 ) and direct stochastic optical reconstruction microscopy ( dSTORM ) ( Mattila et al . , 2013; Owen et al . , 2013 ) .", "In contrast to the BCR which is predominantly oligomeric , the T cell antigen receptor ( TCR ) seems to exist in an equilibrium between monomeric and oligomeric forms ( Schamel et al . , 2005 ) .", "This may be the reason why TCR nanoclusters are more variable in size than the BCR nanoclusters ( Lillemeier et al . , 2010; Sherman et al . , 2011; Mattila et al . , 2013 ) .", "However , common to the two cell types is the finding that upon activation the nanoclusters can form microclusters or cap structures ( Lillemeier et al . , 2010 ) .", "Receptor dissociation and aggregation are thus not mutually exclusive events but rather processes that occur at different size levels and time points during B cell activation .", "Nanoclusters containing opened BCR presumably aggregate to form the microclusters previously detected by live cell imaging studies of activated lymphocytes .", "By activating the PI-3 kinase signaling module , the CD19 coreceptor , together with the B cell adaptor for PI3K ( BCAP ) , plays an important role as an amplifier of the BCR signal in activated B cells ( Aiba et al . , 2008 ) .", "Indeed , CD19-deficient mice are defective in B cell development and function ( Rickert et al . , 1995; Sato et al . , 1997 ) .", "It has been shown that CD19 and IgM co-cap on activated B cells ( Pesando et al . , 1989 ) and it thus was no surprise to find the two receptors in close proximity to each other on these cells .", "Remarkably , however , we found that on resting B cells , CD19 and CD20 are in close proximity to the IgD-BCR but not the IgM-BCR .", "The resting IgD-BCR seems to be also associated with lipid ordered domains , as indicated by the proximity of this receptor to GM1 gangliosides and the GPI-linked protein CD52 .", "The co-localization of the IgM-BCR with CD19 and lipid ordered domains is a hallmark of B cell activation but this is apparently not the case for the IgD-BCR on resting B lymphocytes .", "Thus , the IgD-BCR protein islands are likely to contain inhibitory factors which prevent signaling and we are currently searching for such molecules by Fab-PLA .", "These studies may help to elucidate the still enigmatic function of IgD on the B cell surface ( Geisberger et al . , 2006 ) .", "The finding that Syk is not only a downstream signaling element of the BCR , but directly involved in the opening of the oligomeric BCR , is one of the most surprising results of our nanoscale receptor studies .", "This discovery was only possible because Fab-PLA allows us , for the first time , to specifically monitor the oligomeric organization of the IgM molecule either in solution ( Figure 2 ) or on the B cell surface .", "Our results indicate that Syk opens the BCR by an inside-out-signaling mechanism and makes B cells highly sensitive to antigen engagement by a feed-forward amplification process .", "An inside-out-signaling mechanism has previously been found to play a role in the activation of CD28 by the TCR ( Sanchez-Lockhart et al . , 2011 ) and the regulation of integrin receptors such as LFA-1 ( Wang , 2012 ) .", "In the latter case it is the binding of talin to the cytosolic tail of the β-chain that displaces the α-tail from its complex with the β-tail , thus moving the two tails apart .", "This intracellular movement is translated into an extracellular conformational change that opens the integrin for ligand binding .", "Similarly , the binding of Syk to the phosphorylated ITAM sequences may distort the Igα/Igβ tails in such a way that it destabilizes the BCR oligomer , thus resulting in BCR opening and activation .", "In line with this scenario , we found that a mutation of the disulfide bridge between Igα and Igβ , in combination with a transmembrane mutation of mIgD , prevents the formation of the IgD-BCR oligomer ( Yang and Reth , 2010b ) .", "For antibody production to occur in infected or immunized animals , the antigen must bind to the B cell carrying the cognate BCR .", "With the inside-out Syk/BCR signaling process described here only a few antigen/BCR/Syk complexes can activate all antigen receptors on the B cell surface , thus leading to signal amplification and full B cell activation ( Figure 5H ) .", "This mechanism may allow B cells to become active even under limited antigen conditions .", "Our findings on the BCR/Syk signal amplification could be important for a better understanding of several human diseases .", "Recent data show that BCR components are frequently mutated in human leukemia ( Davis et al . , 2010 ) and that an autonomously signaling BCR can act as a tumor promoter for B cell chronic lymphocytic leukemia ( Duhren-von Minden et al . , 2012 ) .", "Clearly , it is important to learn more about the nanoscale BCR organization and its regulation in different disease settings ." ], [ "For FACS analysis of mouse spleen B cells or transfected TKO cells , following fluorophore- conjugated anti-mouse antibodies were used: Anti-CD45R-PerCP-Cy5 . 5 ( RA3-6B2 ) , Anti-IgM-APC , IgM-PE , IgM-FITC ( II/41; all eBioscience , Frankfurt , Germany ) , Anti-CD20-APC , Anti-CD19-PE-Cy7 , anti-IgD-AF647 , Anti-IgD-APC , Anti-IgD-PE ( 11-26c . 2a; all eBioscience ) .", "For PLA probes against specific targets , the following unlabelled antibodies were used: IgD ( 11-26c . 2a , SouthernBiotech , Birmingham , AL ) , IgD ( AMS9 . 1; Santa Cruz Biotechnology , Dallas , TX ) , IgM ( R33 . 24 . 12 , in house hybridoma culture ) , IgM ( rabbit anti-mouse µHC; Rockland Immunochemicals , Gilbertsville , PA ) , IgM ( 1B4B1; SouthernBiotech ) , Lambda light chain ( JC5-1; SouthernBiotech ) , Kappa light chain ( 187 . 1; SouthernBiotech ) , CD19 ( 6D5; AbD Serotec , Düsseldorf , Germany ) and CD20 ( AISB12; eBioscience ) .", "Igα ( HMK7/A9; abcam , Cambridge , UK ) , Syk ( Syk-01; BioLegend , San Diego , CA ) .", "For PLA probes against human BCR , the following unlabelled antibodies were used: IgD ( IA6-2; BioLegend ) , IgD ( IADB6 ) and IgM ( SA-DA4 ) from Acris Antibodies ( Herford , Germany ) , and IgM ( Fc5u ) from Genway Biotech ( San Diego , CA ) .", "The triple deficient pro B cell line ( Rag2−/− , λ5−/− , SLP65−/− ) ( TKO , Meixlsperger et al . , 2007 ) is a kind gift of Hassan Jumaa .", "To generate the TKO-MD cells , TKO cells were retrovirally co-transfected with vectors encoding the λ1 light chain , B1-8 μm and δm HC carrying selection markers for puromycin and complemented YFP ( Köhler et al . , 2008; Infantino et al . , 2010 ) .", "The resulting cells were stained with 1NIP-peptide-DyLight649 ( custom synthesized from Biosyntan GmbH , Berlin , Germany ) for surface NIP-specific BCR and sorted for cYFP+ and Dylight649+ on BD ( San Jose , CA ) Influx FACS sorter .", "To transfect TKO cells , supernatants containing viral particles were collected from transfected Phoenix retrovirus packaging cells 2 days after plasmid transfection and used as described before ( Duhren-von Minden et al . , 2012 ) .", "Both the TKO cells and the TKO-MD cells were cultured in Iscove's medium ( Biochrom , Berlin , Germany ) supplemented with 10 mM L-glutamine , 100 unit/ml penicillin/streptomycin ( all Gibco , Life Technologies , Darmstadt , Germany ) , 50 μM β-mercaptoethanol ( Sigma-Aldrich , Munich , Germany ) , 10% FCS ( PAN Biotech , Aidenbach , Germany ) and supernatant of cultured J558L mouse plasmacytoma cells stably transfected with a murine IL-7 expression vector .", "Total spleen cells were isolated from 8–12 week old B1-8 transgenic mice harboring NIP-specific B cells .", "Naϊve B cells were enriched by MACS depletion method using anti-CD43 magnetic beads ( Miltyeni Biotech , Bergisch Gladbach , Germany ) according to manufacturer's protocol .", "Before their stimulation , the enriched B cells were rested overnight in complete Iscove's medium as described above and their activation status was monitored by flow cytometry analysis after staining with an anti-CD86 antibody .", "Total PBMCs ( peripheral blood mononuclear cells ) were prepared from freshly withdrawn anti-coagulated peripheral blood about 15–20 ml from a healthy donor using Leucosep 50 tube ( Greiner Bio One , Frickenhausen , Germany ) and Pancoll , 1 . 077 g/l ( PAN Biotech ) according to manufacturers' instruction .", "PBMC washed with PBS and naïve B cells fraction was enriched by MACS depletion method using Naive B cell isolation kit II ( Miltyeni Biotech ) .", "To verify the purity and identify subpopulations such as immature , plasma and memory B cells , isolated fractions were analyzed in FACScan using different B cell surface markers that includes IgD , IgM , CD10 , CD19 , CD20 , CD27 , CD43 .", "Thereafter these B cells , prior to PLA experiment , were cultured at 37°C in 5% CO2 , in RPMI 1640 media supplemented with 25 mM Hepes , and 10 units/ml penicillin/streptomycin ( all from Invitrogen , Life Technologies , Karlsruhe , Germany ) and 2% of filter sterilized self plasma ( separated during PBMC preparation ) for 8–12 hr to rescue from the stress occurred during isolation .", "To activate the BCR , cells were treated with 1 μM Latrunculin A ( Invitrogen ) , 0 . 5 mM of pervanadate , or 40 ng/ml NIP-BSA ( Biosearch , Petaluma , CA ) for the indicated times .", "Pervanadate was freshly prepared for each experiment with equal molar amounts of orthovanadate and H2O2 .", "For kinase inhibition , cells were pre-treated with 5 μM Syk inhibitor R406 ( Selleckchem , Houston , TX ) or 10 μM Lyn inhibitor PP2 ( Sigma-Aldrich ) for 60 min before the stimulation .", "Fab fragments were prepared from the corresponding antibodies with Pierce-Fab-Micro preparation kit ( Thermo Fisher Scientific , Bonn , Germany ) using immobilized Papain or Ficin according to manufacturer's protocol .", "After desalting ( Zeba spin desalting columns , Thermo Fisher Scientific ) , the resulted Fab-fragments were coupled with PLA-probemaker plus or minus oligonucleotides according to the manufacturer's instructions ( Olink Bioscience , Uppsala , Sweden ) to generate Fab-PLA probes .", "For in situ PLA , cells were settled on PTFE-slides ( Thermo Fisher Scientific ) for 30 min at 37°C .", "Resting and activated cells were fixed for 15 min with 2% paraformaldehyde containing 0 . 02% glutaraldehyde in PBS for faster crosslinking at room temperature .", "Reduction of the aldehyde groups was achieved by incubation for 10 min with 0 . 5 mg/ml NaBH4 .", "For intracellular PLA , cells were permeabilized after fixation with 0 . 5% saponin ( quillaja bark , Sigma ) in PBS for 30 min .", "PLA reactions were performed based on a previously described protocol ( Soderberg et al . , 2008 ) .", "Briefly , blocking solution contains 25 μg/ml sonicated salmon sperm DNA and 250 µg/ml BSA in PBS .", "Any treatment with EDTA or tween was avoided .", "After blocking , cells were incubated with appropriate PLA probes for Fab-PLA , 1-PLA or with primary antibody for 2-PLA in probemaker diluent .", "In the case of 2-PLA , cells received further incubation with secondary PLA probes Duolink II kit ( Olink Biosience ) binding to the corresponding primary antibody .", "PLA signal amplification was performed following manufacturer's instruction .", "Resulting samples were directly mounted on slides with DAPI-Fluoromount-G ( Southern Biotech ) to visualize the PLA signals in relation to the nuclei .", "Carboxylate modified latex ( CML ) beads with the diameter of 10 µm ( Invitrogen ) were coupled with NIP ( 15 ) BSA-biotin ( Biosearch ) following manufacturer's instruction .", "Briefly , 250 µl of CML beads ( 40 mg/ml ) were first washed twice with 1 ml MES buffer ( 25 mM , pH6 ) and then resuspended in 500 µl MES .", "200 µl of freshly prepared EDAC ( 1 Ethyl 3- ( 3-Dimethyl Amino Propyl ) Carbodiimide HCl , Sigma-Aldrich , 50 mg/ml in MES ) and 300 µl NIP ( 15 ) BSA-biotin ( Biosearch , 6 . 6 μg/ml in MES ) were added to the latex suspension and incubated at room temperature with gentle mixing for 4 hr .", "The resulting beads were then washed three times with 1 ml PBS and resuspended in 1 ml storage buffer ( 1X PBS with 0 . 1% glycine and 0 . 1% NaN3 ) .", "Purified monomeric and pentameric IgM were loaded on beads by incubating 100 , 000 beads with 0 . 05 µM ( monomeric ) or 0 . 01 µM ( pentameric ) of IgM in 30 µl volume at room temperature for 30 min under mild shaking .", "The equal loading of the monomeric and pentameric IgM was verified by staining the beads with anti-IgM-APC ( eBioscience ) antibody and monitoring the staining by FACScan .", "After loading , the beads were washed with PBS and PLA were performed as described .", "The PLA signal was quantified by a FACScan on LSRFortessa ( Becton Dickinson , Franklin Lakes , NJ ) using the filter set for PE .", "Data were exported and analyzed and plotted using FlowJo software ( TreeStar , Ashland , OR ) .", "All microscopic images were acquired on a Zeiss 780 Meta confocal microscope ( Carl Zeiss , Jena , Germany ) using a Zeiss Plan-Apochromat 63X oil immersion objective lens .", "For each sample , a minimal of five images were captured from randomly chosen regions .", "All recorded images were analyzed by single cell analysis using the BlobFinder ( Centre for Image Analysis , Uppsala university ) software .", "Raw data produced by BlobFinder were exported to Prism ( Graphpad , La Jolla , CA ) software .", "For each sample in each experiment , the average PLA signal counts per cell was calculated from the corresponding images and then normalized to the average PLA signal counts per cell of the reference sample in the same experiment .", "These normalized PLA signal counts from a minimum of three independent experiments were used for the plot and to calculate the difference between samples .", "A one-tailed paired t test was used to determine the p value .", "Fab-PLA probes were labeled by annealing with fluorescence-coupled complementary oligonucleotides and size separated to remove excess unbound oligos .", "Resting and activated B1-8 cells were fixed for 15 min with 2% paraformaldehyde in PBS at room temperature .", "Thereafter , the fixed cells were incubated with fluorescence labeled Fab-PLA probes in blocking solution containing 250 μg/ml BSA , 2 . 5 μg/ml sonicated salmon sperm DNA , washed with PBS and subjected to flow cytometry analysis using a FACScan instrument .", "Resting cells treated with matching concentration of dsDNA prepared by annealing free plus or minus oligo with the corresponding fluorescence coupled complementary oligo were used as a control .", "Schneider S2 cells were cultured and transfected as described previously ( Yang and Reth , 2012 ) .", "To induce the protein expression of the transfected plasmids , cells were treated with 1 mM CuSO4 for 24hr .", "Cells were co-transfected with plasmids encoding BCR and GFP tagged Syk ( wt or mutant ) were sorted for GFP-expression .", "Cells without the co-transfection of Syk were stained by anti-λ-FITC and FITC-positive cells were purified by cell sorting ." ] ]

[ "Binding of antigen to the B cell antigen receptor ( BCR ) initiates a multitude of events resulting in B cell activation .", "How the BCR becomes signaling-competent upon antigen binding is still a matter of controversy .", "Using a high-resolution proximity ligation assay ( PLA ) to monitor the conformation of the BCR and its interactions with co-receptors at a 10–20 nm resolution , we provide direct evidence for the opening of BCR dimers during B cell activation .", "We also show that upon binding Syk opens the receptor by an inside-out signaling mechanism that amplifies BCR signaling .", "Furthermore , we found that on resting B cells , the coreceptor CD19 is in close proximity with the IgD-BCR and on activated B cells with the IgM-BCR , indicating nanoscale reorganization of receptor clusters during B cell activation ." ]

[ "Our immune system protects us against diseases by recognizing invading pathogens , such as bacteria and viruses , and launching a response to eliminate them .", "In vertebrates , like mice and humans , this immune response often involves white blood cells called B cells , which make antibodies .", "B cells can recognize a huge number of different molecules called antigens , including those from pathogens , with the help of their antigen receptors .", "These receptors are proteins that span the surface membrane of the B cells , such that most the receptor is outside of the cell , with the rest being inside the cell .", "When an antigen binds to the outside portion of a B cell receptor , that B cell becomes activated .", "The B cell then starts to multiply , and to produce antibodies that bind to that antigen and hence mark a pathogen for attack by the immune system .", "For many years it was thought that two copies of the receptors had to be brought together for the B cell antigen receptor to activate the B cell .", "However , other research revealed that the receptors tend to cluster together in the membrane , even before an antigen is recognized .", "Now , Kläsener , Maity et al . have used techniques that can essentially measure the distance between two B cell antigen receptors , even when they are just a few billionths of a meter ( or nanometers ) apart .", "This revealed that the receptors start very close together , and actually move further away from each other when the B cells are activated .", "Kläsener , Maity et al . also found that an enzyme , called spleen tyrosine kinase ( Syk ) , is needed to separate the receptors .", "Further experiments revealed that Syk does this by binding to the so-called ‘signaling motif’ of the receptors , which is inside the cell: this causes the receptors to change shape , forcing the parts outside the cell to move apart .", "Furthermore , Kläsener , Maity et al . found that other proteins in the surface membrane called co-receptors—which cooperate with the B cell antigen receptors to activate a B cell—were also re-organized when B cells became activated .", "It is likely that most other membrane proteins are also organized in clusters that are only nanometers across .", "As such , the techniques described by Kläsener , Maity et al . will now allow the study of membrane organization at the nanoscale; which , as yet , has remained largely unexplored ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "chromosomes and gene expression", "cell biology" ]

[ [ "Meiosis is a specialized form of cell division that reduces the number of chromosomes in germ cells by half .", "This is achieved by coupling one round of DNA replication with two rounds of chromosome segregation .", "During the first meiotic division , homologous chromosomes segregate away from each other .", "At the second ( mitosis-like ) meiotic division sister chromatids segregate from each other , producing four meiotic products .", "Successful completion of the first meiotic division requires the proper completion of several key events , each of which must occur at a specific time and place during prophase .", "For instance , programmed double-strand breaks ( DSBs ) , required for the initiation of meiotic recombination , are spatially and temporally controlled .", "Failure to initiate recombination , to create the correct number of DSBs , or to position the DSBs properly can lead to aneuploidy ( Murakami and Keeney , 2008 ) , which in humans can result in disorders of chromosome number such as Down , Klinefelter , or Turner syndrome .", "The reason a failure in initiating recombination induces chromosome missegregation is because a subset of DSBs are repaired into crossovers , and , in most cases , it is the physical linkage ( chiasmata ) of the homologs by crossovers that ensures chromosomes segregate properly at the first meiotic division ( Page and Hawley , 2003 ) .", "Crossovers are formed within the context of the synaptonemal complex ( SC ) , a highly conserved proteinaceous structure formed between homologs during early meiotic prophase ( Zickler and Kleckner , 1999; Page and Hawley , 2004 ) .", "The SC consists of two lateral elements ( LEs ) and a central region that contains both the central element ( CE ) and transverse filament ( TF ) proteins .", "Although crossover formation almost universally requires the presence of SC , the degree to which DSB formation depends on SC formation , or vice versa , differs between organisms .", "In yeast ( Roeder , 1997 ) , SC formation is dependent on DSBs , as DSB sites appear to be the location for the initiation of SC synthesis ( Chua and Roeder , 1998 ) ; and in mammals , SC formation between homologs is dependent on DSB formation ( Baudat et al . , 2000 Romanienko and Camerini-Otero , 2000 ) .", "However , in flies ( McKim et al . , 1998; Jang et al . , 2003 ) and worms ( Dernburg et al . , 1998 ) , SC formation is not dependent on DSB formation , and in fact , DSBs are formed after full-length SC is constructed .", "Moreover , in the absence of SC formation in flies , DSB formation in the oocyte is significantly reduced ( Mehrotra and McKim , 2006; Collins et al . , 2014 ) .", "Although DSBs are induced by the evolutionarily conserved topoisomerase-like protein Spo11 ( Keeney et al . , 1997 ) , many poorly conserved accessory proteins have been identified that are required either to facilitate the formation of the DSBs themselves or position the DSBs within the euchromatin ( de Massy , 2013 ) .", "Indeed , the process of DSB formation is tightly controlled , both in terms of DSB number and position .", "Recently , a feedback mechanism has been proposed that links the process of DSB repair to the DSB formation process in both yeast and worms ( Rosu et al . , 2013; Thacker et al . , 2014 ) .", "In addition , the position of DSBs within the genome is nonrandom , and in many organisms is often controlled by specific sequence motifs that create recombinational hotspots ( de Massy , 2013 ) .", "In most organisms the number of DSBs far exceeds the number of crossover events ( de Massy , 2013 ) .", "For example , the ratio of DSBs to crossovers is 10 to one in mice ( Moens et al . , 2002 ) , while in flies there are at least three times more DSBs than there are crossovers ( Lindsley et al . , 1977; Mehrotra and McKim , 2006 ) .", "Therefore , there must be a selection process that differentiates those DSBs that become crossovers from those that will be repaired by processes that create noncrossover gene conversions .", "Recent studies have identified components of the multistep process that selects those DSBs that will become crossover-competent DSBs .", "These steps appear to be controlled by the ever-growing Zip3 family of proteins and their regulators .", "Zip3 was first identified in yeast ( Ouspenski et al . , 1999; Agarwal and Roeder , 2000 ) , and homologs have now been identified in many other model organisms .", "Recently it has been suggested that there are two subgroups within the Zip3 family: the Zip3/RNF212 group and the Hei10 group ( Chelysheva et al . , 2012; De Muyt et al . , 2014 ) .", "All the members within both subgroups are required for the formation of crossovers and are similar in terms of protein structure; they contain a RING-type zinc finger domain , an internal coiled-coil domain , and a C-terminal domain that tends to be serine rich ( Reynolds et al . , 2013 ) .", "However , not all organisms possess members of both subgroups .", "Both budding yeast ( Agarwal and Roeder , 2000 ) and worms ( Jantsch et al . , 2004; Bhalla et al . , 2008 ) are predicted to carry only a single member of the Zip3/RNF212 group , whereas the Arabidopsis ( Chelysheva et al . , 2012 ) , rice ( Wang et al . , 2012 ) and Sordaria ( De Muyt et al . , 2014 ) genomes are thought to encode only a member of the Hei10 group .", "The genomes of mammals , like humans ( Toby et al . , 2003; Kong et al . , 2008 ) and mice ( Strong and Schimenti , 2010; Reynolds et al . , 2013; Qiao et al . , 2014 ) , appear to encode members from each subgroup .", "The two subgroups display key differences in their overall enzymatic activity .", "Zip3/RNF212 group members appear to act solely as SUMO E3 ligases , whereas some members of the Hei10 group appear to possess both ubiquitin E3 ligase and SUMO E3 ligase activity .", "Yeast Zip3 , which is required to regulate SUMO modification along meiotic chromosomes , has SUMO E3 ligase activity in vitro ( Cheng et al . , 2006 ) , and genetic studies have implicated Zhp-3 , the C . elegans Zip3 homolog , in the SUMO pathway as well ( Bhalla et al . , 2008 ) .", "Conversely , human Hei10 has been shown biochemically to have ubiquitin E3 ligase activity in vitro ( Toby et al . , 2003 ) .", "However , recent studies suggest that mouse Hei10 may also function as a SUMO E3 ligase ( Strong and Schimenti , 2010 ) .", "These observations suggest that the relationship between SUMOylation and ubiquitination of proteins in the vicinity of the DSB determines which DSBs become competent to crossover ( Qiao et al . , 2014 ) .", "Very little is known about how DSBs become crossover-competent DSBs in Drosophila .", "Prior to this study , homologs for most of the proteins required for this process in other organisms ( Msh4/Msh5 ( Yokoo et al . , 2012 ) , RNF212 , Hei10 ( Strong and Schimenti , 2010 ) , Mlh1/Mlh3 ) had not been identified in Drosophila .", "In this manuscript we describe a new meiosis-specific gene that we have named vilya .", "Vilya is a Zip3-like RING-containing protein that is required for programmed DSB formation .", "Vilya interacts with another DSB accessory protein , Mei-P22 , and these proteins localize to sites of DSBs as identified by the chromatin modification γH2AV ( Mehrotra and McKim , 2006 ) .", "When an epitope-tagged version of Vilya is expressed in the female germline , it shows a dynamic localization pattern that is dependent on DSB formation .", "In early pachytene , Vilya localizes both to the central region of the SC and to discrete foci .", "As the oocyte matures into early/mid-pachytene , Vilya is primarily found at discrete foci .", "The number and distribution of these foci along the euchromatic SC of each chromosome arm parallels the number and position of crossover events .", "Indeed , we show that Vilya is a component of recombination nodules ( RNs ) by immuno-electron microscopy ( immuno-EM ) , making it the first RN protein component identified in Drosophila .", "We speculate that Vilya has functions that have recently been described for several members of the Zip3 group , such as DSB fate determination and crossover formation ." ], [ "Drosophila females provide an excellent system to analyze the progression of very early events of the first meiotic division because egg chambers within each ovariole of the ovary are arranged according to developmental age ( King et al . , 1956 ) .", "Figure 1A shows a schematic of the Drosophila germarium , which is the structure at the very tip of the ovariole and is where meiosis begins .", "In region 1 , the germline stem cell ( GSC ) divides to produce a cystoblast , which undergoes four rounds of incomplete cell division to produce a 16-cell interconnected cyst .", "These early divisions are known as the premeiotic divisions .", "Known components of the Drosophila SC , which are thought to be exclusively on meiotic chromosomes , associate with centromeres in the early premeiotic divisions and are required for the pairing and clustering of centromeres that begins at the eight-cell cyst ( Takeo et al . , 2011; Tanneti et al . , 2011; Christophorou et al . , 2013 ) . 10 . 7554/eLife . 08287 . 003Figure 1 . vilya encodes a RING domain-containing protein required for DSB formation .", "( A ) Schematic diagram of a germarium showing the timing of SC and DSB formation .", "( B ) vilya826 homozygotes and Df/vilya826 transheterozygotes cause high levels of X chromosome nondisjunction .", "The high level of X nondisjunction in vilya826 is almost completely rescued by expressing vilya3XHA in the female germline .", "The deficiency that uncovers vilya used in the analysis was Df ( 1 ) ED6630 .", "vilya826 + rescue refers to the genotype y w vilya826 nos-Gal4/vilya826; PUASp-vilya3XHA/+ .", "Wild type and vilya826 nondisjunction rates are from ( Collins et al . , 2012 ) .", "( C ) vilya826 and Df/vilya826 are defective in DSB formation in early pachytene oocytes as identified by an antibody against γH2AV and compared to wild type .", "DSBs in region 2A nurse cells are also significantly reduced in vilya mutants ( see Figure 1—figure supplement 4 ) .", "( D ) Region 2A oocyte nuclei stained with Corolla ( red ) and γH2AV ( green ) in wild type , vilya826 , vilya826exposed to X-ray and vilya826 + vilya3XHA germline rescue construct .", "Images are maximum intensity projections of deconvolved z-series through the selected nuclei .", "Scale bar , 1 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 08287 . 00310 . 7554/eLife . 08287 . 004Figure 1—figure supplement 1 . vilya , CG2709 , encodes a RING domain-containing protein .", "( A ) mei-826 ( Collins et al . , 2014 ) was mapped to CG2709 ( Materials and Methods ) and renamed vilya826 .", "( B ) vilya is predicted to encode a 237 amino acid protein with a RING domain and a potential internal coiled-coil domain .", "vilya826 allele is predicted to truncate the protein at amino acid 213 .", "( C ) Shown is the structural RING domain consisting of Cys3HisCys4 binding to two zinc ( Zn ) cations .", "( D ) Vilya is predicted to contain an internal coiled-coil region based on the COILS program ( Lupas et al . , 1991 ) .", "( E ) Vilya protein sequence is shown with cysteine and histidine residues of the RING domain ( yellow and red ) , the mutation ( R213STOP ) in vilya826 ( blue ) , a predicted SUMO-interacting motif ( green ) , three potential RXL motifs for mediating cyclin binding ( purple ) , and the serines in the serine-rich C-terminal domain ( underlined ) .", "The last quarter of Vilya is 25% serines . DOI: http://dx . doi . org/10 . 7554/eLife . 08287 . 00410 . 7554/eLife . 08287 . 005Figure 1—figure supplement 2 . Centromere clustering and homolog pairing is not affected in vilya826 .", "( A ) Using an antibody to the CENP-A homolog , CID , clustering of centromeres is unaffected in vilya826 compared to wild type in region 2A .", "100% of region 2A oocytes analyzed ( n ) for both wild type and vilya826 contain two or less centromere clusters .", "( B ) FISH analysis of an X chromosomal probe at region 14A-C indicates that homolog pairing is normal throughout pachytene in vilya826 when compared to wild type .", "Nuclei with either a single focus or foci separated by less than 0 . 75 µm were defined as paired .", "Those foci with centers separated by more than 0 . 75 µm were considered unpaired . DOI: http://dx . doi . org/10 . 7554/eLife . 08287 . 00510 . 7554/eLife . 08287 . 006Figure 1—figure supplement 3 . C ( 3 ) G and Orb staining appears normal in vilya826 .", "Immunofluorescence analysis of wild-type ( A ) and vilya826mutant ( B ) germaria showing the timing of SC formation , SC structure and oocyte determination .", "The SC is labeled with an antibody to C ( 3 ) G ( red ) .", "By region 2B the cytoplasm of the oocyte becomes concentrated with Orb ( green ) .", "The position of regions 1 through 3 are labeled to the right of each germarium .", "Scale bar , 15 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 08287 . 00610 . 7554/eLife . 08287 . 007Figure 1—figure supplement 4 . Vilya plays a direct role in DSB formation in early pachytene .", "Immunofluorescence analysis comparing the induction and location of DSBs as marked by an antibody to γH2AV ( green ) in both surrounding nurse cells and oocyte nuclei ( identified with an antibody to Corolla ( red ) ) of wild type , c ( 3 ) G , mei-W68 , mei-P22 , vilya826 and Df/vilya826 .", "In each genotype , region 2A is identified by the white bar on the merged image .", "γH2AV foci are readily identifiable in region 2A in both wild type and c ( 3 ) G indicating that DSBs are induced .", "No , or very few , DSBs can be identified with the γH2AV antibody in any region 2A nuclei ( oocytes or surrounding nurse cells ) in mei-W68 , mei-P22 or in the vilya mutants , suggesting that vilya’s function is required for the induction of DSBs during early pachytene .", "Images are maximum intensity projections of deconvolved z-series through the entire germarium .", "Scale bar , 5 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 08287 . 007 Zygotene of prophase I begins in the 16-cell cyst , in region 2A , which is best defined by the presence of additional punctate SC staining throughout the euchromatin in up to four nuclei .", "As the cyst progresses in region 2A , it enters into pachytene where full-length SC is formed .", "In Drosophila , meiotic DSBs are formed by the Spo11 homolog , Mei-W68 , after the SC is fully formed ( McKim and Hayashi-Hagihara , 1998; Mehrotra and McKim , 2006 ) .", "DSBs can be visualized in Drosophila by the rapid phosphorylation of the histone 2A variant ( γH2AV ) at DSB sites that occur in all 16 nuclei within the cyst ( in both the pro-oocytes and surrounding nurse cells ) in region 2A ( Mehrotra and McKim , 2006 ) .", "As the cyst progresses into region 2B ( early/mid-pachytene ) , only two nuclei have complete SC , and DSB numbers are reduced from those found in early pachytene .", "By region 3 , or mid-pachytene , the oocyte nucleus has been selected and most of the γH2AV staining at DSB sites is removed , indicating that repair is either in progress or complete .", "A germline clone screen for EMS-induced meiotic mutations on the X chromosome produced a novel meiotic mutation , known initially as mei-826 , that caused high levels of nondisjunction at the first meiotic division ( Collins et al . , 2012 ) .", "This fully recessive mutation resulted in a C–T transition within a previously uncharacterized gene known as CG2709 ( Figure 1—figure supplement 1A ) and is predicted to truncate the protein 24 amino acids from the end ( R213STOP ) ( Materials and methods ) ( Figure 1—figure supplement 1B ) .", "We have named this gene vilya and have therefore subsequently renamed the mutant , vilya826 .", "A transgene construct expressing a tagged version of the wild-type vilya gene ( denoted vilya3XHA ) in the germline fully rescued the chromosome segregation defect seen in vilya826 homozygotes ( Figure 1B ) .", "In addition , the meiotic nondisjunction phenotype of vilya826 homozygotes was very similar to Df ( vilya ) /vilya826 transheterozygotes , suggesting that vilya826 is a null allele ( Figure 1B ) .", "vilya is predicted to encode a protein with several identifiable domains .", "In the N-terminal region there is a Cys3HisCys4 Really Interesting New Gene ( RING ) domain ( Figure 1—figure supplement 1B , C ) .", "RING domains are structural domains that bind two zinc cations and are typically found in E3 ligases ( Metzger et al . , 2014 ) .", "In the middle of the protein there is a predicted coiled-coil domain ( Figure 1—figure supplement 1D ) ( Lupas et al . , 1991 ) .", "Coiled-coil domains are often involved in protein–protein interactions and are commonly found in proteins that localize to the SC ( Sym et al . , 1993; Page and Hawley , 2004; Smolikov et al . , 2009; Collins et al . , 2014 ) .", "Additionally , the C-terminal region of Vilya is serine rich , with the last quarter of the protein being approximately 25% serines ( Figure 1—figure supplement 1E ) .", "These characteristics are typical of members of the Zip3 protein family ( Reynolds et al . , 2013 ) ( see Discussion ) .", "Since vilya826 causes very high levels of chromosome missegregation and encodes a protein with a potential coiled-coil domain , we asked whether this mutant was disrupting the early events in meiotic prophase .", "Specifically , we wondered if it affected SC formation or two processes that depend on the SC: the pairing and clustering of centromeres and the pairing of homologous chromosomes .", "We first assayed the processes of centromere clustering ( Figure 1—figure supplement 2A ) and homolog pairing ( Figure 1—figure supplement 2B ) in early pachytene nuclei .", "Unlike mutants that fail to pair and/or cluster their centromeres properly and thus display greater than three centromere foci ( Takeo et al . , 2011 ) , oocytes homozygous for vilya826 showed no defects in centromere pairing/clustering when compared to wild type .", "Similarly , vilya826 was not defective in euchromatic homolog pairing as assayed for the X chromosome by fluorescence in situ hybridization ( FISH ) .", "Moreover , immunofluorescence analysis of early pachytene nuclei did not reveal defects in the ability of the SC protein Corolla to localize properly in vilya826 germaria ( Figure 1D–a , b ) .", "As well , we did not detect defects in timing or localization of the TF SC protein , C ( 3 ) G , or Orb , a cytoplasmic marker for oocyte determination ( Figure 1—figure supplement 3 ) .", "Taken together , we were unable to detect significant defects in any of the processes that occur prior to the initiation of DSBs .", "However , the formation of DSBs , as assayed with an antibody recognizing γH2AV , was greatly reduced in vilya826 and Df/vilya826 oocytes .", "Specifically , we assayed the initiation of DSBs that occur in all nuclei in region 2A cysts within the germarium ( see Figure 1A ) by comparing the timing and presence of γH2AV foci of vilya826 homozygotes and Df/vilya826 transheterozygotes to that observed in wild type , in an SC mutant ( c ( 3 ) G ) that initiates DSBs albeit at reduced levels in the oocyte ( Mehrotra and McKim , 2006 ) , and in two DSB-defective mutants ( mei-W68 and mei-P22 ) ( Figure 1—figure supplement 4 ) .", "We found that unlike wild-type and c ( 3 ) G females , where γH2AV foci are readily observed in region 2A cysts , vilya826 and Df/vilya826 females show an almost complete absence of γH2AV staining , similar to mei-W68 and mei-P22 .", "These observations strongly suggest that vilya826 and Df/vilya826 oocytes are defective in DSB formation .", "Immunofluorescence analysis of vilya826 also reveals a severe failure to initiate programmed DSBs in oocytes in early pachytene compared to wild type ( Figure 1C and Figure 1D–a , b ) .", "This defect is not caused by a delay in DSB formation , as no γH2AV foci were detected in later stages of pachytene in the germarium ( Figure 1—figure supplement 4 ) .", "The near complete absence of the γH2AV foci in vilya826 oocytes was also not due to an inability of vilya826 to modify the histone at DSB sites , as γH2AV was detected in vilya826 oocytes when DSBs were artificially induced by X-ray treatment ( Figure 1D , C ) .", "Finally , the failure to induce DSBs in vilya mutant females is solely due to the lack of functional Vilya because germline expression of vilya3XHA is able to rescue DSB formation ( Figure 1D–d ) .", "To rule out the possibility that the observed reduction of DSBs by this assay was not due to an increased rate of DSB repair in vilya826 oocytes , we analyzed the ability of vilya826 to rescue the defects associated with the DSB repair-deficient mutant , okra ( homolog of yeast Rad54 ) .", "In okra mutant-bearing oocytes , DSBs are left unrepaired , leading to the activation of a DNA damage checkpoint ( Ghabrial et al . , 1998 ) .", "Activation of this checkpoint induces several observable phenotypes , which are bypassed by mutants that fail to form DSBs ( Ghabrial and Schupbach , 1999; Liu et al . , 2002; Lake et al . , 2011 ) .", "We examined the effect of the vilya mutant on two of these phenotypes .", "First , in okra mutant-bearing oocytes , the presence of unrepaired breaks leads to sterility ( Figure 2A ) ( Ghabrial et al . , 1998 ) .", "In vilya826/+ oocytes , which carry one wild-type copy of vilya , homozygosity for okra causes near complete sterility , producing only 0 . 2 progeny per female on average .", "However , in the vilya826 okra double mutant , the fertility was similar to vilya826 alone , averaging 14 . 4 and 16 . 3 progeny per female , respectively , and the rate of X and 4th chromosome nondisjunction in the double mutant was similar to the vilya826 single mutant ( Figure 2A ) .", "Therefore , the reduction of DSBs due to the vilya mutation resulted in the rescue of fertility ( about 30% as fertile as wild type ) caused by the okra mutation .", "Second , in okra mutant oocytes the presence of unrepaired DSBs results in an inability to form the spherical meiotic chromosome mass , known as the karyosome , in late pachytene ( Ghabrial et al . , 1998 ) .", "In the presence of DSBs , but in the absence of repair , the karyosome structure is fragmented ( Figure 2—figure supplement 1 ) .", "vilya826 was also able to rescue the karyosome defect seen in okra mutants ( Figure 2B and Figure 2—figure supplement 1 ) .", "Although we cannot rule out the possibility that DSBs are formed and repaired in a rad54/okra-independent manner so quickly that we are unable to detect them in our assay , these studies strongly support the conclusion that programmed DSBs are rarely formed in vilya826 oocytes . 10 . 7554/eLife . 08287 . 008Figure 2 . vilya826 rescues the fertility and karyosome defects of the DSB repair-deficient mutant okra .", "( A ) vilya826 rescues the fertility defect of the DSB repair-deficient mutant okra and displays an increase in chromosome nondisjunction in the double mutant , similar to that of the single vilya826 mutant .", "The fertility of vilya826 is only about 30% of the wild-type control , likely due to the high levels of chromosome missegregation .", "The high levels of 4th chromosome nondisjunction observed in the vilya mutant are due to the inability of the achiasmate segregation system to withstand the effects of a global reduction in recombination ( Zitron and Hawley , 1989; Hawley et al . , 1992 ) .", "Average number of progeny per female ( gray line ) is shown .", "Number of adjusted progeny scored in the nondisjunction assay: wild type ( 330 ) , vilya826/+; okra ( 20 ) , vilya826; okra ( 1587 ) and vilya826 ( 195 ) .", "Number of females tested in the fertility assay: wild type ( 9 ) , vilya826/+; okra ( 90 ) , vilya826; okra ( 110 ) and vilya826 ( 12 ) .", "Wild type and vilya826 data collected independently from other genotypes .", "ND , nondisjunction .", "( B ) vilya826 rescues the karyosome defect seen in the okra mutant to 89 . 5% of normal .", "Number of karyosomes analyzed: okra ( 38 ) , vilya826/+; okra ( 20 ) , vilya826; okra ( 12 ) , and vilya826 ( 6 ) .", "( A , B ) ( + ) indicates wild-type copy of vilya present on FM7 balancer chromosome . DOI: http://dx . doi . org/10 . 7554/eLife . 08287 . 00810 . 7554/eLife . 08287 . 009Figure 2—figure supplement 1 . vilya826 rescues the karyosome defect of the DSB repair-deficient mutant okra .", "The karyosome structure of a wild-type stage 8 egg chamber is shown for comparison purposes .", "A schematic of the egg chamber is shown below each image to identify the region highlighted in the image .", "The karyosome structure is fragmented in the DSB repair-deficient mutant okra when one copy of wild-type vilya is present ( vilya826/FM7; okra ) .", "vilya826 rescues the karyosome defect seen in okra mutants ( vilya826; okra ) , indicating that DSB formation is suppressed or abolished .", "Images are maximum intensity projections of deconvolved z-series from a DeltaVision microscope through the selected nuclei .", "Stage 8 egg chambers are stained with DAPI ( white ) only .", "Karyosome is identified by a red dashed box .", "Scale bar , 15 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 08287 . 00910 . 7554/eLife . 08287 . 010Figure 2—figure supplement 2 . vilya826 is defective in meiotic recombination .", "( A ) vilya826 is defective in meiotic recombination as assayed for intervals cv-v and v-f on the X chromosome .", "( B ) Recombination frequency across the entire 3rd chromosome in vilya826 is reduced over 50-fold compared to wild type . DOI: http://dx . doi . org/10 . 7554/eLife . 08287 . 010 Finally , if vilya826 oocytes are unable to initiate the formation of the majority of DSBs , we would predict a severe defect in the process of meiotic recombination .", "An analysis of meiotic recombination in two intervals that span the majority of the X chromosome shows a complete failure of recombination in vilya826 ( Figure 2—figure supplement 2A ) .", "Germline expression of vilya3XHA was able to fully rescue the frequency and distribution of recombination in the vilya826 mutant .", "We also analyzed the frequency of recombination across the entire 3rd chromosome and found that the frequency of recombination was reduced over 50-fold in vilya826 compared to wild type ( Figure 2—figure supplement 2B ) .", "Taken together , the chromosome missegregation , the lack of recombination , the near absence of γH2AV staining in all nuclei in each cyst in region 2A , and the ability of vilya826 to rescue the defects of a DSB repair mutant indicate that vilya826 is defective in the ability to initiate programmed DSB formation .", "We analyzed the localization of Vilya throughout pachytene using the epitope-tagged germline expression construct described above that fully rescued both the nondisjunction and meiotic recombination phenotype of the vilya826 mutant .", "The tagged Vilya construct was expressed in the female germline using the Gal4-UAS system under the control of the nanos ( nos ) promoter .", "Using this system , proteins are expressed throughout most stages of oogenesis at high levels ( Van Doren et al . , 1998 ) .", "Immunofluorescence analysis coupled with structured illumination microscopy ( SIM ) allowed us to precisely determine the localization of Vilya during pachytene .", "We find that during early pachytene , Vilya3XHA localizes to the central region of the SC in both linear stretches and discrete foci ( Figure 3A-a ) .", "The Vilya3XHA linear tracks appear within the central region of the SC , as the fluorescence is seen in between the two lateral sides of the SC using an antibody that localizes to the C terminus of the TF protein C ( 3 ) G ( Anderson et al . , 2005 , Collins et al . , 2014 ) .", "In addition , the discrete Vilya3XHA foci can also be seen within the central region ( Figure 3A-b ) .", "As the cyst progresses to early/mid-pachytene , the linear tracks of Vilya become less apparent and the foci become more discrete ( Figure 3A-c , d ) .", "We counted the number of discrete foci throughout each stage of pachytene in the germarium and found that similar to the trend in γH2AV foci number ( see Figure 1C ) , Vilya3XHA foci are most abundant in region 2A ( average 8 foci , SD = 2 ) ( the stage at which programmed DSBs are being induced ) and then decline gradually throughout early/mid-pachytene ( region 2B average 4 . 4 , SD = 0 . 8 ) .", "In mid-pachytene ( region 3 ) , a stage in which DSB repair is underway or complete , we still see an average of 3 . 2 Vilya3XHA foci ( SD = 0 . 9 ) ( compare Figure 1C and Figure 3B ) .", "The observation that Vilya3XHA persists at discrete foci after DSB repair has begun and crossovers are forming suggests that Vilya plays a role in the completion of actual crossovers , such as in the breaking and exchange of LEs . 10 . 7554/eLife . 08287 . 011Figure 3 . Vilya localizes to the central region of the SC in both linear elements and discrete foci .", "( A ) Localization of Vilya3XHA throughout early pachytene as assayed by germline expression of vilya3XHA using antibodies to the transverse filament protein C ( 3 ) G ( green ) and an antibody to HA ( red ) .", "Images are maximum intensity projections of deconvolved z-series from a DeltaVision OMX microscope through the selected nuclei .", "Scale bar , 1 µm .", "( A-a )", "Early pachytene ( region 2A ) oocyte nucleus showing that Vilya localizes to the central region of the SC in both linear strands and discrete foci .", "( A-b )", "Higher magnification of the white dashed box in A showing Vilya3XHA clearly positioned in the central region between the two tracks of C ( 3 ) G ( yellow line ) and a discrete Vilya3XHA focus sitting within and above a stretch of SC ( arrowhead ) .", "( A-c , d )", "Localization of Vilya3XHA in region 2A and 2B showing the discrete foci and SC staining .", "Note in region 2B the SC shortens .", "( B ) Analysis of the number of Vilya3XHA foci throughout early/mid-pachytene .", "( C-a , b )", "Traces of SC between homologous chromosome arms in early/mid-pachytene ( region 2B ) nuclei expressing vilya3XHA .", "Images are maximum intensity projections of deconvolved z-series from a DeltaVision OMX microscope through the selected nuclei .", "( * ) Indicates the chromosome center containing pericentric heterochromatin and is the location of the centromeres .", "Scale bar , 1 µm .", "Individual tracks of SC between homologous chromosome arms were identified and each labeled with a separate color .", "The corresponding Vilya3XHA foci associated with each stretch of SC between homologous chromosome arms are labeled by a ( v ) in the same color as the stretch of SC it is on .", "( C-a )", "The oocyte nucleus is labeled with antibodies to Corolla ( green ) and HA ( red ) .", "This nucleus has five colored chromosome arms and four Vilya3XHA foci .", "Each chromosome arm has been linearized in Figure 3—figure supplement 1D .", "( C-b )", "Oocyte nucleus is labeled with antibodies to C ( 3 ) G ( green ) and HA ( red ) .", "This nucleus has five colored chromosome arms and five Vilya3XHA foci .", "( C-c )", "The chromosome arm outlined with the white dashed line in ( C-b ) has been linearized .", "( D ) The majority ( 92% ) of Vilya3XHA foci in the five nuclei that have been identified as having five clearly identifiable chromosome arms each localize to one strand .", "One chromosome arm contains two foci , and two chromosome arms contain no Vilya3XHA foci . DOI: http://dx . doi . org/10 . 7554/eLife . 08287 . 01110 . 7554/eLife . 08287 . 012Figure 3—figure supplement 1 . Localization of Vilya3XHA within pachytene nuclei .", "( A ) Immunolocalization of Vilya3XHA in a mid-pachytene ( region 3 ) nucleus that did not have any distinct Vilya3XHA foci and a late pachytene ( Stage 6 ) nucleus showing that once the discrete foci disappear in pachytene , the localization of Vilya3XHA is exclusively uniform throughout the central region of the SC .", "Nuclei are stained with antibodies to C ( 3 ) G ( green ) and HA ( red ) .", "Images are maximum intensity projections of deconvolved z-series from a DeltaVision OMX microscope through the selected nuclei .", "Scale bar , 1 µm .", "( B ) Immunofluorescence analysis showing the specificity of the anti-HA antibody to Vilya3XHA protein .", "A region 2A image is shown for both wild-type and vilya3XHA-expressing oocytes , as well as a stage 4 oocyte that has only the linear staining pattern .", "Images are maximum intensity projections of deconvolved z-series from a DeltaVision microscope through the selected nuclei .", "See Materials and Method for details regarding image acquisition on wild-type tissue .", "Scale bar , 1 µm .", "( C ) Traces of SC between homologous chromosome arms in an early/mid-pachytene region 2B nucleus expressing vilya3XHA .", "Image is a maximum intensity projection of a deconvolved z-series from a DeltaVision OMX microscope through the selected nucleus .", "Scale bar , 1 µm .", "Individual chromosome arms were identified and each labeled with a separate color .", "The corresponding Vilya3XHA foci associated with each chromosome arm are labeled by a ( v ) in the same color as the stretch of SC they are on .", "Notice in this nucleus the chromosome arm labeled in white contains two Vilya3XHA foci spaced some distance apart from one another .", "( D ) Linearized chromosome arms from Figure 3C-a . DOI: http://dx . doi . org/10 . 7554/eLife . 08287 . 01210 . 7554/eLife . 08287 . 013Figure 3—figure supplement 2 . Vilya3XHA foci are not found at centromeres in early/mid-pachytene . Immunolocalization of Vilya3XHA and CID in vilya3XHA expressing early/mid-pachytene oocytes showing the absence of foci at centromeres .", "Pachytene nuclei in the specified regions were labeled with antibodies to HA ( mouse ) ( red ) , Corolla ( green ) and CID ( blue ) .", "Images are maximum intensity projections of deconvolved z-series from a DeltaVision microscope through the selected nuclei .", "Boxes mark the centromere clusters in each nucleus shown .", "Scale bar , 1 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 08287 . 013 In those mid-pachytene region 3 oocytes that lack discrete Vilya3XHA foci , Vilya3XHA is localized exclusively throughout the entire central region of the SC ( Figure 3—figure supplement 1A ) .", "This localization pattern is also observed in late pachytene egg chambers , those that have matured past the germarium ( Figure 3—figure supplement 1A–B ) .", "The absence of the discrete Vilya3XHA foci at mid-pachytene may suggest that the foci seen earlier have disassembled; however , the significance of this relocalization of Vilya3XHA to the central region at the later stages of pachytene is unclear .", "The specificity of the anti-HA antibody for both types of Vilya3XHA staining ( discrete foci and linear tracks ) can be seen in Figure 3—figure supplement 1B where there is a complete absence of staining on wild type tissue .", "To further characterize the localization of Vilya3XHA foci , we analyzed the number and distribution of Vilya3XHA foci within the SC in early/mid-pachytene region 2B oocytes .", "At this stage , the Vilya3XHA foci are readily visible , and the SC becomes shorter and thicker than in early pachytene nuclei .", "Using SIM , 3D visualization , and the spot function in Imaris , we were able to trace five independent tracks of SC in five oocytes and determine the distribution of Vilya3XHA foci within each SC track .", "Examples of the traced SC in oocytes can be seen in Figure 3C-a , b; Figure 3—figure supplement 1C and Video 1 , and linearized traces can be seen in Figure 3C-c and Figure 3—figure supplement 1D .", "We presume that each of the linear tracks correspond to the euchromatic SC ( the well-defined SC which is visibly more structured in immunofluorescence assays than is the less-defined heterochromatic/pericentromeric SC ) of the five major chromosome arms ( 2L , 2R , 3L , 3R and the X chromosome ) .", "We cannot discern the SC of the small 4th chromosomes , nor can we trace through the less-distinct SC near the pericentromeric heterochromatin ( Carpenter , 1975a ) to the other chromosome arm of the same chromosome .", "This analysis was designed to tell us whether the distribution of foci within the SC was consistent between oocytes , and whether or not the position of the foci within the SC of each chromosome arm might suggest a possible link to the position of crossovers ( an average of one crossover per chromosome arm within the euchromatic SC ) .", "While a strong correlation between crossover position and the distribution of Vilya foci would strongly support the hypothesis that the establishment of discrete Vilya foci plays a role in crossover formation , the finding of a lack of consistency for the distribution and/or in the position of the foci might indicate the foci are an artifact from using this overexpression system .", "A summary of the number and distribution of Vilya3XHA foci from the five early/mid-pachytene nuclei in which we could clearly identify all five SCs between homologous chromosome arms is shown in Figure 3D .", "Examining these five oocyte nuclei , which contain a total of 25 stretches of SC , we observed 24 Vilya3XHA foci or an average of 4 . 8 foci per oocyte .", "The majority ( 22/24 ) of the SC between homologous chromosome arms were associated with only one Vilya3XHA focus .", "A small fraction ( 1/22 ) contained two foci ( corresponding nucleus shown in Figure 3—figure supplement 1C ) , and 2/22 were not associated with any Vilya3XHA foci .", "These numbers correspond well to the observed distribution of crossovers in Drosophila melanogaster .", "In addition , as is seen in the images in Figures 3C-a , b and corresponding Video 1 , the Vilya3XHA foci were found exclusively within the euchromatic SC , and no Vilya3XHA foci were detected in the less-defined heterochromatic SC .", "Consistent with this observation we failed to detect any colocalization of Vilya3XHA foci with the histone variant ( CID , the CENP-A homolog ) that localizes in the pericentromeric heterochromatin throughout early/mid-pachytene ( Figure 3—figure supplement 2 ) .", "Thus the number of Vilya3XHA foci in early/mid-pachytene oocytes are consistently found and correspond well to the known number and position of crossover events in flies , with each stretch of euchromatic SC between homologous chromosome arms primarily containing one focus ( Lindsley et al . , 1977 ) .", "Due to the position and distribution of Vilya3XHA foci on the SC between homologous chromosome arms during early/mid-pachytene , as well as the persistence of these foci into mid-pachytene , we speculated that Vilya might be localizing to sites of crossovers .", "Unlike many model organisms where crossover-specific proteins have been identified and reagents have been made to analyze their localization , no such proteins or reagents exist in Drosophila .", "However , early studies by Carpenter in Drosophila show that sites of crossovers form large electron-dense structures known as RNs within the central region of the SC ( Carpenter , 1975a; Carpenter , 1975b ) .", "We performed immuno-EM using a secondary antibody labeled with gold particles on oocytes expressing vilya3XHA .", "In the analysis of 50 nm sections we frequently observed gold particles localizing to electron-dense RNs .", "Examples are shown in Figure 4 .", "In one image in Figure 4B we have captured what we believe to be a lateral view of a Vilya3XHA-associated RN sitting in and above the SC .", "In addition to the localization at RNs , we were able to detect gold particles throughout the entire central region of the SC , as well as small clusters of gold particles in what appear to be small electron-dense regions of the central region ( Figure 4C ) .", "From these studies , we conclude that the discrete Vilya3XHA foci we detect within the central region of the SC by immunofluorescence correspond to the EM structure of the RNs , and/or their precursors , and are the sites of crossing over . 10 . 7554/eLife . 08287 . 015Figure 4 . Immuno-EM of Vilya3XHA shows localization to both the RNs and to the central region of the SC .", "Immuno-gold labeling of Vilya3XHA from germline-expressed PUASp-vilya3XHA ovaries .", "( A ) A low magnification image of a section from a single nucleus with two RNs ( outlined with red dashed box ) .", "A higher magnification of each RN with associated gold particles is also shown .", "( B ) Four additional immuno-EM images showing gold particles associated with RNs .", "A lateral view of an RN is also shown .", "( C ) Three immuno-EM images showing gold particles distributed throughout the central region of the SC , as well as at RNs .", "Arrowheads point to cluster of gold particles in what appears to be a small electron-dense region in the central region .", "NE , nuclear envelope; RN , recombination nodule; LE , lateral element .", "Scale bar , 100 nm . DOI: http://dx . doi . org/10 . 7554/eLife . 08287 . 015 Our data above indicate that Vilya is required for programmed DSB formation and localizes to the sites of crossing over , therefore we next wanted to determine whether these discrete foci were forming at sites of DSBs .", "We first analyzed the localization of Vilya3XHA in the absence of mei-P22 or mei-W68 , two genes whose function is absolutely required for DSB formation ( McKim and Hayashi-Hagihara , 1998 , Liu et al . , 2002 ) .", "Unlike in the presence of one wild-type copy of mei-P22 ( Figure 5A ) or mei-W68 ( Figure 5B ) , in the homozygous mutant backgrounds ( Figure 5C , D ) , Vilya3XHA is found exclusively and uniformly throughout the central region of the SC and fails to localize to discrete foci , indicating that DSB formation is required for the localization of Vilya3XHA to discrete foci but not for the linear localization to the central region of the SC . 10 . 7554/eLife . 08287 . 016Figure 5 . Localization of Vilya3XHA to discrete foci is dependent on the process of DSB formation .", "( A-D )", "Immuno-localization of Vilya3XHA foci in the presence and absence of DSB formation .", "Pachytene nuclei in the specified regions were labeled with antibodies to HA ( red ) , C ( 3 ) G ( green ) and Corolla ( blue ) .", "Images are maximum intensity projections of deconvolved z-series from a DeltaVision microscope through the selected nuclei .", "Scale bar , 1 µm .", "( A-B )", "Germline expression of PUASp-vilya3XHA in the presence of DSB formation .", "( A ) y w nos-Gal4/w; PUASp-vilya3XHA/+; mei-P22103/+ .", "( B ) y w nos-Gal4/w; PUASp-vilya3XHA/mei-W68z4572 .", "( C , D )", "Germline expression of PUASp-vilya3XHA in the absence of DSB formation .", "( C ) y w nos-Gal4/w; PUASp-vilya3XHA/+; mei-P22103 .", "( D ) y w nos-Gal4/w; PUASp-vilya3XHA mei-W68z4572/mei-W68z4572 . DOI: http://dx . doi . org/10 . 7554/eLife . 08287 . 016 Since Vilya3XHA foci do not form in the absence of DSBs , we next examined whether these discrete foci are specifically forming at DSB sites .", "We performed immunofluorescence analysis to determine if Vilya3XHA foci are associated with γH2AV , the histone modification that occurs immediately following DSB formation .", "We find that 60 . 5% of the Vilya3XHA foci are closely associated with γH2AV ( 49 of the 81 Vilya3XHA foci from 11 early pachytene nuclei ) .", "The immunofluorescence signals for Vilya3XHA and γH2AV can be seen in region 2A as foci that colocalize or foci that are adjacent to , but cannot be separated from , each other in a single z-section ( Figure 6A-a’ ) ( see Materials and methods ) .", "As the γH2AV modification at the DSB site can spread some distance ( Rogakou et al . , 1999; Downs et al . , 2004; Shroff et al . , 2004 ) , in this experiment we considered both types of localization for Vilya3XHA and γH2AV as being associated . 10 . 7554/eLife . 08287 . 017Figure 6 . A subset of Vilya3XHA foci localize near γH2AV staining at DSB sites .", "( A , B )", "Immunofluorescence analysis of Vilya3XHA ( red ) localization at the sites of programmed DSBs that are recognized with the γH2AV modification ( green ) and Corolla ( blue ) in region 2A ( A ) or region 3 ( B ) pachytene nuclei .", "Images are maximum intensity projections of deconvolved z-series from a DeltaVision microscope through the selected nuclei .", "( A-a’ , B-b’ , )", "Higher magnification of a single z-section from a small region outlined in the yellow box of the corresponding image A-B , respectively , showing the close association of Vilya3XHA with γH2AV marks .", "Genotype for ( A ) wild type in this figure refers to the genotype y w nos-Gal4/+; PUASp-vilya3XHA/+; mei-P22103/+ .", "mei-P22 is not haploinsufficient .", "( B ) y w nos-Gal4/+; PUASp-vilya3XHA/+; spnB .", "Scale bar , 1 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 08287 . 017 Since the process of DSB formation and repair is a dynamic one , we wanted to verify that the degree of association between Vilya3XHA and the γH2AV modification at DSB sites was significant .", "We performed randomized controls , rotating the Vilya3XHA image stack , to determine the degree of colocalization that would occur by chance for the 11 oocytes analyzed above ( see Materials and Methods for details ) .", "In the 11 early pachytene oocytes that showed an association frequency of 60 . 5% , only 8 . 6% ( 7 of the 81 Vilya3XHA foci ) remained associated after rotation of the Vilya3XHA channel , suggesting the observed degree of association between Vilya3XHA foci and the γH2AV modification cannot be explained by coincidence ( p < 0 . 0001 , binomial test ) .", "We also analyzed the number of Vilya3XHA foci and their association with DSBs in a DNA repair-deficient mutant .", "For this experiment we chose to use the mutant spnB ( Ghabrial et al . , 1998 ) , which is located on a separate chromosome from both the expression construct and the germline driver and could easily be combined genetically for this analysis .", "SpnB , the XRCC3 or Rad51-like protein , is required for programmed DSB repair , and therefore in the absence of spnB function , DSBs fail to be repaired and can be seen by immunofluorescence as γH2AV foci accumulating in mid-pachytene oocytes ( region 3 ) .", "We find that in the absence of DSB repair , the number of Vilya3XHA foci in region 3 increases from an average of 3 . 2 ( SD = 0 . 9 ) in an otherwise wild-type background ( Figure 3B ) to 7 . 5 ( SD = 1 . 6 , n = 10 oocytes ) .", "The frequency of Vilya3XHA foci associated with γH2AV marks was 68% ( 51 of the 75 Vilya3XHA foci in 10 oocytes ) , comparable to the 60 . 5% seen in early pachytene when DSB repair is progressing normally ( p = 0 . 93 , binomial test ) ( Figure 6B and 6B-b’ ) .", "In addition , similar to the DSB repair-proficient background above , in the absence of DSB repair the frequency of association between Vilya3XHA and γH2AV was reduced to 10 . 6% ( 8 of the 75 Vilya3XHA foci ) upon rotation of the Vilya3XHA channel ( p < 0 . 0001 , binomial test ) .", "In order to determine whether the SC was required for Vilya3XHA to localize properly at pachytene , we expressed vilya3XHA in the absence of the TF protein C ( 3 ) G and assayed for the presence of both linear staining and discrete Vilya3XHA foci .", "The SC is not required for the temporal induction of DSBs in nurse cells in early pachytene ( see Figure 1—figure supplement 4 ) , however it is required for wild-type levels of DSB formation in oocytes ( Mehrotra and McKim , 2006 ) .", "Although it is difficult to distinguish oocyte nuclei from nurse cell nuclei in the absence of c ( 3 ) G , occasionally the oocyte can be located by the weak haze of nuclear Corolla staining ( Collins et al . , 2014 ) .", "As shown in Figure 7 , we find that in the absence of C ( 3 ) G , Vilya3XHA is able to localize in early pachytene oocytes to discrete foci that are often associated with γH2AV marks ( 75% of Vilya3XHA foci , 34 of 45 , colocalize with γH2AV marks in 14 oocyte nuclei analyzed ) .", "As the number of DSBs in oocytes of a c ( 3 ) G mutant are reduced to 15–20% of normal ( Mehrotra and McKim , 2006 ) , as expected , Vilya3XHA foci are also reduced in number compared to wild type in region 2A ( an average of 3 . 2 Vilya3XHA foci per oocyte compared to 8 . 0 in wild type ) .", "Interestingly , we failed to see a persistence of Vilya3XHA in region 2B oocytes that we could identify by Corolla staining .", "Of the seven region 2B oocytes we analyzed that no longer contained γH2AV staining , only two had a single Vilya3XHA focus while the remaining had none .", "We speculate that the absence of Vilya3XHA foci at this stage is a consequence of a failure to form RNs and thus repair those DSBs into crossovers .", "At this time , however , we cannot rule out the possibility that the levels of expression of vilya3XHA using the Gal4-UAS system in the c ( 3 ) G mutant is less than in our wild-type background .", "In addition , we never observed linear Vilya3XHA staining in the absence of c ( 3 ) G , indicating that the SC is required to localize Vilya to the central element but not to DSBs . 10 . 7554/eLife . 08287 . 018Figure 7 . Localization of Vilya3XHA to discrete foci in early pachytene is not dependent on the SC . Immunofluorescence analysis of vilya3XHA-expressing region 2A oocytes in the presence and absence of c ( 3 ) G .", "Early pachytene nuclei in region 2A were labeled with antibodies to HA ( red ) , γH2AV ( green ) and Corolla ( blue ) .", "Images are maximum intensity projections of deconvolved z-series from a DeltaVision microscope through the selected nuclei .", "Scale bar , 1 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 08287 . 018 These results strongly support the view that Vilya plays a crucial role in DSB formation , and its localization to discrete foci requires genes whose products are known to be involved in the induction of DSBs .", "In the absence of DSB formation , localization of Vilya3XHA to the central region in linear tracks , which is dependent on the SC , is not disrupted; however discrete Vilya3XHA foci do not form .", "Interestingly , in the absence of DSB repair , the number of discrete Vilya foci increases at unrepaired DSB sites in mid-pachytene .", "It is currently unclear as to whether this increase in Vilya3XHA foci in the absence of DSB repair indicates more DSBs are selected to become crossovers , or whether we may be underestimating the number of Vilya foci throughout pachytene due to the temporal nature of DSB formation and repair .", "To determine whether exogenous DSBs can recruit Vilya to them , which would support a downstream role for Vilya in addition to its role in DSB formation , we analyzed whether the localization pattern of Vilya3XHA in a mei-W68 mutant changed after X-ray treatment .", "We speculated that if Vilya only plays a role in DSB formation , exogenous DSBs would fail to recruit Vilya3XHA .", "However , if Vilya was also required for a downstream function at the RNs , these lesions may indeed recruit and concentrate Vilya3XHA to them , and thus we would observe the exclusive linear staining change in this mutant background after X-ray treatment .", "In the presence of germline expressed PUASp-vilya3XHA in a mei-W68 mutant , where discrete Vilya3XHA foci are not detected ( see Figure 5D and Figure 8 ) , we exposed females to X-rays and looked for the appearance of Vilya3XHA foci after 5 hr , a timeframe previously shown to have γH2AV signal present at X-ray-induced lesions throughout early/mid-pachytene oocytes ( Mehrotra and McKim , 2006 ) .", "We find that in some instances , Vilya3XHA foci could be detected at γH2AV marks ( Figure 8 ) .", "These Vilya3XHA foci appear to be discrete foci and are different from the often observed , more concentrated areas of linear Vilya3XHA staining associated with dense regions of SC ( based on Corolla staining ) in the mei-W68 mutant background in the absence of X-ray ( Figure 8 ) .", "However , in the X-ray-treated females , we also observed instances of Vilya3XHA foci that were not associated with γH2AV signals and many γH2AV marks not associated with Vilya3XHA foci .", "We should also note that X-ray-induced DSBs did not appear to significantly alter the localization of linear tracks of Vilya3XHA in this mutant background , in that we did not see significant removal of Vilya3XHA from the central region of the SC in oocytes with high levels of X-ray-induced breaks . 10 . 7554/eLife . 08287 . 019Figure 8 . Some DSBs created by X-ray can recruit Vilya3XHA to discrete foci . Immunofluorescence analysis of germline expression of PUASp- vilya3XHA in the absence of functional mei-W68 with and without X-ray treatment .", "Ovaries were stained with antibodies to HA ( red ) , γH2AV ( green ) and Corolla ( blue ) .", "Four examples of early/mid-pachytene oocytes are shown for each treatment .", "Yellow boxes in the no X-ray treatment show examples of concentrated regions of linear Vilya3XHA that are associated with dense Corolla staining .", "Yellow boxes in the X-ray treatment show examples of discrete Vilya3XHA foci associated with γH2AV marks that are not associated with dense Corolla staining .", "Scale bar , 1 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 08287 . 019 Although we do not know at this time whether the X-ray-induced DSBs that recruit Vilya3XHA to them can be processed into crossovers , the ability of X-ray-induced lesions to concentrate and form discrete Vilya3XHA foci at some of them suggests that Vilya is playing an active role in the processing of DSBs .", "Mei-P22 is a relatively small protein that localizes to discrete foci prior to DSB formation , is required for DSB formation , and partially colocalizes with γH2AV ( Liu et al . , 2002 , Mehrotra and McKim , 2006 ) .", "Because of this , we wondered whether Vilya , which is also required for DSB formation , and Mei-P22 directly interact .", "In the yeast two-hybrid system , we found that Vilya and Mei-P22 strongly interact as assayed on the reporter plate ( Figure 9 ) .", "We determined that the Vilya826 form of the protein could also interact with Mei-P22 in this assay , although this interaction appeared to be weaker than with full-length Vilya when controlled for plating .", "We also tested whether a mutant form of Mei-P22 , Mei-P22103 , a nonsense mutation resulting in a premature stop codon truncating the Mei-P22 protein by 32 amino acids ( Liu et al . , 2002 ) , can interact with Vilya .", "We found that this mutation , which abolishes Mei-P22 function and DSB formation in vivo , is still able to bind to both Vilya , and Vilya826 , albeit to a lesser extent for Vilya826 when controlling for plating . 10 . 7554/eLife . 08287 . 020Figure 9 . Vilya and Mei-P22 interact by yeast two-hybrid . Yeast two-hybrid was used to test for an interaction between Vilya and Mei-P22 .", "All diploid strains , where the OD600 was equalized before plating , grow equally well under selection for both the bait and prey plasmids ( SD -Leu-Trp ) .", "Six two-fold dilutions for each diploid were plated on each selection plate .", "Vilya and Mei-P22 strongly interact on the reporter plate ( SD -Leu-Trp-Ade-His + X-αgal ) .", "Vilya826 and Mei-P22 also interact , but it appears to be a weaker interaction than with full-length Vilya on the reporter plate .", "Vilya and Mei-P22103 interact , as well as Vilya826 and Mei-P22103 , although this interaction is also weaker than with full-length Vilya .", "No interaction was detected with empty construct for any of the plasmids used .", "The control plasmids are pGBKT7-53 and pGADT7-T supplied by Clontech . DOI: http://dx . doi . org/10 . 7554/eLife . 08287 . 02010 . 7554/eLife . 08287 . 021Figure 9—figure supplement 1 . A functional RING domain is required for Vilya to interact with Mei-P22 in yeast-two hybrid assay .", "( A ) Mutations in any of the key residues in the RING domain ablate the ability of Vilya to interact with Mei-P22 .", "( B ) Western blot analysis showing that the RING domain mutants are expressed in the Y187 strain used as the bait in ( A ) .", "GAL4-BD-cMyc-Vilya protein is predicted to be 49 kDa .", "GAL4-BD-cMyc protein ( empty vector ) is predicted to be 22 kDa . DOI: http://dx . doi . org/10 . 7554/eLife . 08287 . 021 We then determined whether the RING domain of Vilya is required for this interaction by generating a series of mutations in Vilya that disrupt critical residues in the RING domain and testing each for the ability to interact with Mei-P22 in a yeast two-hybrid system .", "We substituted each of the cysteines for serines and the histidine for a tyrosine in the RING domain ( see Figure 1—figure supplement 1 ) .", "Each of the mutations ablates the ability of Vilya to interact with Mei-P22 ( Figure 9—figure supplement 1A ) .", "The failure of the RING domain mutants to interact with Mei-P22 is not due to altered protein expression levels or degradation , as we do not observe any obvious differences in the amount or size of the RING domain mutant proteins compared to wild type ( Figure 9—figure supplement 1B ) .", "However , as is the case for virtually all yeast two-hybrid studies , we cannot fully rule out the possibility that mutating key residues of the RING domain may alter the protein conformation , thus creating a failed interaction .", "These studies , however , do suggest that a functional RING domain within the Vilya protein is critical for Vilya and Mei-P22 interaction .", "Based on these studies , Vilya and Mei-P22 likely interact in vivo , and their interaction is dependent on the RING domain of Vilya .", "Previous studies using the expression construct hsp83:mei-P223XHA have shown that Mei-P223XHA localizes to discrete foci , which are found on chromatin closely associated with the SC and are not dependent on DSB formation ( Liu et al . , 2002 ) .", "Therefore , we anticipate that Mei-P22 localization is also not dependent on Vilya .", "The persistence of Vilya3XHA at discrete foci into early/mid-pachytene , and the absence of Mei-P223XHA staining at this stage ( Liu et al . , 2002; Mehrotra and McKim , 2006 ) , suggests that Vilya may have additional functions at the DSB site , such as in crossing over , that are independent from Mei-P22 ." ], [ "Vilya , in conjunction with another DSB accessory protein , Mei-P22 , acts to facilitate the initiation of recombination during meiotic prophase .", "As shown in our model ( Figure 10 ) , DSBs are not formed in the absence of Mei-P22 , Vilya , or Mei-W68 ( Dm Spo11 ) , resulting in the absence of crossovers ( Liu et al . , 2002 , Mehrotra and McKim , 2006 ) ( this study ) .", "Unlike Mei-P22 , whose localization to discrete foci in early pachytene is not dependent on DSBs ( or at least mei-W68 function ) but is dependent on the SC ( Liu et al . , 2002 ) , Vilya’s ability to localize to discrete foci appears to require only the formation of DSBs but not the SC .", "In the absence of either Mei-P22 or Mei-W68 , the discrete Vilya3XHA foci , which are first apparent in early pachytene and often persist throughout mid-pachytene , do not form .", "Our studies also suggest that the localization of Vilya3XHA along the central region of the SC is not required for its localization to discrete foci , as we did not detect any linear staining of Vilya in a c ( 3 ) G mutant , but we did detect discrete foci often colocalizing with γH2AV marks ( see Figure 7 ) .", "Therefore , Vilya is not only required for the formation of DSBs , but it’s localization to discrete foci , which can be found at a significant number of DSBs ( based on γH2AV staining ) , is dependent on DSB formation .", "Based on these observations , along with the finding that Mei-P22 and Vilya interact in a yeast two-hybrid assay , we propose that Mei-P22 acts upstream of Vilya , and recruits Vilya and Mei-W68 , which catalyzes DSBs .", "Vilya is recruited to at least a subset of DSBs , which are then visualized as discrete foci in early pachytene often colocalizing with γH2AV marks . 10 . 7554/eLife . 08287 . 022Figure 10 . Model of DSB formation in Drosophila female oocytes .", "( Left )", "In wild-type oocytes , Mei-P22 , which localizes to discrete foci prior to the time γH2AV foci are present , is located at chromatin adjacent to the SC ( Liu et al . , 2002 ) .", "Vilya localizes to the central region of the SC and is required along with its binding partner , Mei-P22 , and Mei-W68 ( the Spo11 homolog ) for formation of DSBs .", "Although initially Vilya may localize to a vast majority of , if not all , DSBs , as the oocytes mature into early/mid-pachytene , Vilya is retained and/or recruited to form discrete foci at sites of crossing over .", "Failure to accumulate Vilya at DSB sites would direct that DSB to a noncrossover fate .", "( Right )", "In the absence of Mei-P22 or Mei-W68 , and thus in the absence of DSB formation , Vilya fails to localize to discrete foci and is found exclusively along the central region of the SC .", "In the absence of Vilya , we speculate , based on the fact that Mei-P22 can localize to discrete foci in the absence of Mei-W68 ( Liu et al . , 2002 ) , that Mei-P22’s localization is unaffected .", "However , DSB formation would fail due to the absence of vilya function .", "In the absence of Mei-W68 , Mei-P22 is able to bind normally ( Liu et al . , 2002 ) , however , due to the absence of DSBs , Vilya does not form discrete foci .", "In all these instances , crossovers do not form . DOI: http://dx . doi . org/10 . 7554/eLife . 08287 . 02210 . 7554/eLife . 08287 . 023Figure 10—figure supplement 1 . Protein alignment of Vilya to Zip3 and HEI10 homologous proteins .", "( A ) Protein alignment of Drosophila melanogaster ( Dm ) Vilya ( AAF45818 ) , Caenorhabditis elegans ( Ce ) ZHP-3 ( NP_001250801 ) , Saccharomyces cerevisiae ( Sc ) Zip3 ( NP_013498 ) , Mus musculus ( Mm ) RNF212 ( F6TQD1 ) and HEI10 ( NP_001104589 ) , Arabidopsis thaliana ( At ) HEI10 ( NP_175754 ) , Homo sapiens ( Hs ) HEI10 ( NP_878269 ) , Oryza sativa ( Os ) HEI10 ( EEE56612 ) , Zea mays ( Zm ) HEI10 ( NP_001152027 ) , Physcomitrella patens ( Pp ) HEI10 ( XP_001769363 ) and Penicillium marneffei ( Pm ) HEI10 ( XP_002145282 ) .", "Proteins were aligned and visualized using Muscle and ClustalX programs in Jalview ( http://www . jalview . org ) .", "Black box corresponds to the region surrounding the RING domain .", "( B ) Maximum likelihood tree constructed from the sequences above using LG/G + I model ( best fit model identified with MEGA 6 ( http://www . megasoftware . net ) ) ( Hall , 2013 ) .", "This maximum likelihood tree appears to have similar grouping to that reported in the BLAST similarity network by Chelysheva et al . ( Chelysheva et al . , 2012 ) showing the HEI10 proteins as one group and the Zip3-like proteins ( including Zip3 , ZHP-3 and RNF212 ) as the other group .", "In this analysis Vilya is positioned within the Zip3-like group . DOI: http://dx . doi . org/10 . 7554/eLife . 08287 . 023 Our model further suggests that only those DSBs that recruit sufficient Vilya to form foci are converted into crossovers and can be visualized by the discrete prominent foci seen in early/mid pachytene .", "We base this proposal on the observation in budding yeast that the differential enrichment of Zip3 at DSBs positively biases the DSB toward the crossover pathway ( Serrentino et al . , 2013 ) .", "We know from our studies that exogenous DSBs have the ability to recruit or concentrate Vilya to a subset of them , in the absence of SC there appears to be a failure to maintain Vilya3XHA at discrete foci in early/mid-pachytene oocytes , and our immuno-EM analysis of oocytes expressing vilya3XHA indicate that Vilya is indeed a component of the RN .", "Together , these data suggest that Vilya plays an active role at the sites of crossing over .", "Although we do not have direct evidence that Vilya controls DSB fate by promoting crossover maturation , our demonstration that Vilya is recruited to the sites of DSBs in a similar number and position to that of RNs strongly supports this hypothesis .", "It is very tempting to place Vilya within the Zip3 family of homologs based on findings presented here .", "DIOPT , the Drosophila RNAi Screening Center Integrative Ortholog Prediction Tools ( Hu , et al . , 2011 ) , predicts that Vilya is orthologous to S . cerevisiae Zip3 ( also known as Cst9 ) and C . elegans Zhp-3 , however , no homologs for Vilya were identified in other organisms including mice and humans using this tool ( FlyBase ) .", "Here , we show in Figure 10—figure supplement 1A the sequence alignment of Vilya to Zip3 homologs of selected plants , fungi , worms , and vertebrates .", "These selected sequences were used to generate a maximum likelihood tree to show the relationship of Vilya to these homologs ( Figure 10—figure supplement 1B ) .", "Consistent with the BLAST similarity network findings of Chelysheva et al . ( Chelysheva et al . , 2012 ) , two groups could be identified .", "One contains the Hei10-like homologs of plants , fungi and vertebrates; and one contains the Zip3-like members including Zip3 , Zhp-3 and RNF212 .", "This analysis suggests that evolutionarily Vilya is more closely related to the Zip3-like members than Hei10 members .", "In addition , based on predicted protein structure and domains , there is an overall similarity between members within the Zip3 group members and Vilya .", "These similarities include an N-terminal RING domain ( see Figure 10—figure supplement 1A ) , which predicts E3 ligase activity , an internal coiled-coil domain , and a C-terminal serine-rich domain , which many Zip3 members possess ( Reynolds et al . , 2013 ) .", "We have shown above that Vilya’s RING domain is required for the interaction with Mei-P22 in a yeast two-hybrid assay , and its coiled-coil domain suggests Vilya would localize to the central region of the SC .", "In addition to these three domains , Vilya also has a putative SUMO-interacting motif ( SUMO-IM ) and three putative RXL cyclin-binding domains that are also common to Zip3 family members ( Figure 1—figure supplement 1E ) ( Cheng et al . , 2006; Ward et al . , 2007; De Muyt et al . , 2014 ) .", "In budding yeast , the SUMO-IM in Zip3 has been shown to be required , along with the RING domain , for its interaction with the E2 enzyme Ubc9 .", "These domains are also thought to be required for Zip3’s SUMO E3 ligase activity in vivo ( Cheng et al . , 2006 ) .", "Studies in Sordaria have shown that the RXL motif of Hei10 , along with the RING domain , are required to modulate the levels of SUMOylation along the SC ( De Muyt et al . , 2014 ) .", "Although we have not specifically determined whether the SUMO-IM and RXL motifs in Vilya are required for its function , in preliminary studies we have not been able to detect an interaction between Vilya and the Drosophila Ubc9 in a yeast two-hybrid assay .", "Nor have we been able to identify SUMOylation of proteins at the SC in Drosophila oocytes using an antibody to Drosophila SUMO ( Abgent AP1287b , San Diego , CA ) .", "Therefore , although structurally Vilya bears a strong resemblance to the Zip3 family , we have no evidence Vilya possess SUMO E3 ligase activity , interacts directly with an E2 enzyme , or that SUMOylation plays a role at the SC in Drosophila at this time .", "Vilya is also similar to some members of the Zip3 family in terms of its dynamic localization pattern .", "In early pachytene , using the expression system shown in this manuscript , Vilya3XHA localizes along linear tracks as well as discrete foci .", "As the oocyte progresses into early/mid pachytene , the discrete foci predominate and the signal along the central region of the SC appears to diminish ( see Figure 3A ) .", "In oocytes that have progressed past mid-pachytene , the discrete foci disappear and the signal along the central region becomes uniform ( see Figure 3—figure supplement 1A–B ) .", "This dynamic localization is unlikely to be a result of the overexpression system we are using because in this same system in the absence of meiotic recombination initiation we fail to observe this pattern and only linear central region staining is present .", "Moreover , similar dynamic localization patterns have been seen for C . elegans Zhp-3 , mouse RNF212 , and Arabidopsis and rice Hei10 ( Jantsch et al . , 2004; Bhalla et al . , 2008; Chelysheva et al . , 2012; Wang et al . , 2012; Reynolds et al . , 2013 ) .", "The general dynamic trend seems to begin with the localization along the SC as continuous linear tracks or arrays of discrete foci , and culminates with localization at discrete foci that mark the sites of crossing over .", "In the absence of meiotic recombination initiation in C . elegans ( Bhalla et al . , 2008 ) and mice ( Reynolds et al . , 2013 ) , Zhp-3 and RNF212 , respectively , were also found to localize along the SC , albeit the SC of nonhomologous chromosomes in mice , and failed to coalesce into discrete foci .", "However , we cannot rule out that the reappearance of Vilya3XHA to the central region of the SC in late pachytene is not a consequence of this ectopic expression since we have not discerned a later function for Vilya .", "Although Vilya clearly has a unique function not found in any other Zip3 family member so far ( i . e . being required for meiotic DSB formation ) , other unique functions for some Zip3 family members have been identified as well .", "For example , Zip3 in budding yeast appears to be the only member required for SC assembly ( Agarwal and Roeder , 2000; Bhalla et al . , 2008 ) , and Hei10 in Sordaria is uniquely required for processes involving spindle pole body dynamics ( De Muyt et al . , 2014 ) .", "Because DSB accessory proteins are highly divergent and Drosophila appear to lack homologs of the meiosis-specific MutS complex ( Yokoo et al . , 2012 ) , which are required to stabilize strand invasion during crossing over , and the crossover-specific complex MutLγ , to which Zip3 family members bind , perhaps then it is not surprising that Drosophila has found an unique way to couple the events of DSB formation to those of crossing over ." ], [ "All Drosophila strains were maintained on standard food at 25°C unless otherwise noted .", "Descriptions of genetic markers and chromosomes can be found at http://www . flybase . org .", "Wild-type strains used in the manuscript were y w FRT19A , the parent chromosome used to generate vilya826 , or Canton-S .", "Deficiency strains used for mapping were obtained from the Bloomington Drosophila Stock Center .", "Deficiency Df ( 1 ) ED6630 ( BL8948 ) uncovers vilya .", "Other stocks used in this study include Pnos-Gal4::VP16 ( Van Doren et al . , 1998 ) , Pw +; UASp-vilya3XHA ( this study ) , net dp ho b mei-W684572 ( Lake , et al . , 2013 ) mei-W684572 ( Bhagat et al . , 2004 ) , st spnBBU sr e/TM6B ( Ghabrial et al . , 1998 ) , okraAA cn bw/CyO and okraRU cn bw/CyO ( Ghabrial et al . , 1998 ) , mei-P22103 st/TM3 , Sb and mei-P22103 th st cu e ca/TM3 , ry Sb ( Liu et al . , 2002 ) and c ( 3 ) G68 e and c ( 3 ) G68 e ca ( Jeffress et al . , 2007 ) .", "okra refers to the genotype okraRU/okraAA .", "mei-W68 refers to the genotype net dp ho b mei-W684572/mei-W684572 .", "mei-P22 refers to the genotype mei-P22103 st/mei-P22103 th st cu e ca .", "c ( 3 ) G refers to the genotype c ( 3 ) G68 e/c ( 3 ) G68 e ca .", "The meiotic mutation mei-826 ( Collins et al . , 2012 ) was mapped by standard genetic assays .", "Recombination mapping placed the lesion between sc ( 1A8 ) and w ( 3B6 ) , and deficiency mapping placed the mutation within the interval 3B1–3C5 due to the failure of mei-826 to complement the Bloomington deficiency Df ( I ) ED6630 ( BL8948 ) .", "Several genes within this region were selected as potential candidates , PCR amplified , and sequenced to look for potential EMS-induced lesions .", "One nonsense mutation ( C640T ) was identified in gene CG2709 that resulted in a stop codon at amino acid 214 ( R214STOP ) .", "CG2709 was renamed vilya , and the mei-826 mutant was subsequently renamed vilya826 .", "We named this gene vilya , as vilya encodes a protein with a RING domain , and Vilya is considered to be the mightiest of the Three Rings of Power given by the Elves of Eregion .", "To obtain the coding sequence ( CDS ) of vilya , cDNA was made using ImProm-II Reverse Transcription Kit System ( Promega , Madison , WI ) and Trizol extracted RNA from y w FRT19A egg chambers ( stage 1–10 ) with oligo-dT primer supplied with the kit .", "PCR was performed on the cDNA with gene-specific primers ( 5’-taccatggcgaaatcacaagcagg-3’ and 5’-atcgctagctcacagatcgaacga-3’ ) , directly cloned into TOPO-TA vector ( Life Technologies , Grand Island , NY ) , and confirmed by Sanger sequencing , resulting in the plasmid pTOPO-vilya .", "vilya was amplified from pTOPO-vilya and cloned into pBS-KS + ( Clontech , Mountain View , CA ) using primers containing XbaI restriction sites on both 5’ and 3’ ends and an internal NheI engineered restriction site immediately upstream of the stop codon ( 5’--ggcgtctagaatggcgaaatcacaagcaggtc-3’ and 5’-ctggtctagatcagctagccagatcgaacgagttgttcggc-3’ ) .", "The NheI site was used to clone in the 3X hemagglutinin ( 3XHA ) tag that had been previously amplified from the pPFHW vector ( DGRC , Bloomington , IN ) with primers containing flanking NheI sites ( 5’-tcgcgctagctacccatacgatgttcc-3’ and 5’-gctcgctagcagcgtaatctggaacg-3’ ) to create the vector pBS-vilya3XHA .", "After confirmation of sequence and orientation of 3XHA , vilya3XHA was digested out of pBS-vilya3XHA with XbaI and cloned into pUASp-attB ( Takeo et al . , 2010 ) at the XbaI site and sequenced for directionality .", "pUASp-attB-vilya3XHA was introduced into Drosophila by targeted integration using the attP-40 line ( Genetic Services , Boston , MA ) .", "To measure the frequency of nondisjunction and meiotic recombination on the X chromosome , virgin females of the listed genotype were crossed individually to y sc cv v f / B[S]Y males ( Zimmering , 1976; Matsubayashi and Yamamoto , 2003 ) .", "To assay meiotic recombination , only female progeny resulting from the above cross were analyzed for the markers cv , v and f .", "To obtain the frequency of nondisjunction when using Df ( 1 ) ED6630 , normal male progeny were doubled due to the inability to recover Df ( 1 ) ED6630 males in the assay .", "To assay both X and 4th chromosome nondisjunction , tester female virgins were crossed to X^Y , In ( 1 ) EN , v f B; C ( 4 ) RM , ci eyR males .", "Calculations were performed as previously described ( Zitron and Hawley , 1989; Hawley et al . , 1992 ) .", "To assay meiotic recombination on the 3rd chromosome , tester female virgins ( +/+; ru h th st cu sr e ca/+ or vilya826; ru h th st cu sr e ca/+ ) were crossed to +/Y; ru h th st cu sr e ca males and the resulting female progeny were scored for all markers .", "To verify vilya was not haploinsufficient , we assayed as above for a meiotic defect of the X chromosome deficiency ( BL8948 ) with the original y w FRT19A chromosome that the mutation was induced on ( Collins et al . , 2012 ) .", "No meiotic phenotype was observed , indicating that vilya is not haploinsufficient ( data not shown ) .", "The Matchmaker Gold Yeast Two-Hybrid System User Manual ( Clontech , Mountain View , CA ) was followed for yeast transformation and for yeast two-hybrid assays .", "AH109 yeast were used in place of Y2Hgold .", "AH109 genotype is as follows: MATa , trp1-901 , leu2-3 , 112 , ura3-52 , his3-200 , gal4Δ , gal80Δ , LYS2: : GAL1UAS-GAL1TATA-HIS3 , GAL2UAS-GAL2TATA-ADE2 , URA3: : MEL1UAS-MEL1 TATA-lacZ .", "Y187 genotype is as follows: MATα , ura3-52 , his3-200 , ade2-101 , trp1-901 , leu2-3 , 112 , gal4Δ , met– , gal80Δ , URA3: : GAL1UAS-GAL1TATA-lacZ .", "Bait and prey vectors used were pGBKT7 and pGADT7 , and cDNAs were cloned into the vectors using compatible restriction sites within the vector and contained within the primers .", "The CDS for vilya was obtained as above using primers 5’-gcggcatatggcgaaatcacaagcaggtc-3’ and 5’-tcgcctgcagtcacagatcgaacgagttg-3’ for full-length vilya or 5’-gcggcatatggcgaaatcacaagcaggtc-3’ and 5’-tcgcctgcagtcagcgtcgactggaggac-3’ for vilya826 .", "The CDS for mei-P22 was obtained from the DNA of Canton-S and is identical to the sequence on FlyBase ( D . melanogaster Release 6 ) .", "Primers used for cloning full-length mei-P22 were 5’-ggcgtcgcatatggacaggacaacagttgt-3’ and 5’-ggcgctcgagctaaggtacttccaattc-3’ , and primers used for cloning mei-P22103 were 5’-ggcgtcgcatatggacaggacaacagttgt-3’ and 5’-ggcgctcgagtcactccaagtcaacgttcaacatgg-3’ .", "Mutations in vilya cDNA were made using the Quik Change II XL Site-Directed Mutagenesis Kit ( Stratagene , CA ) .", "The vector pTOPO-vilya ( above ) was used to generate the site-directed mutants , and each was cloned into the yeast expression vector ( pGBKT7 ) using the primers above .", "Protein expression of each of the Vilya point mutants , as well as full-length wild-type protein , was verified by Western blotting .", "Briefly , a 50 mL culture of transformed Y187 yeast cells was inoculated from an over-day 5 mL culture in minimal media lacking Trp .", "After 7 hr , the OD600 was determined , and equal amounts of yeast based on OD600 were pelleted before being frozen .", "Cells were thawed into 0 . 1 M sodium hydroxide containing 1X protease inhibitor ( Sigma , MO ) and incubated at RT for 5 min .", "Equal amounts of 2X SDS loading dye containing β-mercaptoethanol was added , samples were boiled 5 min , pelleted , and lysate loaded onto a 12% SDS-PAGE gel .", "Protein was transferred onto PVDF membrane .", "For detection of expressed protein , an antibody to the c-Myc epitope , found within the pGBKT7 vector upstream and in-frame , was used ( anti-c-Myc clone 9E10 , Abcam , MA ) at 1:1000 dilution overnight in PBS containing 0 . 1% Tween20 and 4% dry powdered milk .", "After washing , a secondary alkaline phosphatase-conjugated goat anti-mouse antibody at 1:5000 was added for 2 hr .", "The bound antibodies were detected by reacting with substrate solution containing 5-bromo-4-cholor-indolyl-phosphase and 4-Nitro Blue Tetrazolium chloride .", "Germarium preparations for whole mount immunofluorescence were prepared as according to ( Page and Hawley , 2001 ) with minor exceptions .", "Three- to five-day-old females were collected and yeasted overnight in the presence of males .", "Ovaries were dissected in PBS for no longer than 20 min prior to fixing ( 200 µL of PBS containing 2% formaldehyde ( Ted Pella , Redding , CA ) and 0 . 5% Nonidet P-40 plus 600 µL heptane ) at room temperature .", "Ovaries were then washed three times for 10 min in PBS with 0 . 1% Tween ( PBST ) .", "Late stage egg chambers were removed by cutting the ovaries with forceps , and the ovarioles containing the germarium tips were teased apart before being blocked in PBST with 1% bovine serum albumin ( BSA ) ( EMD Chemicals , San Diego , CA ) for one hr .", "Primary antibody diluted in PBST was incubated with germarium tips overnight at 4°C while nutating .", "After washing three times for 20 min in PBST the secondary antibodies were added for 4 hr followed by the addition of 4’6-diamididino-2-phenylindole ( DAPI ) at a concentration of 1µg/ml for the final 10 mins of incubation .", "Ovary material was washed as before and the samples were mounted in ProLong Gold ( Life Technologies , Grand Island , NY ) .", "For immuno-EM samples , three- to five-day-old mated females were yeasted overnight .", "Ovaries from five to seven females were dissected in cold Ringer’s solution and fixed at 20°C for 30 min ( inverting every 10 min ) in 200 µL of PBS containing 3% EM-grade formaldehyde ( Ted Pella , Redding , CA ) and 0 . 5% Nonidet P-40 , plus 600 µL of hexane .", "Ovaries were washed in PBST as above and quenched with fresh 0 . 1 M ammonium chloride in PBS .", "Following another set of washes , ovaries were blocked for 1 hr at RT in 1% BSA in PBST .", "Primary antibodies were added and incubated in 1% BSA PBST overnight at 4°C .", "The ovaries were washed 6 x 10 min each in PBST and incubated with secondary for 4 hr in 1% BSA , 0 . 1% cold water fish gelatin ( Electron Microscopy Sciences , Hatfield , PA ) , and 2% normal goat serum in PBST .", "Secondary antibodies used were anti-rabbit Alexa-488 and anti-rat ultra-small gold ( Electron Microscopy Sciences , Hatfield , PA ) .", "After washing 6 x 10 min , ovaries were post-fixed as before except at RT .", "Ovaries were then washed in distilled water for 3 x 20 min and gold was enhanced with Aurion R-GENT SE-EM ( Electron Microscopy Sciences , Hatfield , PA ) for 1 hr 15 min .", "Following silver enhancement , samples were treated with 0 . 03 M sodium thiosulfate in distilled water for 5 min , followed by 3 x 10 min washes with distilled water .", "Ovaries were then post-fixed in 1% OsO4 in PBS for 30 min at RT , washed as before with water and dehydrated in ethanol .", "Samples were embedded in epoxy resin at RT for two days followed by polymerization at 60° for two days .", "Serial sections ( 50 nm thick ) were cut and transferred to formvar-carbon-coated slot grids and stained with aqueous uranyl acetate and lead citrate .", "Sections that contained SC were first identified by immunofluorescence , and those sections were then imaged on an FEI transmission electron microscope ( 80 kv ) .", "Primary antibodies used include mouse anti-C ( 3 ) G 1A8-1G2 ( 1:500 ) ( Anderson et al . , 2005 ) , affinity-purified rabbit anti-Corolla ( animal 210 ) ( 1:2000 ) ( Collins et al . , 2014 ) , rat anti-CID ( 1:2000 ) ( gift of Sunkel Laboratory ) ( Martins et al . , 2009 ) , mouse anti-Orb antibodies 4H8 and 6H4 ( 1:40 each ) ( Developmental Studies Hybridoma Bank , Iowa ) , mouse anti-γ-H2AV ( 1:1000 ) ( Lake et al . , 2013 ) , mouse anti-HA . 11 ( Covance , Princeton , NJ ) , and high-affinity rat anti-HA clone 3F10 ( 1:100 IF or 1:50 immuno-EM ) ( Roche , Indianapolis , IN ) .", "Secondary goat anti-mouse , rabbit or rat Alexa-488 , Alexa-555 and Alexa-647 IgG H&L chain conjugated antibodies were used at 1:500 ( Molecular Probes , Life Technologies , Grand Island , NY ) , and secondary goat anti-rat ultra-small gold IgG H&L chain conjugated antibody ( 1:50 ) ( Electron Microscopy Sciences , Hatfield , PA ) .", "Images were acquired with a DeltaVision microscopy system ( GE Healthcare , Piscataway , NY ) consisting of a 1x70 inverted microscope with a high-resolution CCD camera or an Applied Precision OMX Blaze microscope ( Issaquah , WA , USA ) equipped with a PCO Edge sCMOS camera .", "Images were deconvolved ( DeltaVision and OMX ) and reconstruction was performed ( OMX ) using SoftWoRx v . 6 . 1 software ( Applied Precision/GE Healthcare ) following Applied Precision protocols .", "To analyze the specificity of the rat HA antibody for Vilya3XHA protein we compared staining of anti-HA on vilya3XHA-expressing tissue to staining on wild-type tissue .", "We prepared samples for each in parallel .", "We acquired five germarium and five late stage images of vilya3XHA-expressing tissue using a target intensity value of 3000 on the DeltaVision microscopy system for each filter ( DAPI , TRITC ( anti-HA ) and Cy5 ( anti-Corolla ) .", "We recorded the percent transmission ( which stayed consistent ) and exposure time in each channel for each of the acquired images .", "We averaged the five exposure times for each filter .", "We fixed these as values for our wild-type image acquisition .", "The images were then deconvolved as above .", "In addition , we recorded the exposure time on wild type for each of the filters when using a target intensity value of 3000 .", "These were then averaged as before and compared to the averages of vilya3XHA-expressing tissue as a ratio of average exposure time in vilya3XHA-expressing ovaries to average exposure time in wild type .", "For the DAPI the ratio was 1 . 0:0 . 9 , TRITC ( HA ) 1 . 0:5 . 78 and Cy5 ( Corolla ) 1 . 0:1 . 2 .", "Thus in order to reach the same intensity value , the exposure time had to be increased on wild-type tissue by almost six times .", "The analysis of centromere clustering was performed as previously described ( Takeo et al . , 2011 ) , where individual oocyte nuclei were scored for the number of CID foci by analyzing CID staining throughout each section of the nucleus in SoftWoRx .", "To determine the number of γH2AV foci or Vilya3XHA foci , we used Imaris software 7 . 7 . 2 ( Bitplane , Zurich , Switzerland ) to crop in 3D each oocyte using the SC to define the sections pertaining to each nucleus .", "Using Imaris software , we displayed each z-section using the gallery function and only clearly defined foci were counted manually in the corresponding z-series .", "To determine the colocalization frequency of γH2AV and Vilya3XHA foci , we performed 3D crop of the selected nuclei as above .", "We identified the number of Vilya3XHA foci for each nucleus analyzed as described above .", "We then rotated the images in 3D to verify that the γH2AV signal was not simply above or below the Vilya3XHA focus .", "We scored those signals that overlapped , as well as those signals that were adjacent ( no apparent gap between the foci ) , but not separated above or below in 3D , as being associated .", "To verify the relevance of their association , we took each 3D cropped oocyte nucleus and rotated the channel for the Vilya3XHA foci by 180 degrees using ImageJ software .", "First , we split each of the channels of the image .", "We selected the channel with the Vilya3XHA foci , and used the transform function to flip the z-series horizontally .", "We then used the stack tool to reverse the stack .", "Together these two manipulations are equivalent to a 180 degree rotation of the Vilya3XHA channel .", "After merging the channels back together , we again analyzed the association of γH2AV and Vilya3XHA foci as before .", "3D projections and tracing of SC between homologous chromosome arms was performed using Imaris software , and maximum intensity projections were made unless otherwise noted .", "Image J custom plugins for straightening of Imaris spot profiles are available at http://research . stowers . org/imagejplugins .", "For immunofluorescence analysis of DSBs created by X-ray , three- to five-day-old mated females were exposed to 1000 rad of X-ray at a dose of 112 rad/min .", "Ovaries from treated ( or non-treated control females ) were collected and fixed as above 5 hr after X-ray treatment .", "Ovaries from three- to five-day mated females that had been yeasted for one day were dissected .", "FISH and immunofluorescence was performed as previously described ( Blumenstiel et al . , 2008 ) using amine-labeled probes made with ARES Alex Fluor DNA labeling kit ( Invitrogen Life Technologies , Grand Island , NY ) for euchromatic region 14 .", "Overlapping region 14 BACs were labeled and used ( BACR03G18 , BACR06P10 and BACR13G13 ) ( CHORI ) .", "Pairing was determined as previously described ( Page et al . , 2008; Joyce et al . , 2013 ) ." ] ]

[ "Meiotic recombination begins with the induction of programmed double-strand breaks ( DSBs ) .", "In most organisms only a fraction of DSBs become crossovers .", "Here we report a novel meiotic gene , vilya , which encodes a protein with homology to Zip3-like proteins shown to determine DSB fate in other organisms .", "Vilya is required for meiotic DSB formation , perhaps as a consequence of its interaction with the DSB accessory protein Mei-P22 , and localizes to those DSB sites that will mature into crossovers .", "In early pachytene Vilya localizes along the central region of the synaptonemal complex and to discrete foci .", "The accumulation of Vilya at foci is dependent on DSB formation .", "Immuno-electron microscopy demonstrates that Vilya is a component of recombination nodules , which mark the sites of crossover formation .", "Thus Vilya links the mechanism of DSB formation to either the selection of those DSBs that will become crossovers or to the actual process of crossing over ." ]

[ "DNA in animal cells is arranged into structures called chromosomes .", "Usually , a cell divides in such a way that both daughter cells inherit a complete set of chromosomes .", "However , the sex cells ( sperm and egg cells ) are formed in a different process – called meiosis – that results in these cells having only half the number of chromosomes that the parent cell had .", "This ensures that when animals reproduce , an egg cell and a sperm fuse together to make a new cell that contains a full set of chromosomes .", "During meiosis , sections of chromosomes are rearranged so that the sperm and egg cells will end up with different combinations of the DNA inherited from the animal's mother and father .", "Matching chromosomes from the mother and father pair up with each other and the DNA - which is made of two strands - breaks at precise locations throughout the chromosomes .", "Then , sections of DNA around the double-strand breaks are exchanged between the matching chromosomes by a process known as crossover .", "An incorrect number of double-strand breaks , or a failure to position them properly , can lead to genetic abnormalities like Down's syndrome , in which cells contain the wrong number of chromosomes .", "Cells tightly regulate the formation of double-strand breaks , but in most organisms the number of breaks formed exceeds the number of crossovers .", "This implies that there must be a process that selects certain double-strand breaks to form crossovers .", "Although fruit flies are often used as a model to study animal cells , we do not know how they select which double-strand breaks will form crossovers .", "Lake et al . studied meiosis in fruit flies and identified a new protein called Vilya that is required for double-strand breaks to form .", "This protein is similar to a group of “Zip3-like” proteins that act on double-strand breaks in other animals .", "Vilya is found at the double-strand break sites that go on to form crossovers and interacts with a small protein called Mei-P22 , which is known to be involved in the formation of double-strand breaks .", "Lake et al . apos;s findings show that Vilya links the process of forming double-strand breaks to the selection of the breaks that will undergo crossover .", "Future studies will focus on understanding the molecular details of how Vilya works ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "computational and systems biology", "microbiology and infectious disease" ]

[ [ "The human gut microbiome is a complex , spatially heterogeneous and dynamic ecosystem consisting of hundreds of species interacting with each other and with the human host .", "It is a daunting task to develop predictive models for such a system , yet the potential rewards are high and would , for instance , enable targeted interventions to shift dysbiotic communities towards more healthy states .", "Two conditions need to be fulfilled for predictive models to be successful: first , the system has to be sufficiently well characterized to build the model; and , second , the dynamics should be generally deterministic .", "First successes in modeling the behavior of gut microbial communities give reason for cautious hope ( Buffie et al . , 2015; Cremer et al . , 2017; Muñoz-Tamayo et al . , 2016; Stein et al . , 2013 ) .", "Most of these studies took a top-down approach , in which the change in composition of an entire community in vivo is modeled .", "For instance , Cremer et al . ( 2017 ) predicted the ratio of Firmicutes and Bacteroidetes in fecal samples as a function of estimated water content and nutrient influx using a diffusion model .", "Others have fitted population models to time series of taxon ( mostly genus ) abundances obtained from 16S rRNA gene sequencing .", "For instance , one study fitted a variant of the generalized Lotka-Volterra ( gLV ) model to a cecal gut time series of mice exposed to the pathogen Clostridium difficile , an antibiotic or both , thereby inferring the interactions between different genera ( Stein et al . , 2013 ) .", "The same approach was also used to predict species that inhibit C . difficile growth in murine and human microbiota , one of which significantly lowered mortality when transferred to mice before infection with C . difficile ( Buffie et al . , 2015 ) .", "Despite these successes , the gLV model and its variants have several drawbacks that limit their widespread application .", "gLV-type models describe species dynamics as a function of their growth rates and pairwise interactions , without taking the concentrations of exchanged metabolites into account .", "Thus , they assume that community dynamics can be predicted from pair-wise interactions and that the interaction mechanisms can be ignored .", "These assumptions have recently been tested both experimentally and computationally: Friedman et al . ( 2017 ) experimentally quantified the accuracy reached when predicting the behavior of more complex soil communities from species pairs , whereas Momeni et al . ( 2017 ) systematically compared LV models of metabolite-mediated species interactions to their mechanistic counterparts .", "While the authors in the former case concluded that the behavior of larger communities could , to a considerable extent , be predicted from that of smaller ones , the latter study showed that the ( extended ) gLV model cannot accurately describe several common types of interaction mechanisms .", "An alternative to the gLV model and its variants are mechanistic models , which in contrast to gLV models account for metabolite-mediated interactions by explicitly describing the dynamics of the produced and consumed compounds ( see Momeni et al . , 2017 and references therein ) .", "They thus require more system knowledge than generic gLV and related models do .", "However , most members of the gut community have not been thoroughly characterized , and little is known about their responses to different nutrients , pH values and interaction partners , even for those that have been studied more closely .", "It is challenging to obtain this type of biological knowledge and to resolve interaction mechanisms in vivo .", "However , in vitro studies allow the acquisition of detailed knowledge not only of the microorganisms' pH and nutrient preferences but also of their behavior in the presence of other microorganisms .", "In vitro studies of human gut microorganisms have a long tradition and have been carried out in several different ways .", "Classical mono- and co-culture studies in batch and chemostat fermentors have explored nutrient preferences and interaction mechanisms ( Falony et al . , 2006; Falony et al . , 2009a; Moens et al . , 2016; Moens et al . , 2017; Rivière et al . , 2016 ) .", "Artificial gut systems , such as the TNO In Vitro Model of the Colon ( TIM-2 ) ( Venema , 2015 ) and the Simulation of the Human Intestinal Microbial Ecosystem ( SHIME ) ( Van de Wiele et al . , 2015 ) , seek to reproduce the conditions of the human gastro-intestinal tract as closely as possible and in a well-controlled manner .", "The gut community has also been studied in vitro at smaller scales , in minibioreactor arrays ( Auchtung et al . , 2015 ) and with gut-on-chip microfluidic devices ( Kim et al . , 2012; Shah et al . , 2016 ) .", "In most cases , however , gut simulators are inoculated with fecal material .", "In the range from top-down to bottom-up approaches that explore gut microbial community dynamics , these can be considered as intermediate cases , in which the host is eliminated but the community is not further simplified .", "The goal of these studies is usually to quantify the behavior of the entire community under different conditions .", "In the cases of HuMiX and of SHIME's HMI module , the interaction of particular gut microorganisms with epithelial cells is targeted ( Marzorati et al . , 2014; Shah et al . , 2016 ) .", "As the exact composition of fecal material ( which also includes bacteriophages and fungi ) is difficult to resolve , it is hard to track each member in such a community .", "Although the in vitro dynamics of colon ( Kettle et al . , 2015 ) and rumen ( Muñoz-Tamayo et al . , 2016 ) communities has been described with mechanistic models previously , these models did not account for the behavior at species level , and instead grouped species with similar metabolic activities into guilds .", "While it is of interest to model guild dynamics , the resolution of guild-level models may be insufficient to provide an understanding of microbial community dynamics in the gut .", "Species in the same guild do not necessarily respond in the same manner to altered environments and perturbations .", "Guild definitions are arbitrary to an extent , and gut bacteria with flexible metabolic strategies may change their guild membership .", "In addition , the concepts of tipping elements ( Lahti et al . , 2014 ) and strongly interacting species ( Gibson et al . , 2016 ) suggest that particular species can have a disproportionate impact on gut community dynamics .", "In our opinion , experiments using defined communities of known composition , grown under well-controlled conditions , are crucial to learn more about the interactions of gut species and how these shape community dynamics .", "Well-controlled in vitro experiments are also necessary for the development and validation of predictive models of gut microbial communities .", "However , only a few in vitro experiments with defined gut communities have been reported to date ( Newton et al . , 2013; Trosvik et al . , 2008; Trosvik et al . , 2010 ) and only one study has , to our knowledge , employed mechanistic models to predict community dynamics in the infant gut microbiome ( Pinto et al . , 2017 ) .", "The objective of the present study was therefore to establish a defined community composed of human strains that are representative of the adult gut microbiome , to study their interactions under well-controlled conditions in vitro and to validate a quantitative mechanistic model by predicting community behavior in a tri-culture with parameters from mono-culture data .", "Mechanistic models have been tested in this manner before for a cystic fibrosis community ( Schmidt et al . , 2011 ) but such an approach has not yet been applied to a synthetic gut community .", "To reach our objective , we created a synthetic community composed of three abundant and typical members of the human gut microbiome: Faecalibacterium prausnitzii A2-165 ( Duncan et al . , 2002b ) , Roseburia intestinalis L1-82 ( Duncan et al . , 2002a ) and Blautia hydrogenotrophica S5a33 ( Bernalier et al . , 1996 ) .", "All three strains were isolated from human feces and their draft genomes are available .", "Furthermore , they are of particular medical relevance because of the ability of two of these strains ( R . intestinalis L1-82 and F . prausnitzii A2-165 ) to produce butyrate , a beneficial short chain fatty acid that is an important energy source for gut epithelial cells ( Geirnaert et al . , 2017; Rivière et al . , 2016 ) .", "Butyrate producers are often depleted in dysbiotic gut microbiota relative to healthy controls ( Antharam et al . , 2013; Rivera-Chávez et al . , 2016 ) .", "Thus , high butyrate production will probably be a quality criterion for bacterial cocktails designed for therapeutic purposes .", "In R . intestinalis L1-82 , fermentation of carbohydrates results in the production of butyrate as well as hydrogen gas and carbon dioxide ( Duncan et al . , 2002a; Falony et al . , 2009c ) , whereas F . prausnitzii A2-165 produces formate in addition to butyrate and requires acetate for growth ( Duncan et al . , 2002b; Moens et al . , 2016 ) .", "B . hydrogenotrophica S5a33 is able to grow on carbon dioxide and hydrogen gas , but also on glucose and fructose , in all cases generating acetate ( Bernalier et al . , 1996 ) .", "Therefore , as Figure 1 illustrates , our community contains multiple cross-feeding and competitive interactions .", "For instance , all three strains compete for fructose .", "B . hydrogenotrophica S5a33 can use the hydrogen gas generated by R . intestinalis L1-82 as well as the carbon dioxide and formate produced by both R . intestinalis L1-82 and F . prausnitzii A2-165 .", "In turn , B . hydrogenotrophica S5a33 provides acetate that is beneficial to R . intestinalis L1-82 and F . prausnitzii A2-165 .", "This system thus constitutes a rare example of two strain pairs that simultaneously compete and mutually cross-feed .", "The three strains were grown as mono- , bi- , or tri-cultures in 2 L laboratory fermentors in batch mode .", "We monitored the dynamics of each combination by quantifying bacteria through optical density ( OD ) , flow cytometry and qPCR and by measuring the concentration of substrates and fermentation products , including short chain fatty acids and gasses .", "Finally , we sequenced the total RNA in selected samples .", "Figure 2 summarizes our approach .", "To our knowledge , this is the first study to investigate a synthetic gut community with a combination of mono- and co-cultures , mechanistic modeling and gene expression analysis ." ], [ "We first confirmed the cross-feeding interactions postulated for B . hydrogenotrophica S5a33 with small-volume screening experiments , in which the pH was not kept constant and the atmosphere contained 10% carbon dioxide and 10% hydrogen gas .", "We found that under these conditions , B . hydrogenotrophica S5a33 was able to grow heterotrophically on formate , which was entirely consumed .", "Although we did not quantify gasses during screening and therefore could not ascertain the consumption of carbon dixoide and hydrogen gas , we observed growth in the absence of an added carbon source , indicating autotrophic growth as described previously ( Bernalier et al . , 1996 ) .", "Presumably , both formate and carbon dioxide are assimilated via the Wood-Ljungdahl pathway , of which all required genes are present in the genome of B . hydrogenotrophica S5a33 according to the AGORA database ( Magnúsdóttir et al . , 2017 ) .", "We also found that B . hydrogenotrophica S5a33 grew on fructose , oligofructose and glucose , as reported by Rey et al . ( 2010 ) for B . hydrogenotrophica S5a36 , and documented partial consumption of these saccharides .", "For glucose and fructose , the maximal OD tended to be lower than for formate ( mean maximal ODs for glucose: 0 . 3 , fructose: 0 . 6 , formate: 1 . 3 ) .", "In agreement with ( Bernalier et al . , 1996 ) , we detected lactate in addition to acetate for these substrates and confirmed lactate production in the presence of fructose in the fermentor .", "Notably , when growing B . hydrogenotrophica S5a33 on formate but without fructose in the fermentor , carbon dioxide and hydrogen gas were produced besides acetate , but lactate was absent .", "B . hydrogenotrophica S5a33 also consumed small concentrations of galactose , but did not consume fucose , inulin or lactate .", "In conclusion , we confirmed the potential competition between B . hydrogenotrophica S5a33 and the two primary fermenters for fructose as well as the potential cross-feeding of formate .", "We employed pH-controlled mono-cultures to characterize the properties and growth kinetics of the individual strains in our model .", "Table 1 provides an overview of all of the fermentation experiments carried out , whereas Supplementary file 1 gives additional information for each experiment .", "When grown in monoculture , R . intestinalis L1-82 consumed fructose and produced butyrate , carbon dioxide and hydrogen gas , as described previously ( Falony et al . , 2009c ) , as well as small amounts of lactate and formate ( Figure 3A ) .", "Interestingly , there was no net consumption of acetate when more fructose than acetate was provided .", "Net acetate consumption has been found to correlate negatively with hydrogen gas production ( Falony et al . , 2009c ) , but here we saw that it also depended on the ratio of initial fructose and acetate .", "When given in equal concentrations , R . intestinalis L1-82 partially consumed acetate .", "Consequently , in all further experiments , when acetate was added , it was added at the same concentration as fructose .", "F . prausnitzii A2-165 in monoculture produced formate , less carbon dioxide and butyrate than R . intestinalis L1-82 and no hydrogen gas , but did not entirely consume fructose ( Figure 3B ) .", "After having excluded a number of explanations — exposure to oxygen ( by adding oxygen gas via sterile water ) , redox potential ( by continuously adding the oxidizing agent potassium ferrocyanide trihydrate ) , pH ( lowered to 5 . 8 ) , a threshold requirement for fructose ( halving the fructose concentration did not stop its consumption ) or end-product inhibition ( by adding initial butyrate ) — we found that doubling the concentration of yeast extract lowered residual fructose concentrations .", "Adding fresh but autoclaved medium during the fermentation did not lower residual fructose concentrations , so we assumed that F . prausnitzii A2-165 was growth-limited by one or several heat-labile co-factor ( s ) present in the yeast extract .", "A recent flux balance analysis with a manually curated metabolic reconstruction suggests that the growth of F . prausnitzii A2-165 requires several amino acids ( L-alanine , L-cysteine , L-methionine , L-serine and L-tryptophan ) and the co-factors biotin ( vitamin B7 ) , cobalamin ( vitamin B12 ) , folic acid ( vitamin B9 ) , hemin , nicotinic acid , pantothenic acid and riboflavin ( vitamin B2 ) ( Heinken et al . , 2014 ) .", "With the exceptions of cobalamin and externally supplied hemin , these nutrients should be present in yeast extract according to the metabolic reconstruction of Saccharomyces cerevisiae iMM904 ( Mo et al . , 2009 ) and , furthermore , the amino acids should be present in other medium components ( bacteriological peptone , soy peptone and tryptone ) .", "According to previous experimental findings as well as the flux balance analysis , F . prausnitzii A2-165 can grow in the presence of oxygen gas ( Heinken et al . , 2014; Khan et al . , 2012 ) , which is in agreement with our observation that the addition of low concentrations of oxygen gas does not alter its growth curve .", "F . prausnitzii A2-165 is assumed to transfer electrons to oxygen through extracellular redox mediators such as riboflavin ( Khan et al . , 2012; Prévoteau et al . , 2015 ) .", "B . hydrogenotrophica S5a33 produced acetate , hydrogen gas , carbon dioxide and small concentrations of lactate , while consuming formate almost entirely ( Figure 3C ) .", "It also consumed fructose , but did not deplete it .", "While the carbon recovery for F . prausnitzii A2-165 and R . intestinalis L1-82 monocultures was close to 100% , it only reached 60% for B . hydrogenotrophica S5a33 in monoculture on formate and fructose .", "These unexpected behaviors defy simple kinetic models typically based on additive Monod functions and necessitate adjustment of the equations .", "We designed a model that described the dynamics of each strain and of key compounds ( including fructose , formate , acetate , butyrate , hydrogen gas and carbon dioxide ) with ordinary differential equations implementing a combination of additive and multiplicative Monod functions ( see 'Materials and methods' ) .", "The model differentiates between substrates required for growth and co-substrates such as acetate that enhanced growth but were not required .", "It also took strain-specific differences in lag phases into account .", "As we observed that F . prausnitzii A2-165 did not deplete fructose , presumably because of a lack of co-factors , we introduced a dependency on an undefined metabolite referred to as ‘unknown compound’ .", "We parameterized this model on selected monoculture experiments and then predicted monoculture dynamics ( Figure 4A–C , Figure 4—figure supplement 1 ) .", "The model reached high prediction accuracy for F . prausnitzii A2-165 and R . intestinalis L1-82 , but did not describe well the experimental data for B . hydrogenotrophica S5a33 ( see Table 1 ) .", "More precisely , the model showed that B . hydrogenotrophica S5a33 did not consume formate and fructose as quickly as would be expected if its growth follows Monod kinetics .", "We confirmed culture homogeneity by analyzing the16S rRNA gene sequencing data of the last sample ( Figure 3—figure supplement 1 ) .", "A yeast contaminant ( S . cerevisiae S288c ) that was detected in the RNA-seq data for the B . hydrogenotrophica S5a33 monoculture samples ( Figure 3—figure supplement 2 ) does not explain the incongruence between growth and energy source consumption , since", "( i ) no contamination was observed on plates inoculated with bioreactor samples and incubated under anaerobic and aerobic conditions ,", "( ii ) S . cerevisiae would consume fructose , and", "( iii ) no ethanol production was measured .", "We also found only small concentrations of potential peptide degradation products ( isobutyric acid and isovaleric acid ) .", "We therefore assumed that B . hydrogenotrophica S5a33 in monoculture initially grew on undefined medium components and only later switched to formate and fructose , but the time resolution was insufficient to take this potentially biphasic growth into account .", "We also compared model performance for R . intestinalis L1-82 with and without product inhibition by hydrogen gas .", "As we found no differences in model performance , we removed an initial hydrogen gas inhibition term .", "When growing F . prausnitzii A2-165 and B . hydrogenotrophica S5a33 together , we observed that fructose was entirely depleted and that acetate , butyrate , hydrogen gas , carbon dioxide and small concentrations of lactate were produced ( Figure 3F ) .", "Interestingly , there was an initial production of formate , which was then consumed , confirming that formate was cross-fed from F . prausnitzii A2-165 to B . hydrogenotrophica S5a33 .", "Formate consumption was also observed without initial acetate ( Figure 3H ) .", "In the bi-culture of R . intestinalis L1-82 and B . hydrogenotrophica S5a33 , carbon dioxide , hydrogen gas , butyrate and small concentrations of lactate were produced , whereas fructose and a small amount of acetate were consumed ( Figure 3D ) .", "The same fermentation products were also obtained in the absence of initial acetate ( Figure 3G ) .", "In contrast to R . intestinalis L1-82 in monoculture , no formate was detected in this bi-culture , suggesting that it was entirely cross-fed to B . hydrogenotrophica S5a33 .", "It was unclear whether the carbon dioxide and hydrogen gas produced by R . intestinalis L1-82 reached concentrations that were sufficient to be cross-fed to B . hydrogenotrophica S5a33 .", "Finally , when R . intestinalis L1-82 and F . prausnitzii A2-165 were co-cultivated , fructose and acetate were consumed and butyrate , formate , hydrogen gas and carbon dioxide were produced ( Figure 3E ) .", "The finding that formate reached lower concentrations in this co-culture than in F . prausnitzii A2-165 monoculture already hints at a negative effect of R . intestinalis L1-82 on the growth of F . prausnitzii A2-165 .", "Since Gause's early work on competition between yeast and Paramecium species ( Gause , 1932; Gause , 1934 ) , growth rates in mono- and bi-culture experiments have been compared to determine ecological interactions ( e . g . de Vos et al . , 2017; Freilich et al . , 2011; Wang et al . , 2017 ) .", "The rationale is that the growth rates of mutualistic organisms grown in bi-culture should increase compared to their growth rates in monoculture , whereas the bi-culture growth rates of competitors should decrease compared to their growth rates in monoculture .", "When comparing maximal abundances , cross-feeding and competitive interactions were already apparent .", "Both F . prausnitzii A2-165 and B . hydrogenotrophica S5a33 reached significantly higher maximal bacterial counts in F . prausnitzii A2-165/B .", "hydrogenotrophica S5a33 bi-cultures and in tri-cultures with F . prausnitzii A2-165 dominance ( Figure 3F , H and J ) than they did in monoculture ( Figure 3B and C ) , suggesting a mutualistic relationship ( unpaired two-sided Wilcoxon F . prausnitzii A2-165: shift 0 . 4 , 95% confidence interval 0 . 12–0 . 55 , p-value 0 . 03; B . hydrogenotrophica S5a33: shift 0 . 5 , 95% confidence interval 0 . 33–0 . 69 , p-value 0 . 017 ) .", "The maximal cell number of F . prausnitzii A2-165 tended to be lower when competing with R . intestinalis L1-82 ( Figure 3E ) than when grown alone ( unpaired two-sided Wilcoxon: shift 0 . 47 , 95% confidence interval −0 . 03 and 1 . 42 , p-value 0 . 11 ) .", "Interestingly , there was no difference in maximal bacterial counts for R . intestinalis L1-82 alone versus R . intestinalis L1-82 grown with F . prausnitzii A2-165 in bi-cultures or in tri-cultures with R . intestinalis L1-82 dominance ( unpaired two-sided Wilcoxon: shift 0 . 07 , 95% confidence interval −0 . 39 and 0 . 31 , p-value 0 . 69 ) , so that formally , their relationship could be described as amensalism ( one organism is affected negatively whereas the other is not affected ) .", "Finally , according to the maximal bacterial counts , B . hydrogenotrophica S5a33 benefited more from the presence of F . prausnitzii A2-165 than from that of R . intestinalis L1-82 ( unpaired two-sided Wilcoxon: shift 0 . 29 , 95% confidence interval 0 . 06 and 0 . 93 , p-value 0 . 008 ) .", "When growing all three gut bacterial strains together , fructose was consumed and butyrate , acetate , carbon dioxide , hydrogen gas and lactate were produced .", "Formate was produced initially , peaked between 10 and 15 hr and was below the detection limit after 18 hr of fermentation ( Figure 3I and J ) .", "We performed the tri-culture six times with varying species proportions in the inoculum and found that in all tri-cultures , B . hydrogenotrophica S5a33 was always dominant , together with either R . intestinalis L1-82 or F . prausnitzii A2-165 as co-dominant partner .", "In two out of the six cases , R . intestinalis L1-82 was co-dominant , whereas F . prausnitzii A2-165 was co-dominant in the remaining four .", "The result mattered for the final butyrate concentrations , which averaged 37 . 5 mM when R . intestinalis L1-82 won and 23 . 5 mM when F . prausnitzii A2-165 won .", "We attempted to describe tri-culture dynamics with the model parameterized on monocultures , but failed to obtain a good fit ( see Table 1 and Figure 4—figure supplements 2 and 3 ) .", "After a series of tests , we concluded that incorporating bi-culture data was necessary to describe tri-culture dynamics .", "We finally selected two F . prausnitzii A2-165 monocultures and the R . intestinalis L1-82/B .", "hydrogenotrophica S5a33 and F . prausnitzii A2-165/B .", "hydrogenotrophica S5a33 bi-cultures with initial acetate to parameterize our model .", "As a validation , we predicted the behavior of R . intestinalis L1-82/B .", "hydrogenotrophica S5a33 and F . prausnitzii A2-165/B .", "hydrogenotrophica S5a33 bi-cultures without initial acetate , which resulted in a good fit ( Figure 5G and H , Figure 5—figure supplement 2 ) .", "The model parameterized on mono- and bi-cultures fitted the tri-culture data better than the model parameterized on monocultures only ( Table 1 , Figure 4I and J , Figure 5I and J , Figure 4—figure supplement 3 and Figure 5—figure supplement 3 ) .", "When inspecting the differences between the two parameterizations , we found that the model parameterized on monocultures predicted lower abundances for all three species in bi- and tri-cultures than they actually reached ( Figure 4D–J , Figure 4—figure supplements 2 and 3 ) .", "Vice versa , the model parameterized on mono- and bi-cultures predicted too high abundances for R . intestinalis L1-82 and B . hydrogenotrophica S5a33 in monoculture ( Figure 5A and C , Figure 5—figure supplement 1; the F . prausnitzii A2-165 monoculture was included in the parameterization ) .", "According to the difference in maximal tri-culture cell counts predicted with the two parameterizations , B . hydrogenotrophica S5a33 did significantly better in tri-culture than expected on the basis of its monoculture growth ( unpaired two-sided Wilcoxon: shift 83 , 95% confidence interval 30–92 , p-value: 0 . 002 ) .", "The fact that a single model parameterization could not describe well both mono- and tri-culture dynamics is a sign of emergent behavior in the presence of interaction partners .", "When looking at the parameters inferred from mono- and bi-cultures ( given in Supplementary file 2 ) , B . hydrogenotrophica S5a33's consumption rates for formate and fructose and R . intestinalis L1-82's consumption rate for fructose were lower than their values obtained from mono-culture parameterization , whereas their maximal growth rates were not much affected ( B . hydrogenotrophica S5a33 ) or increased ( R . intestinalis L1-82 ) .", "Thus , according to this analysis , less of the energy source is needed in the presence of an interaction partner than in monoculture .", "Next , we tested whether dominance in tri-culture could be predicted from lag phase and initial abundance .", "Towards this aim , we computed the F . prausnitzii A2-165/R .", "intestinalis L1-82 ratio in simulations with varying lag phase and initial abundance .", "Experimental observations of dominance agreed well with the model predictions ( Figure 6A and D ) .", "Our systematic investigation also showed that there was a non-linear relationship between initial F . prausnitzii A2-165 abundance and R . intestinalis L1-82 dominance ( Figure 6F ) .", "Thus , even when initial abundances , lag phases and species interactions were known , it is hard to predict the winner ( and hence the resulting butyrate concentration ) intuitively without a model in hand .", "The final abundances of the three strains in simulations were also non-linearly dependent on other parameters , including B . hydrogenotrophica S5a33's growth rate , its fructose consumption rate and its fructose half-saturation constant ( Figure 6—figure supplement 1 ) .", "These results underline that , in addition to kinetic parameters , initial conditions and lag phase can determine strain abundances in co-culture in a non-linear way .", "To further investigate the emergent behavior , we sequenced RNA for three time points and two biological replicates for each of the three monocultures and for the tri-culture with F . prausnitzii A2-165 co-dominance , and assessed significantly differential gene expression across all samples in mono- versus tri-cultures for all three strains ( Supplementary file 3 ) .", "In total , 9 . 3% , 10 . 9% and 7 . 0% of R . intestinalis L1-82's , F . prausnitzii A2-165's and B . hydrogenotrophica S5a33's protein-coding genes were significantly differentially expressed ( protein numbers taken from UniProt; The UniProt Consortium , 2017 ) .", "Interestingly , in tri-culture , F . prausnitzii A2-165 downregulated a series of enzymes needed for vitamin B12 coenzyme biosynthesis .", "Cobalamin ( vitamin B12 ) was one of the co-factors suspected to limit F . prausnitzii A2-165 growth in monoculture , and this finding could mean that F . prausnitzii A2-165 benefited from greater co-factor availability in tri-culture .", "We tested whether F . prausnitzii A2-165 grown in test tubes benefited from added vitamin B12 ( Supplementary file 4 ) but did not see a significant increase in cell numbers .", "Although this indicates that F . prausnitzii A2-165 downregulates the B12 production pathway upon presumably higher cobalamin availability in the tri-culture , this does not explain the change in growth characteristics .", "In tri-culture , F . prausnitzii A2-165 also upregulated enzymes that are involved in amino acid and oligopeptide transport and amino acid and protein biosynthesis .", "B . hydrogenotrophica S5a33 likewise upregulated amino acid biosynthesis in tri-culture .", "For R . intestinalis L1-82 , which reached lower abundances in the selected tri-cultures than in monoculture , the transcription response was mixed: some amino acid biosynthesis enzymes were downregulated , others upregulated ( including enzymes involved in ornithine biosynthesis ) .", "However , the expression of ribosomal proteins was lower than that in R . intestinalis L1-82 mono-culture , in agreement with its long lag phase in the selected tri-cultures .", "In summary , the analysis of differential gene expression uncovered a number of metabolic changes in the presence of interaction partners , thus further supporting the altered behavior detected through modeling ." ], [ "Here , we investigated the dynamics of a well-defined , small , but representative synthetic gut microbial community , consisting of the three strains B . hydrogenotrophica S5a33 , F . prausnitzii A2-165 and R . intestinalis L1-82 .", "We found that B . hydrogenotrophica S5a33 is metabolically versatile and grew as fast as primary fermenters such as R . intestinalis L1-82 .", "We demonstrated experimentally that formate was cross-fed between B . hydrogenotrophica S5a33 on the one hand and F . prausnitzii A2-165 and R . intestinalis L1-82 on the other , and confirmed mutualistic as well as competitive interactions between these three bacterial strains .", "When growing on formate , we identified B . hydrogenotrophica S5a33 as a net producer of both hydrogen gas and carbon dioxide , in contrast with its traditionally assumed role in the gut ecosystem .", "Although formate is rarely highlighted as a key intermediate in gut cross-feeding interactions , it has been reported to be an end-product of primary polysaccharide degradation by both Bifidobacterium and Lactobacillus species ( Falony et al . , 2009b; Moens et al . , 2017 ) .", "Hence , our results invite a re-evaluation of the ecological niche of B . hydrogenotrophica in relation to its potential for microbial formate production .", "Although only one strain was tested for each of the three colonic species considered , the relationships described here probably generalize to species level .", "Each of the strains in the species descriptions ( including four additional R . intestinalis strains and one additional strain each of F . prausnitzii and B . hydrogenotrophica ) are reported to produce and consume the same key metabolites as the strain selected for our experiments ( Bernalier et al . , 1996; Duncan et al . , 2002a; Duncan et al . , 2002b ) .", "In addition , three further F . prausnitzii strains ( SL3/3 , KLE1255 and M21/2 ) have been predicted to produce formate ( Magnúsdóttir et al . , 2017 ) and two additional R . intestinalis strains ( M50/1 and XB6B4 ) contain pyruvate ferredoxin oxidoreductase , an enzyme assumed to be crucial for hydrogen gas production in Clostridial cluster XIVa ( Falony et al . , 2009c ) .", "In addition , Rey et al . ( 2010 ) demonstrated that in gnotobiotic mice , B . hydrogenotrophica S5a36 grows better in the presence of a primary fermenter ( Bacteroides thetaiotaomicron ) than alone .", "However , further experiments are required to test whether the potential for cross-feeding is realized in other strain combinations .", "The model , which encodes our knowledge of the system , is important not only for predictions but also as a reference .", "We gained insights from its agreements as well as from its disagreements with our observations .", "For instance , we assumed initially that R . intestinalis L1-82 would be inhibited by the hydrogen gas it generated .", "However , a hydrogen gas inhibition term did not improve the accuracy of predicted R . intestinalis L1-82 behavior in monoculture , which suggested that hydrogen gas inhibition did not affect R . intestinalis L1-82 growth at the concentrations reached in our experiments .", "We also needed the model to ascertain that changes from mono- to bi- or tri-cultures were not just the result of variations in the inoculum composition or the lag phase , but that there was a true change in the dynamics that the model parameterized on monocultures alone could not capture .", "We confirmed this emergent behavior with RNA-seq , which revealed significantly different gene expression in tri-culture than in mono-culture , especially for F . prausnitzii A2-165 and B . hydrogenotrophica S5a33 .", "The downregulation of F . prausnitzii A2-165's vitamin B12 coenzyme biosynthesis pathway in tri-culture is of particular interest , as it suggests that dependency on co-factors changes with interaction partners .", "It has been posited that the majority of gut microorganisms in need of B12 precursors are unable to synthesize them ( Degnan et al . , 2014 ) .", "If this need is altered by the presence of interaction partners , it cannot be exploited as easily for selective manipulation as suggested by Degnan et al . ( 2014 ) .", "Although kinetic models parameterized on monocultures may in some cases describe bi-culture dynamics correctly ( Van Wey et al . , 2014 ) , our example shows that this is not a general property .", "This means that models of microbial communities will have to take the internal metabolism of community members and their response to interaction partners into account .", "Gut bacteria such as B . hydrogenotrophica S5a33 have flexible metabolic strategies that they employ according to circumstances .", "Emergent metabolism in the presence of interaction partners has been described in theoretical work before ( Chiu et al . , 2014 ) , but has been rarely investigated experimentally ( for example , in Aharonovich and Sher , 2016 ) .", "Constraint-based modeling approaches , which can take emergent metabolism into account ( Orth et al . , 2010 ) , require high-quality metabolic reconstructions for each community member , which take months of curation effort to obtain ( Thiele and Palsson , 2010 ) .", "Thus , scaling strain-level quantitative models to larger communities will be a formidable challenge .", "Mono- and bi-cultures are increasingly carried out in batch in a high-throughput fashion to determine ecological interactions and to quantify their strengths ( de Vos et al . , 2017; Sher et al . , 2011 ) .", "Such systematic quantification is an important step forward , but there are challenges to tackle .", "Our work showed that dominance in batch may sensitively ( i . e . , non-linearly ) depend on initial conditions such as the lag phase and the initial abundance , both of which are hard to control experimentally .", "Thus , a growth experiment performed in biological replicates but with the same inoculum may identify one strain as the winner and another as the loser .", "However , a replicate with a slightly different inoculum composition may provide results that support the opposite conclusion .", "Such a dependency on the initial conditions ( albeit with larger abundance differences ) has also been reported in several competition experiments for Streptomyces species ( Wright and Vetsigian , 2016 ) and may thus be a common case .", "To ascertain that bacteria change their behavior in response to an interaction partner , RNA-seq is carried out on mono- and bi-cultures ( Aharonovich and Sher , 2016; González-Torres et al . , 2015; Plichta et al . , 2016 ) .", "Here , we showed that a model can also reveal emergent behavior by its failure to describe co-culture dynamics when parameterized on monocultures only .", "How such approaches could be scaled up to achieve the high-throughput needed for systematic measurements of interaction strengths is an open question .", "Although the combination of mutualism and competition has been explored in vitro previously ( Rivière et al . , 2015 ) , to the best of our knowledge , this is the first investigation of a defined bacterial community in which two strain pairs mutually cross-feed and compete .", "In the case of F . prausnitzii A2-165 and B . hydrogenotrophica S5a33 , mutualism appears to supersede competition , leading to increased maximal bacterial numbers coupled with upregulation of biosynthesis for both interaction partners .", "For R . intestinalis L1-82 and B . hydrogenotrophica S5a33 , we had no such clear experimental evidence , as RNA-seq was performed on tri-cultures dominated by F . prausnitzii A2-165 , but the comparison of maximum bacterial numbers across mono- , bi- and tri-cultures suggested that R . intestinalis L1-82 and B . hydrogenotrophica S5a33 did not benefit as much from each other as F . prausnitzii A2-165 and B . hydrogenotrophica S5a33 did .", "As the model described R . intestinalis L1-82/B .", "hydrogenotrophica S5a33 bi-culture dynamics well without taking carbon dioxide and hydrogen gas cross-feeding into account , we assume that because of their low partial pressure , gasses were less efficiently cross-fed to B . hydrogenotrophica S5a33 than formate , although both are probably metabolized via the same pathway ( Wood-Ljungdahl ) .", "Thus , interactions that look similar on paper can play out differently , depending on the environment .", "In the two replicates of the R . intestinalis L1-82/F .", "prausnitzii A2-165 bi-culture , R . intestinalis L1-82 and F . prausnitzii A2-165 both survived ( albeit F . prausnitzii A2-165 in far lower numbers ) despite competing for the same substrate .", "This is presumably because in our experimental set-up , the time until nutrient depletion was too short for the competitive exclusion principle to apply .", "Our tri-culture experiments also demonstrated the importance of initial conditions in determining fermentation end-products .", "According to our model , the initial abundance and lag phase determined whether butyrate reached high or low concentrations in the tri-culture fermentations .", "Since these are likely to be relevant parameters in the gut environment and difficult to control , cocktail communities will have to be designed such that they will carry out their function across a wide range of initial conditions .", "Although our work highlighted a number of challenges to microbial community modeling , the model's ability to predict tri-culture dynamics from bi-cultures gives hope that , with sufficient knowledge , we will ultimately be able to model more complex microbial communities ." ], [ "Human isolates of Roseburia intestinalis A2-165 ( DSM 14610T ) , Faecalibacterium prausnitzii L1-82 ( DSM 17677T ) and Blautia hydrogenotrophica S5a33 ( DSM 10507T ) were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen ( DSMZ , Germany ) and stored at −80°C in reinforced clostridial medium ( RCM; Oxoid Ltd . , Basingstoke , United Kingdom ) , supplemented with 25% ( vol/vol ) glycerol as a cryoprotectant .", "A recently published medium for colon bacteria ( mMCB ) that allows growth of F . prausnitzii A2-165 ( Moens et al . , 2016 ) was modified by adding nitrogen sources and trace elements as detailed below .", "This medium had the following composition ( concentrations in g L−1 ) : bacteriological peptone ( Oxoid ) , 6 . 5; soy peptone ( Oxoid ) , 5 . 0; yeast extract ( VWR International , Darmstadt , Germany ) , 3 . 0; tryptone ( Oxoid ) , 2 . 5; NaCl ( VWR International ) , 1 . 5; K2HPO4 ( Merck , Darmstadt , Germany ) , 1 . 0; KH2PO4 ( Merck ) , 1 . 0; Na2SO4 ( VWR International ) , 2 . 0; MgSO4·7H2O ( Merck ) , 1 . 0; CaCl2·2H2O ( Merck ) , 0 . 1; NH4Cl ( Merck ) , 1 . 0; cysteine-HCl ( Merck ) , 0 . 4; NaHCO3 ( VWR International ) , 0 . 2; MnSO4·H2O ( VWR International ) , 0 . 05; FeSO4·7H2O ( Merck ) , 0 . 005; ZnSO4·7H2O ( VWR International ) , 0 . 005; hemin ( Sigma-Aldrich , Steinheim , Germany ) , 0 . 005; menadione ( Sigma-Aldrich ) , 0 . 005; and resazurin ( Sigma-Aldrich ) , 0 . 001 .", "The medium was supplemented with 1 mL L−1 of selenite and tungstate solution ( NaOH ( Merck ) , 0 . 5; Na2SeO3·5H2O ( Merck ) , 0 . 003; Na2WO4·2H2O ( Merck ) , 0 . 004 and 1 L of distilled water ) and 1 mL L−1 of trace element solution SL-10 ( HCl ( Merck , 25% , vol/vol; 7 . 7 M ) , FeCl2·4 H20 ( Merck ) , 1 . 5; ZnSO4·7H2O ( VWR International ) , 0 . 148; MnSO4·H2O ( VWR International ) , 0 . 085; H3BO3 ( Merck ) , 0 . 006; CoCl2·6 H20 ( Merck ) , 0 . 19; CuSO4·5 H20 ( VWR International ) , 0 . 0034; NiCl2·6 H20 ( Merck ) , 0 . 024; and Na2MoO4·2 H20 ( Merck ) , 0 . 036 ) .", "Acetate ( 50 mM or 6 . 8 g L−1 of CH3COO−Na+3H2O ( Merck ) ) was added to the medium for the mono-culture fermentations with R . intestinalis L1-82 and F . prausnitzii A2-165 , whereas formate ( 50 mM or 3 . 4 g L−1 of HCOO−Na+ ( VWR International ) ) was added to the medium for the mono-culture fermentations with B . hydrogenotrophica S5a33 .", "The pH of the medium was adjusted to 6 . 8 and the medium was autoclaved at 210 kPa and 121°C for 20 min .", "After sterilization , D-fructose ( Merck ) was added as the sole energy source aseptically , at a final concentration of 50 mM fructose using sterile stock solutions obtained through membrane filtration using Minisart filters ( pore size , 0 . 2 μm ( Sartorius , Göttingen , Germany ) ) .", "Mono-culture cultivation experiments for B . hydrogenotrophica S5a33 were performed in stationary glass bottles without controlling the pH ( screening ) .", "The bottles contained 50 mL of heat-sterilized pH 6 . 8 mMCB medium , supplemented with 50 mM of D-fructose ( Merck ) , D-glucose ( Merck ) , D-galactose ( Merck ) , L-fucose ( Merck ) , sodium formate ( VWR International ) , sodium acetate trihydrate ( Merck ) , DL lactic acid ( VWR International ) , oligofructose ( Raftilose P95; Beneo-Orafti NV , Tienen , Belgium ) or inulin ( OraftiHP; Beneo-Orafti ) as energy sources ( Falony et al . , 2009b ) .", "Additional cultivation experiments were performed in medium devoid of any main energy source to test autotrophic growth .", "For the cultivation experiments in bottles , stock solutions of fructose , glucose , galactose , fucose , sodium formate , sodium acetate trihydrate , and lactic acid were initially made anaerobically through autoclaving at 210 kPa and 121°C for 20 min .", "The solutions were subsequently filter-sterilized and transferred into glass bottles , which were sealed with butyl rubber septa that were pierced with a Sterican needle ( VWR International ) connected with a Millex-GP filter ( Merck ) to assure sterile conditions .", "For the cultivation experiments with lactate , the pH was adjusted to 6 . 8 under anaerobic conditions , using sterile solutions of sodium hydroxide ( Merck ) .", "Stock solutions of oligofructose and inulin were made sterile by membrane filtration .", "The inocula were prepared as follows .", "Cells of the strains under study were transferred from −80°C to test tubes containing 10 mL of RCM that were incubated anaerobically at 37°C for 24 hr .", "Subsequently , the strains were propagated for 12 hr in glass bottles containing 50 mL of heat-sterilized pH 6 . 8 mMCB medium , supplemented with the energy source under study , always at a final concentration of 50 mM of fructose equivalents .", "These pre-cultures were finally added to the glass bottles aseptically .", "During the inoculum build-up , the transferred volume was always 5% ( vol/vol ) .", "All bottles were incubated anaerobically at 37°C in a modular atmosphere-controlled system ( MG anaerobic work station; Don Withley Scientific Ltd . , West Yorkshire , United Kingdom ) that was continuously sparged with a mixture of 80% N2 , 10% CO2 , and 10% H2 ( Air Liquide , Paris , France ) .", "Samples for further analyses were withdrawn after 0 , 6 , 12 , 24 , 48 , and 100 hr .", "All experiments were performed at least in duplicate .", "To prepare inocula , cells were transferred from −80°C to test tubes containing 10 mL of RCM , and incubated at 37°C for 24 hr .", "Subsequently , the strains were propagated twice for 12 hr in glass bottles containing 100 mL of mMCB medium ( with acetate in the case of R . intestinalis L1-82 and F . prausnitzii A2-165 , and with formate in the case of B . hydrogenotrophica S5a33 ) , supplemented with fructose .", "All incubations were performed anaerobically in a modular atmosphere-controlled system ( MG anaerobic workstation ) that was continuously sparged with a mixture of 80% N2 , 10% CO2 , and 10% H2 ( Air Liquide ) .", "The inocula were finally added aseptically to the fermentors .", "During the inoculum build-up , the transferred volume was always 5% ( vol/vol ) .", "Fermentations were carried out in 2 L Biostat B-DCU fermentors ( Sartorius ) containing 1 . 5 L of mMCB medium supplemented with the co-substrates ( acetate and/or formate ) if necessary and 50 mM of D-fructose as the energy source .", "Anaerobic conditions during fermentations were assured by continuously sparging the medium with N2 ( PraxAir , Schoten , Belgium ) at a flow rate of 70 mL min−1 .", "The fermentation temperature was kept constant at 37°C .", "A constant pH of 6 . 8 was imposed and controlled automatically , using 1 . 5 M solutions of NaOH and H3PO4 .", "To keep the medium homogeneous , a gentle stirring of 200 rpm was applied .", "Temperature , pH , and agitation speed were controlled online ( MFCS/win 2 . 1 software , Sartorius ) .", "Fermentations were followed for 48 hr , with samples taken at 10 min and 2 hr , 3 hr , 5 hr , 6 hr , 7 hr , 9 hr , 10 hr , 11 hr , 13 hr , 14 hr , 15 hr , 17 hr , 18 hr , 24 hr , 30 hr and 48 hr after inoculation .", "At selected time points ( 3 hr , 9 hr and 15 hr after inoculation ) , subsamples were treated for RNA extraction by adding 5 vol of RNAlater ( Thermo Fisher Scientific ) .", "All mono- and tri-culture fermentations were performed in triplicate .", "All bi-culture fermentations were performed in duplicate , except for the bi-culture fermentation using medium lacking acetate with F . prausnitzii A2-165 and B . hydrogenotrophica S5a33 , which was performed only once .", "F . prausnitzii A2-165 was grown in test tubes containing 10 mL of RCM each .", "Then 10 , 50 and 100 μL of filter-sterilized ( 0 . 22 μm , Merck Millipore ) 0 . 1 g/L vitamin B12 solution ( Sigma-Aldrich ) was added to reach a final concentration of 0 . 1 , 0 . 5 and 1 mg/L , respectively , in the test tubes .", "For each of the three concentrations as well as for the control ( without added B12 ) , bacterial abundance was followed in three test tubes .", "Samples were taken after 24 hr and 48 hr and cell counts were obtained via flow cytometry as described below .", "During all experiments , the optical density at 600 nm ( OD600 ) was measured against ultrapure water as blank with a VIS spectrophotometer ( Genesys 20; Thermo Scientific , Waltham , MA , USA ) .", "Each measurement was performed in triplicate .", "Total bacterial abundance was also measured by flow cytometry , using an Accuri C6 flow cytometer ( BD Biosciences , Erembodegem , Belgium ) , as described previously ( Moens et al . , 2016 ) .", "All samples were diluted in filter-sterilized water ( Vittel , France ) to obtain a concentration between 1 . 0 × 103 and 5 . 0 × 106 cells mL−1 .", "Flow cytometric analysis was performed by mixing 500 μL of sample with 5 μL of a 100 × SYBR Green I solution ( Sigma-Aldrich ) and 5 μL of a 500 mM ethylenediaminetetra acetic acid ( EDTA ) solution ( Sigma-Aldrich ) .", "Afterwards , samples were left in the dark at room temperature for 15 min .", "Flow cytometric counts were obtained using an Accuri C6 flow cytometer ( BD Biosciences ) , equipped with a 50 mW solid state laser ( 488 nm ) .", "Green fluorescence was measured in the FL1 channel ( 530 ± 15 nm ) and all data were processed with the Cflow Plus software ( Accuri ) .", "Gating was performed to distinguish signals from noise .", "All data were collected as a FL1/SSC density plot with a primary threshold of 10 , 000 on the FL1 channel .", "Measurements were performed in triplicate .", "qPCR assays with strain-specific TaqMan primers and probes were performed to quantify the abundance of each strain separately .", "For this , 2 mL of fermentation sample was centrifuged at 20 , 570 × g for 20 min .", "Cells were washed in 2 mL of physiological solution ( NaCl , 8 . 5 g L−1 ) and centrifuged again at 20 , 570 × g for 20 min to obtain washed cell pellets .", "Subsequently , these cell pellets were resuspended in 2 mL of physiological solution and diluted 20 times for DNA extraction .", "Direct DNA extractions by alkaline thermal lysis were performed on the basis of the methods used by Girish et al . ( 2013 ) and Rudbeck and Dissing ( 1998 ) , modified as follows: 100 μL of the sample was mixed with 100 μL of 0 . 2 M NaOH in a sterile microcentrifuge tube .", "The mixture was vortexed and heated at 90°C for 10 min , after which eight volumes ( 1600 μL ) of 0 . 04 M Tris HCl pH 7 . 5 ( Thermo Fisher Scientific ) was added for pH neutralization .", "4 μL of the final mixture was used for qPCR .", "The extracted genomic DNA was stored at −20°C until qPCR amplification .", "Calibration curves were obtained by initially growing all strains in RCM for 24 hr , and two-fold propagation in medium for 12 hr , as described above .", "From each of these grown cultures , separate four-fold decimal and nine-fold binary dilution series were prepared .", "The generation of cell pellets , direct extraction of DNA , and subsequent quantification of cell concentrations by flow cytometry were performed as described above , with the exception that , prior to DNA extraction , samples for calibration were diluted less than the fermentation samples , to accommodate a wider qPCR quantification range .", "Primers and oligoprobes ( listed in Supplementary file 5 ) were manually designed using the online Primer3Plus software ( Untergasser et al . , 2007 ) and the genome sequences of the strains .", "Melting temperatures and the presence of hairpins , self-dimers , and pair-dimers were double-checked using the online OligoCalc software ( Kibbe , 2007 ) .", "Secondary structures of the generated amplicons were investigated using the online Mfold program ( Zuker , 2003 ) .", "Primers and probes were synthesized by Thermo Fisher Scientific .", "The strain specificities of primers and probes were confirmed in silico by Primer-BLAST ( Ye et al . , 2012 ) and in vitro by PCR and qPCR analysis on genomic DNA of the strains ( Supplementary file 5 ) .", "qPCR assays were carried out using a 7500 FAST Real-Time PCR system ( Applied Biosystems , Carlsbad , CA , USA ) , equipped with 96-well plates .", "Each qPCR assay mixture of 20 μL contained 10 . 0 μL of TaqMan Fast Universal PCR Master Mix ( 2X ) , no AmpErase UNG ( Thermo Fisher Scientific ) , 2 . 0 μL of each primer ( 3 . 0 μM ) , 2 . 0 μL of the TaqMan probe ( 1 . 5 μM ) , and 4 . 0 μL of extracted genomic DNA solution or sterile nuclease-free water ( Thermo Fisher Scientific ) .", "The qPCR amplification program consisted of an initial denaturation step at 95°C for 20 s , followed by 45 two-step cycles at 95°C for 3 s and at 60°C for 30 s .", "In each run , negative ( sterile nuclease-free water without genomic DNA ) and positive controls ( with extracted genomic DNA from the relevant strains ) were used .", "The cycle threshold ( Ct ) values were determined using the automatically determined thresholds from the 7500 software v2 . 0 . 6 ( Applied Biosystems ) .", "Finally , during a re-analysis of all qPCR runs , Ct values were normalized using an inter-plate calibrator to account for differences among qPCR runs .", "The above-described generation of cell pellets , direct extraction of DNA , and qPCR assays were performed in triplicate .", "Contamination was checked by aerobic and anaerobic plating on RCM agar and 16S rRNA gene amplicon sequencing of end point fermentation samples ( 48 hr ) .", "Sequencing was performed as described previously ( D’hoe et al . , 2018 ) .", "Concentrations of fructose , as well as concentrations of formate , acetate , butyrate , lactate and ethanol , were determined through high-performance liquid chromatography ( HPLC ) with refractive index detection , using a Waters chromatograph ( Waters , Milford , MA , USA ) equipped with an ICSep ICE ORH-801 column ( Transgenomic North America , Omaha , NE , USA ) , and applying external standards , as described previously ( Falony et al . , 2009b ) .", "Briefly , the mobile phase consisted of 5 mM H2SO4 at a flow rate of 0 . 4 mL min−1 .", "The column temperature was kept constant at 35°C .", "Sample preparation involved a first centrifugation ( 4618 x g for 20 min at 10°C ) for removal of cells and debris , followed by the addition of an equal volume of 20% ( mass/vol ) trichloroacetic acid for protein removal .", "For determining oligofructose and inulin consumption , samples were incubated at room temperature for 24 hr to assure complete hydrolysis of the polysaccharides .", "Subsequently , the samples were centrifuged ( 21 , 912 x g , 20 min , 4°C ) and filtered ( pore size of 0 . 2 μm; Uniflo 13 Filter Unit; GE Healthcare , Little Chalfont , UK ) , prior to injection ( 30 μL ) into the column .", "Samples were analyzed in triplicate .", "Concentrations of hydrogen gas and carbon dioxide in the fermentor gas effluents were determined online through gas chromatography ( GC ) with thermal conductivity detection ( TCD ) , using a CompactGC ( Interscience , Breda , The Netherlands ) equipped with a 10 m Molsieve 5A column ( hydrogen gas ( Varian , Palo Alto , CA , USA ) ) and a 10 m PoraBOND Q column ( carbon dioxide ( Varian ) ) , and applying external standards , as described previously ( Falony et al . , 2009b ) .", "For an additional screening experiment with B . hydrogenotrophica S5a33 grown in the presence of 350 mM formate , the concentrations of ethanol , acetoin , acetic acid , propionic acid , butyric acid , isobutyric acid and isovaleric acid produced were determined by gas chromatography with flame ionization detection ( GC-FID ) , using a FocusGC chromatograph ( Interscience ) equipped with a Stabilwax-DA column ( Restek , Bellefonte , PA , USA ) , and applying external standards , as described previously ( Moens et al . , 2014 ) .", "The samples were analyzed in triplicate .", "We modeled change of species abundances over time with the following three ordinary differential equations:dXRIdt=ΦRI ( QRI , Sfructose , Sacetate ) XRIdXFPdt=ΦFP ( QFP , Sunknown , Sfructose , Sacetate ) XFPdXBHdt=ΦBH ( QBH , Sfructose , Sformate ) XBHwhere X denotes species abundance , S metabolite concentration and Q a lag phase parameter .", "The growth rates are then defined as non-linear growth functions as described by Grivet ( 2001 ) and Smith and Waltman ( 1995 ) , and assuming Monod kinetics ( Monod , 1950 ) :ΦRI ( QRI , Sfructose , Sacetate ) =ΓRI ( QRI ) μRISfructoseKRI−fructose+Sfructose⟮1+ωRISacetateKRI−acetate+Sacetate⟯ΦFP ( QFP , Sunknown , Sfructose , Sacetate ) =ΓFP ( QFP ) μFPSunknownKFP−unknown+SunknownSfructoseKFP−fructose+ Sfructose⟮1+ωFPSacetateKFP−acetate+Sacetate⟯ΦBH ( QBH , Sfructose , Sformate ) =ΓBH ( QBH ) μBH⟮SfructoseKBH−fructose+Sfructose+ωBHSformateKBH−formate+Sformate⟯where K is the Monod ( half-saturation ) constant , μ is the maximal specific growth rate and ω a weight parameter .", "Nutrient dependency can be either obligatory ( growth without nutrient is not possible ) or facultative ( growth without nutrient is possible ) .", "For instance , the fructose uptake is multiplied with R . intestinalis L1-82's maximal growth rate , whereas its acetate uptake is modeled with an additive term .", "Therefore , in the absence of fructose , R . intestinalis L1-82's growth rate is zero , but this is not the case when acetate is absent .", "The weight parameter adjusts how strongly a facultative substrate contributes to the overall growth rate .", "The unknown compound models the dependency of F . prausnitzii A2-165 on an undetermined co-factor .", "The lag phase function is defined as in Baranyi and Roberts ( 1994 ) : Γi ( Qi ) =Qi1+Qi , where i stands for R . intestinalis L1-82 , F . prausnitzii A2-165 or B . hydrogenotrophica S5a33 .", "The Qi variables follow exponential growth:dQidt=μiQi Thus , the larger the initial value of Qi , the shorter the lag phase .", "The changes of metabolite concentrations are then modeled as follows:dSfructosedt=−νRI , fructoseΦRIXRI−νFP , fructoseΦFPXFP−νBH , fructoseΦBH , fructoseXBHdSformatedt=αRI , formateΦRIXRI+αFP , formateΦFPXFP−νBH , formateΦBH , formateXBHdSacetatedt=−νRI , acetateΦRI , acetateXRI−νFP , acetateΦFP , acetateXFP+αBH , acetateΦBHXBHdSbutyratedt=αRI , butyrateΦRIXRI+αFP , butyrateΦFPXFPdSunknowndt=−νFP , unknownΦFPXFPdSH2dt=αRI , H2ΦRIXRI+αRI , H2ΦBHXBHdSCO2dt=αRI , CO2ΦRIXRI+αFP , CO2ΦFPXFP+αBH , CO2ΦBHXBHΦRI , acetate=ΓRI ( QRI ) μRIwRISfructoseKRI , fructose+SfructoseSacetateKRI , acetate+SacetateΦFP , acetate=ΓFP ( QFP ) μFPwFPSunknownKFP , unknown+SunknownSacetateKFP , fructose+SfructoseSacetateKFP , acetate+SacetateΦBH , fructose=ΓBH ( QBH ) μBHSfructoseKBH , fructose+SfructoseΦBH , fructose=ΓBH ( QBH ) μBHwBHSformateKBH , formate+Sformate The α and ν parameters are production and consumption rates , respectively .", "Species abundance is measured in 108 bacterial counts/mL , metabolite concentration in mM , the unit of μ is 1/h , the unit of K is mM , the unit of α and of ν is mM/ ( 108 bacterial counts/mL ) and ω is dimensionless .", "The model assumes that death rates are negligible , that metabolites are produced in proportion to the growth rate of the strains , that metabolites are not transformed in the bioreactor except through the strains themselves and , crucially , that the strains do not alter their metabolism in the presence of interaction partners .", "Furthermore , Monod kinetics assumes that bacteria grow exponentially at low abundances , that bacterial growth is limited only by the limiting substrate concentration and that the maximal specific growth rate and the Monod constant do not change over time .", "A number of these assumptions are met by taking the lag phase into account , by omitting data points from the stationary phase and by including the unknown compound for F . prausnitzii A2-165 growth .", "Carbon dioxide and hydrogen gas consumption by B . hydrogenotrophica S5a33 is not included in the final version of the model .", "We tried to account for carbon dioxide consumption with a multiplicative term in B . hydrogenotrophica S5a33's growth rate .", "However , this did not improve the model fit .", "As the model without carbon dioxide describes R . intestinalis L1-82/B .", "hydrogenotrophica S5a33 bi-culture dynamics well , we assume that B . hydrogenotrophica S5a33 grew mostly heterotrophically on fructose and formate , and that the hydrogen gas and carbon dioxide produced by R . intestinalis L1-82 did not reach sufficient concentrations in the head space to allow autotrophic growth .", "The model definition is available as Source code 1 in Python ( Model definition ) .", "We parameterized our model on monocultures alone ( parameterization", "1 ) and then on mono- and bi-cultures ( parameterization 2 ) .", "The model was fitted using the function fmin ( ) from the scipy Python package ( Jones et al . , 2001 ) , to minimize the normalized root mean square error ( RMSE ) .", "During fitting , the biological replicate ( s ) of a mono- or bi-culture that gave the best overall fit were selected by trial and error .", "An initial estimate of the parameters was obtained by manually fitting the data iteratively .", "The initial concentration of the unknown compound was set to 30 mM .", "Samples taken after the end of the log phase , when the bacterial counts started to decline , were omitted from the fitting .", "Parameterization 2 consisted of several steps , as fitting all parameters at once did not lead to convergence , because of the nonlinear growth rates .", "First , parameters for F . prausnitzii A2-165 were obtained from two F . prausnitzii A2-165 monocultures .", "The consumption parameters of B . hydrogenotrophica S5a33 were obtained from F . prausnitzii A2-165/B .", "hydrogenotrophica S5a33 bi-cultures with initial acetate; afterwards , the maximal specific growth rates and half-saturation ( Monod ) constants were obtained from the same bi-cultures .", "The parameters for R . intestinalis L1-82 were obtained from a R . intestinalis L1-82/B .", "hydrogenotrophica S5a33 bi-culture with acetate .", "Lag phases were calculated as the time to reach Γi ( Qi ) =0 . 5:lagphase=−ln⁡ ( Qi ( 0 ) ) /μi Qi ( 0 ) was estimated by visual inspection of the log plots .", "Model parameters obtained and maximal abundances predicted with both parameterizations as well as estimated lag phases and experiment-specific RMSE values are provided in Supplementary file 2 .", "Data and model fits were plotted with Python's matplotlib ( Hunter , 2007 ) .", "Total RNA was extracted from RNAlater-treated samples using the phenol-free total RNA purification kit coupled with DNase I treatment ( VWR International ) according to the manufacturer’s protocol for Gram-positive bacteria .", "A secondary DNAse digestion was performed using the Ambion TURBO DNA-free DNase Treatment and Removal Reagents Kit ( Thermo Fisher Scientific ) , after which the samples were purified using the RNA Clean and Concentrator−25 kit ( Zymo Research , Irvine , CA , USA ) according to the manufacturer’s instructions .", "The eluted RNA was stored at −80°C .", "The absence of DNA contamination was evaluated using PCR ( 35 or 40 cycles ) and gel electrophoresis .", "The concentrations of the samples were determined with a Nanodrop , and with a Qubit 2 . 0 fluorometer using the Qubit dsDNA HS Assay Kit ( Thermo Fisher Scientific ) .", "RNA integrity , expressed as the RNA integrity number ( RIN ) , and yield were determined using RNA Nano/Pico 6000 LabChips ( Agilent Technologies , Santa Clara , CA , USA ) , which were run in an Agilent 2100 Bioanalyzer ( Agilent Technologies ) .", "Most of the RINs were above 7 , but the RINs of three B . hydrogenotrophica S5a33 monoculture samples at 3 hr , 9 hr and 15 hr were around 2 . 6 , and in four cases ( B . hydrogenotrophica S5a33 monoculture at 15 hr , F . prausnitzii A2-165 monoculture at 9 hr , and for both tri-culture replicates at 3 hr ) , the RINs could not be determined .", "By pooling over three extraction rounds , however , sufficient RNA for sequencing ( minimum of 536 ng and median of 2800 ng ) was obtained for all samples .", "Library preparation encompassed the use of Ribozero rRNA removal for Gram-positive bacteria and the Illumina TruSeq stranded mRNA Library preparation kit ( IIlumina , San Diego , CA , USA ) .", "Library preparation was performed without the mRNA purification step , according to the manufacturer’s instructions .", "The enriched libraries were sequenced on an Illumina NextSeq 500 instrument ( paired-end , 2 × 76 bp reads , Mid output kit , Illumina ) .", "From the Illumina platform , paired-end reads in FASTQ format ( CASAVA 1 . 8 , Phred + 33 ) were obtained and separated into distinct files for each single-end read and for each sample .", "The analysis of the raw sequencing reads was performed as follows: reads were trimmed using Trimmomatic ( Bolger et al . , 2014 ) with the following parameters ‘CROP:74 HEADCROP:10 SLIDINGWINDOW:4:15 MINLEN:51’ , to remove initial and last bases which had biases in their nucleotide content as reported by FastQC ( Andrews , 2010 ) , to remove stretches of low-quality bases and to keep reads with at least 51 bases after trimming .", "FastQC was re-run on the trimmed data to ensure that the previous biases were corrected .", "SortMeRNA ( Kopylova et al . , 2012 ) was used with default parameters and included databases to remove rRNA reads .", "With the remaining non-rRNA reads , we ran MetaPhlAn2 ( Truong et al . , 2015 ) with default parameters and database , and mash screen ( Ondov and Philippy , 2017; Ondov et al . , 2016 ) with default parameters against the complete RefSeq genomes and plasmids database , to search for potential contaminants .", "Both MetaPhlAn2 and the top hits from mash screen found the correct bacterial genomes from the three strains used in this study , together with reads from yeast ( S . cerevisiae S288c ) .", "In addition , low amounts of the phage PhiX174 were reported by mash screen .", "To quantify the presence of these potential contaminants in our samples accurately , and to quantify gene expression from the cultured bacteria , we mapped the non-rRNA reads to these five strains using Bowtie2 ( Langmead and Salzberg , 2012 ) , with default parameters except for ‘–X 800’ to allow for longer insert sizes .", "The reference genomes used are the following: GCF_000156535 . 1_ASM15653v1_genomic . fna ( R . intestinalis L1-82 ) , GCF_000157975 . 1_ASM15797v1_genomic . fna ( B . hydrogenotrophica S5a33 ) , GCF_000162015 . 1_ASM16201v1_genomic . fna ( F . prausnitzii A2-165 ) , CF_000146045 . 2_R64_genomic . fna ( yeast ) and NC_001422 . 1 ( PhiX174 ) .", "Gene expression was quantified using the htseq-count Python script ( Anders et al . , 2015 ) ( with parameter –a 2 to exclude multimapping reads ) for all species using their available * .", "gff reference annotation files .", "Given the small size of the PhiX174 , we quantified the reads mapping to its entire genome rather than its gene expression .", "Differential gene expression analysis of the three cultured strains was performed with DESeq2 ( Love et al . , 2014 ) .", "To remove the effect of the different bacterial compositions in the tri-culture samples , we extracted the reads from each strain prior to the differential expression analysis and analyzed each strain separately .", "In the DESeq2 design formula , we included two factors: type of culture ( mono- or tri-culture ) and time ( 3 hr , 6 hr and 15 hr ) .", "The results of the differential expression analyses were computed using a Wald test of the tri-culture versus the mono-culture samples .", "For each strain , we extracted the genes whose expression changed significantly ( with Benjamini-Hochberg adjusted p-value<0 . 05 ) in tri-culture and mapped them to different functional annotations downloaded from the IMG database ( Markowitz et al . , 2012 ) : COG categories , COG numbers and KO numbers .", "The RNA-seq data-processing code is available on GitHub ( Lloréns-Rico , 2018; copy archived at https://github . com/elifesciences-publications/syntheticGutCommunity ) .", "RNA-seq results have been deposited in the Short Read Archive under the study identifier SRP136465 ( https://www . ncbi . nlm . nih . gov/sra/SRP136465 ) .", "Fermentation data have been submitted to Dryad ( doi:10 . 5061/dryad . g83f29f ) .", "The model definition is provided in Source code 1 ( Model definition ) .", "The RNA-seq data processing code is provided on GitHub ( Lloréns-Rico , 2018; copy archived at https://github . com/elifesciences-publications/syntheticGutCommunity ) ." ] ]

[ "The composition of the human gut microbiome is well resolved , but predictive understanding of its dynamics is still lacking .", "Here , we followed a bottom-up strategy to explore human gut community dynamics: we established a synthetic community composed of three representative human gut isolates ( Roseburia intestinalis L1-82 , Faecalibacterium prausnitzii A2-165 and Blautia hydrogenotrophica S5a33 ) and explored their interactions under well-controlled conditions in vitro .", "Systematic mono- and pair-wise fermentation experiments confirmed competition for fructose and cross-feeding of formate .", "We quantified with a mechanistic model how well tri-culture dynamics was predicted from mono-culture data .", "With the model as reference , we demonstrated that strains grown in co-culture behaved differently than those in mono-culture and confirmed their altered behavior at the transcriptional level .", "In addition , we showed with replicate tri-cultures and simulations that dominance in tri-culture sensitively depends on the initial conditions .", "Our work has important implications for gut microbial community modeling as well as for ecological interaction detection from batch cultures ." ]

[ "Our gut is home to trillions of microorganisms , most of them bacteria , which have an important impact on our body .", "During healthy periods , these microorganisms help our digestion , protect our cells , and compete against disease-causing bacteria .", "But specific communities of gut bacteria are linked to many diseases .", "We already have a good knowledge of the bacterial composition present in a wide range of human guts , but how the different bacterial species within such communities affect each other , has so far been unclear .", "Future disease treatments may be able to steer ‘bad’ communities to healthier mixtures .", "For this to happen we need to know how species interact and how these interactions change the behavior of the whole community .", "To investigate this further , D'hoe , Vet , Faust et al . studied three common species of gut bacteria under controlled conditions in the laboratory .", "The different species were either grown alone , in pairs or together , and the number of bacteria and the concentration of nutrients were measured over time .", "The results showed that when grown alone or together , their behavior changed .", "D'hoe et al . then used a mathematical model to estimate the rates at which species multiplied and consumed nutrients .", "This model was able to predict the dynamics of each of the species grown alone .", "However , the data from bacteria grown in pairs was needed to predict the dynamics of bacteria grown as a group of three .", "Next , D'hoe et al . compared the activity of genes between bacteria grown alone or together , and discovered several differences .", "This suggests that bacterial species affect each other greatly , and community behavior cannot be predicted from knowledge of its members alone .", "Therefore , studying bacteria in isolation is not enough to understand the complex environments of our guts , which are inhabited not by three but hundreds of bacterial species .", "In future , interactions between bacteria will need to be studied to ultimately be able to shift the gut community into better shapes ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "microbiology and infectious disease" ]

[ [ "Arboviruses contribute to a substantial portion of the global disease burden , which can be effectively transmitted by insect vectors ( Eigenbrode et al . , 2018 ) .", "Among the plant-virus-vector associations , insect vectors are the only organisms that can freely disperse and closely interact with both host plants and plant viruses ( Han et al . , 2015 ) .", "Over the past decades , many studies on virus-induced changes on the behavior of insect vectors have been published , where the underlying mechanisms were typically attributed to modifications of plant nutritive and defensive metabolites in response to virus infection ( Mauck , 2016; Mauck et al . , 2016 ) .", "In particular , the persistently transmitted viruses ( PTVs ) , rather than non-persistently or semi-persistently transmitted viruses , manage to cross the physical barriers , circulate within the hemolymph , and even replicate in insect vectors .", "Therefore , it has been speculated that PTVs utilize a direct neuromanipulation mechanism to induce behavioral changes in insect vectors; however , the molecular mechanism remains poorly understood ( Eigenbrode et al . , 2018; Jia et al . , 2018 ) .", "Olfactory signaling , the most extensively investigated pathway , may be hijacked by plant viruses to modulate the behavior of insect vectors and their associations with host plants .", "For example , Rice stripe virus and Southern rice black-streaked dwarf virus were able to directly regulate the gene transcripts of odorant receptor coreceptor ( ORco ) and odorant-binding protein ( OBP ) of their insect vectors Laodelphax striatellus and Sogatella furcifera , respectively , and reversed their odorant preferences between virus infected and uninfected host plants ( Hu et al . , 2019; Li et al . , 2019 ) .", "Some rhabdoviruses have been reported to cross the salivary gland barrier through neurotrophic routes possibly due to neuroinvasion of the virus into the brain and nerve ganglia of its insect vectors ( Ammar and Nault , 1985; Ammar and Hogenhout , 2008; Chen et al . , 2011 ) .", "Furthermore , studies on insect viruses demonstrate that the virus infection in the brain could impair the learning and memory function leading to sensitivity deficit and response lag in its insect host ( Han et al . , 2015 ) .", "These relevant behavioral changes in infected insect hosts , including a reduction in mating and locomotor activities , were typically defined as sickness behavior resulting from immune activation and nervous system dysfunction ( Han et al . , 2015; Jenkins et al . , 2011; Patot et al . , 2009 ) .", "For instance , replication of the deformed wing virus ( DWV , Iflaviridae ) in the brain of Apis mellifera especially in the regions associated with vision and olfaction resulted in an insensitive responsiveness to food ( Iqbal and Mueller , 2007; Shah et al . , 2009 ) .", "Likewise , some plant viruses induced a similar sickness behavior in insect vectors , possibly believing that sickness behavior could be also utilized by plant viruses ( Eigenbrode et al . , 2018; Han et al . , 2015; Mauck et al . , 2016 ) .", "In mammals , the sickness behavior is the result of neuroinflammation induced by activation of the innate immune system ( Godbout et al . , 2005 ) .", "The toll-like receptor ( TLR ) and nod-like receptor ( NLR ) cascades that are triggered by pathogens amplify the signal of several inflammatory programmed cell death formats in the central nervous system ( CNS ) ( Dantzer et al . , 2008; Heneka et al . , 2014; Heneka et al . , 2018; LeBlanc and Saleh , 2009; Man et al . , 2017 ) .", "With the loss of neurons , the motor and sensory deficits become irreversible in the case of permanent tissue damage to the host of pathogens ( Glass et al . , 2010; Shattuck and Muehlenbein , 2015 ) .", "By contrast , plant viruses theoretically cause limited or no physical damage to insect vectors , but some cases of severe lesions leading to slower behavior and recognition dysfunction have been reported previously , suggesting that nerve damage and immune activation may be involved in the interaction between plant virus and its insect vector ( Ingwell et al . , 2012; Mauck et al . , 2012; Moreno-Delafuente et al . , 2013 ) .", "Intriguingly , it has been shown that some intracellular immune responses of insect vectors , such as apoptosis and autophagy , benefit virus propagation and transmission , which is contrary to the established antiviral function in the virus-host interaction ( Chen et al . , 2019; Chen et al . , 2017; Huang et al . , 2015; Wang et al . , 2012 ) .", "Tomato yellow leaf curl virus ( TYLCV , Geminiviridae ) , a type member of the genus begomovirus , is exclusively transmitted by Bemisia tabaci ( whitefly ) in a persistent circulative manner , and causes epidemic outbreaks worldwide resulting in extensive crop yield losses ( Czosnek et al . , 2017; Pakkianathan et al . , 2015 ) .", "TYLCV could enhance the odorant attractiveness of the plant to non-viruliferous Mediterranean ( MED , also named as biotype Q ) whitefly by suppressing the repellent volatile emission .", "After acquiring TYLCV , the whitefly displays no special preference for TYLCV-infected relative to uninfected tomato plants .", "This suggests that the loss of host preference in whitefly is possibly due to direct manipulation of TYLCV , but the underlying mechanism has not been fully elucidated ( Fang et al . , 2013 ) .", "Therefore , we aimed to understand the neural mechanisms of viruliferous whitefly underlying this direct behavioral manipulation of host preference , as well as the resulting virus transmission .", "Here , we present the evidence that TYLCV induces caspase-dependent apoptotic neurodegeneration in the brain of whitefly , leading to sensory deficits in whitefly and promotes TYLCV transmission among the plant community ." ], [ "Our previous field experiments have shown that non-viruliferous whitefly prefers to settle on TYLCV-infected plants , while the viruliferous whitefly does not exhibit a preference for TYLCV-infected relative to uninfected plants ( Figure 1A ) .", "Due to the complexity of the host seeking behavior of whitefly , a short-term free-choice assay was used to compare the host preference between viruliferous and non-viruliferous whiteflies , and the result showed that whiteflies tended to prefer TYLCV-infected plants before acquiring the virus , while they exhibited an equal preference for virus-infected and uninfected plants after acquiring TYLCV ( Figure 1B ) .", "Two sets of dual-choice experiments were conducted to separate the vision and olfaction cues from host plants , and the results revealed that a less number of viruliferous whiteflies preferred yellow light and odors released from virus-infected plants , suggesting TYLCV acquisition defected both the visual and olfactory sensitivities of whiteflies ( Figure 1C–D ) .", "To avoid the plant effect and directly detect the effect of virus acquisition on whiteflies , virions were purified and added to the artificial diet feed of whiteflies .", "Similarly , with the rearing on plants , and after the 2-day acquiring of virions , fewer whiteflies preferred virus-infected plants , suggesting that TYLCV impaired both the visual and olfactory preference of whiteflies to virus-infected plants ( Figure 1E–G ) .", "The mTYLCV , a coat protein mutant of TYLCV in which a partial sequence of CP was substituted by the Papaya leaf curl China virus and hardly penetrated the whitefly gut barrier , was used to determine whether the gut barrier could prevent the impairment of host preference of viruliferous whitefly ( Guo et al . , 2018; Wei et al . , 2017 ) .", "Compared with the wild-type TYLCV virions , mTYLCV virions failed to change the host preference of whitefly in the free-choice and odorant dual-choice experiments , suggesting that a successful crossing midgut was necessary for TYLCV to modify the host preference of whitefly ( Figure 1E–G ) .", "In order to determine the effect of TYLCV on the host selection of whitefly , the responding time of viruliferous whitefly to plant volatiles were monitored , and it was found that the reaction of viruliferous whitefly to plant odors became slower as the feeding time increased ( Figure 1H ) .", "It has been believed that the insensitivity of olfactory recognition was probably due to the impairment of odorant signaling in viruliferous whiteflies; however , none of the 8 OBPs and 12 chemosensory proteins ( CSPs ) significantly changed between viruliferous and non-viruliferous whiteflies ( Figure 1I ) .", "Merely the ORco , which is an indispensable transmembrane receptor located in olfactory sensory neurons , was downregulated by TYLCV ( Figure 1I ) .", "To determine the difference of gene expression profile in the nervous system between viruliferous and non-viruliferous whiteflies , the transcriptome of four head samples of each treatment ( eight in total ) were sequenced ( RNA-Seq ) .", "About 6 . 06 Gb data on average were generated for each sample .", "About 40 , 058 , 194 to 41 , 030 , 800 clean reads were obtained and mapped to the MED whitefly reference genome , and the mapping rates ranged from 69 . 15% to 71 . 55% .", "A total of 332 ( 203 upregulated , 129 downregulated ) genes were differentially expressed ( log2 fold change >1 , adjust p-value<0 . 05 ) in the head of viruliferous whitefly compared with those of non-viruliferous whitefly ( Supplementary file 2 , Supplementary file 3 ) .", "The enrichment analysis of KEGG pathway showed that 17 differentially expressed genes ( DEGs ) were enriched in the neurodegeneration pathway ( Figure 1—figure supplement 1 ) .", "The GO analysis showed that 13 DEGs were related to neurotransmitter receptors , synaptic vascular transporter proteins , and ion channel components located on neuron membranes .", "Furthermore , qRT-PCR assays confirmed that 10 out of 13 DEGs were downregulated by TYLCV , indicating that the TYLCV infection had substantial effects on the nervous system of whitefly ( Figure J ) .", "Neurodegenerative diseases , including Alzheimer's disease , Parkinson's disease , amyotrophic lateral sclerosis , and multiple sclerosis , have been well delineated in mammals , and are attributed to neuroinflammation , along with neuronal dysfunction and death ( Glass et al . , 2010 ) .", "The morphology symptom in insects exhibits vacuolar neuropathological lesions in the brain , which are caused by pathogen infection or aging ( Cao et al . , 2013; Kounatidis et al . , 2017 ) .", "In regard to vacuolar lesions in the brain of whitefly , TYLCV induced severe neurodegeneration in whitefly despite acquiring virions from plants vs . artificial diets ( Figure 2A–B ) .", "The progressive cell loss in specific neuronal populations during neurodegenerative disorders is mostly inducted by caspase-dependent cell death , which includes apoptosis , pyroptosis , necrosis , etc . ( Bredesen et al . , 2006; Fan et al . , 2017; Jellinger , 2001 ) .", "Only two caspase genes have been characterized in the genome of whitefly , namely , BtCaspase1 and BtCaspase3b , which were demonstrated to be involved in cell death to initiate apoptotic response to UV stress ( Wang et al . , 2018 ) .", "Acquiring virions from tomato plants and tobacco plants , and artificial diet , respectively , upregulated the gene transcripts of BtCaspase1 and BtCaspase3b , both in the whole body and head of whitefly ( Figure 2C–D , Figure 2—figure supplement 1C ) .", "Canonical effector caspase cleavage in model species has been well characterized and represents the initiation of apoptosis .", "Full-length effector caspase can be cleaved by initiator caspase into three subunits including a short prodomain , a p10 domain subunit , and a p20 domain subunit .", "The p20 and p10 subunits closely associate with each other to form a caspase monomer , and then , two monomers combine into an active homodimer that executes the apoptosis activation ( McIlwain et al . , 2013; Riedl and Shi , 2004 ) .", "However , it remains unclear whether Caspase3b of whitefly has the same/similar function with mammals or Drosophila melanogaster because whitefly loses many apoptosis-related genes ( McIlwain et al . , 2013; Nishide et al . , 2019; Riedl and Shi , 2004; Zhang et al . , 2014 ) .", "Using a time-course UV treatment experiment and non-reduced denaturing western blot assay with a p10 antibody , we found that inactive Caspase3b existed as 49 KDa monomer in whitefly ( most effector caspases usually exist as homodimer ) , and the cleavage process is the same as the canonical manner ( Figure 2E ) .", "Once apoptosis was induced by UV , cytoplasmic inactive Caspase3b rapidly decreased in 5 min , indicating cleavage initiation .", "Afterward , the full-length inactive Caspase3b was recovered in 15–30 min , and increased in 60–120 min , since the transcriptional expression was also induced .", "Cleaved p10 and monomer increased during apoptosis activation in 5–30 min , and afterward , these monomers formed the active homodimer ( 60–120 min ) ( Figure 2E ) .", "Together , these findings suggest that the decrease of full-length Caspase3b , and increase of p10 and monomer , as well as the increase of active homodimer represent the apoptosis activation in whitefly; however , they are shown in different stages of Caspase3b cleavage .", "This Caspase3b cleavage was also confirmed by another anti-Caspase3b p20 antibody , which displayed a similar result using p10 antibody ( Figure 2—figure supplement 2 ) .", "These results demonstrate that TYLCV virions directly induced Caspase3b cleavage in whitefly ( Figure 2F ) .", "TUNEL assay , which is the most frequently used method to detect cell death by labeling the end of the deoxynucleotidyl transferase dUTP , was performed to determine apoptosis in the head of viruliferous whitefly .", "Compared to the regular programmed cell death in non-viruliferous whitefly , TYLCV highly induced broad apoptosis in the brain of viruliferous whitefly , which strongly accumulated protein product of BtCaspase3b ( Figure 2G–H ) .", "Considering the difficulty in dissecting the pure brain tissue of whitefly , FISH and immunofluorescence were used to detect virus DNA and coat protein in the head of viruliferous whitefly .", "The results revealed that the DNA and coat protein of TYLCV was located in the brain , eyes , and antennas of viruliferous whitefly ( Figure 2—figure supplement 3 ) .", "These results suggest that TYLCV induced the BtCaspase1 and BtCaspase3b cascade , and caused apoptotic neurodegeneration in the brain of whitefly .", "The BtCaspase1 and BtCaspase3b in whitefly were silenced with double-stranded RNA ( Figure 2—figure supplement 1A–B ) .", "For dsCaspase1 or dsCaspase3b-silenced whitefly , TYLCV failed to impair the preference of whitefly to TYLCV-infected plants in the free-choice and dual-choice assays ( Figure 3A–B ) .", "The silence of BtCaspase1 and BtCaspase3b at the transcription level suppressed the cleavage of full-length BtCaspase3b into the homodimer at the protein level ( Figure 3C ) , and subsequently ameliorated the DNA fragmentation and neurodegeneration in the brain of whitefly ( Figure 3D–F ) , leading to the decreased accumulation of BtCaspase3b in the brain of viruliferous whitefly ( Figure 3G ) .", "These data demonstrate that Btcaspase3b is required for the virus-induced neurodegeneration of whitefly , and the preference change induced by TYLCV .", "In mammals , neurodegenerative disorders are usually conducted by NLRs associated with neuroinflammation ( Glass et al . , 2010 ) .", "NLRs can recognize the pathogen-associated molecular patterns ( PAMPs ) or damage-associated molecular patterns ( DAMPs ) , triggers the inflammasome assembly , and activates the downstream caspase cascades or cytokines release ( Glass et al . , 2010; Guo et al . , 2015; Heneka et al . , 2018 ) .", "Four NLR-like genes had been annotated in the present transcriptome data , which were named as NLRLs .", "Furthermore , six Spaetzles , which are homologous to cytokines in mammals , were also found in the transcriptome data , based on sequence similarity ( Figure 4A–B ) .", "The qRT-PCR confirmation revealed that only BtNLRL4 and BtSpaetzle1 and 2 ( two genes were quantified by a pair of primers due to high sequence similarity ) were upregulated in viruliferous vs . non-viruliferous whitefly ( Figure 4C–D ) .", "Cytoplasm NLRs are structurally and functionally conserved , and widely expanded from lower metazoans to mammals , except for insects , indicating that distinct domain architectures might be specially evolved in the NLRs of insects ( Meunier and Broz , 2017 ) .", "The architecture of BtNLRL4 was analyzed using website tools ( InterPro , Prosite , and SMART ) , and the similarity to model species was compared using PSI-BLAST .", "These revealed that the leucine-rich repeat domain ( LRR ) of BtNLRL4 , which generally function as the sensor of PAMPs and DAMPs , was highly similar to NOD3 in Homo sapiens or NLRC3 in Mus musculus .", "However , the NOD or PYRIN/CARD/DED domains were not identified ( only predicted by SMART with a substandard significance score ) ( Figure 4E ) .", "In order to further determine the roles of BtNLRL4 and BtSpaetzle1 and 2 , which involved the TYLCV-induced neurodegeneration in whitefly , BtNLRL4 , and BtSpaetzle1 and 2 were silenced with RNA interference ( Figure 2—figure supplement 1A–B ) .", "The TYLCV-induced neurodegeneration was ameliorated in fewer and smaller vacuolar lesions in the brain when BtNLRL4 and BtSpaetzle1 and 2 were silenced ( Figure 2 4 F-G ) .", "The silencing of BtNLRL4 and BtSpaetzle1 and 2 also suppressed the cleavage of full-length BtCaspase3b , and reduced the apoptosis cells , and ameliorated the accumulation of Caspase3b in the brain of whitefly ( Figure 4H–J ) .", "The qRT-PCR experiments revealed that TYLCV failed to upregulate BtCaspase1 and BtCaspase3b when BtNLRL4 and BtSpaetzle1 and 2 were silenced in whitefly , respectively ( Figure 4K–L ) .", "Likewise , TYLCV failed to suppress the host selection ability of viruliferous whitefly when BtNLRL4 and BtSpaetzle1 and 2 were silenced ( Figure 4M–N ) .", "These results demonstrate that the NLRL4-Spaetzle1 and 2 signaling was required for the TYLCV-induced caspase-dependent neurodegeneration in whitefly .", "TYLCV induced a typical sickness behavior and impaired the host selection ability of viruliferous whitefly .", "In order to determine the effects of TYLCV-induced sickness behavior on the virus transmission ability of whitefly in the plant community , infected or uninfected plants were alternatively placed in a 1 m diameter circle , and 150 whiteflies for each treatment were released at the center for the 4 hr virus transmission experiment ( Figure 4—figure supplement 1 ) .", "A total of 16 previously uninfected plants for each treatment were marked and detected after 7 days to evaluate the virus transmission ability of viruliferous whitefly .", "The PCR results revealed that 15 out of 16 plants were successfully infected with TYLCV , but fewer plants were infected by viruliferous whitefly when BtCaspase1 , BtCaspase3b , BtNLRL4 , and BtSpaetzle1 and 2 were , respectively , silenced ( Figure 4O ) .", "These results indicate that TYLCV-induced sickness behavior promoted TYLCV transmission in the plant community ." ], [ "Since all known geminiviridae viruses are plant viruses , the modification of insect activities that facilitate virus transmission was largely attributed to the quality of virus-infected plants ( Eigenbrode et al . , 2018; Liu et al . , 2013; Moreno-Delafuente et al . , 2013 ) .", "The changes in defensive metabolites and nutritive values of host plants were empirically responsible for improving the feeding efficiency and oviposition of viruliferous whitefly ( Liu et al . , 2013; McKenzie , 2002; Moreno-Delafuente et al . , 2013 ) .", "The disease symptoms of TYLCV-infected plants , such as yellow curl leaves and less repellent volatiles , enhanced the attractiveness to non-viruliferous whitefly to acquire TYLCV virions ( Fang et al . , 2013 ) .", "It has been reasonably speculated that sustaining a strong attractiveness to viruliferous insect vectors was a disadvantage for the virus spread .", "In this study , we found that TYLCV was able to eliminate the unfavorable attractiveness from virus-infected plants by directly impairing the host selection ability of viruliferous whitefly .", "This virus-induced sickness behavior of whitefly was triggered by caspase-dependent apoptotic neurodegeneration , which was the outgrowth of the activation of innate immune response in whitefly .", "Our finding reveals the virus-induced immune-neuro-behavior communication in viruliferous insect vector , which promoted the virus transmission among plants .", "In most cases , host preferences between viruliferous and non-viruliferous vectors are opposite , in which the non-viruliferous vector preferred virus-infected plants , while the viruliferous vector preferred uninfected plants ( Eigenbrode et al . , 2018 ) .", "These opposite preferences benefit the virus spread during both the virus acquisition and transmission stages , and these have been reported to be regulated by the olfaction signaling cascades of insect vectors , including the transcriptional regulation of ORs and OBPs ( Hu et al . , 2019; Li et al . , 2019 ) .", "By contrast , our results show that viruliferous whitefly exhibits an unbiased preference between TYLCV-infected and uninfected plants , suggesting that the manipulation of TYLCV on the host preference of whitefly could differ in the modifications of olfaction signaling .", "Likewise , it appears that TYLCV suppressed the perception of whitefly to olfactory or visional cues from host plants , in terms of behavioral choice assays .", "The TYLCV-induced sensory deficits in viruliferous whitefly were similar to typical sickness behavior , even though this was a coordinated symptom of behavioral changes that responded to the infection .", "Furthermore , the sickness behavior was considered as an adaptive means of redirecting energy from disadvantageous behavior to an effective immune response ( Shattuck and Muehlenbein , 2015 ) .", "The insect virus-induced sickness behavior reduced the sexual activity and feeding of the insect host , which were beneficial for insect population , thereby deterring the virus spread from healthy individuals but negatively affecting the sexually transmitted pathogens ( Han et al . , 2015 ) .", "In this study , viruliferous whitefly that exhibited the sickness behavior efficiently transmitted the TYLCV , when compared to those without sickness behavior , suggesting that the sickness behavior of viruliferous whitefly facilitates the spread of PTVs .", "Neurodegenerative disorders accompanied by CNS cell loss are responsible for the sickness behavior in insects ( Godbout et al . , 2005; McCusker and Kelley , 2013; Ransohoff , 2016 ) .", "Pathogen infection triggers the innate immune response in the CNS of Drosophila resulting in inflammatory signaling and neurodegeneration ( Cao et al . , 2013; Delorme-Axford and Klionsky , 2019; Liu et al . , 2018 ) .", "Interestingly , we found that the acquisition of PTVs induced neuropathological lesions in the brain of insect vectors , suggesting that whitefly could be directly infected by TYLCV ( Figure 2A–B , Figure 1—figure supplement 1 ) .", "It is noteworthy that apart from insect viruses , few plant viruses have been found to infect or replicate in the brain of insects .", "However , in this study , both the coat protein and DNA of TYLCV can be stained by fluorescence confocal microscopy in the brain , eyes , and antenna of whitefly , indicating that an undiscovered nerve route of whitefly may exist for the infection of TYLCV ( Figure 2—figure supplement 3 ) .", "Similar to mammals , caspase-dependent CNS cell death is also responsible for TYLCV-induced neurodegeneration ( Wang et al . , 2018 ) .", "For virus-vector interactions , apoptosis is an effective weapon of the innate immunity to eliminate the virus infection , replication , and dissemination within the insect vector ( Chen and Wei , 2019 ) .", "These results revealed that the silencing of apoptosis-related caspases was behaviorally unfavorable to TYLCV transmission ( Figure 4O ) .", "Likewise , for leafhoppers , Rice gall dwarf virus activates the caspase-dependent apoptosis by targeting the mitochondria , and inducing mitochondrial degeneration to promote viral infections within the insect vector ( Chen et al . , 2019 ) .", "These findings suggested that vector apoptosis could be utilized by PTVs to promote the virus spread .", "Furthermore , compared with wild-type TYLCV , the mutant TYLCV was unable to induce the sickness behavior in whitefly , suggesting that the successful entry into the hemolymph was a preliminary step for the stimulation of whitefly innate immunity .", "Furthermore , the immune response was activated by TYLCV only after 12 hr of virus acquisition ( Figure 2—figure supplement 1D ) , while 24–48 hr is usually required for TYLCV to cross the salivary gland barrier ( Brown and Czosnek , 2002 ) .", "It has been suggested that the initiation of the innate immune response in whitefly is essential for TYLCV to cross the physical barrier of the midgut .", "In addition , the immune deficiency ( IMD ) pathway is involved in the neurodegeneration of D . melanogaster , but this is exceptionally absent in some hemipteran insects , such as Acyrthosiphon pisum , Diaphorina citri , and B . tabaci , suggesting that whitefly quite differs from D . melanogaster , in terms of regulation of neurodegeneration ( Myllymäki et al . , 2014; Nishide et al . , 2019 ) .", "NLRs , which cascade the inflammatory signaling , are required to amplify the immune activation , and causes neurodegeneration in human .", "Remarkably , conserved NLRs are the most abundant cytoplasmic innate immune receptors in plants and animals , other than insects .", "In the present study , a putative NLR , BtNLRL4 , was found to be necessary for the TYLCV-induced neurodegeneration of whitefly , even though the architecture differed from NLRs in mammals or plants ( Figure 4F–G ) .", "Considering the DD domain of initiator BtCaspase1 and the putative DD/DED domain of BtNLRL4 , the direct interaction of BtCaspase1 and BtNLRL4 might exist in whitefly rather than the constructing inflammasome in mammals .", "Spaetzles are homologous to cytokines in mammals which have been well characterized in terms of its inflammatory function .", "In these present results , BtSpaetzle1 and 2 was also essential to the TYLCV-induced neurodegeneration of whitefly ( Figure 4F–G ) .", "TYLCV was unable to upregulate the gene transcripts of BtCaspase1 and BtCaspase3 when BtNLRL4 and BtSpaetzle1 and 2 were , respectively , silenced , indicating that BtNLRL4-BtSpaetzle1 and 2 was possibly the upstream signaling of the caspases cascade , which is consistent with the inflammatory signaling in mammals ( Glass et al . , 2010; Heneka et al . , 2018 ) .", "The behavioral manipulation on insect vector preference is one of the most effective strategies of plant viruses to enhance their spread ( Roosien et al . , 2013 ) .", "A mathematic model concluded that the host preference of insect vectors could lead to a dramatic difference in plant virus epidemic ( Roosien et al . , 2013 ) .", "For single species of plants , viruliferous whitefly preferably chooses uninfected plants , which appears to be beneficial to the TYLCV spread , while the unbiased host preference is more practical and has more efficacy to the plant community , in terms of the complexity and diversity of the volatile components released from a broad range of host plants of TYLCV .", "Although TYLCV-induced immune responses are favorable for virus spread , it has been shown that the activation of autophagy leads to degradation of the coat protein and genome DNA of TYLCV ( Chen et al . , 2017 ) .", "It has been suggested that merely the optimal degree of immune responses triggered by the virus could maximize the benefits of TYLCV in balancing the virus replication and transmission .", "In summary , TYLCV changes the host preference of whitefly to promote its spread by inducing the caspase-dependent apoptotic neurodegeneration in the insect vector .", "A deep understanding of the innate immunity and utilization of the antagonistic effect between apoptosis and autophagy in insect vectors could be an efficient approach to control the transmission and spread of PTVs in the future ." ], [ "The Mediterranean ( MED ) /Q whitefly ( mtCOI GenBank accession no: GQ371165 ) of the Bemisia tabaci species complex were reared on cotton plants ( Gossypium hirsutum cv Guo-Shen 7886 ) placed in insect-proof cages between 26°C and 28°C , with a photoperiod of 16:8 hr ( light/dark ) .", "Adult male or female whiteflies were randomly obtained using a sucking device for the experiments .", "The infectious clone of TYLCV isolate SH2 ( GenBank accession no: AM282874 ) was provided by Professor Xueping Zhou ( State Key Laboratory for Biology of Plant Diseases and Insect Pests , Institute of Plant Protection , Chinese Academy of Agricultural Sciences ) .", "The infectious clone of mutant TYLCV was provided by Xiaowei Wang ( Institute of Insect Sciences , Zhejiang University ) .", "Tomato plants ( Solanum lycopersicum cv Moneymaker ) were used as the natural host for both TYLCV and whitefly in behavioral assays .", "Virus-infected Nicotiana benthamiana was used to purify the TYLCV virions .", "All plants were reared at 26–28°C , with 60% relative humidity and a photoperiod of 16:8 hr ( light/dark ) .", "The infectious clone was inoculated into plants at the 3–4 true leaves stage , and plants with both obvious symptoms and a positive result in the PCR analysis were used in the experiments as TYLCV infected plants .", "A total of 30 whiteflies from the same treatment were collected in a clean pipet tip as one biological replicate for all behavioral assays .", "The free-choice assay was performed in an insect-proof cage ( 40 × 40 × 40 cm3 ) , with two TYLCV infected and two uninfected tomato plants placed diagonally .", "Then , the number of whiteflies on each plant was counted after a 10 min choosing .", "Any whitefly that settled on the ground or hung on the cage was recorded as ‘no choice’ .", "For the olfactory related dual-choice assay , two hermetical-sealed glass chambers ( 40 cm in height , 23 cm in diameter ) that contained the test plants were connected to two branch arms of a glass Y-tube olfactometer ( 24 cm in length for each arm ) , respectively .", "A purified airflow was equally and continually pumped from the glass chamber to the olfactometer at 300 ml/min .", "For the visual-related dual-choice assay , two branch arms of the olfactometer were connected to the same glass chamber and equally illuminated by green and yellow light , respectively .", "For the responding test , one branch arm of the Y-tube was connected to the glass chamber that contained the tomato plant , and another branch arm was connected to the clean airflow .", "The Y-tube olfactometer was rotated at 180° after each replicate to avoid positional bias , and all tests were conducted between 15:00 and 20:00 , in case of the circadian difference .", "Whiteflies were released in the main olfactometer arm for up to 10 min .", "A choice for one of the two branch arms was considered as valid when the whitefly moved >5 cm onto either arm , and stayed in that arm for at least 15 s .", "This experiment design has been modified from Fereres’ and Li’s works ( Fereres et al . , 2016; Li et al . , 2014 ) .", "Whitefly preference was quantified with an attraction index ( AI ) , calculated as: AI = ( V - N ) /30 , where V is the number of viruliferous whiteflies , N is the number of non-viruliferous whiteflies , and 30 is the sum of whiteflies was used in one test .", "The AI calculation method was also performed in previous study ( Keesey et al . , 2017 ) .", "TYLCV particles were isolated from young leaves of Nicotiana benthamiana plants after post-inoculation 3 weeks of the TYLCV infectious clone .", "The brief protocol was previously described and modified in the present experiment ( Pakkianathan et al . , 2015 ) .", "One gram of fresh weight leaf tissue was homogenized in 2 . 4 ml of ice-cold lysis buffer ( pH 8 . 0 , 100 mM trisodium citrate , 18 . 5 mM ascorbic acid , 60 mM sodium sulfite , 5 mM EDTA , and 1% [wt/vol] β-mercaptoethanol ) and produced in 2 . 5% ( vol/vol ) Triton X-100 , stirred overnight , filtered through four layers of cheesecloth , and clarified by 10 min centrifugation at 8000 g .", "The supernatant was filtered through 0 . 2 μm of Supor membrane Non-Pyrogenic ( PALL ) and centrifuged for 3 hr at 90 , 000 g in a SW41Ti rotor with a coulter optima XPN-80 ultracentrifuge ( Beckman ) .", "The pellet was resuspended in buffered ( pH 8 . 0 ) CEM buffer ( 10 mM trisodium citrate , 1 mM EDTA , and 0 . 1% β-mercaptoethanol ) , and loaded onto a 10 . 5 ml linear 10–50% sucrose gradient in CEM buffer .", "The sucrose gradient was fractionated ( 1 ml per fraction ) after 3 hr of centrifugation at 90 , 000 g .", "The positive fractions ( determined by PCR ) were diluted with CEM buffer , and centrifuged for 3 hr at 90 , 000 g , and the final pellet was suspended in 15% sucrose in CEM buffer ( without β-mercaptoethanol ) .", "The presence of viral particles was confirmed by staining with 2% phosphotungstic acid , and observed using a Tecnai G2 F20 TWIN transmission electron microscope .", "Then , about 12 g of fresh weight leaves was finally suspended in 1 ml of CEM buffer for whitefly artificial diet feeding .", "A total of eight whitefly samples were respectively collected from TYLCV infected or uninfected tomato plants for sequencing , and each sample contained approximately 300 dissected heads .", "The RNA was extracted using an Absolutely RNA Nanoprep Kit ( Agilent ) .", "The concentration and quality of the total RNA were determined using a NanoDrop spectrophotometer ( Thermo ) and by gel electrophoresis .", "Beads that contained oligo ( dT ) were used to isolate the poly ( A ) mRNA from the total RNA .", "The purified mRNA was fragmented in the fragmentation buffer , and used as templates to synthesize the first-strand cDNA .", "Then , the second-strand cDNA was synthesized using a buffer , dNTPs , RNase H , and DNA polymerase I .", "The short fragments with additional ‘A’ base were ligated to the Illumina sequencing adaptors .", "The selected size DNA fragment was amplified by PCR , and sequenced on an Illumina HiSeq 2000 sequencing machine .", "The dirty raw reads were removed before analyzing data .", "Then , the resulting reads were aligned to the MED whitefly reference genome ( http://gigadb . org/dataset/100286 ) from the Giga Database , and the fragments per kilobase of transcript per million fragments mapped ( FPKM ) were estimated .", "DEseq2 was used to filtrate the differentially expressed genes ( DEGs ) .", "Then , the enrichment analysis of KEGG was performed to identify the regulation pathways represented by these DEGs .", "The transcriptome raw data has been released already with ID: PRJNA606896 .", "Whiteflies were collected and placed in 4% paraformaldehyde fixative overnight at 4°C , washed in 70% ethanol and processed into the Tissue-Tek O . T . C . Compound ( Sakura ) .", "After freezing at −20°C , the embedded whiteflies were sectioned at 10 μm using a Leica CM1950 freezing microtome and stained with H & E according to the standard protocol .", "Images were taken using a Nikon light microscope , equipped with a DS-Fi1c camera ( Nikon ) , and the images were generated using the NIS-Element D software ( Nikon ) .", "The appearance of vacuolar lesions in the brain neuropil was the typical symptom of neurodegeneration , and six levels of neurodegeneration ( 0 , 1 , 2 , 3 , 4 , and 5 ) were defined for quantification in previous research ( Cao et al . , 2013 ) .", "The same standard was applied to quantify the neurodegeneration of whiteflies .", "The total DNA of plants or whiteflies was extracted using the RoomTemp Sample Lysis Kit ( Vazyme ) , according to manufacturer’s protocol , and a 412 bp fragment of TYLCV was amplified with the standard PCR protocol using 2 × Taq PCR MasterMix ( Tiangen ) .", "The primers , which were named V61 and C473 , as previously described ( Atzmon et al . , 1998 ) .", "The total RNA of the whitefly samples for the RT-qPCR analysis were extracted by TRIzol Reagent ( Ambion ) , and reverse transcribed using the FastQuant RT Kit with gDNase ( Tiangen ) .", "The RT-qPCR reactions were carried out on the PikoReal 96 Real-Time PCR System ( Thermo ) using the PowerUp SYBR Green Master Mix ( Applied Biosystems ) .", "Three technical replicates were applied for each biological replicate .", "The data was analyzed by relative quantification with the 2-△△CT method .", "For the RT-qPCR , the sequences and partial primers of caspases , OBPs and CSPs were obtained from previous studies ( Wang et al . , 2017; Wang et al . , 2018 ) , while others were obtained from the MED whitefly genome data ( Xie et al . , 2017 ) , and primers were designed by Primer Premier 6 .", "Actin was used as the housekeeping gene in the experiments .", "The availability of each pair of primers had been tested in the preliminary experiments .", "The oligonucleotides are listed in Supplementary file 1 .", "except CSP9 .", "CSP9 was not detected in our study .", "A total of 100 whole whiteflies were pooled per condition per experiment and lysed in 150 μl of RIPA buffer ( CST ) supplemented with a protease inhibitor cocktail ( CST ) .", "Protein samples were separated by non-reduced denaturing polyacrylamide gel electrophoresis with 8–20% or 15% precast Tris-glycine gel ( EZBiolab ) and transferred onto 0 . 22 μm polyvinylidene difluoride membranes ( Millipore ) .", "Then , these membranes were blocked with SuperBlock T20 blocking buffer ( Pierce ) , and incubated with the primary antibody .", "After incubation with the secondary antibody ( CST ) , the signals were visualized using the SuperSignal West Pico PLUS Chemiluminescent Substrate ( Pierce ) .", "Using the synthetic peptides YFRPKRPAIDL*C ( Caspase3b 427-437aa , p10 domain ) or LSQEDHSDADC ( Caspase3b 252-262aa , p20 domain ) as the immunogen , the rabbit-anti Caspase3b polyclonal antibody was prepared by Beijing Genomics Institute ( BGI ) .", "The commercial primary antibody mouse anti-GAPDH ( 60004–1-Ig ) was purchased from Proteintech , and the commercial anti-mouse ( ab6789 ) and anti-rabbit ( ab6721 ) were purchased from Abcam .", "After the Caspase3b ( dilution 1:1 , 000 ) signals were visualized , the same blot was stripped by Restore Western Blot stripping buffer ( Pierce ) , and incubated with anti-GAPDH ( dilution 1:10 , 000 ) after blocking , in order to ensure that these two different signals ( Caspase3b and GAPDH ) came from the same protein sample .", "Three independent experiments were performed .", "The dsRNA was synthesized using the T7 RiboMAX Express RNAi System ( Promega ) , according to the manufacturer’s protocol .", "Approximately 150 adult whiteflies were placed in 30 mm in diameter , by 60 mm in height cylindrical dark containers .", "Each container provided a 500 μl diet with 400 μl of 15% sucrose , 50 μl purified virions ( or CEM buffer as control ) , and 50 μl 10 μg/μl dsRNA ( dsGFP as control ) .", "All RNA and protein samples were collected after a 48 hr feeding .", "Each treatment was replicated for at least three times .", "The frozen sections were rinsed for three times in TBST ( TBS with 0 . 05% Tween-20 ) and blocked with SuperBlock T20 ( Pierce ) .", "Then , the samples were incubated with the primary antibody ( anti-Caspase3b , 1:500; anti-TYLCV CP , 1:500 ) overnight at 4°C , rinsed three times in TBST , and incubated with the secondary antibody ( 1:1000 ) at room temperature for 2 hr .", "Negative controls had been performed in each independent experiment .", "The monoclonal antibody mouse anti-TYLCV CP was kindly provided by Professor Jianxiang Wu ( Institute of Biotechnology , Zhejiang University ) .", "The anti-mouse conjugate Alexa 488 ( ab150113 ) and anti-rabbit conjugate Alexa 555 ( ab150078 ) were purchased from Abcam .", "The samples were rinsed for three times in TBST , and mounted in Fluoroshield Mounting Medium with DAPI ( Abcam ) .", "Sections were imaged using a Zeiss LSM710 confocal microscope .", "The apoptotic cell death of the frozen sections with different treatments were analyzed using a One Step TUNEL Apoptosis Assay Kit ( Beyotime ) .", "The sections were rinsed twice in PBS , and incubated with PBST ( PBS with 0 . 5% Triton-X ) for 5 min at room temperature .", "Then , these sections were rinsed twice in PBS , and incubated with the TUNEL mixture ( Enzyme Solution: Label Solution = 1:9 ) for 1 hr at 37°C .", "After rinsing thrice in PBS , these sections were mounted and imaged using a microscope .", "The whole whitefly was fixed in Carnoy’s fixative ( chloroform-ethanol-glacial acetic acid [6:3:1 , vol/vol] ) overnight at 4°C , and rinsed for three times in TBS .", "After washing by TBST ( TBS with 0 . 2%Triton-X ) for 10 min , the whitefly was rinsed for three times in hybridization buffer ( 20 mM Tris-HCl , pH 8 . 0 , 0 . 9 M NaCl , 0 . 01% [wt/vol] sodium dodecyl sulfate , 30% [vol/vol] formamide ) for pre-hybrid ( without the probe ) .", "Then , 10 pmol of the fluorescent DNA probe ( conjugated with Cy5 ) was added into 500 μl of hybridization buffer , and the whitefly was hybridized overnight at room temperature in the dark .", "Afterward , the hybridized whitefly was rinsed for three times in TBS , and mounted before imaging by microcopy .", "The probe was described in a previous study and is listed in Supplementary file 1 Table S1 ( Pakkianathan et al . , 2015 ) .", "Statistical analyses were performed using SPSS ( Chicago , IL ) .", "The Wilk-Shapiro test was used to determine the normality of each data set .", "Normally distributed data were then analyzed using two-tailed , paired t-test .", "Nonparametric distributed data were assessed using Mann–Whitney test .", "An asterisk denotes statistical significance between two groups ( *p<0 . 05 , **p<0 . 01 , ***p<0 . 001 ) ." ] ]

[ "The mechanism by which plant viruses manipulate the behavior of insect vectors has largely been described as indirect manipulation through modifications of the host plant .", "However , little is known about the direct interaction of the plant virus on the nervous system of its insect vector , and the substantial behavioral effect on virus transmission .", "Using a system consisting of a Tomato yellow leaf curl virus ( TYLCV ) and its insect vector whitefly , we found that TYLCV caused caspase-dependent apoptotic neurodegeneration with severe vacuolar neuropathological lesions in the brain of viruliferous whitefly by inducing a putative inflammatory signaling cascade of innate immunity .", "The sensory defects caused by neurodegeneration removed the steady preference of whitefly for virus-infected plants , thereby enhancing the probability of the virus to enter uninfected hosts , and eventually benefit TYLCV spread among the plant community .", "These findings provide a neuromechanism for virus transmission to modify its associated insect vector behavior ." ]

[ "When a plant becomes infected by a virus , its defenses get weakened , which attracts insects that are looking for an easy meal .", "Insects detect which plants are infected based on the color of the sickened plant and the smell of chemicals it releases .", "Once an insect leaves the infected plant , it may carry the virus to new plants , allowing the virus to spread .", "Insects , however , prefer the easy pickings of plants that are already infected , making them less likely to spread the virus .", "Plant viruses have found ways to overcome this preference , but how they do this was not fully understood .", "Learning more about how plant viruses manipulate insects into helping them spread could allow scientists to develop new ways of protecting food crops from viral diseases .", "Viruses that infect insects can trigger excessive immune system responses that damage insects’ nerves and cause them to behave differently .", "For example , their senses may become impaired , they may move less , or be less able to remember things .", "This has led scientists to wonder whether plant viruses that use insects to spread might manipulate the insects’ behaviors using a similar mechanism .", "Now , Wang et al . have investigated whether the tomato yellow leaf curl virus –TYLCV for short – changes the behavior of whiteflies , which are known to spread the virus .", "The experiments showed that whiteflies typically prefer tomato plants infected with the virus , but after carrying TYLCV , they displayed equal preference for both infected and uninfected plants .", "Analyzing which genes were active in the whiteflies revealed that TYLCV triggers a harmful immune response which turns on genes that cause cells in the brain to die .", "This impairs the whiteflies' sight and sense of smell , making it harder for them to distinguish between infected and uninfected plants .", "These findings suggest that the immune response triggered by the virus may be essential for the spread of TYLCV .", "It also identified a protein that causes the death of brain cells , leading to behavioral changes in the whiteflies .", "This suggests that targeting this protein , or other steps in this process , could help stop the spread of TYLCV in tomato plants ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "physics of living systems" ]

[ [ "The Frank-Starling law of the heart describes the relationship between cardiac stroke volume and end-diastolic volume and operates on a beat-to-beat basis .", "At the cellular level , the Frank-Starling relationship implies that increasing cardiac sarcomere length results in enhanced performance of cardiac muscle cells during the subsequent contraction ( Allen and Kentish , 1985; de Tombe et al . , 2010 ) .", "This myofilament length-dependent activation ( LDA ) is two-fold: with an increase in sarcomere length ( SL ) , there are increases in ( 1 ) the maximum force developed by the myofilaments at high Ca2+ and ( 2 ) their sensitivity to Ca2+ .", "Impaired LDA and hence a defective Frank–Starling relationship may also contribute to the pathogenesis of hypertrophic cardiomyopathy and heart failure ( Schwinger et al . , 1994; Sequeira et al . , 2013 ) .", "Although the Frank-Starling law of the heart is one of its fundamental properties and has been investigated for over a century , the detailed molecular mechanism for the LDA in heart muscle remains elusive .", "Over the last forty years , the main focus has been on the myofilament Ca2+-sensitivity component of LDA .", "Contraction of cardiac muscle is driven by an interaction between myosin and actin that is controlled by the transient binding of Ca2+ ions to troponin in the actin-containing thin filament on a beat-to-beat basis ( Tobacman , 1996; Gordon et al . , 2000; Kobayashi and Solaro , 2005 ) .", "An increase in SL leads to a decreased inter-filament spacing ( Irving et al . , 2000; Lee et al . , 2013 ) and it has been suggested that this leads directly to an increase in Ca2+ sensitivity of force generation ( McDonald and Moss , 1995; Fuchs and Smith , 2001 ) .", "However , when osmotic compression of inter-filament spacing matches that induced by SL , the myofilament Ca2+ sensitivity is only affected by SL ( Konhilas et al . , 2002 ) , indicating that the reduction of inter-filament spacing is unlikely to mediate LDA .", "Titin-based passive tension in the heart muscle cells seems to play an important role in LDA ( Cazorla et al . , 2001; Fukuda et al . , 2001; Lee et al . , 2010 ) .", "Such a mechanism might involve a strain sensor and a signal transduction pathway that conveys the strain signal to the contractile machinery .", "A single titin molecule runs from the Z-disc to the M-line in the A-band contributing to muscle assembly and passive tension ( Labeit and Kolmerer , 1995 ) .", "The fact that titin is largely responsible for the length-dependent passive tension of cardiac muscle and also possesses both actin and myosin binding domains ( Linke et al . , 2002; Granzier and Labeit , 2004 ) , makes it a candidate for the link between SL and force generating apparatus .", "Small-angle X-ray diffraction studies suggest that length-dependent changes in the myosin head orientation in diastole may be a factor in LDA ( Farman et al . , 2011 ) .", "Moreover , a recent X-ray study shows that length-dependent structural changes are also observed in the thin filament in diastole ( Ait-Mou et al . , 2016 ) , supporting the notion that thin filament activation may also play an important role in LDA .", "The length-dependent changes in the structure of both thick and thin filaments are related to the titin strain ( Ait-Mou et al . , 2016 ) .", "To identify the molecular mechanism ( s ) underlying LDA , we measured the structural changes in the thin and thick filaments of intact sarcomeres in heart muscle induced by SL changes using bifunctional rhodamine ( BR ) probes on the cardiac troponin C ( cTnC ) and myosin regulatory light chain ( cRLC ) ( Sun et al . , 2009; Kampourakis et al . , 2014 ) .", "These probes allowed us to differentiate the structural changes in both types of filament caused by calcium activation , force-generating myosin heads and SL changes in the native environment of the cardiac sarcomere .", "The results presented here show that LDA is accompanied by distinctive structural changes in both the thin and thick filaments ." ], [ "Ca2+-activated force development was determined in demembranated ventricular trabeculae from rat heart .", "Maximum active force at pCa 4 . 5 increased by ~40% when SL was increased from 1 . 9 to 2 . 3 μm ( Figure 1 ) .", "Passive force increased in the same SL range by ~30% of maximum Ca2+-activated force at SL 1 . 9 μm ( Figure 1a ) .", "Increasing SL also produced a leftward shift of the Ca2+-force relationships , indicating an increase in myofilament Ca2+ sensitivity ( Figure 1b ) .", "These length-dependent changes in force are consistent with previous reports ( Kentish et al . , 1986; Dobesh et al . , 2002 ) . 10 . 7554/eLife . 24081 . 003Figure 1 . Sarcomere length-force relationships in rat cardiac trabeculae .", "( a ) Passive force ( ○ ) and maximum Ca2+-activated force ( ● ) at five sarcomere lengths ( n = 6 trabeculae ) .", "Force is normalised to maximum force measured at SL 1 . 9 μm ( T/T0 ) .", "( b ) pCa-force relationships at sarcomere lengths 1 . 9 μm ( ○ ) and 2 . 3 μm ( ● ) .", "The data were summarised from experiments for Figure 2a and b and fit to the Hill Equation ( n = 10 ) .", "Dotted line is the Hill curve at SL 2 . 3 μm normalised to its maximum force .", "Increases in SL resulted in increases in maximum Ca2+-activated force and Ca sensitivity ( leftward shift of pCa-force curve ) .", "Error bars denote SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 24081 . 003 Contraction of heart muscle is initiated by Ca2+ binding to the regulatory domain of troponin in the actin-containing thin filaments , that triggers a cascade of structural changes in the thin filament and leads to an azimuthal movement of tropomyosin around the filament that allow myosin to interact with actin and generate force ( Tobacman , 1996; Gordon et al . , 2000; Kobayashi and Solaro , 2005 ) .", "The IT arm of troponin , a rigid domain containing the C-terminal lobe of TnC and a coiled-coil formed by α-helices from troponin I ( TnI ) and troponin T ( TnT ) , anchors the troponin complex to the thin filament .", "To determine whether the changes in SL-dependence of force are accompanied by structural changes in the thin filaments of cardiac muscle cells , we measured the orientation of the regulatory head and IT arm of troponin .", "Cardiac troponin C with bifunctional rhodamine ( BR ) cross-linking residues 55 and 62 along the C helix ( BR-cTnC-C ) in the regulatory head or 95 and 102 along the E helix ( BR-cTnC-E ) in the IT arm of troponin was incorporated into demembranated trabeculae ( Sun et al . , 2009 ) .", "The polarized fluorescence intensities from trabeculae containing BR-labelled cTnC were used to calculate the order parameter <P2> that describes the orientation of the cTnC helix to which BR probe is attached with respect to the trabecular axis ( for details , see Materials and methods ) .", "The Ca2+-dependence of the <P2> changes was described using the Hill equation , which gave a good fit to the data for both the C and E helix probes in the range of [Ca2+] between pCa 7 and 4 . 5 ( Figure 2 ) .", "Here pCa50 , the pCa for half-maximal changes in <P2> , is a measure of Ca2+ sensitivity; nH , the Hill coefficient , describes the steepness of the Ca2+ dependence and is a measure of the cooperativity of the Ca2+-dependent change . 10 . 7554/eLife . 24081 . 004Figure 2 . Ca2+-dependence of force and troponin orientation , <P2> , in cardiac trabeculae .", "( a ,", "c ) BR-cTnC-C; ( b ,", "d ) BR-cTnC-E .", "Open symbols , SL 1 . 9 μm; filled symbols , SL 2 . 3 μm .", "Error bars denote SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 24081 . 004 It is generally thought that force-generating myosin heads sensitise the thin filament to calcium ( Moss et al . , 2004; Smith et al . , 2009 ) , and this led to the idea that LDA might be a consequence of the higher force or number of myosin heads attached to actin at longer SL .", "We tested the role of force-generating myosin heads in the length-dependent structural changes of cTnC by inhibiting force generation using blebbistatin ( Straight et al . , 2003 ) .", "In the presence of 25 μM blebbistatin , the active force was reduced to 1 . 5% ± 0 . 4% ( n = 12 ) of the control value .", "It should be noted that blebbistatin may inhibit active force generation by stabilising the OFF structure of the myosin thick filament in which the myosin heads are folded back on the surface of the thick filaments and unavailable for interacting with actin in the thin filaments ( Zhao et al . , 2008; Xu et al . , 2009 ) .", "The Ca2+-sensitivity and steepness of the cTnC C helix orientational changes were reduced after addition of blebbistatin at SL 1 . 9 μm ( Figure 4a , short-dashed line; Table 2 ) , and pCa50 decreased by ~0 . 08 pCa units ( Figure 4c ) .", "The change in <P2> associated with activation ( from pCa 6 . 0 to 4 . 5; Δ<P2> ) was ~16% smaller after addition of blebbistatin ( p<0 . 05; Table 2 ) , although the effect of blebbistatin at each of these pCa values was not significant at the 5% level .", "Similar results were obtained for the E-helix probe ( Figure 4b ) , except that <P2> increased significantly on addition of blebbistatin at both pCa 6 and pCa 4 . 5 ( Table 2 ) .", "The addition of blebbistatin also reduced the steepness of the Ca2+ dependence for both the C and E helix orientations ( nH , Table 2 ) .", "Similar results were obtained previously ( Sun et al . , 2009; Robertson et al . , 2015 ) , indicating that either active force increases myofilament Ca2+ sensitivity or stabilising the OFF structure of the thick filament decreases the Ca2+ sensitivity . 10 . 7554/eLife . 24081 . 007Figure 4 . Effects of active force inhibition by 25 μM blebbistatin on the orientation of cTnC probes and their length-dependence .", "( a ,", "c ) BR-cTnC-C; ( b ,", "d ) BR-cTnC-E .", "( a–b )", "Continuous lines denote Hill fits to data at SL 1 . 9 μm ( data not shown for clarity ) .", "Circles denote <P2> in the presence of blebbistatin at SL of 1 . 9 μm ( ○ ) and 2 . 3 μm ( ● ) .", "( c–d )", "Fitted Hill parameter , pCa50 , for the control at 1 . 9 μm SL ( white ) and in the presence of blebbistatin at 1 . 9 μm SL ( gray ) and 2 . 3 μm ( black ) .", "Mean ± SEM ( n = 5–7 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 24081 . 00710 . 7554/eLife . 24081 . 008Table 2 . Effects of force inhibition by 25 μM blebbistatin on Ca2+-dependence of force and the cTnC orientation parameter <P2> .", "Mean ± SEM .", "pCa50 and nH are fitted parameters of Hill equation .", "Δ<P2> , changes in <P2> during Ca2+-activation from pCa 6 to 4 . 5 .", "Comparisons ( paired t test , two-tailed ) : before and after addition of blebbistatin ( *p<0 . 05 ) ; between sarcomere lengths 1 . 9 and 2 . 3 μm in the presence of blebbistatin ( #p<0 . 05 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 24081 . 008BR-cTnC-CBR-cTnC-ESL ( μm ) 1 . 91 . 92 . 31 . 91 . 92 . 325 μM Blebbistatin−++−++ForcepCa505 . 39 ± 0 . 03 5 . 39 ± 0 . 04 nH3 . 72 ± 0 . 23 4 . 23 ± 0 . 20 <P2>pCa505 . 40 ± 0 . 015 . 32 ± 0 . 02 *5 . 41 ± 0 . 03 #5 . 35 ± 0 . 025 . 28 ± 0 . 03 *5 . 36 ± 0 . 02 #nH3 . 29 ± 0 . 102 . 74 ± 0 . 20 *2 . 49 ± 0 . 153 . 57 ± 0 . 222 . 90 ± 0 . 19 *2 . 60 ± 0 . 17at pCa 6 . 00 . 091 ± 0 . 0040 . 088 ± 0 . 0050 . 086 ± 0 . 0040 . 276 ± 0 . 0100 . 282 ± 0 . 010 *0 . 305 ± 0 . 009 #at pCa 4 . 50 . 009 ± 0 . 0020 . 016 ± 0 . 0020 . 024 ± 0 . 0030 . 168 ± 0 . 0080 . 202 ± 0 . 007 *0 . 225 ± 0 . 006 #Δ<P2>0 . 082 ± 0 . 0040 . 069 ± 0 . 004 *0 . 062 ± 0 . 003 #0 . 098 ± 0 . 0070 . 080 ± 0 . 004 *0 . 080 ± 0 . 005n = 5n = 7 Increasing SL from 1 . 9 to 2 . 3 μm after addition of blebbistatin increased pCa50 by ~0 . 08 pCa units for both the C and E helix probes ( Figure 4c and d , Table 2 ) , similar to the ca 0 . 11 pCa unit increase observed before the addition of blebbistatin ( p>0 . 05 , two-tailed t-test ) .", "The decrease in <P2> for the C-helix probe associated with activation ( Δ <P2> ) was smaller at the longer SL in the presence of blebbistatin ( p<0 . 05 , Table 2 ) .", "For the E helix probe , on the other hand , increasing SL changed <P2> over the whole range of [Ca2+] in a direction characteristic of lower activation , with no change in Δ<P2> ( Figure 4b ) .", "These results show that the length-dependent increase in the Ca2+-sensitivity of cTnC structural changes is retained after complete force inhibition and stabilisation of the OFF structure of the thick filament .", "Thus , the SL seems to directly modulate thin filament activation , and the higher force/number of force-generating myosin heads at longer SL is NOT the main cause of higher Ca2+ sensitivity in LDA .", "The results described above show that the higher active force at longer SL is not due to increased thin filament activation .", "We therefore tested the possibility that it might be due to a different structural or regulatory state of the thick filament using a probe on the RLC region of the myosin motors .", "The orientation of this probe has previously been shown to be sensitive to both the change in orientation of the myosin heads associated with force generation per se and to interventions like RLC phosphorylation that alter the regulatory or structural state of the thick filament ( Kampourakis et al . , 2014 , 2015 , 2016 ) .", "This bifunctional sulforhodamine ( BSR ) probe cross-links helices B and C in the N-terminal lobe of the cardiac myosin regulatory light chain ( BSR-cRLC-BC ) , and is roughly perpendicular to the long axis of the myosin head .", "<P2> for the BSR-cRLC-BC probe increased when the trabeculae were activated at SL 1 . 9 μm and was further increased by stretching to SL 2 . 3 μm ( Figure 5 ) .", "Although <P2> increases with SL at both low ( pCa 6 . 6 ) and high Ca2+ ( pCa 4 . 5 ) , the effect was larger at high Ca2+ ( Figure 5 ) .", "These results suggest that the higher active force at longer SL is associated with length-dependent changes in the orientation of the myosin heads . 10 . 7554/eLife . 24081 . 009Figure 5 . Force and orientation ( <P2> ) of the BSR-RLC-BC probe in relaxing ( pCa 6 . 6 ) and activating ( pCa 4 . 5 ) solution at sarcomere lengths of 1 . 9 ( white ) and 2 . 3 μm ( gray ) .", "Mean ± SEM ( n = 5 ) .", "Statistical significance was assessed using a two-tailed paired t-test . DOI: http://dx . doi . org/10 . 7554/eLife . 24081 . 009" ], [ "Increasing SL induced a change in the orientation of both cTnC probes in the direction associated with lower activation ( Figure 2c and d ) .", "In particular , the SL-dependent change in the orientation of the E helix probe in the IT arm of troponin occurred over the whole range of [Ca2+] , independent of the presence of force generating myosin heads ( Figure 4b ) .", "These results show that the changes in troponin structure induced by increased SL are distinct from those induced by Ca2+ activation or force generation .", "A recent study by time-resolved small-angle X-ray diffraction also showed that increasing SL induces changes in troponin conformation in relaxed cardiac muscle cells ( Ait-Mou et al . , 2016 ) .", "In addition , FRET measurements showed that the conformation of the switch region of cTnI relative to cTnC is also sensitive to SL in relaxed cardiac muscle ( Li et al . , 2016 ) .", "These SL-dependent changes in troponin structure could be related to the increased passive force at long SL ( Figure 1a ) .", "In cardiac muscle cells , the passive force is attributed to the giant protein titin , which runs from the Z-disc to the M-line ( Labeit and Kolmerer , 1995; Granzier and Labeit , 2004; Patel et al . , 2012 ) .", "Titin-based passive force plays an important role in LDA ( Cazorla et al . , 2001; Fukuda et al . , 2001 , 2003 ) .", "The length-dependent passive force is reduced in cardiac muscle from transgenic rats that express a giant splice isoform of titin , and the length-dependent change in the X-ray troponin reflection is also reduced in these muscle cells ( Ait-Mou et al . , 2016 ) .", "The combination of the present results with those of the X-ray and FRET studies mentioned above suggests that length-dependent changes in the structure of the thin filament of cardiac muscle can be one of the mechanisms underlying LDA , and hence the Frank-Starling law of the heart .", "As discussed above , the increase in maximum active force in LDA is likely to be determined by processes downstream of thin filament activation in the signalling pathway .", "Our previous study described the SL-induced structural changes in the myosin filament over the whole range of [Ca2+] ( Kampourakis et al . , 2016 ) .", "However , it made no direct comparison of <P2> for the BSR-cRLC-BC probe at different SLs as the primary focus of that work was on the effects of cRLC phosphorylation .", "The present results have shown that <P2> for the BSR-cRLC-BC probe increased during Ca2+ activation and increased even further when SL was increased ( Figure 5 ) .", "Moreover , it has been shown that the length-dependent increase of isometric force in cardiac muscle is due to the change in the number of force-generating myosin heads ( Caremani et al . , 2016 ) .", "Together , these results show that the higher active force at longer SL is associated with a more ON state of the thick filament .", "X-ray measurements on resting cardiac muscle showed a close correlation between the orientation of myosin heads before activation and enhanced force generation as SL increases , further supporting the idea of a direct effect of SL on thick filament structure ( Farman et al . , 2011 ) .", "Again , titin is implicated in the length-dependent structural changes in the thick filament .", "In resting cardiac muscles from rats that express the mutant titin described above , the SL-dependent changes in the intensities of the myosin-based X-ray reflections were greatly reduced , suggesting that titin-based passive force mediates the effect on thick filament structure ( Ait-Mou et al . , 2016 ) .", "It has long been established that an increase in SL in cardiac muscle results in increases in both the myofilament Ca2+ sensitivity and the maximum Ca2+-activated force ( Kentish et al . , 1986; Dobesh et al . , 2002 ) .", "The main focus has been on myofilament Ca2+ sensitivity as a predominant component in LDA ( Allen and Kentish , 1985 ) and myosin crossbridges as a main mediator ( Fitzsimons and Moss , 1998; Smith et al . , 2009 ) .", "The present results provided evidence that two distinct pathways are involved in LDA .", "Increasing SL exerts a direct effect on the structure of troponin in the thin filament , which is the main contributor to the length-dependent increase of myofilament Ca2+ sensitivity in LDA .", "A structural change of myosin in thick filament is responsible for the increase of maximal force generation in LDA .", "It is well known that cTnI plays an important role in the LDA of cardiac muscle ( Arteaga et al . , 2000; Konhilas et al . , 2003 ) .", "It has also been shown that phosphorylation of Ser23/Ser24 at its cardiac-specific N-terminal region enhances the length-dependent increase in Ca2+ sensitivity while having no effect on the length-dependent increase in maximum force ( Wijnker et al . , 2014 ) .", "This is in support of our conclusion that the increase in Ca2+ sensitivity in LDA is mediated through a change in thin filament structure .", "The thin filament mediated length-dependent change in Ca2+ sensitivity may play an important role in the development of hypertrophic cardiomyopathy by missense mutations in the sarcomeric proteins ( Sequeira et al . , 2013 ) .", "The LDA occurs immediately after a length change and independently of the increase in the intracellular [Ca2+] ( Allen and Kurihara , 1982; Mateja and de Tombe , 2012 ) .", "Recent evidence has suggested that the passive force originated by titin plays a critical role in the transduction of the length signal to contractile apparatus in cardiac LDA ( Fukuda et al . , 2001; Methawasin et al . , 2014 ) .", "Increasing SL increases the passive force of cardiac muscle by stretching the titin that links the thick filament to the Z-disk and also relays its mechanical strain to the thick filament ( Linke , 2008 ) .", "A recent X-ray study on skeletal muscle showed a connection between increased thick filament stress and its transition to ON state ( Linari et al . , 2015 ) .", "This mechano-sensing mechanism may also exist in cardiac muscle as the X-ray measurement of cardiac muscle has indicated a structural rearrangement within the thick filament associated with titin strain and LDA ( Ait-Mou et al . , 2016 ) .", "The results presented here , together with the previous studies described above , also demonstrated that increasing SL in cardiac muscle changes the structure of troponin in the thin filament .", "This length-dependent change in troponin structure is correlated with the titin strain and is present in the absence of active force-generating myosin heads .", "It is not known how either titin strain or the ON state of the thick filament can be transmitted to the thin filament , although MyBP-C may be involved in this interfilament signal transmission .", "MyBP-C is localised to the central region of each half-thick filament ( Bennett et al . , 1986; Luther et al . , 2011 ) .", "It is bound to the thick filament via its C-terminal region ( Flashman et al . , 2004 ) and its N-terminal region can activate the thin filament to trigger force generation in the absence of Ca2+ ( Herron et al . , 2006; Kampourakis et al . , 2014; Mun et al . , 2014 ) .", "In the absence of MyBP-C , while the length-dependent increase in the maximum force generation is preserved , the length-dependent increase in Ca2+ sensitivity is blunted ( Mamidi et al . , 2014 ) .", "cMyBP-C is one of the key components in modulating cardiac contractility in a manner that depends on its phosphorylation state ( Gautel et al . , 1995; Kunst et al . , 2000; Pfuhl and Gautel , 2012 ) .", "Phosphorylation of MyBP-C , which inhibits the interaction of its N-terminal region with the thin filament ( Shaffer et al . , 2009; Kampourakis et al . , 2014 ) , has been shown to modulate LDA in cardiac muscle ( Kumar et al . , 2015; Mamidi et al . , 2016 ) .", "Thus , MyBP-C is a strong candidate for transmitting the signal of titin strain generated by SL change from the thick to the thin filament ." ], [ "All animal procedures were in accordance with Schedule 1 of the UK Animal ( Scientific Procedures ) Act 1986 .", "Adult Wistar rats ( 200–250 g ) were sacrificed by cervical dislocation .", "The hearts were excised and rinsed free of blood with Krebs-Henseleit solution ( K3753 , Sigma , St . Louis , MO ) oxygenated with a carbogen mixture of 95% O2% and 5% CO2 .", "Suitable trabeculae ( free running , unbranched , diameter <250 µm ) were dissected from the right ventricle in Krebs-Henseleit solution containing 25 mM 2 , 3-butanedione-monoxime , permeabilised in relaxing solution ( see below ) containing 1% Triton X-100 for 30 min , and stored in relaxing solution for experiments .", "Double cysteine mutants E55C/D62C and E95C/R102C of human cTnC were obtained by site-directed mutagenesis and expressed in E . coli .", "The native cysteines C35 and C84 were replaced by serine ( Sia et al . , 1997 ) .", "The difference between human and rat isoforms of cTnC is one amino acid residue at 119 , Isoleucine ( human ) vs Methionine ( rat ) .", "Ile and Met belong to the same group of amino acids with nonpolar , aliphatic side chains .", "TnC was expressed , purified and labelled as described previously ( Ferguson et al . , 2003; Sun et al . , 2009 ) .", "Each pair of cysteines was cross-linked with bifunctional rhodamine ( BR ) to form 1:1 BR:TnC conjugates ( Corrie et al . , 1998 ) , and purified by reverse-phase HPLC ( Agilent 1200 HPLC System ) .", "Stoichiometry and specificity of BR-labelling were confirmed by electrospray mass spectrometry ( Agilent 6120 Quadrupole LCMS System ) .", "Endogenous cTnC was exchanged by incubation of trabeculae in relaxing solution containing 25–30 μM BR-cTnC overnight at 4°C .", "The fraction of cTnC replaced by BR-TnC was about 80% ( Sevrieva et al . , 2014 ) .", "BSR-cRLCs were exchanged into demembranated trabeculae by extraction in CDTA-rigor solution ( mM: 5 CDTA , 50 KCl , 40 Tris-HCl pH 8 . 4 , 0 . 1% ( v/v ) Triton X-100 ) for 30 min followed by reconstitution with 40 mM BSR-cRLC in relaxing solution for 1 hr , replacing 30–50% of the endogenous cRLCs ( Kampourakis et al . , 2014 ) .", "Following exchange of cTnC or cRLC , the trabeculae were mounted horizontally via aluminum T-clips between a force transducer ( SI-KG7 , World Precision Instruments ) and a fixed hook in a 60 μl trough containing relaxing solution .", "The experimental temperature was 20–22°C .", "Experimental solutions contained 25 mM imidazole , 5 mM MgATP , 1 mM free Mg2+ , 10 mM EGTA ( except pre-activating solution ) , 0–10 mM total calcium , 1 mM dithiothreitol and 0 . 1% ( v/v ) protease inhibitor cocktail ( P8340 , Sigma ) .", "Ionic strength was adjusted to 200 mM with potassium propionate; pH was 7 . 1 at 20°C .", "The concentration of free Ca2+ was calculated using the program WinMAXC V2 . 5 ( http://www . stanford . edu/~cpatton/maxc . html ) .", "The calculated free [Ca2+] , expressed as pCa ( i . e . , −log[Ca2+] ) , was in the range 1 nM ( pCa", "9 ) to 32 µM ( pCa 4 . 5 ) .", "In pre-activating solution , [EGTA] was 0 . 2 mM and no calcium was added .", "When required , 25 µM blebbistatin ( B0560 , Sigma ) was added from a 10 mM stock solution in DMSO .", "Measurement of fluorescence polarization was similar to previously described methods ( Sun et al . , 2009; Sevrieva et al . , 2014 ) .", "A central 0 . 5 mm segment of a trabecula exchanged with BR-cTnC was briefly illuminated from below with 532 nm light polarised either parallel or perpendicular to the trabecular axis .", "BR fluorescence at 610 nm was collected in line with the illuminating beam that was propagating perpendicular to the trabecular axis .", "The emitted fluorescence was then separated into parallel and perpendicular components .", "Together with the order parameter <P2d> that describes the amplitude of rapid ( sub-nanosecond ) probe motion and has been measured in our previous study ( Sun et al . , 2009 ) , two independent orientation order parameters were calculated from these measured intensities: <P2> and <P4> , describing the distribution of angles between BR fluorescence dipole and trabecular axis averaged over slower timescales ( Dale et al . , 1999 ) .", "<P2> would be +1 if every BR dipole were parallel to the trabecular axis , and −0 . 5 if they were all perpendicular .", "<P4> provides higher resolution angular information , and orientational disorder decreases the absolute values of both <P2> and <P4> .", "The detailed results are presented here only for <P2> , which can be measured with greater signal-to-noise ratio .", "If there are multiple populations of BR dipoles with distinct orientations , the observed <P2> is linearly related to the fraction of dipoles in each population .", "Each trabecular activation was preceded by a 1 min incubation in pre-activating solution .", "Isometric force and fluorescence intensities were measured after steady-state force had been established in each activation .", "Maximum force was recorded before and after each series of activations at sub-maximal [Ca2+] .", "If the maximum force decreased by >15% , the trabecula was discarded .", "The experiment was completed when full Ca2+-force relationships at both short and long SLs were obtained from the same trabecula .", "The dependence of force and <P2> on [Ca2+] was fitted to data from individual trabeculae using nonlinear least-squares regression to the Hill equation:Y=1/ ( 1+10nH ( pCa−pCa50 ) ) where pCa50 is the pCa corresponding to half-maximal change in either force or <P2> , and nH is the Hill coefficient .", "The steepness of the Ca2+-force relationships in the present study ( nH = ~4 , Table", "1 ) was less than that in a study where SL was kept constant during the contraction by adjusting overall muscle length ( Dobesh et al . , 2002 ) .", "This is likely due to the end-compliance of trabecular preparations that allows SL shortening during activation ( ter Keurs et al . , 1980; Kentish et al . , 1986 ) .", "As the trabecula generates more force at higher concentrations of Ca2+ , the central sarcomeres shorten more .", "Consequently , the Ca2+-force relationship shifts to the right at higher [Ca2+] and becomes less steep .", "Therefore , both pCa50 and nH of the Ca2+-force relationship at a given SL were likely to be underestimated in fixed end conditions as in the present study .", "To minimise the impact of SL shortening during activation on the interpretation , only the data from experiments in which full Ca2+-force relationships at both short and long SLs were obtained from the same trabecula were fit to the Hill equation .", "In addition , paired-comparisons were used throughout the present study .", "Thus , the differential shortening of the fixed-end contractions at the different lengths would not affect the interpretation of the results .", "All values are given as mean ± standard error except where noted , with n representing the number of trabeculae ." ] ]

[ "The Frank-Starling relation is a fundamental auto-regulatory property of the heart that ensures the volume of blood ejected in each heartbeat is matched to the extent of venous filling .", "At the cellular level , heart muscle cells generate higher force when stretched , but despite intense efforts the underlying molecular mechanism remains unknown .", "We applied a fluorescence-based method , which reports structural changes separately in the thick and thin filaments of rat cardiac muscle , to elucidate that mechanism .", "The distinct structural changes of troponin C in the thin filaments and myosin regulatory light chain in the thick filaments allowed us to identify two aspects of the Frank-Starling relation .", "Our results show that the enhanced force observed when heart muscle cells are maximally activated by calcium is due to a change in thick filament structure , but the increase in calcium sensitivity at lower calcium levels is due to a change in thin filament structure ." ]

[ "The heart needs to pump out the same volume of blood that enters it .", "This is not as simple as it sounds , as changes in heart rate – for example , in response to exercise – alter how hard the heart must pump .", "When blood flows into the heart it stretches the heart muscle , which consists of units called sarcomeres .", "Sarcomeres contain two types of protein filament , known as thick filaments and thin filaments .", "When a heartbeat is triggered by calcium ions flowing into the heart muscle cells , the thick filaments slide over the thin filaments .", "This causes the heart muscle cell to contract .", "The Frank–Starling mechanism helps to regulate the contraction of the heart .", "This mechanism has two aspects .", "Firstly , as the sarcomere lengthens , its protein filaments are able to contract with more force for a given high level of calcium ions .", "Secondly , the lengthening of the sarcomere makes the filaments more sensitive to calcium ions , which again causes the heart to contract more forcefully .", "However , the molecular mechanisms that underlie these effects were not clear .", "Zhang et al . have now studied rat heart muscle cells using a new fluorescence-based method that can detect structural changes in the thick and thin filaments .", "The results show that the increased force that is generated when sarcomeres are stretched can be accounted for by changes in the structure of the thick filament .", "In contrast , the increase in calcium sensitivity that occurs as the sarcomere lengthens is largely due to structural alterations in the thin filament .", "These two processes can be controlled independently , but work together in the Frank–Starling mechanism .", "Now that we better understand the molecular basis of the Frank–Starling mechanism , further work could investigate new strategies for designing and testing treatments for heart disease ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "neuroscience" ]

[ [ "State-of-the-art basal ganglia ( BG ) computational models ( Gurney et al . , 2015; Schultz et al . , 1997 ) divide the BG network into two functionally related subsystems .", "First , the main axis ( or 'actor' in machine learning terminology ) which corresponds to the BG structures that connect state-encoding thalamo-cortical areas to cortical and brainstem motor centers .", "Second , the neuromodulators ( machine learning's 'critics' , e . g . , the midbrain dopaminergic neurons and striatal cholinergic interneurons ) that adjust activity along the BG main axis by encoding a prediction error signal capable of modulating the efficacy of cortico-striatal transmission ( Deffains and Bergman , 2015; Reynolds et al . , 2001; Shen et al . , 2008 ) .", "The input structures of the BG main axis ( the striatum and subthalamic nucleus , STN ) receive considerable glutamatergic inputs from the cortex and the thalamus .", "The striatum and STN provide major inhibitory GABAergic and excitatory glutamatergic drive respectively to the external segment of the globus pallidus ( GPe ) and the BG output structures ( internal segment of the globus pallidus and substantia nigra reticulata , GPi/SNr ) ( Parent and Hazrati , 1995a , 1995b ) .", "In return , the GPe emits feedback GABAergic projections to the STN ( Carpenter et al . , 1981 ) and the striatum ( Hegeman et al . , 2016; Mallet et al . , 2012 ) as well as massive feedforward GABAergic projections to the GPi/SNr ( Parent and Hazrati , 1995b ) .", "Thus , aside from the action of the BG neuromodulators and lateral connectivity , the increase-decrease balance of spiking activity ( I/D balance ) of pallidal and nigral neurons is fined-tuned by the inhibitory and excitatory drives exerted by the striatum and STN , respectively .", "However , how these antagonistic drives operate to convey relevant information from the state-encoding thalamo-cortical areas through the central ( GPe ) and output ( GPi and SNr ) BG structures to brain motor centers is still unknown .", "Many human disorders are caused by malfunctions of the BG neuromodulators which impact neuronal activity along the BG main axis .", "Traditionally , in Parkinson's disease ( PD ) , it is assumed that degeneration of midbrain dopaminergic neurons leads to striatal dopamine depletion which provokes a cascade of physiological disturbances in the BG main axis , notably the emergence of synchronized oscillatory activity in the BG and cortical networks ( Levy et al . , 2002; Nini et al . , 1995; Oswal et al . , 2013 ) .", "These abnormal oscillations likely compromise information flow through the BG main axis and result in the release of abnormal commands by BG output structures .", "Despite evidence of subthalamic dopamine depletion in PD and its role in the pathophysiology of the disease ( Francois et al . , 2000; Galvan et al . , 2014; Rommelfanger and Wichmann , 2010 ) , the striatum remains the main site of dopamine depletion in human patients and animal models of PD .", "In addition , the striatum is much larger than the STN ( 107 vs . 105 neurons in non-human primates , respectively , Hardman et al . , 2002 ) .", "Nevertheless , the STN , not the striatum , is the prime target for deep brain stimulation ( DBS ) of human patients with advanced PD ( Limousin et al . , 1998; Odekerken et al . , 2016 ) .", "Moreover , it has been shown that STN-DBS abolishes abnormal synchronized oscillations in the BG network of animal models of PD ( Meissner et al . , 2005 ) and human PD patients ( Kühn et al . , 2008; Wingeier et al . , 2006 ) .", "These findings suggest that STN plays a pivotal role in the release of commands by BG output structures , but the respective influence of the striatum and STN activity on the activity of the BG central and output structures in PD are still unknown .", "To tackle these issues , we recorded the neuronal activity of the BG input and downstream ( central and output ) structures of two monkeys engaged in a classical temporal discounting conditioning task ( i . e . , normal/healthy state , Figure 1A ) .", "In the task , we manipulated the value of 2-s cues ( predicting future appetitive , aversive or neutral outcomes ) and the delivery time of the outcome ( immediate or 6-s delayed ) .", "Then , once we completed the recordings in the normal state , we proceeded to record neuronal activity in the BG network of the same two monkeys after systemic induction of PD symptoms ( i . e . , parkinsonian state ) with 1-methyl-4-phenyl-1 , 2 , 3 , 6-tetrahydropyridine ( MPTP ) .", "These multi-site recordings in both the normal and parkinsonian states served us to reveal how BG activity propagates along the BG main axis in health and parkinsonism .", "Moreover , it sheds light on which BG input structure ( striatum or STN ) is more influential in shaping the activity of the BG downstream structures in the recorded conditions . 10 . 7554/eLife . 16443 . 003Figure 1 . Task design and behavioral monitoring .", "( A ) Temporal discounting classical conditioning task .", "Each trial started with the presentation of a visual cue ( 2 s ) that predicted the delivery of food ( reward/appetitive trials ) , airpuff ( aversive trials ) or sound only ( neutral trials ) .", "Cue offset was immediately followed by the outcome period ( immediate outcome condition ) or by a 6-s delay period which preceded the outcome period ( delayed outcome condition ) .", "The outcome period ( 0 . 15 s ) was followed by a variable inter-trial interval ( ITI ) of 6–10 s .", "Trial order and ITI duration were randomized .", "( B ) Animals' task performance .", "Frequency of licking ( top ) and blinking ( bottom ) movements over time are aligned to cue onset ( time = 0 ) .", "Time 2 and 8 s correspond to outcome delivery in the immediate and delayed conditions , respectively .", "Data were averaged for each session ( hundreds of trials ) and then across sessions ( N = 41 and 30 , monkey K and S , respectively ) .", "Data were grouped since no significant differences were found between the two monkeys .", "Solid line and shaded envelope represent mean ± standard error of the mean ( SEM ) , respectively .", "Color code indicates the cue/outcome value: blue-appetitive , green-neutral and red-aversive . DOI: http://dx . doi . org/10 . 7554/eLife . 16443 . 003" ], [ "After an intensive training period in the task ( Figure 1A ) , a recording chamber was attached to the animal's skull to allow access through a burr-hole to all BG structures ( Figures 2A and B ) .", "Then , neuronal activity was recorded in the normal/healthy state while the monkeys were engaged in the task ( Figures 2C and D ) and in the parkinsonian state ( Figures 8 , 9 and 10A ) . 10 . 7554/eLife . 16443 . 004Figure 2 . Recordings sites and neuronal activities in the BG network .", "( A ) Schematic model of BG functional connectivity .", "Glutamatergic and GABAergic connections are shown in red and green , respectively .", "D1-/D2-MSNs: striatal medium spiny ( projection ) neurons expressing D1/D2 dopamine receptors; STR: striatum; STN: subthalamic nucleus; GPe-i: external or internal segment of the globus pallidus; SNr: substantia nigra reticulata .", "( B ) Representative coronal section - 6 mm from the anterior commissure ( AC - 6 ) , adapted from Martin and Bowden ( 2000 ) .", "( C ) Left , examples of 4 s of multi-unit activity , online filtered between 250 and 6000 Hz .", "Right , 100 randomly chosen superimposed waveforms of the extracellular action potentials of a cell sorted from its multi-unit activity .", "IS indicates the isolation score of the sorted cell .", "( D ) Peri-stimulus time histograms ( PSTHs ) and raster plots of 6 cells ( single units ) recorded in the different structures of BG , aligned to cue onset ( time = 0 ) .", "Time 2 and 8 s indicate outcome delivery in the immediate and delayed outcome conditions , respectively .", "PSTHs were built by summing activity across trials at a 1-ms resolution and then smoothing with a Gaussian window ( SD = 20 ms ) .", "Each line of dots in the raster plots corresponds to activity during a trial and each dot represents a single spike .", "Color code indicates trial type according to outcome: appetitive trials in blue , neutral trials in green and aversive trials in red . DOI: http://dx . doi . org/10 . 7554/eLife . 16443 . 004 In the striatum , we targeted the phasically active medium spiny neurons ( MSNs ) that correspond to the striatal projection neurons and the tonically active neurons ( TANs , presumably the striatal cholinergic interneurons , but see Beatty et al . , 2012 ) .", "The striatum is composed of the posterior putamen ( motor area ) , the caudate nucleus ( associative area ) and ventral striatum/nucleus accumbens ( limbic area ) ( Parent and Hazrati , 1995a ) .", "In this study , striatal ( MSN and TAN ) spiking activity was preferentially collected within the posterior putamen .", "The remaining striatal recordings were made in the caudate nucleus .", "As previously reported ( Adler et al . , 2013a; Graybiel et al . , 1994 ) , we did not find significant differences in the activity of MSNs and TANs between these two striatal sub-regions ( data not shown ) .", "Therefore , we grouped each striatal cell-type recorded from the putamen and caudate nucleus for further analysis .", "We also recorded the activity of the neurons of the STN and the high-frequency discharge ( HFD ) neurons of the GPe , GPi and SNr .", "HFD neurons of the GPe , GPi and SNr comprise >85% of the neurons in these structures and , like striatal MSNs and STN neurons , are probably the projection neurons of these structures .", "Thus , a unique , and very advantageous for the current study , feature of the BG network is that each BG nucleus forms a single layer network; in other words , the BG projection neurons are innervated by the projections of the upward structures .", "Namely , the activity of striatal and STN projection neurons directly affects the activity of the recorded GPe , GPi and SNr projection neurons .", "We used this property to infer the relative influence of the BG input structures ( striatum and STN ) on the activity of the central ( GPe ) and output ( GPi and SNr ) structures of the BG network .", "All recorded neurons were analyzed offline for discharge rate stability and isolation quality ( Joshua et al . , 2007 ) to guarantee the data quality ( see Materials and methods ) .", "Neuronal database details are given in Table 1 . 10 . 7554/eLife . 16443 . 005Table 1 . Neuronal database .", "N is the number of recorded neurons that passed inclusion criteria .", "The recording span represents the stable recording period ( in seconds ) for each neuron .", "The isolation score ranges from 0 to 1 .", "The recording span and isolation score were averaged for each structure and state .", "Values are means ± standard deviation ( SD ) .", "For each neuronal assembly , statistics were calculated and are presented for both monkeys . DOI: http://dx . doi . org/10 . 7554/eLife . 16443 . 005Neuronal assemblybefore MPTPafter MPTPNRecording span ( s ) Isolation scoreNRecording span ( s ) Isolation scorestriatum ( MSN ) 1501096 . 8 ± 564 . 80 . 85 ± 0 . 10128950 . 6 ± 616 . 10 . 85 ± 0 . 12striatum ( TAN ) 1161568 . 8 ± 907 . 60 . 89 ± 0 . 08811217 . 8 ± 708 . 80 . 89 ± 0 . 09STN1031080 . 0 ± 411 . 10 . 77 ± 0 . 12111780 . 0 ± 317 . 70 . 80 ± 0 . 12GPe1821760 . 4 ± 978 . 20 . 91 ± 0 . 08105877 . 7 ± 332 . 70 . 91 ± 0 . 09GPi1191437 . 0 ± 607 . 90 . 89 ± 0 . 08noneSNr1101205 . 2 ± 366 . 40 . 90 ± 0 . 08121874 . 7 ± 445 . 40 . 90 ± 0 . 09 During the task , we systematically recorded licking and blinking movements to assess the animals' performance ( Figure 1B ) .", "In both conditions ( immediate and delayed ) of the behavioral task , the frequency of the licking and blinking movements increased in response to food and airpuff delivery , respectively .", "Remarkably , the animals responded with appropriate anticipatory licking and blinking behavior to the outcome delivery in the immediate condition .", "In contrast , in the delayed condition , the animals did not display any robust anticipatory licking and blinking movements during the cue and delay periods .", "Together , these behavioral results indicate that the animals learned the cue values ( appetitive , aversive or neutral ) and differentiated between cues predicting an immediate or a delayed outcome .", "However , the introduction of a 6-s delay period after cue offset probably compromised the temporal predictability of the outcome delivery in the delayed condition .", "Figure 3 depicts the mean ( for each BG neuronal assembly ) relative ( gray ) and absolute ( green ) peri-stimulus histograms ( PSTHs ) .", "The mean relative PSTH is calculated as the arithmetic average of the single neurons' PSTHs ( Figure 2D ) and is more standard in neuroscience .", "The mean relative PSTH reflects the common assumption that the on-going activity of the studied population is close to zero and that the studied neurons excite their neuronal targets that act as integrate-and-fire neurons ( Izhikevich , 2007 ) .", "On the other hand , most BG neurons fire at tonic high frequencies ( Figures 2C and D ) and a large fraction of their responses to behavioral events consists of decreases in discharge rate ( Adler et al . , 2012; Espinosa-Parrilla et al . , 2013; Joshua et al . , 2009b; Turner and Anderson , 2005 ) .", "Thus , neurons of the BG network might follow resonance rather than integrate-and-fire rules when they are activated by their afferents ( Izhikevich , 2007 ) .", "We therefore calculated the mean absolute PSTH ( absolute deviation from the baseline of the firing rate , see Materials and methods ) of the BG neurons recorded in our study .", "These absolute PSTHs revealed that the average population response to the cue of each neuronal assembly of the BG main axis persisted until the outcome delivery in both conditions ( immediate and delayed ) ( Figure 3 , green lines ) .", "Therefore , these persistent modulations of activity along the BG main axis temporally bridged sensory visual information ( provided by cue presentation ) and the actions ( licking and blinking movements ) wired to outcome delivery and continued even when the visual information was no longer available during the 6-s delay period in the delayed condition .", "The persistent responses in the BG main axis contrast with the transient response of the TANs ( i . e . , one of the BG neuromodulators ) to the cue onset ( Figure 3 ) . 10 . 7554/eLife . 16443 . 006Figure 3 . Relative and absolute population responses to the task behavioral events . The response of a single neuron to the cue was calculated as the relative or absolute deviation from the baseline of the firing rate .", "The baseline firing rate was calculated during the 500 ms prior to cue onset .", "For each condition ( immediate/delayed ) , each neuron was tested three times ( appetitive , neutral and aversive trials ) .", "Relative ( black ) and absolute ( green ) population responses to the cue were defined as the average of the relative or absolute responses of all neurons of the same structure , respectively .", "The shaded areas mark SEMs . DOI: http://dx . doi . org/10 . 7554/eLife . 16443 . 006 For each neuronal assembly of the BG main axis , the absolute response of the population was systematically larger than the relative response ( Figure 3 ) , thus suggesting in line with previous studies ( Adler et al . , 2012; Espinosa-Parrilla et al . , 2013; Joshua et al . , 2009b; Turner and Anderson , 2005 ) that single neuronal responses in the BG main axis consist of either increases or decreases in the firing rate .", "We found that neurons of the BG main axis exhibited diverse patterns in timing and polarity ( increase/decrease ) of discharge modulations rather than coordinated sustained modulations of the discharge rate ( Figure 4 ) . 10 . 7554/eLife . 16443 . 007Figure 4 . Temporal patterns of increases and decreases in activity of the BG neuronal assemblies . In the ( red/blue ) surface plot , each row represents the time course of increases ( red ) and decreases ( blue ) in activity of a single neuron from cue onset to outcome delivery .", "Only modulated neurons are displayed .", "A neuron was considered modulated if at least one responsive bin was detected past the cue onset ( for each neuronal assembly , >80% of the neurons were modulated whatever the cue value , in both conditions ) .", "The temporal patterns of increases and decreases in activity are ordered such that modulatory activities consisting of longer increases are at the top and ones consisting of longer decreases are at the bottom . DOI: http://dx . doi . org/10 . 7554/eLife . 16443 . 007 In both conditions , we also found that the fraction of neurons that modulated their activity ( regardless of the polarity of the modulation ) at each time bin ( 20 ms ) , from cue onset to outcome delivery , systematically exceeded chance level ( i . e . , p<0 . 05 , the values lay beyond two standard deviations of the mean , empirical 68-95-99 . 7 rule ) for all neuronal assemblies of the BG main axis ( Figure 5 , left and center histograms ) .", "Except for striatal MSNs , analysis of variance ( ANOVA ) revealed a significant effect of task period ( cue in the immediate condition and cue and delay in the delayed condition ) on the fraction of responsive bins ( one-way ANOVA , p<0 . 001 ) for all other neuronal assemblies of the BG main axis .", "The fractions of responsive bins in the STN and downstream structures decreased during the delay period ( p<0 . 001 , post hoc comparisons , Bonferroni corrected , Figure 5 , right bar graphs ) .", "Thus , while the fraction of modulated MSNs remained unchanged over time , STN , GPe , GPi and SNr neurons exhibited comparable changes following cue offset in the delayed condition . 10 . 7554/eLife . 16443 . 008Figure 5 . The BG main axis exhibits persistent modulations of activity . For each neuronal assembly of the BG main axis , the black histograms ( left ) represent the fraction of responsive neurons ( in the appetitive , neutral and aversive trials ) at each time bin ( 20 ms ) from cue onset to outcome delivery in both conditions .", "The bar plot ( right ) depicts the mean fraction of responsive neurons ( average of the fractions of responsive neurons over a task period ) in the different task periods ( cue period of the immediate condition and cue and delay periods of the delayed condition ) compared to the mean fraction of responsive neurons in the cue period of the immediate condition . DOI: http://dx . doi . org/10 . 7554/eLife . 16443 . 00810 . 7554/eLife . 16443 . 009Figure 5—source data 1 . Fraction of responsive neurons during the different task periods . DOI: http://dx . doi . org/10 . 7554/eLife . 16443 . 009 Theoretical ( van Vreeswijk and Sompolinsky , 1996 ) and intracellular recording ( Taub et al . , 2013 ) studies usually quantify changes in membrane potential as an excitation/inhibition balance ( E/I balance ) .", "However , using extracellular recording method , we can only record changes in the discharge rate of neurons but not the corresponding changes in synaptic and membrane potentials .", "Since both an increase in excitation and an abolition of inhibition can lead to an increase in discharge rate ( and vice versa for decreases in discharge rate ) , we used the more conservative nomenclature of the increase/decrease discharge rate balance ( I/D balance ) .", "To assess the time-varying I/D balance within the different neuronal assemblies of the BG main axis , we calculated the fraction ( Figure 6—figure supplement", "1 ) and the magnitude ( Figure 6—figure supplement", "2 ) of increases and decreases in spiking activity .", "This was done for each neuronal assembly , from cue onset to outcome delivery , in the two behavioral conditions ( immediate and delayed ) .", "This way , we examined the I/D balance ( i . e . , relative increase vs . decrease dominance of activity as measured by the fraction weighted by the magnitude ) over time along the BG main axis ( Figure 6 , left and central histograms ) .", "We found that during the cue periods in both conditions , increases in activity ( positive I/D balance ) strongly predominated in all BG structures ( Figure 6 , right bar plots ) .", "In contrast , during the delay period , although the I/D balance was still strikingly biased towards increases in activity for the MSNs , this imbalance was significantly reduced in the STN and reversed in the GPe , GPi and SNr ( one-way ANOVA , p<0 . 001 , with Bonferroni correction for multiple comparisons , Figure 6 , right bar plots ) .", "Thus , whereas the inhibitory GABAergic drive from the striatum was unchanged over the different task periods , the STN excitatory glutamatergic drive was reduced when moving from the cue to the delay periods .", "This reduced STN excitatory drive was concurrent with a reduction in the activity in the BG downstream structures ( Figures 3 , 5 and 6 ) and with the animals' modified behavioral policy ( Figure 1B ) . 10 . 7554/eLife . 16443 . 010Figure 6 . Modifications in subthalamic activity are concomitant with reversal of the increase/decrease balance of activity in the BG downstream structures . Left , relative dominance of the increases and decreases in spiking activity ( I/D balance ) from cue onset ( time = 0 ) to outcome delivery ( time = 2 or 8 s in the immediate and delayed conditions , respectively ) , along the BG main axis .", "On the y-axis , as values approach 1 , increases prevail over decreases and vice-versa as values approach -1 .", "Values close to 0 indicate equal weight of increases and decreases .", "Right , the mean value of the I/D balance for the different task periods in each neuronal assembly .", "Error bars represent SEMs . DOI: http://dx . doi . org/10 . 7554/eLife . 16443 . 01010 . 7554/eLife . 16443 . 011Figure 6—source data 1 . Increase/decrease balance of activity during the different task periods . DOI: http://dx . doi . org/10 . 7554/eLife . 16443 . 01110 . 7554/eLife . 16443 . 012Figure 6—figure supplement 1 . Fraction of increases and decreases in activity along the BG main axis . Left , fractions of increases ( red ) and decreases ( blue ) in activity out of the total number of responsive neurons at each time bin ( 20 ms ) from cue onset to outcome delivery in the immediate and delayed task conditions .", "Right , mean fraction of increases and decreases in activity in the different task periods .", "Error bars ( difficult to see because of their small size ) represent SEMs . DOI: http://dx . doi . org/10 . 7554/eLife . 16443 . 01210 . 7554/eLife . 16443 . 013Figure 6—figure supplement 2 . Magnitude of increases and decreases in activity along the BG main axis . Left , magnitudes of the modulations in activity ( in spikes/s ) for the neurons exhibiting increases ( red ) and decreases ( blue ) in activity ( compared to the baseline firing rate calculated during the 500 ms prior to cue onset ) at each time bin ( 20 ms ) from cue onset to outcome delivery in the immediate and delayed task conditions .", "Right , mean magnitude of increases and decreases in activity in the different task periods .", "Same conventions as in Figure 6—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 16443 . 013 In the previous paragraph , we have analyzed the I/D balance of the different populations of BG neurons and concluded that the I/D balance of STN neurons , not MSNs , resembled the I/D balance of the BG downstream neurons .", "However , we have not used formal similarity/dissimilarity test ( e . g . , cross-correlation analysis ) .", "Here , the cross correlation function was calculated between the neuronal responses ( binned relative PSTHs ) of ( non-simultaneously recorded ) BG input-downstream neuron pairs .", "For this purpose , the BG neuronal responses were categorized as a function of their principal polarity as either increases or decreases ( see Materials and methods ) , and the cross-correlation ( similarity/dissimilarity ) was tested between neuronal responses with similar and opposite principal polarities .", "We found that increases dominated during the cue periods in both conditions ( χ2 test , p<0 . 05 , for each BG neuronal population , Figure 7A ) .", "During the delay period , except for striatal MSNs where increases remained preponderant ( χ2 test , p<0 . 001 , Figure 7A ) , this dominance of the increases disappeared for STN , GPe and GPi neurons and decreases even became dominant for SNr neurons ( χ2 test , p<0 . 001 , Figure 7A ) . 10 . 7554/eLife . 16443 . 014Figure 7 . Subthalamic , not striatal , activity fluctuations correlate with modulations in BG downstream activity .", "( A ) Fraction of neuronal responses depending on their principal polarity along the BG main axis for the different task periods .", "The sum of the fractions of increases ( red ) and decreases ( blue ) is shown in black .", "( B ) Mean similarity coefficient of the neuronal responses of striatal ( MSN ) or subthalamic - BG downstream neuron pairs exhibiting similar ( Increase-Increase or Decrease-Decrease pairs ) and opposite ( Increase-Decrease or Decrease-Increase pairs ) polarity , for the different task periods .", "The similarity coefficient ranges from −1 ( i . e . , opposite neuronal activity ) to +1 ( i . e . same neuronal activity ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 16443 . 014 The similarity coefficients ( i . e . , the zero lag coefficients of the cross correlation functions ) of the neuronal responses of the BG input-downstream neuron pairs exhibiting similar or opposite polarity are shown in Figure 7B .", "Remarkably , for each task period , two-way ANOVA revealed a significant and systematic effect of the BG input ( striatum vs . STN ) on the similarity coefficient of the neuronal responses of the BG input-downstream neuron pairs ( two-way ANOVA , p<0 . 001 ) .", "Nevertheless , this significant BG input effect varied depending on the polarity of the neuronal response of the BG input neurons that composed the BG input-downstream pairs .", "For each task period , we found that subthalamic , rather than striatal ( MSN ) , increases correlated with BG downstream increases ( Inc-Inc pairs ) and decreases ( Inc-Dec pairs ) , while striatal and subthalamic decreases ( although there was a significant BG input effect ) correlated almost to an equal extent with BG downstream neuronal responses , regardless of their principal polarity ( both Dec-Dec and Dec-Inc pairs ) .", "However , given the relatively low fraction of striatal decreases ( Figure 7A ) , we concluded that subthalamic , but not striatal MSN , neuronal responses ( increases or decreases ) were more strongly correlated to the BG downstream neuronal responses , regardless of their polarity .", "We used systemic MPTP injections ( see Materials and methods ) to induce a severe parkinsonian state .", "F-18 FDOPA positron emission tomography ( Monkey K , Figure 8A , right ) and post-mortem tyrosine hydroxylase immunohistochemistry ( Monkey S , Figure 8B , right ) revealed large dopamine depletion in the dorsal striatum ( i . e . , caudate nucleus and putamen ) following the MPTP treatment .", "Our animals developed all major motor signs of PD , including bradykinesia/akinesia , rigidity , abnormal flexed posture and low-frequency tremor ( Figure 9 ) .", "In the parkinsonian state , given the severity of the motor symptoms , the animals were unable to execute the behavioral task and all recordings were made in a quiet alert state .", "After the initiation of dopamine-replacement therapy , neuronal recordings were made off dopaminergic medication ( see Materials and methods ) in order to mimic the recording conditions in human parkinsonian patients undergoing DBS surgery .", "Data collected before and after initiation of dopamine-replacement therapy ( in the OFF state ) were grouped since no significant difference was detected between these two parkinsonian conditions . 10 . 7554/eLife . 16443 . 015Figure 8 . Dopamine depletion assessment in parkinsonism .", "( A ) Dynamic positron emission tomography ( PET ) of healthy monkey's brain ( left ) and MPTP-treated monkey's brain ( Monkey K , right ) .", "The top panels are PET images showing the regional distribution of F-18 FDOPA at the carotid level during the first 30 s following the injection of the radioligand .", "The color bar ( top panels ) indicates the radioactivity concentration in Bq/ml .", "The bottom panels are PET images ( acquired from 50 to 60 min after the injection of the radioligand ) showing the regional distribution of F-18 FDOPA at the striatal level .", "Radioactivity concentration was Z-normalized , using the radioactivity concentration in the region of interest ( ROI ) for the cerebellum ( i . e . , region of non-specific F-18 FDOPA uptake ) .", "The color bar ( bottom panels ) indicates the relative radioactivity concentration in z-score .", "( B ) Photomicrographs of tyrosine hydroxylase ( TH ) staining demonstrating the loss of the midbrain dopaminergic neurons of the ventral tier of the substantia nigra compacta in the MPTP-treated monkey ( Monkey S , right ) compared with a healthy animal ( left ) .", "The top and bottom photomicrographs illustrate the midbrain and ( rostral ) striatum levels , respectively .", "After MPTP intoxication , TH-positive cells were lost in the ventral tier of the substantia nigra compacta ( see arrow ) and relatively spared in the ventral tegmental area .", "Note the lack of TH-positive staining in the dorsal striatum ( caudate nucleus and putamen ) compared to the ventral striatum ( particularly the shell of nucleus accumbens ) in the MPTP-treated monkey .", "Cd: caudate nucleus; IC: internal capsule; Pu: putamen; SN: substantia nigra; VTA: ventral tegmental area . DOI: http://dx . doi . org/10 . 7554/eLife . 16443 . 01510 . 7554/eLife . 16443 . 016Figure 9 . Long term and persistent parkinsonian motor symptoms following MPTP intoxication .", "( A ) Evolution of the sum of the scores of the major parkinsonian motor symptoms ( bradykinesia , rigidity , posture and tremor ) in each monkey before and after MPTP intoxication , up to the completion of the recordings .", "Day 0 indicates the first MPTP injection .", "Arrows mark the first day of dopamine-replacement therapy for each monkey .", "After initiation of dopamine-replacement therapy , scoring of the parkinsonian motor symptoms was made off dopaminergic medication ( overnight washout >12 hr ) .", "The level of parkinsonism was assessed by using a primate parkinsonism scale that rates each motor parkinsonian symptom ( bradykinesia , rigidity , posture and tremor ) from 0 ( normal ) to 3 ( severe ) .", "Hence , the minimum score is 0 and the maximum is 12 .", "( B ) Mean scores for the four motor symptoms ( ranges from 0-normal to 3-severe ) during the complete recording period in each MPTP-treated monkey .", "Error bars represent SEMs . DOI: http://dx . doi . org/10 . 7554/eLife . 16443 . 016 Theoretical studies have shown that neural oscillations can emerge at the population level in networks of neurons exhibiting an irregular ( i . e . , non-oscillatory ) discharge pattern and a low firing rate ( Brunel and Hakim , 2008; Kopell and LeMasson , 1994 ) .", "In addition , it has been reported that multi-unit cross-correlations might be a more sensitive detector of neuronal relationships than single-unit cross-correlations ( Bedenbaugh and Gerstein , 1997; Gerstein , 2000 ) .", "Indeed , most intra-operative human physiological studies during DBS procedures are conducted at the level of multi-unit activity ( MUA ) .", "For these reasons and given the very low firing rate of the MSNs , we investigated multi-unit oscillatory activity recorded in the vicinity of the recorded BG cells rather than single-unit oscillatory activity ( Figure 10A ) .", "Using the MUA recording in the vicinity of a well-isolated single unit better guarantees the location of the recording and rules out shifts in electrode position during the recording . 10 . 7554/eLife . 16443 . 017Figure 10 . Abnormal oscillatory spiking activity is not shared by all BG neuronal components in parkinsonism .", "( A ) Examples of 1 s traces of multi-unit activity recorded in the vicinity of an isolated neuron ( striatal MSN , striatal TAN , STN , GPe and SNr neurons ) in the normal ( before MPTP , left ) and parkinsonian ( after MPTP , right ) state and PSDs of the respective multi-unit activity .", "Multi-unit activity was online filtered between 250 and 6000 Hz .", "PSD was calculated over the complete recording span of the multi-unit activity constrained by the period of stable discharge rate and satisfactory isolation quality of the isolated neuron .", "Abscissas of the PSDs are in log scale .", "( B ) Average PSDs of activity of MSN , STN ( BG input stage , top panels ) , GPe and SNr ( BG downstream stages , bottom panels ) recordings , before and after MPTP intoxication ( left and right , respectively ) .", "Average PSD of activity of striatal TANs is in the inset of the top panels .", "Abscissas are in log scale .", "The shaded areas mark SEMs .", "N is the number of recording sites averaged . DOI: http://dx . doi . org/10 . 7554/eLife . 16443 . 01710 . 7554/eLife . 16443 . 018Figure 10—source data 1 . Power spectral densities of the multi-unit activity before and after MPTP . DOI: http://dx . doi . org/10 . 7554/eLife . 16443 . 018 Comparison of the mean power spectral densities ( PSDs ) of the MUA at the different stages of the BG main axis before and after MPTP intoxication revealed the emergence of 8–15 Hz oscillations in the STN , GPe and SNr ( GPi activity was not recorded in the MPTP-treated monkeys ) , but not in the recordings from the areas surrounding the striatal MSNs ( Figure 10B ) .", "The distributions of the oscillation frequencies were similar for the STN , GPe and SNr , and peaks of the distribution were found at 10 Hz for the three structures ( Figure 10B ) .", "Despite the absence of significant oscillations in MSN spiking activity ( comprising > 95% of the striatal cells ) in the dopamine-depleted striatum , we also found 10 Hz oscillations of striatal TANs after MPTP treatment ( Figure 10B , insets ) , indicating that abnormal oscillatory activity did not spare the striatum .", "Accordingly , we found that the peak values of the PSD of the spiking activity ( in the 8–15 Hz range , see Materials and methods ) were significantly higher in parkinsonism ( after MPTP ) than in healthy state ( before MPTP ) for TANs , STN , GPe and SNr neurons ( two-sample t-test , p<0 . 01 , Figure 11 ) , but not for MSNs .", "Thus , although low beta ( 8–15 Hz ) oscillations were found in the spiking activity of the STN , downstream BG structures , and even striatal TANs , MSNs ( recorded in striatal areas with TAN oscillations ) did not exhibit such abnormal oscillatory spiking activity . 10 . 7554/eLife . 16443 . 019Figure 11 . Mean PSD peak value in the 8–15 Hz range for the BG neuronal components before and after MPTP intoxication . Mean PSD peak value in the 8–15 Hz range is defined as the average of the PSD peak values in the 8–15 Hz range ( Z normalized ) of all the PSDs .", "Error bars represent SEMs . DOI: http://dx . doi . org/10 . 7554/eLife . 16443 . 019 After MPTP intoxication , we also found exaggerated 8–15 Hz oscillations of local field potentials ( LFPs ) recorded in all BG structures , including the striatum in the vicinity of both TANs and MSNs ( Figure 12 ) .", "LFPs are commonly attributed to the sub-threshold ( synaptic ) activity of the recorded structure .", "Therefore , we further performed a spike-field coherence analysis to determine whether the spiking activity of the neurons was synchronized ( phase locked ) to the LFP recorded in their surrounding areas . 10 . 7554/eLife . 16443 . 020Figure 12 . Exaggerated 8–15 Hz oscillations of LFPs within all structures of the BG network in parkinsonism . Superimposed PSDs of the LFPs recorded in the different BG nuclei ( Striatum , STN , GPe and SNr ) in the normal ( before MPTP ) and parkinsonian ( after MPTP ) state .", "A custom-made artifact removal procedure was used to clean sharp peak artifacts recorded by the high impedance microelectrodes .", "Abscissas are in log scale .", "N is the number of recording sites . DOI: http://dx . doi . org/10 . 7554/eLife . 16443 . 02010 . 7554/eLife . 16443 . 021Figure 12—source code 1 . Custom-made artifact removal procedure . DOI: http://dx . doi . org/10 . 7554/eLife . 16443 . 02110 . 7554/eLife . 16443 . 022Figure 12—source data 1 . Power spectral densities of the local field potential before and after MPTP . DOI: http://dx . doi . org/10 . 7554/eLife . 16443 . 022 Figure 13 depicts an example of STN spiking activity after MPTP intoxication that coincides with the negative waves of the 8–15 Hz oscillatory LFP recorded in the vicinity of the neuron .", "Estimates of the coherence between spike trains of the isolated units ( single-unit activity ) and LFPs revealed that except for MSNs , peak coherence values in the 8–15 Hz range increased significantly after MPTP intoxication ( two-sample t-test , p<0 . 001 ) for all other BG neuronal assemblies ( Figure 14 , left plots ) .", "To guarantee that these spike-field coherence results were not confounded by the slow discharge rate of the striatal MSNs , we also examined the spike-field coherence between the MUA recorded in the striatum ( and elsewhere ) and the LFP ( Figure 14—figure supplement 1 , left plots ) .", "We found that using spike train of the isolated units or MUA for the calculation of the spike-filed coherence yielded the same qualitative results . 10 . 7554/eLife . 16443 . 023Figure 13 . Recording of the subthalamic spiking and LFP activity after MPTP intoxication . The traces reflect the different filter properties applied to the signal recorded by the electrode .", "Upper trace depicts the broad ( 1–8000 Hz ) band-pass filtered trace .", "The second trace is band-pass filtered at the low beta range ( 8–15 Hz ) and depicts LFP oscillations .", "In the third trace , a 250–6000 Hz band-pass filter is used and reveals the multi-unit activity ( MUA ) that exhibits a periodic oscillatory pattern synchronized to the 8–15 Hz LFP oscillation .", "Below the 250–6000 Hz filtered MUA is the digital display of the spikes detected online from the MUA using the online template-matching algorithm . DOI: http://dx . doi . org/10 . 7554/eLife . 16443 . 02310 . 7554/eLife . 16443 . 024Figure 14 . Single-unit spiking activity and LFP synchronization for the BG neuronal assemblies before and after MPTP intoxication . Left , average spike-field coherences between the spike train of the neurons ( single-unit spiking activity ) and the LFPs recorded in their vicinity before and after MPTP .", "Abscissas are in log scale .", "Right , phase histograms of the phases of the single-unit spiking activity relative to the LFP at the frequency of coherence peak values calculated in the 8–15 Hz range , after MPTP intoxication .", "The red arrows represent the mean phase of the spiking activity relative to the LFP peak ( phase = 0 degree ) .", "Numbers in bold indicate the numbers of spike-LFP pairs .", "Given the absence of spike-field coherence for the MSNs after MPTP intoxication , the phase histogram of the MSN spike-LFP pairs was not calculated . DOI: http://dx . doi . org/10 . 7554/eLife . 16443 . 02410 . 7554/eLife . 16443 . 025Figure 14—source data 1 . Spike-LFP synchronization before and after MPTP . DOI: http://dx . doi . org/10 . 7554/eLife . 16443 . 02510 . 7554/eLife . 16443 . 026Figure 14—figure supplement 1 . Multi-unit spiking activity and LFP synchronization for the BG neuronal assemblies before and after MPTP intoxication . Same convention as Figure 14 , except that the spiking activity corresponds to multi-unit activity rather than single-unit activity . DOI: http://dx . doi . org/10 . 7554/eLife . 16443 . 026 After MPTP intoxication , despite the emergence of exaggerated 8–15 Hz LFP oscillations , not all LFPs expressed 8–15 Hz oscillatory activity ( Figure 12 ) .", "Therefore , one might suggest that MSN spike-striatal field coherence ( Figure", "14 ) could materialize only when the striatal LFP ( recorded in the vicinity of the MSNs ) oscillated .", "In other words , the current average analysis might not reflect a small population of MSNs that synchronized their activity with the robust 8–15 Hz oscillatory LFPs .", "To test this hypothesis , we correlated the strength of the LFP oscillations and the degree of spike-field coherence in the striatum ( and elsewhere ) of the parkinsonian monkeys .", "We found that regardless of the BG neuronal component ( i . e . , including striatal MSNs ) the degree of synchronization between the single-unit ( Figure", "15 ) or multi-unit ( Figure 15—figure supplement", "1 ) spiking activity and the LFP oscillations was not ( linearly ) related to the strength of the 8–15 Hz LFP oscillations .", "This rules out the possibility that synchronization between MSN spiking activity and striatal LFP emerged only when LFP recorded in the vicinity of the neuron exhibited robust 8–15 Hz oscillatory activity . 10 . 7554/eLife . 16443 . 027Figure 15 . Strength of LFP oscillations is not related to the degree of synchronization between single-unit spiking activity and LFP oscillations along the BG network after MPTP intoxication . Scatter plots of the 8–15 Hz LFP PSD peak values and the 8–15 Hz spike-field coherence peak values for the different BG neuronal components .", "Red line represents the linear regression line between the LFP PSD peak values and the spike-field coherence peak values .", "r is the Pearson correlation coefficient and p indicates the probability that r = 0 . DOI: http://dx . doi . org/10 . 7554/eLife . 16443 . 02710 . 7554/eLife . 16443 . 028Figure 15—figure supplement 1 . Strength of LFP oscillations is not related to the degree of synchronization between multi-unit spiking activity and LFP oscillations along the BG network after MPTP intoxication . Same convention as Figure 15 , except that multi-unit spiking activity , rather than single-unit spiking activity , was used to calculate the spike-field coherence . DOI: http://dx . doi . org/10 . 7554/eLife . 16443 . 028 Calculation of the phase of the spiking activity relative to the LFP at the frequency of the coherence peak values in the 8–15 Hz range revealed that LFP relative phase values of the single-unit ( Figure 14 , right polar histograms ) and multi-unit ( Figure 14—figure supplement 1 , right polar histograms ) spiking activity of TAN , STN , GPe and SNr neurons mainly ranged from 180 to 270º .", "Namely , TAN , STN , GPe and SNr neurons tended to fire around the negative peak of the 8–15 Hz oscillatory LFP - but see Nelson and Pouget ( 2010 ) for possible confounding effects of electrode and instrument filter properties on the exact phase relationships between spikes and LFPs .", "We verified the results obtained with the coherence/phase analysis by examining the population spike-triggered averages of the LFP ( STAs LFP ) .", "The STA LFP analysis revealed that except for MSNs , the coincidence between the spikes of the STN , GPe , SNr neurons , as well as those of the TANs , and the negative waves of the LFP oscillations increased after MPTP intoxication ( Figure 16 , left and central columns ) .", "Finally , to rule out the possibility that differences in the population STAs LFP were not a mere consequence of the differences in discharge rate ( and the total number of triggers ) of the different BG neuronal components ( Figure 17 ) , we also calculated the STAs LFP after random dilution of the spike trains of the neurons ( see Materials and methods ) .", "We found that the random dilution of the spike trains did not affect the STA-LFP results ( Figure 16 , right column ) .", "Therefore , in the parkinsonian state , spike-LFP synchronization was not influenced by the firing rate of BG neuronal components and more importantly , the absence of phase locking between MSN spiking activity and striatal LFPs was probably not due to the confounding effects of the slow discharge rate of the MSNs . 10 . 7554/eLife . 16443 . 029Figure 16 . Population spike-triggered averages of LFP after MPTP intoxication show larger fluctuations around spike times of STN , BG downstream neurons and striatal TANs , but not around spike times of striatal MSNs . For each BG neuronal assembly , population STA LFP was defined as the average of the STAs LFP of all neurons .", "After MPTP intoxication , STAs LFP were calculated before and after random dilution of the spike trains of the neurons .", "LFP was recorded in the vicinity of the neuron ( i . e . , spiking activity and LFP were recorded on the same electrode ) .", "Dashed vertical lines indicate the time of the spikes ( time = 0 ) .", "The shaded areas mark SEMsDOI: http://dx . doi . org/10 . 7554/eLife . 16443 . 02910 . 7554/eLife . 16443 . 030Figure 17 . Firing rate in the BG network before and after MPTP intoxication .", "( A ) Distribution of the firing rate of the neuronal components of the BG network .", "Gray - before MPTP; light green - after MPTP; dark green - overlapping bins .", "Inset , mean firing rate before and after MPTP intoxication .", "Error bars represent SEMs .", "Numbers in parentheses indicate the number of neurons .", "( B ) Percentage of change in the firing rate after MPTP intoxication , for the neuronal components of the BG network .", "Gray line ( 100% ) represents the healthy ( before MPTP ) firing rate . DOI: http://dx . doi . org/10 . 7554/eLife . 16443 . 030" ], [ "The BG main axis connects the brain areas encoding the current state of the subject and the brain areas controlling action .", "However , current BG computational models do not explicitly describe how the BG main axis ( actor ) holds and evaluates transient sensory information ( i . e . , current state ) until it can be used later by the cortical and brainstem motor centers that guide the optimal action/response .", "Therefore , it is crucial to show that persistent modulatory activity ( i . e . , during prolonged delays between sensory perception and action , especially when sensory information is no longer available ) is not a unique property of the frontal cortex and other cortical areas ( Curtis and Lee , 2010; Durstewitz et al . , 2000; Goldman-Rakic , 1995 ) , but can also be found along the BG main axis .", "Unlike the cortex , many projection neurons of the BG use the inhibitory transmitter GABA as a carrier of information within and between BG nuclei .", "Nevertheless , the STN is a major source of glutamate ( an excitatory transmitter ) to the BG downstream structures ( GPe , GPi and SNr ) .", "Accordingly , in most BG structures , modifications of spiking activity by behavioral events consist of either increases or decreases in firing rate ( Espinosa-Parrilla et al . , 2013; Joshua et al . , 2009b; Turner and Anderson , 2005 ) .", "Due to their low baseline firing rate ( Figure 17A ) , it might be posited that like other low-discharge rate neurons ( e . g . , in the cortex ) , the neuronal responses of the striatal MSNs would be restricted to increases in activity .", "Nevertheless and in line with earlier studies ( Adler et al . , 2012; Báez-Mendoza et al . , 2016; Samejima et al . , 2005 ) , we found task-related decreases ( albeit less frequent than increases ) in the discharge rate of MSNs .", "This suggests that neurons in the BG main axis ( including striatal MSNs ) exhibited diverse timing and directional ( increase or decrease ) patterns of discharge modulations from cue onset to outcome delivery .", "These diverse patterns of BG discharge modulations resulted in persistent population activity , even in the absence of sensory stimulation ( after cue offset ) ( Figures 3 , 4 and 5 ) .", "Hence , in each neuronal assembly of the BG main axis , a modulation of the population activity may form a persistent internal representation ( Curtis and Lee , 2010; Durstewitz et al . , 2000; Goldman-Rakic , 1995 ) .", "Our working hypothesis holds that the BG persistent population activity reflects the neuronal coupling between the current state ( defined by cue presentation ) and the upcoming contingent behavioral response ( licking or blinking movement in our task ) .", "The heterogeneity of the BG main axis responses ( Adler et al . , 2012; Deffains et al . , 2010; Joshua et al . , 2009a ) contrasts with the homogeneity of the transient responses of the BG neuromodulators ( Graybiel et al . , 1994; Joshua et al . , 2009a ) and corroborates the view that the BG main axis has a large information capacity that can encode the enormous and diverse possibilities of state-to-action mapping ( Bar-Gad et al . , 2003 ) .", "For many years , the STN was considered to be a relay station of the BG indirect pathway ( striatum-GPe-STN-GPi/SNr ) under the exclusive influence of cortico-striatal transmission , whereas the role of the ( hyper ) direct projections from the cortex to the STN ( Haynes and Haber , 2013; Nambu , 2004 ) was neglected ( Bergman et al . , 1990 ) .", "Here , we found that in the normal awake monkey engaged in a temporal discounting classical conditioning task , the fluctuations in STN activity were concurrent with the modifications of activity in the BG downstream network , without apparent parallel modulations of striatal activity ( Figures 5 and 6 ) .", "Our experimental methods ( i . e . , extracellular recording of spiking activity in behaving animals ) did not allow us to differentiate between the activity of MSNs of the BG direct ( D1 MSNs-GPi/SNr ) and indirect ( D2 MSNs-GPe-STN-GPi/SNr ) pathways ( Albin et al . , 1989; Gerfen et al . , 1990; Figure 2A ) .", "However , examination of the temporal patterns of changes in activity in the MSN assembly did not indicate the presence of two distinct sub-populations ( Figures 3 , 4 , 5 and 6 ) , which is consistent with recent studies revealing the co-activation of the striatal direct and indirect pathways ( Cui et al . , 2013; Oldenburg and Sabatini , 2015 ) .", "In addition , a formal clustering analysis of the MSN responses from cue onset to outcome delivery in both conditions ( data not shown ) found no evidence for two distinct MSN sub-groups ( e . g . , one group that responded during cue presentation and another group that responded during the extended delay period ) that could account for the reversal of I/D balance in the BG downstream network during the delay period in the delayed condition .", "Although there is an ongoing debate on the distribution of STN output in the BG downstream network ( Mathai and Smith , 2011; Sato et al . , 2000 ) , a compelling functional model of action selection process in the BG network claims that information transfer via the hyper-direct ( cortex-STN-GPi/SNr ) pathway is faster and more broadly distributed in the BG output structures than the information flowing along the direct and indirect ( striatum-GPi/SNr ) pathways ( Mink , 1996; Nambu et al . , 2002 ) .", "Given that the striatum and the STN are the two input structures of the BG network and that STN neuronal responses were more strongly correlated with BG downstream neuronal responses , compared to striatal MSN responses ( Figure 7B ) , we suggest that the STN might orchestrate the time-varying I/D balance in the BG downstream network .", "Thus , the STN would exert a powerful glutamatergic and apparently GABAergic striatal-free control over the release of BG output commands ( Baunez et al . , 2001; Frank et al . , 2007a; Jahanshahi et al . , 2015 ) prior to their fine modulation by the direct and indirect pathways .", "Nonetheless , this specific correlation between the divergent excitatory STN projections and BG downstream neurons was only shown here for a classical conditioning task with immediate and delayed outcomes and should be tested in the future under a range of behavioral conditions and paradigms .", "In this study , we used a harsh MPTP intoxication procedure ( see Materials and methods ) rather than a slower regimen of MPTP intoxication ( i . e . , that would have led to a L-DOPA responsive parkinsonian monkey capable of executing behavioral tasks ) .", "This decision was made to induce a large striatal dopamine depletion ( Figure 8 ) and guarantee the emergence of the full spectrum of pathological synchronous oscillations in the BG .", "Indeed , earlier studies have failed to find abnormal BG oscillatory activity at the early stage of chronic MPTP treatment ( Leblois et al . , 2007 ) .", "In addition , the stability of the parkinsonian symptoms during the complete duration of the recordings was critical in our study ( Figure 9A ) to enable the comparison of activities at the different stages of the BG .", "Therefore , we favored severe MPTP-treated monkeys that usually develop stable clinical symptoms ( Potts et al . , 2014 ) .", "The emergence of synchronized oscillatory activity in the BG network of animal models of PD and human PD patients has been well-described ( Brown , 2003; Wichmann and DeLong , 2003 ) .", "These abnormal oscillations are thought to alter the information flow through the BG main axis , thus leading to the release of abnormal commands by BG output structures ( Bergman et al . , 1998; Brown et al . , 2001 ) .", "However , the frequency range of these oscillations varies across species ( Stein and Bar-Gad , 2013 ) .", "In line with earlier studies conducted on MPTP-treated monkeys ( Bergman et al . , 1994; Raz et al . , 2001; Soares et al . , 2004 ) , we found that abnormal spiking oscillatory activity after MPTP intoxication of African green monkeys ( vervets ) occurred in the low beta ( 8–15 Hz ) range .", "In addition , we showed that this pathological oscillatory activity , at least at the level of spiking activity , was not shared by all BG neuronal components in parkinsonism ( Figures 10 and 11 ) .", "Even though earlier rodent ( Costa et al . , 2006 ) and primate ( Singh et al . , 2015 ) studies have shown that MSN activity patterns are altered in parkinsonism , we found that MSNs apparently failed to express pathological 8–15 Hz neuronal oscillations , as did the STN and BG downstream structures ( Figures 10 and 11 ) .", "The lack of oscillatory activity of MSNs in the MPTP-treated monkey is consistent with our unpublished observations of no oscillatory spiking activity in the striatum of parkinsonian human patients undergoing DBS procedures .", "However , not all components of striatal activity were deprived of abnormal oscillatory activity after MPTP intoxication .", "Indeed , as already described in earlier studies of TANs in MPTP-treated monkeys ( Raz et al . , 1996 ) , we found that TANs also exhibited 10 Hz oscillatory activity .", "This MSN-TAN incongruity is in line with the view of a weak functional in vivo connectivity between MSNs and TANs ( Adler et al . , 2013b ) and probably reflects their different synaptic drives .", "In each structure of the BG main axis , including the striatum , we found exaggerated 8–15 Hz oscillations of LFP after MPTP intoxication ( Figure 12 ) .", "Nevertheless , 8–15 Hz LFP oscillations ( although sparser ) were also observed in the healthy state ( before MPTP ) .", "In normal behavior control , beta oscillatory activity is known to play a role in the maintenance of the current sensorimotor or cognitive state ( the status quo ) ( Brittain and Brown , 2014; Engel and Fries , 2010 ) .", "Therefore , the 8–15 Hz LFP oscillations observed in the healthy state likely correspond to one of the neuronal processes involved in state-to-action mapping .", "Consequently , exaggeration of 8–15 Hz oscillatory activity after induction of parkinsonism would compromise the ability of human PD patients to adjust their behavior to situational demands ( Brown and Marsden , 1988; Cools et al . , 1984; Frank et al . , 2007b ) due to an abnormal persistence of the BG 'status quo' signal .", "The LFP probably represents a global signature of the synaptic input and dendritic processing , whereas MUA reflects the efferent ( output ) activity of the local neuronal population ( Buzsáki et al . , 2012; Logothetis , 2003 ) .", "Thus , as for other structures in the closed loop of the BG network , we found that the dopamine-depleted striatum was also bombarded with pathological oscillations by its major afferent neurons ( Figure 12 ) .", "However , these striatal oscillating synaptic inputs apparently only entrained the spiking activity of the TANs , but not the MSNs ( Figures 10 and 11 ) .", "Spike-field coherence analysis revealed that the single-unit ( Figure 14 ) and multi-unit ( Figure 14—figure supplement 1 ) spiking activities of the STN , GPe and SNr neurons after MPTP intoxication were synchronized to the 8–15 Hz oscillatory LFP recorded in their surrounding areas .", "On the other hand , in the dopamine-depleted striatum , only TAN ( single- and multi-unit ) spiking activity was phase locked to the striatal 8–15 Hz LFP oscillations .", "After MPTP intoxication , we found no evidence for a ( linear ) relationship between the strength of the 8–15 Hz LFP oscillations and the degree of spike-field coherence in the 8–15 Hz range ( Figure 15 and Figure 15—figure supplement 1 ) along the BG network .", "Therefore , whatever the strength of the 8–15 Hz LFP oscillations within the striatum , MSN spiking activity failed to lock with these LFP oscillations .", "Unlike the low discharge rate of the isolated MSNs ( lower than the frequency of the 8–15 Hz oscillations , Figure 17 ) , the MUA recorded in the vicinity of the isolated striatal MSNs reflects the discharge of many neurons .", "Therefore , given the consistency of the spike-field coherence results using spike trains of isolated MSNs ( Figure 14 ) or striatal MUAs ( Figure 14—figure supplement 1 ) , the absence of coherence between MSN spiking activity and LFP after MPTP intoxication was most likely not confounded by the slow MSN discharge .", "Indeed , STA LFP analysis ( even after random dilution of the spike trains of the neurons ) also indicated that MSN spikes did not synchronize with LFP oscillations , as did the spikes of TAN , STN , GPe and SNr neurons ( Figure 16 ) .", "Finally , in line with earlier studies ( Goldberg et al . , 2004; Kühn et al . , 2005 ) , we showed that these synchronized spiking activities occurred during the negative wave of the 8–15 Hz oscillatory LFP ( Figures 14 , 16 and Figure 14—figure supplement 1 ) .", "Together , these results rule out the possibility that the absence of synchronization between MSN spiking activity and abnormal oscillating LFP striatal inputs in parkinsonism was due to the low discharge rate of the MSNs .", "Rather , the abnormal striatal synaptic inputs may not have been strong enough to depolarize the membrane potential of the MSNs above the spike threshold .", "Based on the classical rate model of the BG network ( Albin et al . , 1989; Bergman et al . , 1990; Gerfen et al . , 1990 ) , striatal dopamine depletion leads to a reduction of the D1-MSM discharge and an elevation of the D2-MSN discharge .", "In contrast to an earlier study ( Liang et al . , 2008 ) , we did not find any robust increase in the firing rate of the MSNs recorded within the dopamine-depleted striatum ( Figure 17 ) .", "This inconsistency could be due to differences in the experimental approaches , such as the species , the methods of MPTP intoxication , the time elapsed between MPTP treatment and neuronal recordings , and the severity of the parkinsonian symptoms .", "In any case , our methods of extracellular recording in behaving animals cannot discriminate between D1- and D2-MSNs .", "However , in agreement with earlier studies ( Bergman et al . , 1994; Filion and Tremblay , 1991; Miller and DeLong , 1987 ) , we also found that after MPTP intoxication , the firing rate decreased in the GPe and increased in the STN ( Figure 17 ) .", "Therefore , we cannot rule out the possibility that the slight elevation of the striatal GABAergic drive to the GPe ( via D2-MSNs , Figure 2A ) may have induced oscillatory activity in the BG downstream structures ( Terman et al . , 2002 ) , probably through changes in the activity pattern of the GPe-STN closed circuit ( Bevan et al . , 2002; Plenz and Kital , 1999 ) .", "Noticeably , despite the abnormal oscillating synaptic inputs in the striatum , striatal projection neurons ( MSNs ) failed to express spiking ( output ) activity .", "Therefore , abnormal oscillatory activity within dopamine-depleted striatum did not propagate to the BG downstream structures .", "In this study , we did not assess the striatal vs . extra-striatal dopamine depletion after MPTP intoxication ( Figure 8 ) and we cannot exclude the possible role of dopamine degeneration of the STN and BG downstream structures in the emergence of oscillatory activity in the STN and along the BG network ( Francois et al . , 2000; Galvan et al . , 2014; Rommelfanger and Wichmann , 2010 ) .", "Nevertheless , the abnormal oscillatory spiking activity of the STN neurons emerged in tandem with an increase in synchronization between the oscillatory spiking activity of the STN neuronal targets ( i . e . , BG downstream neurons ) and their abnormal LFP oscillations ( i . e . , BG downstream oscillating synaptic inputs ) .", "Therefore , our results are in line with other studies ( Nambu and Tachibana , 2014 ) and suggest that the abnormal oscillations observed in PD resonate across the closed loop of the cortico-BG network through the STN , not the striatum .", "Overall , the current study conducted on both normal and parkinsonian animals support the idea that the STN may be the driving force behind the physiology ( Kitai and Kita , 1987 ) as well as the pathophysiology of the BG .", "Nevertheless , the striatum obviously remains a major player in BG functioning in health and disease .", "Striatal dopaminergic input is involved not only in the modulation of the efficacy of the cortico-striatal synapses ( Reynolds et al . , 2001; Shen et al . , 2008 ) , but also in the excitability of the striatal projection neuron ( Nicola et al . , 2000; Onn et al . , 2000 ) .", "Thus , the coincidence of cortical/thalamic and midbrain dopaminergic activity might be a necessary condition for MSN spike generation .", "Consequently , MSN spiking activity would be shaped by striatal inputs only during brief , but functionally important periods ( probably marked by transient dopaminergic activity ) .", "This refined control of striatal output patterns was probably undetected in the current population analysis .", "Moreover , in parkinsonism , due to the utilization of a harsh MPTP intoxication procedure , our MPTP-treated monkeys developed stable and severe motor symptoms , and were unable to execute the behavioral task .", "Therefore , the study of the neuronal activity along the BG network in similar classical ( but also operant ) conditioning tasks using MPTP-treated monkeys that exhibit moderate parkinsonian symptoms is undoubtedly a necessary step that should enhance our understanding of the physiology and pathophysiology of the BG .", "However , the unique anatomical and physiological features of the STN highlighted in the current study provide a concrete explanation for the efficacy of the inactivation of the STN in MPTP-treated monkeys ( Bergman et al . , 1990 ) , human patients ( Alvarez et al . , 2005 ) and STN-DBS in MPTP-treated monkeys ( Benazzouz et al . , 1993 ) and parkinsonian patients to alleviate BG-related motor ( Odekerken et al . , 2016 ) and non-motor ( Lhommée et al . , 2012 ) clinical symptoms ." ], [ "Two female African vervet green monkeys ( K and S , Cercopithecus aethiops aethiops ) , weighing ~4 kg , were trained in a temporal discounting classical conditioning task ( Figure 1A ) .", "Six different fractal visual cues were presented for 2 s .", "The cues used here were full-screen isoluminant images generated using ChaosPro 3 . 2 program ( www . chaospro . de ) and displayed on a 21 inch LCD monitor , located 50 cm in front of the monkeys' faces .", "Cues predicted either a food outcome , an airpuff outcome ( directed at both eyes ) or neither of them , thus grouping them into three categories: appetitive/rewarding , aversive and neutral cues .", "For each category , depending on the cue , its offset was either immediately followed by the outcome period ( immediate condition ) or by a 6-s delay period which preceded the outcome period ( delayed condition ) .", "This delay period was chosen on the basis of previous studies indicating that monkeys engaged in a delayed-release task maintain central fixation for no more than 4 s ( Slovin et al . , 1999 ) and that trial-over-trial behavioral learning of monkeys during smooth pursuit eye movement task is forgotten within 6 s ( Yang and Lisberger , 2010 , 2014 ) .", "The outcome period ( 0 . 15 s ) was signaled by one of three sounds that differentiated the three possible outcomes ( food , airpuff or no outcome ) and was followed by a variable inter-trial interval ( ITI ) of 6–10 s .", "Fractal images and sounds were swapped between the two monkeys .", "After an intensive training period in the task ( ~3 months , 5–6 days a week , 300–600 trials/day ) , the monkeys underwent a surgical procedure for the implantation of a MRI-compatible Cilux head holder ( Crist Instruments , MD ) and a 27 mm ( inner edge ) square Cilux recording chamber ( AlphaOmega Engineering , Israel ) .", "During surgery , the head of the animal was fixed in a stereotaxic frame ( David Kopf Instruments , CA ) and the recording chamber was implanted over the right hemisphere , above a burr hole in the skull .", "The head holder and the chamber were attached to the skull using titanium screws ( Crist Instruments , MD ) and wires ( Fort Wayne metals , IN ) embedded in acrylic cement .", "The recording chamber was tilted 45° laterally in the coronal plane and stereotaxically positioned to cover most of the basal ganglia ( BG ) nuclei ( Contreras et al . , 1981; Martin and Bowden , 2000 ) .", "Surgery was performed under aseptic conditions and isoflurane and N2O deep anesthesia , following induction with Medetomidine ( 0 . 1 mg/kg IM ) and Ketamine ( 10 mg/kg IM ) .", "The surgical procedure was carried out by a board-certified neurosurgeon and anesthesia induction and level maintenance ( end-tidal CO2 , arterial O2 saturation , ECG , blood pressure and rectal temperature ) were performed by the veterinary team of the animal facility .", "Analgesics ( Carprofen , 4 mg/kg SC ) and antibiotics ( Ceftriaxone , 35 mg/kg IV or IM ) were administered during surgery and the first 2–3 postoperative days .", "Recording began after a postoperative recovery period of 5–7 days .", "During this recovery period , an anatomical MRI scan ( Whole-body 3T , 32-channel head coil , Siemens scanner ) was performed to ascertain the correct placement of the chamber and estimate the stereotaxic coordinates of the neuronal recordings .", "The MRI sequences included a high-resolution T2-weighted TSE sequence: TR = 4232 ms , TE = 63 ms , flip angle = 180° .", "70*1 mm coronal slices ( no gap ) were used with a field of view ( FOV ) of 240*240 mm and matrix 320*313 ( in-plane resolution of 0 . 75*0 . 77 mm ) .", "Four averages were used to improve the signal to noise ratio .", "The coronal slices were positioned on a T2-weighted TSE sagittal sequence ( TR = 2200 ms , TE = 63 ms , flip angle = 180° , 35 slices of 1 mm with 10% gap , FOV = 240*240 mm , matrix = 320*313 ) .", "Before the MRI scan , the chamber was filled with 3% agar and five tungsten electrodes were inserted into the animal's brain at equally spaced coordinates of the recording chamber [Y , X = ( 6 , 0 ) , ( 0 , −6 ) , ( 0 , 0 ) , ( 0 , 6 ) and ( −6 , 0 ) in mm from the chamber center] .", "An additional MRI scan was performed at the final stage of the recordings to re-validate the location of the recording sites and to rule out significant brain shifts .", "For the purpose of MRI examination , the animal was sedated using IM Medetomidine 0 . 1 mg/kg and IM Ketamine 10 mg/kg .", "Throughout the entire course of the experiments , the chambers were washed 1–2 times/day with neomycin-sulfate saline solution followed by saline solution .", "To induce parkinsonism , the monkeys were treated with 1-methyl-4-phenyl-1 , 2 , 3 , 6-tetrahydropyridine ( MPTP-hydrochloride , Sigma , Israel ) .", "Five IM injections of 0 . 35 mg/kg/injection were made over the course of four days ( two injections in the first day ) under Ketamine ( 10 mg/kg IM ) sedation .", "We used a modified Benazzouz primate parkinsonism scale ( Benazzouz et al . , 1995 ) to assess the severity of the level of parkinsonism .", "Neuronal recordings during the parkinsonian state began when the animal exhibited severe parkinsonian symptoms , 7 days after the first injection ( Figure 9 ) .", "Since following the MPTP injections both animals became severely akinetic and lost the ability to feed themselves , they were fed with a high-calorie nutritional supplement ( Ensure Plus , Abbott Labs Nutrition , OH ) through a nasogastric tube 2–3 times/day , 7 days/week .", "In addition , special care was taken such as the use of soft mattresses and frequent changes of position to prevent the development of pressure sores .", "Two or three weeks after the first MPTP injection , we started dopamine-replacement therapy ( Dopicar , Teva Pharmaceutical Industries , Israel; 125 mg L-DOPA + 12 . 5 mg Carbidopa twice a day ) .", "From then on , neuronal recordings were conducted while the animals were off dopaminergic medication ( overnight washout >12 hr ) to mirror the conditions of recording observed in human parkinsonian patients undergoing DBS surgery .", "In the subsequent hours of L-DOPA administration ( at the end of the daily recording session ) , significant clinical improvement occurred in both animals , thus corroborating the diagnosis of parkinsonism .", "Nevertheless , during the neuronal recordings before ( MPTP naive ) and after ( MPTP off medication ) dopamine-replacement therapy , both animals exhibited severe bradykinesia , a reduction in spontaneous blinking frequency , marked rigidity , significantly flexed posture and episodic low ( ~4–6 Hz ) frequency tremor ( Figure 9 ) .", "The data collected during the two states ( MPTP naive and MPTP off medication ) were grouped since no significant difference was detected between them ( data not shown ) .", "F-18 FDOPA positron emission tomography ( PET ) scan and post-mortem tyrosine hydroxylase ( TH ) immunohistochemistry ( Monkey K and S , respectively ) were used to assess the severity of dopamine depletion following the MPTP treatment .", "During the recording sessions , the monkeys' heads were immobilized with a head-holder and eight glass-coated tungsten microelectrodes ( impedance range at 1 kHz: 0 . 3–0 . 8 MΩ ) were advanced separately ( Electrode Positioning System , AlphaOmega Engineering , Israel ) toward and through the targeted BG structures .", "The electrical activity was amplified by 5000 , band-pass filtered from 1 to 8000 Hz using a hardware four-pole Butterworth filter and sampled at 25 kHz by a 12-bit ( ± 5V input range ) Analog/Digital ( A/D ) converter ( Multi Channel Processor , AlphaOmega Engineering , Israel ) .", "Spiking activity was sorted online using a template matching algorithm ( Alpha Spike Detector , AlphaOmega Engineering , Israel ) .", "Up to three different units could be isolated from the same electrode and the detection timestamp of the sorted units was sampled at 40 kHz .", "The animals' task performance was assessed by monitoring licking and blinking behavior .", "Licking movements were recorded using an infra reflection detector ( Dr . Bouis Devices , Germany ) directed toward the animal's mouth .", "The infrared signal was amplified by 6 , filtered between 1 and 100 Hz by a four-pole Butterworth filter and sampled at 1 . 56 kHz ( Multi Channel Processor , AlphaOmega Engineering , Israel ) .", "Blinking movements were monitored using infrared digital video cameras which recorded the animal's face at 50 Hz .", "Eye states ( open or closed ) were automatically determined based on the number of dark pixels in the eye area , using custom software ( Mitelman et al . , 2009 ) .", "Neuronal data , licking movements , task and video synchronization digital signals were stored by the data acquisition system ( Alpha-Map , AlphaOmega Engineering , Israel ) for further analysis .", "Video data were stored on a separate PC .", "A home-made synchronization protocol ensured the synchronization of the behavioral , neuronal and video data .", "The different BG neuronal assemblies were identified according to their stereotaxic coordinates ( based on MRI and primate atlas data ( Contreras et al . , 1981; Martin and Bowden , 2000 ) and the real-time assessment of their electrophysiological features .", "In the striatum , MSNs were differentiated from TANs and the fast spiking neurons ( FSIs , presumably GABAergic interneurons co-expressing parvalbumin ) based on their spike shape , discharge rate and pattern ( Adler et al . , 2013b; Berke et al . , 2004; Deffains et al . , 2010; Sharott et al . , 2009 ) .", "The GPe border was characterized by sudden high frequency discharge activity below the striatum .", "The border between GPe and GPi was identified based on the depth of the electrode within the pallidum , the detection of the pallidal border cells ( characterized by their typical regular firing pattern and broad action potential ) and the differences in activity patterns of GPe and GPi cells ( Bezard et al . , 2001; DeLong , 1971 ) .", "In the GPe , neurons exhibit either a high-frequency discharge interrupted by pauses ( HFD-P neurons ) or a low-frequency discharge with occasional brief high frequency bursts ( LFD-B neurons ) .", "In contrast , in the GPi , nearly all the neurons exhibit a continuous ( without pauses ) high-frequency discharge .", "Beyond the internal capsule ( discernible by the high density of fibers ) , the STN and SNr were differentiated based on the depth of the electrode , the level of background activity ( higher background activity in the STN due to higher neuronal density ) , the electrophysiological features ( narrower spike shape and higher firing rate in SNr ) of the cells ( Bergman et al . , 1994; DeLong et al . , 1985; Schultz , 1986 ) and the detection of the surrounding neurons ( e . g . , neurons of the zona incerta for the STN , and neurons of the substantia nigra pars compacta for the SNr ) .", "Spike detection and sorting were done online ( see 'data collection and physiological recordings' section above ) .", "Each sorted unit was offline subjected to visual inspection of the stability of its firing rate over its recording span and the longest stable period was manually selected for further analysis while the rest of the data was discarded .", "Then , the quantification of its isolation quality ( only for the stable period ) was graded by calculating its isolation score ( Joshua et al . , 2007 ) .", "The isolation score ranged from 0 ( i . e . , multi-unit activity ) to 1 ( i . e . , perfect isolation ) .", "Only cells exhibiting a stable firing rate for ≥15 min ( ≥9 min for MSNs and STN neurons ) and an isolation score ≥0 . 7 ( ≥0 . 6 for MSNs and STN neurons ) were included in the database .", "The adjustment of the inclusion criteria for MSNs and STN neurons was due to the extremely small soma of the MSNs and the highly dense cellular structure of the STN which make cell stability and isolation difficult .", "Analysis of the subset of MSNs and STN neurons that fulfilled the same inclusion criteria of TANs , pallidal and nigral neurons ( stability ≥15 min , isolation score ≥0 . 7 ) yielded similar results to those reported here .", "Neuronal activities for the different task cues were characterized by their relative and absolute peri-stimulus time histograms ( PSTHs , Figures 2D and 3 ) .", "The PSTHs ( i . e . , 6 PSTHs per neuron ) were calculated in 1 ms bins and smoothed with a Gaussian window ( SD = 20 ms ) .", "For each smoothed PSTH , we calculated the relative deviation from the baseline ( i . e . , relative PSTH ) by subtracting the baseline firing rate ( i . e . , mean firing rate in the last 500 ms of the ITI ) from the PSTH ( Figure 3 ) .", "We also calculated the absolute deviation from the baseline ( i . e . , absolute PSTH ) .", "Given that this operation does not provide a natural zero baseline , the average of the absolute deviation from the baseline during the last 500 ms of the ITI was subtracted from this statistic ( Figure 3 ) .", "Next , the relative PSTH was segmented into consecutive and non-overlapped 20 ms bins .", "A bin was considered responsive when the bin activity ≥2 or ≤ – 2 SD of the baseline firing rate ( p<0 . 05 , empirical 68-95-99 . 7 rule ) .", "To examine the time course of the modulations of activity , we calculated the fraction of responsive bins at each time bin ( 20 ms ) .", "The fraction of responsive bins was calculated for all neurons in one of the BG structures , from cue onset to outcome delivery in both conditions ( immediate and delayed ) and for the different trial types ( appetitive , neutral and aversive ) .", "BG main axis neuronal responses to behavioral events are composed of either increases or decreases in discharge rate ( Espinosa-Parrilla et al . , 2013; Joshua et al . , 2009b; Turner and Anderson , 2005 ) .", "For this reason , at each time bin , we determined for each neuron and for each trial condition and type ( immediate/delayed conditions and appetitive/neutral/aversive types ) : ( 1 ) the fraction of increases and decreases in activity out of the total number of responsive bins , depending on whether the bin activity ≥ 2 or ≤ - 2 SD of the baseline firing rate , respectively ( Figure 6—figure supplement 1 ) ; ( 2 ) the mean magnitude of increases and decreases in activity ( Figure 6—figure supplement 2 ) ; ( 3 ) the relative dominance of the increases and decreases in activity [ ( increase - decrease ) / ( increase + decrease ) ] after having weighted the fractions of increases and decreases by their respective magnitude ( i . e . , increase-decrease balance of spiking activity or I/D balance , Figure 6 ) .", "This way , we examined the temporal evolution of the I/D balance over the different task periods in each neuronal assembly of the BG main axis .", "Note that for each condition ( immediate/delayed ) we tested each neuron three times ( for appetitive , neutral and aversive trials ) .", "Grouping all trial types of a single neuron revealed similar results ( data not shown ) .", "Finally , segmentation of the relative PSTH using 50 ms bins yielded the same qualitative results as those reported ( data not shown ) .", "For each neuronal activity ( i . e . , binned relative PSTH ) of the BG main axis , we determined the principal polarity ( increase or decrease ) of the neuronal response during each task period ( Figure 7A ) .", "The neuronal response was defined as an increase or a decrease when at least 75% of the modulated bins exhibited increases or decreases in activity , respectively .", "Then , for each task period and each trial type , we calculated the zero lag coefficient of the cross correlation function ( i . e . , similarity coefficient ) between the neuronal activities of each BG input-downstream neuron pair .", "Neuron pairs were clustered according to the principal polarity of their neuronal responses .", "This yielded four distinct scenarios: neuron pairs exhibiting a similar ( increase-increase or decrease-decrease ) or opposite ( increase-decrease or decrease-increase ) polarity of their neuronal responses ( Figure 7B ) .", "The similarity coefficient ranged from −1 ( i . e . , opposite neuronal activity ) to +1 ( i . e . same neuronal activity ) .", "The similarity coefficients for the three trial types ( appetitive , neutral and aversive trials ) were pooled since no significant difference was detected between them .", "For the power spectral density ( PSD ) calculations , the raw signal ( 1 to 8000 Hz , multi-unit activity , MUA ) recorded in the vicinity of the cells that fulfilled the inclusion criteria of this study ( stable discharge rate duration ≥15 or 9 min and isolation score ≥0 . 7 or 0 . 6 for TANs , pallidal , nigral neurons and MSNs , STN neurons , respectively ) was online band-pass filtered from 250 to 6000 Hz ( four-pole Butterworth filter ) .", "The filtered signal was Z-score normalized to obtain an unbiased estimate ( by the electrode impedance or the amplitude of the recorded neuronal activity ) of the oscillatory activity ( Zaidel et al . , 2010 ) .", "The Z-normalized signal was rectified by the 'absolute' operator ( Deffains et al . , 2014; Moran et al . , 2008; Zaidel et al . , 2010 ) .", "The rectified signal follows the envelope of the MUA and therefore enables the detection of burst frequencies below the range of the online band-pass filter ( 250–6000 Hz ) .", "Since the local field potential ( LFP ) frequency domain was filtered out , the resulting PSD only represented the oscillatory features of the spiking activity .", "The PSD of each signal was calculated using Welch's method with a 3-s Hamming window ( 50% overlap ) and a spectral resolution of 1/3 Hz ( nfft = 75000 , sampling frequency = 25 , 000 Hz ) .", "Any DC component generated by the sampling processes and by the 'absolute' operator was removed by subtracting the mean of every windowed segment of the rectified signal .", "For each neuronal assembly , the mean PSD was defined as the average of the PSDs of all signals ( Figure 10B ) .", "Quantification of the peak value of every PSD between 8–15 Hz was carried out by using a Z-score method ( Moshel et al . , 2013 ) .", "Briefly , for every PSD , the peak value ( maximum value ) of the PSD between 8 and 15 Hz was measured , and a tail mean and standard deviation was defined in the frequency range of 55 to 75 Hz .", "In this tail range , no particular power spectrum phenomena were observed between before and after MPTP intoxication ( Figure 10 ) .", "The PSD peak value in the 8–15 Hz range was defined as the Z-score amplitude of the PSD peak value ( Figure 11 ) .", "Similarly , we also calculated the PSD of the LFP ( 1 to 250 Hz ) recorded in the vicinity of the isolated cells ( Figure 12 ) .", "To do so , the PSD of each Z-normalized LFP was calculated using Welch's method ( see above for the parameters; but nfft = 2344 , sampling frequency = 781 . 3 Hz ) and without prior rectification by the absolute operator .", "Given the use of high-impedance microelectrodes , the LFPs were often contaminated by noise , which manifested as sharp peaks in the PSD .", "For this reason , we applied custom-made artifact removal software on the PSD of each LFP .", "For each peak of the PSD ( PSD fpeak ) , if PSD fpeak > 2*[ ( PSD fpeak−2 + PSD fpeak+2 ) / 2] , where fpeak was the frequency of the peak , PSD fpeak was considered as an artifact and PSD fpeak−2 , PSD fpeak−1 , PSD fpeak , PSD fpeak+1 and PSD fpeak+2 were removed ( replaced with Matlab Not a Number , NaN ) .", "For each sorted unit that fulfilled the inclusion criteria of the study ( Table 1 ) , the spike train ( i . e . time epochs of the detected spikes ) was low-passed filtered ( cutoff frequency = 100 Hz ) and Z-normalized .", "Synchronization between the Z-normalized spiking activity and the Z-normalized LFP recorded in the vicinity of the sorted unit was done by using the magnitude squared ( MS ) coherence method ( Figure 14 , left plots ) .", "Welch's method was utilized ( see PSD calculation for the parameters ) .", "Coherence values ranged ( by definition ) from 0 to 1 .", "For every coherence estimate , we determined the coherence peak value ( the highest coherence ) in the 8–15 Hz range .", "We also calculated the cross power spectrum density for all spike-LFP pairs .", "For each , we computed the cross spectrum phase and determined the phase lag value at the frequency of the 8–15 Hz coherence peak value .", "Then , for every neuronal assembly , we built the phase histogram of the phases of the spiking activity relative to the LFP at the frequency of the 8–15 Hz coherence peak values ( Figure 14 , right polar histograms ) .", "To examine the relationship between the strength of the LFP oscillations and the degree of spike-LFP phase locking in the 8–15 Hz range , we performed a linear regression analysis between the 8–15 Hz LFP PSD peak values ( see calculation of the 8–15 Hz MUA PSD peak values for the details ) and the 8–15 Hz spike-field coherence peak value ( Figure 15 ) .", "Finally , we carried out a similar spike-field coherence analysis using the MUA rather than the spike train ( single-unit activity ) of the sorted units ( Figure 14—figure supplement 1 and Figure 15—figure supplement 1 ) .", "To investigate the spike-LFP relationship in the temporal domain and not only in the frequency domain ( i . e . , spike-field coherence analysis ) , we also calculated the spike-triggered average ( STA ) of the LFP ( Goldberg et al . , 2004 ) for each sorted unit included in the neuronal database .", "To do so , each Z-normalized LFP was offline low-pass filtered ( four-pole Butterworth filter , filtfilt Matlab function , cutoff frequency = 30 Hz ) .", "This low-pass filter removed the ringing artifacts following spike time and the residual waveform of the spikes in the LFP recorded in the vicinity of the sorted unit ( i . e . , from the same electrode ) .", "For each neuronal assembly , the population STA LFP was defined as the average of the STAs LFP of all neurons ( Figure 16 ) .", "Finally , in the parkinsonian state , to rule out the possibility that STA LFP results in the striatum were influenced by the slow discharge rate of the MSNs ( Figure 17 ) , we randomly diluted the spike trains of every sorted units ( MSNs included ) firing at a higher rate than the mean discharge rate of the MSNs .", "Following the random dilution , the discharge rate of these units equaled the mean discharge rate of the MSNs ( Figure 17 ) and then we repeated the STA-LFP analysis ( Figure 16 , right column ) .", "Analysis was conducted identically on the different neuronal assemblies .", "The data from the two monkeys were pooled since no significant difference was detected between them .", "All statistical analyses were done using custom-made MATLAB 7 . 5 routines ( Mathworks , Natick , MA , USA ) .", "Statistical comparisons between the different task periods were tested with one-way ANOVAs .", "If necessary , Bonferroni correction was used to adjust the chosen significance level according to the number of multiple comparisons .", "In addition , differences in fractions of neuronal responses depending on their principal polarity ( increase vs . decrease ) were assessed by using χ2 test and two-way ANOVAs were used to statistically compare the similarity coefficients between BG input ( striatum or STN ) and downstream ( GPe , GPi or SNr ) structures .", "Finally , for every neuronal assembly , two-sample t-test was used for statistical comparisons between the healthy ( before MPTP ) and parkinsonian ( after MPTP ) states .", "The criterion for statistical significance was set at p<0 . 05 ." ] ]

[ "The striatum and the subthalamic nucleus ( STN ) constitute the input stage of the basal ganglia ( BG ) network and together innervate BG downstream structures using GABA and glutamate , respectively .", "Comparison of the neuronal activity in BG input and downstream structures reveals that subthalamic , not striatal , activity fluctuations correlate with modulations in the increase/decrease discharge balance of BG downstream neurons during temporal discounting classical condition task .", "After induction of parkinsonism with 1-methyl-4-phenyl-1 , 2 , 3 , 6-tetrahydropyridine ( MPTP ) , abnormal low beta ( 8-15 Hz ) spiking and local field potential ( LFP ) oscillations resonate across the BG network .", "Nevertheless , LFP beta oscillations entrain spiking activity of STN , striatal cholinergic interneurons and BG downstream structures , but do not entrain spiking activity of striatal projection neurons .", "Our results highlight the pivotal role of STN divergent projections in BG physiology and pathophysiology and may explain why STN is such an effective site for invasive treatment of advanced Parkinson's disease and other BG-related disorders ." ]

[ "The symptoms of Parkinson’s disease include tremor and slow movement , as well as loss of balance , depression and problems with sleep and memory .", "The death of neurons in a region of the brain called the substantia nigra pars compacta is one of the major hallmarks of Parkinson’s disease .", "These neurons produce a chemical called dopamine , and their death reduces dopamine levels in another area of the brain called the striatum .", "This structure is one of five brain regions known collectively as the basal ganglia , which form a circuit that helps to control movement .", "The most effective treatment currently available for advanced Parkinson’s disease entails lowering electrodes deep into the brain in order to shut down the activity of part of the basal ganglia .", "However , the target is not the striatum; instead it is a structure called the subthalamic nucleus .", "The striatum and the subthalamic nucleus are the two input regions of the basal ganglia: each sends signals to the other three structures downstream .", "So why does targeting the subthalamic nucleus , but not the striatum , reduce the symptoms of Parkinson’s disease ?", "To shed some light on this issue , Deffains et al . recorded the activity of neurons in the basal ganglia before and after injecting two monkeys with a drug called MPTP .", "Related to heroin , MPTP produces symptoms in animals that resemble those of Parkinson’s disease .", "Before the injections , spontaneous fluctuations in the activity of the subthalamic nucleus produced matching changes in the activity of the three downstream basal ganglia structures .", "Fluctuations in the activity of the striatum , by contrast , had no such effect .", "Moreover , injecting the monkeys with MPTP caused the basal ganglia to fire in an abnormal highly synchronized rhythm , similar to that seen in Parkinson’s disease .", "Crucially , the subthalamic nucleus contributed to this abnormal rhythm , whereas the striatum did not .", "The results presented by Deffains et al . provide a concrete explanation for why inactivating the subthalamic nucleus , but not the striatum , reduces the symptoms of Parkinson’s disease .", "Further research is now needed to explore how the striatum controls the activity of downstream regions of the basal ganglia , both in healthy people and in those with Parkinson's disease ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "developmental biology", "cell biology" ]

[ [ "Matrix metalloproteinase 14 ( MMP14 , also known as membrane type I-MMP ) is a member of the family of MMPs and contains a transmembrane domain for insertion into the plasma membrane ( Sato et al . , 1994 ) .", "MMP14 has been implicated in cancer cell invasion ( Hotary et al . , 2003 ) and embryonic development ( Holmbeck et al . , 1999; Zhou et al . , 2000 ) because of its ability to degrade extracellular matrix ( ECM ) macromolecules especially type I collagen ( Overall , 2001; Tam et al . , 2002; Lee et al . , 2006; Kessenbrock et al . , 2010; Gialeli et al . , 2011 ) .", "Mice deficient in MMP14 die within a few weeks of birth with generalized connective tissue abnormalities including osteopenia and soft tissue frailty ( Holmbeck et al . , 1999; Zhou et al . , 2000 ) .", "We were curious why absence of MMP14 , which is an efficient collagenase in vitro , leads to connective tissue frailty rather than collagen accumulation .", "Collagens are a large family of triple helical proteins that are widespread throughout the vertebrate body and are critical for tissue scaffolding ( Huxley-Jones et al . , 2007 ) .", "More than 28 collagen and collagen-related proteins occur in vertebrate tissues of which type I collagen is the archetypal member of the subfamily of fibril-forming collagens ( Kadler et al . , 2007 ) .", "The fibrils formed from type I collagen are the largest ( with a mass per unit length up to ∼0 . 3 TDa/µm ) and most size pleomorphic ( from ∼1 µm to >1 mm ) protein polymers in vertebrates and are essential for fibrous tissue development ( Schnieke et al . , 1983 ) .", "Collagen fibril assembly has best been studied in embryonic tendon , which contains narrow diameter ( ∼50 nm ) fibrils in which one end of the fibril is located within actin-dependent ( Canty et al . , 2006 ) invaginations of the plasma membrane called fibripositors ( Canty et al . , 2004 ) .", "Fibripositors are points of fibril assembly and sites of attachment of the cell to the ECM ( Kalson et al . , 2013 ) and exhibit a range of morphologies depending on the presence or absence of a slender finger-like projection of the plasma membrane .", "Protruding fibripositors exhibit the invagination and the finger-like projection whereas recessed fibripositors only exhibit the invagination ( Kalson et al . , 2013 ) .", "The transport of collagen fibrils into fibripositors is powered by non-muscle myosin II and is not part of a fibril degradation process in embryonic tendon ( Kalson et al . , 2013 ) .", "Short collagen fibrils can be found within membrane-sealed compartments termed fibricarriers ( Canty et al . , 2004; Kalson et al . , 2013 ) .", "The transition from a unimodal distribution of narrow ( ∼50 nm ) diameter collagen fibrils in embryonic tendon to a bimodal distribution in adult tissues with means of ∼50 nm and ∼200 nm diameter fibrils is a fascinating phenomenon ( Parry et al . , 1978; Derwin and Soslowsky , 1999 ) .", "The presence of narrow-diameter collagen fibrils in fibripositors during E14 . 5 to birth ( in mice ) signifies stage 1 of tendon development during which fibril number is determined ( Kalson et al . , 2015 ) .", "Stage 2 occurs soon after birth ( in mice ) and is characterized by the disappearance of fibripositors , the release of fibrils to the ECM and the growth of fibrils in length and diameter ( Kalson et al . , 2015 ) .", "We show here that in the absence of MMP14 , progression from stage 1–2 does not occur , fibrils are retained by fibripositors , fibril diameters keep to ∼50 nm , and tendon development stops at stage 1 ." ], [ "Wild type ( WT ) and Mmp14 knockout ( KO ) littermates had similar birth weights but the KO mice were growth retarded at 3 days after birth ( P3 ) ( Figure 1A , as reported previously [Holmbeck et al . , 1999] ) .", "Morphometric analyses at P0 showed that the tendons of the KO mice were thinner than those of WT mice ( Figure 1B ) and had fewer bundles of fibrils ( Figure 1C , D ) .", "In transverse section , Mmp14 KO tendons had fewer fibrils that were organized into fewer and irregular bundles ( Figure 1C , D ) .", "We detected no difference in the number of cells per unit volume of the tendon between WT and Mmp14 KO tendon ( Figure 1—figure supplement 1A–C ) .", "There was no difference in fibril volume fraction ( FVF ) ( Figure 1D ) ; therefore , there was no evidence of abnormal fibril–fibril interactions .", "Mmp14 KO tendons were mechanically weaker than WT tendon ( Figure 1E ) .", "However , after adjustment for size , the KO tendons had similar mechanical properties to those of tendons from WT animals , which was consistent with the normal FVF .", "Quantitative PCR analysis showed no differences in Col1a1 gene expression at P0 ( Figure 1F ) and [14C]-proline labeling showed that procollagen processing was unaffected by loss of MMP14 ( at P7 , Figure 1G ) .", "However , despite no differences in gene expression or procollagen processing with MMP14 deficiency we found decreased collagen synthesis in KO samples , compared to WT animals ( Figure 1—figure supplement 1D ) .", "We next analyzed collagen fibril diameters .", "As shown in Figure 1H , the diameters in P0 KO mouse-tail tendon were significantly larger ( by ∼12% ) than those in WT mice . 10 . 7554/eLife . 09345 . 003Figure 1 . Neonatal Mmp14-deficient mice have small and weak tendons .", "( A ) Weight of wild type ( WT ) and Mmp14 knockout ( Mmp14 KO ) littermates at birth ( P0 ) and at 3 days postnatal ( P3 ) .", "( B ) The cross-sectional transverse area of P0 Mmp14 KO tendons is significantly smaller than WT tendons .", "( C ) TEM images of P0 tail tendon demonstrate that KO tendons are smaller and show dysmorphic , enlarged bundles of collagen fibrils ( arrowhead ) .", "Scale bars 5 μm .", "( D ) KO tendons have fewer , larger fibril bundles , but the FVF is not different to WT tendons .", "( E ) KO tendons are weaker than WT tendons but have normal mechanical properties after adjusting for differences in size .", "( F ) Analysis of Col1a1 mRNA by qPCR in P0 tendons revealed no difference in gene expression .", "( G ) 14C-proline labeling of collagen demonstrated normal collagen processing at P7 in WT and KO tail tendon .", "( H ) Fibril diameter distributions of KO and WT tail tendon at P0 revealed significantly increased fibril diameters in KO mice .", "Bars show SEM .", "*p < 0 . 05 , †p > 0 . 05 ( t-tests ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 09345 . 00310 . 7554/eLife . 09345 . 004Figure 1—figure supplement 1 . Cell number and type I collagen synthesis in neonatal WT and Mmp14 KO tail tendon . 3D reconstruction from SBF-SEM analysis of P0 ( A ) WT and ( B ) Mmp14 KO tendons .", "Green/turquoise , outline of the tendon .", "Red/pink spheres , cell nuclei .", "Scale bars 10 µm .", "( C ) Estimated mean cell number per unit volume of tissue shows that the absence of MMP14 does not affect cell number at birth .", "Bars show SEM .", "†p > 0 . 05 ( t-test ) .", "( D ) Densitometry data for P7 tail tendons were labeled with 14C-proline for 1 hr and separate extracellular and intracellular extracts prepared as described ( Canty et al . , 2004 ) .", "A significant decrease in [14C]-collagen relative to β-actin ( detected by western blotting ) in the intracellular extracts was observed in the KO samples ( *p < 0 . 01 , t-test ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 09345 . 004 We used transmission electron microscopy ( TEM ) , serial block face-scanning electron microscopy ( SBF-SEM ) , and serial section electron tomography to examine embryonic WT and Mmp14 KO tendons .", "We were careful to use tail tendon from anatomical site- and age-matched embryonic WT and KO mice .", "TEM analysis showed E15 . 5 WT tendons contained collagen fibrils in the ECM and a small number of collagen fibrils in fibripositors ( Figure 2A and Figure 2—figure supplement 1A ) .", "In contrast , Mmp14 KO tendons contained conspicuous electron-lucent invaginations characteristic of recessed fibripositors ( Figure 2B and Figure 2—figure supplement 1B ) .", "There were ∼8 times the number of fibripositor cross-sections per nucleus in KO tenocytes ( Figure 3—figure supplement 3C ) .", "Tracing of fibrils in 3D reconstructions from SBF-SEM and electron tomography ( Figure 2—figure supplement 1C , D ) showed the presence of deep recessed fibripositors in Mmp14 KO cells ( Videos 1 , 2 , respectively ) with looped collagen fibrils .", "Figure 2—figure supplement 2 shows a diagrammatic representation of a typical recessed fibripositor containing looped collagen fibrils . 10 . 7554/eLife . 09345 . 005Figure 2 . Fibricarrier analysis of wild-type , Mmp14 KO , and Col-r/r embryonic tail tendon . Tail tendons at E15 . 5 of development from ( A ) wild-type , ( B ) Mmp14 KO , and ( C ) Col-r/r mice .", "Black arrowhead , recessed fibripositor ( electron lucent ) -containing collagen fibrils .", "White arrow , enclosed electron-dense compartment .", "Scale bars 500 nm . DOI: http://dx . doi . org/10 . 7554/eLife . 09345 . 00510 . 7554/eLife . 09345 . 006Figure 2—figure supplement 1 . Mmp14-deficient mice have prominent recessed fibripositors . SBF-SEM of ( A ) WT and ( B ) Mmp14 KO tendons at E15 . 5 showing fibripositors in WT tendons ( blue box ) and intracellular collagen fibril-containing compartments in Mmp14 KO tendons ( red box; arrowheads ) .", "Scale bars 1 µm .", "3D reconstruction of SBF-SEM data from ( C ) WT and ( D ) Mmp14 KO tendon cells .", "Brown , cytoplasm .", "Purple/blue , nucleus .", "Green , internal collagen fibril compartments .", "Yellow circles , fibril ends .", "Lime green ( in WT only ) , Golgi apparatus . DOI: http://dx . doi . org/10 . 7554/eLife . 09345 . 00610 . 7554/eLife . 09345 . 007Figure 2—figure supplement 2 . Schematic showing looping of collagen fibrils in recessed fibripositors . Diagrammatic representation of a recessed fibripositor with looping of a collagen fibril .", "Not drawn to scale . DOI: http://dx . doi . org/10 . 7554/eLife . 09345 . 00710 . 7554/eLife . 09345 . 008Video 1 . Step-through video generated from SBF-SEM images of E17 . 5 embryonic WT mouse-tail tendon . z-depth is 100 µm .", "Scale bar 2 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 09345 . 00810 . 7554/eLife . 09345 . 009Video 2 . Step-through video generated from SBF-SEM images of E17 . 5 embryonic Mmp14 KO mouse-tail tendon . z-depth is 100 µm .", "Scale bar 2 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 09345 . 009 MMP14 can activate proMMP2 ( Sato et al . , 1994 ) and proMMP13 ( Knauper et al . , 1996 ) .", "Also , MMP2 inhibition blocked uptake and subsequent intracellular digestion of collagen fibrils in periosteal tissue explants ( Creemers et al . , 1998 ) .", "However , EM analysis of embryonic Mmp2 KO and Mmp13 KO tail tendons showed no obvious changes to fibripositor occurrence ( Figure 3—figure supplement 1 ) .", "We used SBF-SEM to quantitate the numbers and mean lengths of collagen fibrils in embryonic ( E15 . 5 ) WT and Mmp14 KO tendon , using methods described previously ( Starborg et al . , 2013 ) .", "WT tendon contained numerous fibril tips and short fibrils ( Figure 3A ) .", "In contrast , fibril tips were less frequent in KO tendon ( Figure 3A ) .", "We then calculated the mean length of fibrils based on the relative frequency of tips-to-shaft numbers .", "The average fibril length in E15 . 5 WT tendon was 16 ± 3 μm whereas that in an age-matched and anatomical-site-matched Mmp14 KO tendon was 38 ± 6 μm ( Figure 3B ) .", "At E15 . 5 , the tendon cross-sectional area and the FVF in WT and KO samples were not significantly different ( Figure 3C , D ) .", "Therefore , given the same transverse area occupied by collagen fibrils in WT and KO tissue , the difference in collagen mean fibril length equates to a ∼2 . 5-fold reduction in fibril number in KO tendon .", "Analysis at E16 . 5 confirmed that fibrils were , on average , shorter in WT tendon than KO tendon ( mean length 50 ± 9 μm ) compared with 111 ± 26 μm ( n = 668 WT fibrils , 683 KO fibrils , respectively , each tracked over 10 μm ) . 10 . 7554/eLife . 09345 . 010Figure 3 . Deficiency in MMP14 activity results in fewer collagen fibrils .", "( A ) 10 µm-deep ( z-axis ) slices of 3D reconstructions of SBF-SEM data taken from of WT and Mmp14 KO embryonic tendons at E15 . 5 showing collagen fibrils ( blue ) with a tip ( marked by asterisks ) found within the volume .", "Purple fibrils passed through the volume and so did not have tips in the reconstruction .", "Scale bars 500 nm .", "( B ) Quantification of mean fibril length based on the number of tips identified shows that E15 . 5 WT fibrils are shorter than fibrils in age- and anatomical position-matched tail tendons from KO mice ( 308 and 266 fibrils tracked , respectively ) .", "( C ) Tendon cross-sectional area and ( D ) FVF are not different at E15 . 5 KO tendons .", "( E , F )", "Electron microscopy of tendon-like constructs cultured in the presence of MMP inhibitor GM6001 ( 10 µM in 0 . 1% DMSO ) show increased number of recessed fibripositors ( arrowheads ) compared to vehicle control .", "Scale bars 1 µm .", "( G ) Increase in calculated mean fibril length in GM6001-treated tendon-like constructs .", "Bars show SEM .", "*p < 0 . 05 , †p > 0 . 05 ( t-tests ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 09345 . 01010 . 7554/eLife . 09345 . 011Figure 3—figure supplement 1 . Embryonic tendons deficient in Mmp2 or Mmp13 do not have overt tendon phenotypes . Electron microscopy images of embryonic tendons from ( A ) WT , ( B ) Mmp2 KO , and ( C ) Mmp13 KO mice .", "Arrowheads indicate the locations of fibripositors .", "Scale bars 1 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 09345 . 01110 . 7554/eLife . 09345 . 012Figure 3—figure supplement 2 . Deficiency in MMP14 activity results in fewer collagen fibrils tips at P0 . ( A ) 3D reconstruction from SBF-SEM analysis of P0 WT and Mmp14 KO tendons .", "Scale bars 2 µm .", "( B ) Estimated mean fibril length 645 ± 14 µm WT and 674 ± 24 µm Mmp14 KO shows that the absence of MMP14 does not affect fibril length at birth .", "Bars show SEM .", "†p > 0 . 05 ( t-test ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 09345 . 01210 . 7554/eLife . 09345 . 013Figure 3—figure supplement 3 . Quantitative analysis of Col-r/r embryonic tendons . Measurement of tendon cross-sectional area ( A ) and collagen fibril diameter ( B ) shows no significant difference between WT and Col-r/r mice at E15 . 5 .", "†p > 0 . 05 ( t-tests ) .", "( C ) Quantitation of fibril-containing carriers in WT , Mmp14 KO , and Col-r/r embryonic tenocytes .", "*p < 0 . 05 ( one-way ANOVA ) .", "Bars show SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 09345 . 013 MMP14 has non-proteolytic activities ( Mori et al . , 2013 ) .", "Therefore , we treated cells cultured in 3D tendon-like constructs ( Kapacee et al . , 2008 ) with the broad-spectrum MMP inhibitor GM6001 and performed TEM .", "Previous studies had confirmed that GM6001 was an effective inhibitor of MMPs in the tendon ( Kalson et al . , 2013 ) .", "The addition of GM6001 to tendon-like constructs recapitulated the fibripositor phenotype of the Mmp14-deficient mouse ( Figure 3E , F ) .", "This indicated that the catalytic activity of MMP14 is required for normal collagen fibril transport at fibripositors .", "Estimation of collagen mean fibril lengths in the constructs showed that inhibition of MMPs resulted in increased mean fibril length ( Figure 3G ) , as was observed in embryonic Mmp14 KO tendon .", "SBF-SEM analysis showed that the mean length of fibrils in WT and Mmp14 KO was the same in P0 tendons ( Figure 3—figure supplement 2 ) .", "This was in contrast to what was seen in embryonic tendon in which the fibrils were longer in KO tendons ( Figure 3 ) .", "Therefore , the more abundant but shorter fibrils in WT tendon grow in length during later embryonic development so that at P0 the fibrils are of equal mean length but there are more fibrils in the WT tendon than in Mmp14 KO tendon .", "The Col-r/r mouse carries a mutation in the MMP ¾-¼ cleavage site in the triple helical domain of the α1 chain of type I collagen ( Liu et al . , 1995 ) that renders both the α1 ( I ) and α2 ( I ) chains resistant to cleavage by MMPs ( Wu et al . , 1990 ) .", "Therefore , we were interested to compare collagen fibril transport in Col-r/r and Mmp14 KO mice .", "In contrast to Mmp14 KO , there were no significant differences in tendon size ( Figure 3—figure supplement 3A ) and collagen fibril diameter ( Figure 3—figure supplement 3B ) between embryonic ( E15 . 5 ) WT and Col-r/r mice .", "Analysis of the frequency of fibricarrier profiles per nucleus ( using methods used previously [Canty et al . , 2006] ) showed that Mmp14 KO and Col-r/r tenocytes have significantly more fibripositor profiles than WT , with Mmp14 KO cells having the most ( Figure 3—figure supplement 3C ) .", "SBF-SEM analysis showed that Col-r/r tenocytes contained conspicuous recessed fibripositors , as seen in the Mmp14 KO samples ( Figure 2C and Video 3 ) .", "Electron dense vacuoles were also present , which appeared to contain fibrillar structures in various stages of decomposition ( Figure 2C , white arrows ) .", "Such compartments were rarely seen in WT or Mmp14 KO samples . 10 . 7554/eLife . 09345 . 014Video 3 . Step-through video generated from SBF-SEM images of E17 . 5 embryonic Col-r/r mouse-tail tendon .", "z-depth is 100 µm . Scale bar 2 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 09345 . 014 We wanted to study the requirement of MMP14 on the stage 1 to stage 2 transition in the tendon but were unable to do so because the global Mmp14 KO mice were distressed shortly after birth .", "Therefore , we generated a tendon-specific Mmp14 KO mouse by crossing Scleraxis-Cre ( Scx-Cre ) mice with Mmp14 lox/lox mice ( Figure 4—figure supplement 1 ) .", "We examined the tendons at P0 by TEM and confirmed that the Scx-Cre::Mmp14 lox/lox tendons also contained multiple fibripositors with multiple fibrils , a similar phenotype observed for global Mmp14 KO mice ( Figure 4A , B ) .", "At birth the mice appear normal , however , the Scx-Cre::Mmp14 lox/lox mice exhibited a clear limb phenotype ∼1 week after birth , with dorsiflexion of the fore and hind paws ( Figure 4C , D , arrowhead ) and a dome-shaped skull ( Figure 4D , arrow ) .", "There was no apparent impairment in movement of tail or back muscles as observed in Scx-null mice ( Murchison et al . , 2007 ) .", "Difference in size was apparent from 3 weeks postnatal concurrent with hip dysplasia and reduced bone density ( Figure 4—figure supplement 2A ) , overgrowth of soft tissues in the paws ( Figure 4E ) and severe dorsiflexion of paws which was particularly obvious in the hind paws ( Figure 4F ) .", "Smaller tendon ( tail and Achilles ) size and weaker bones were also observed in 7-week-old Scx-Cre::Mmp14 lox/lox mice .", "The mice became distressed from 7 weeks therefore analyses were performed no later than 7 weeks postnatal .", "Morphometric analysis of x-rays confirmed that Scx-Cre::Mmp14 lox/lox mice had significantly shorter cranium length and stunted skeletal growth ( Figure 4—figure supplement 2B ) . 10 . 7554/eLife . 09345 . 015Figure 4 . Scx-Cre::Mmp14 lox/lox mice have limb and skeletal deformities . Tail tendons from littermates at P0 from ( A ) WT and ( B ) Scx-Cre::Mmp14 lox/lox mice show Mmp14-null tendons have multiple fibripositors-containing multiple fibrils ( red box ) than fibripositors in WT tendons ( blue box ) .", "Black arrowhead , recessed fibripositor ( electron lucent ) -containing collagen fibrils .", "Scale bars 500 nm .", "( C ) Control pups at P8 showed normal limb development but ( D ) Scx-Cre::Mmp14 lox/lox littermates show dorsiflexion of their limbs ( arrowhead ) and dome-shaped skull ( arrow ) .", "Adult ( 7 week old ) Scx-Cre::Mmp14 lox/lox mice have ( E ) enlarged paws ( open arrow ) and ( F ) extreme dorsiflexion of hind limbs ( arrowhead ) compared to control littermates .", "Scale bars 1 cm . DOI: http://dx . doi . org/10 . 7554/eLife . 09345 . 01510 . 7554/eLife . 09345 . 016Figure 4—figure supplement 1 . Genotyping the Scx-Cre::Mmp14 lox/lox colony . A typical genotyping result to identify Scx-Cre::Mmp14 lox/lox mice .", "Wt ctrl , control wild-type DNA .", "Het , heterozygous ( lox/wt , Cre+ ) .", "KO , homozygous knockout ( lox/lox , Cre+ ) .", "WT , wild-type ( no Cre ) .", "Cre+ WT , Cre-expressing WT ( wt/wt , Cre+ ) used as controls . DOI: http://dx . doi . org/10 . 7554/eLife . 09345 . 01610 . 7554/eLife . 09345 . 017Figure 4—figure supplement 2 . Adult Scx-Cre::Mmp14 lox/lox mice have skeletal deformities .", "( A ) Representative x-ray shows adult ( 7 weeks old ) Scx-Cre::Mmp14 lox/lox mice are smaller than control littermates and have reduced bone density .", "White arrow indicates hip dysplasia .", "Scale bars 1 cm .", "( B ) 7-week-old Scx-Cre::Mmp14 lox/lox mice have shorter cranium length , smaller pelvis , and shorter long bones .", "Bars show SEM .", "*p = 0 . 0105; ***p < 0 . 001 ( t-tests; 12 control , 8 Scx-Cre::Mmp14 lox/lox ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 09345 . 017 EM analysis of adult ( 7 weeks old ) WT tendons showed the typical bimodal distribution of collagen fibril diameters ( Figure 5A ) .", "Cells lacked fibripositors and were stellate in cross section ( Figure 5—figure supplement 1A ) .", "In contrast , the tendons of Scx-Cre::Mmp14 lox/lox mice contained a unimodal distribution of small diameter fibrils ( Figure 5B ) , and the cells were engorged with fibrils in membrane-bound compartments ( Figure 5—figure supplement 1B ) .", "The inclusion of many fibrils into compartments made it difficult to delineate the cell–matrix interface .", "In comparison , cells in Col-r/r tendons were similar in appearance to those in WT tendons ( Figure 5—figure supplement 1C ) and the ECM contained fibrils with a broad bimodal distribution of diameters ( Figure 5C ) . 10 . 7554/eLife . 09345 . 018Figure 5 . Deficiency in MMP14 activity inhibits bimodal fibril diameter distribution in tendons from adult mice . Tail tendons from 7 week-old ( A ) WT , ( B ) Scx-Cre::Mmp14 lox/lox , and ( C ) Col-r/r mice .", "Larger diameter fibrils can be observed in the ECM of WT and Col-r/r postnatal tendons but only narrow diameter fibrils are observed in Mmp14-deficient postnatal tendons .", "Scale bars 500 nm . DOI: http://dx . doi . org/10 . 7554/eLife . 09345 . 01810 . 7554/eLife . 09345 . 019Figure 5—figure supplement 1 . Cleavage of the ¾-¼ collagen-I site is not required for release of fibrils in tendons from adult mice . Electron microscopy images of tendons from 7-week-old ( A ) WT , ( B ) Scx-Cre::Mmp14 lox/lox , and ( C ) Col-r/r mice .", "Larger diameter fibrils occur in the ECM of WT and Col-r/r postnatal tendons but only narrow diameter fibrils occur in Mmp14-deficient postnatal tendons .", "Scale bars 2 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 09345 . 01910 . 7554/eLife . 09345 . 020Figure 5—figure supplement 2 . Immuno-electron microscopy of Scx-Cre::Mmp14 lox/lox tendon .", "( A ) WT and ( B ) Scx-Cre::Mmp14 lox/lox tail tendon from P7 ( 7 days ) mice were labeled with anti-MMP14 antibody .", "( C ) Scx-Cre::Mmp14 lox/lox tendon labeled with anti-collagen-I antibody .", "Scale bars 500 nm . DOI: http://dx . doi . org/10 . 7554/eLife . 09345 . 020 ImmunoEM of P7 ( 7 days postnatal ) WT tendon using an anti-MMP14 antibody showed labeling of intracellular compartments without labeling the ECM or plasma membranes in contact with extracellular collagen fibrils ( Figure 5—figure supplement 2A ) .", "As expected , Mmp14-null tendons were negative for MMP14 labeling ( Figure 5—figure supplement 2B ) .", "Labeling using an anti-type I collagen antibody confirmed that the fibrils within the recessed fibripositors of Scx-Cre::Mmp14 lox/lox tendons contained type I collagen ( Figure 5—figure supplement 2C ) .", "To investigate why collagen fibrils were retained in fibripositors in Mmp14-null tendons , we used mass spectrometry LS/MS–MS to perform an unbiased comparison of proteins in Mmp14-deficient and WT Achilles tendons .", "Care was taken to minimize muscle contamination and to remove as much associated loose connective tissue as possible .", "Although not quantitative , the analysis identified specific macromolecules that appear to be more abundant in Scx-Cre::Mmp14 lox/lox and global Mmp14 KO tendons than in WT ( Supplementary file 1 ) .", "Peptides from FN were consistently more abundant in Mmp14-deficient samples .", "We identified a unique peptide from FN that was found in WT tendon in vivo and which contained an additional alanine residue at its N-terminus in Mmp14-deficient tendons ( Figure 6A ) , suggesting that MMP14 is responsible for cleaving FN between Ala ( 1078 ) and Thr ( 1079 ) .", "This was confirmed by LS/MS–MS analysis of recombinant human FN treated with recombinant human MMP14 prior to digestion with trypsin ( data not shown ) .", "Immunofluorescence analysis at E15 . 5 showed accumulation of FN in Scx-Cre::Mmp14 lox/lox tendons compared to WT tendons and the levels of FN appeared to progressively accumulate in KO tendons at P0 and P10 ( Figure 6B ) .", "The LS-MS/MS analyses also identified periostin and integrins including α11β1 ( Supplementary file 1 ) .", "Analysis of periostin in tendons showed similar intensities at E15 . 5 and P0 but was increased in the tendon epithelium at P10 Scx-Cre::Mmp14 lox/lox mice compared to WT mice ( Figure 6—figure supplement 1A ) .", "Subsequent western blot analysis of P7 mice confirmed that levels of FN were higher in Scx-Cre::Mmp14 lox/lox tendons compared to WT littermates ( Figure 6C , D ) .", "We stripped and re-probed the blot for periostin detection and confirmed that it was not accumulated to the extent FN was in Scx-Cre::Mmp14 lox/lox tendons ( Figure 6—figure supplement 1B ) . 10 . 7554/eLife . 09345 . 021Figure 6 . Elevated FN in Mmp14-deficient tendons .", "( A ) Sequence of a unique semi-tryptic peptide of FN identified in neonatal ( P7-10 ) WT tendon and the sequence of the corresponding peptide from Mmp14-deficient tendons without the additional Ala ( 1078 ) -Thr ( 1079 ) cleavage .", "( B ) Immunofluorescence analysis of FN in tendons of WT and Scx-Cre::Mmp14 lox/lox mice at E15 . 5 , P0 , and P10 of development .", "Scale bars 200 µm .", "( C ) Western blot analysis of P7 WT and Scx-Cre::Mmp14 lox/lox tendons show elevated FN in Mmp14-deficient tendons .", "( D ) Ponceau S-stained membrane shows equivalent extractability of WT and Scx-Cre::Mmp14 lox/lox tendons . DOI: http://dx . doi . org/10 . 7554/eLife . 09345 . 02110 . 7554/eLife . 09345 . 022Figure 6—figure supplement 1 . Elevated periostin levels only in postnatal Scx-Cre::Mmp14 lox/lox tendons .", "( A ) Immunofluorescence analysis of periostin in tendons of WT and Scx-Cre::Mmp14 lox/lox mice at E15 . 5 , P0 , and P10 of development show accumulation of periostin in epithelium ( white arrows ) of Scx-Cre::Mmp14 lox/lox tendons .", "Scale bars 200 µm .", "( B ) Western blot analysis of P7 WT and Scx-Cre::Mmp14 lox/lox Achilles tendons show similar levels of periostin in Mmp14-deficient tendons . DOI: http://dx . doi . org/10 . 7554/eLife . 09345 . 02210 . 7554/eLife . 09345 . 023Figure 6—figure supplement 2 . Exogenous FN induces recessed fibripositors in tendon-like constructs . Transmission electron microscopy of tendon-like constructs in the absence ( A ) and presence ( B ) of 200-µg/ml human plasma fibronectin .", "Arrowheads show recessed fibripositors , which were abundant in the treated constructs .", "Scale bars 500 nm . DOI: http://dx . doi . org/10 . 7554/eLife . 09345 . 023 Next , we wanted to determine if elevated levels of FN might account for the fibripositor phenotype and retention of collagen fibrils at the cell surface .", "Thus , we formed tendon-like constructs in the presence of 200 µg/ml exogenous human plasma FN .", "The constructs formed within ∼10 days as previously described ( Kapacee et al . , 2008 ) .", "TEM of the constructs showed pronounced fibripositors in cells incubated in exogenous FN ( Figure 6—figure supplement 2 ) ." ], [ "We show here that MMP14 is essential for the stage 1–stage 2 transition of tendon development ( which occurs around birth in the mouse ) by catalyzing the release of collagen fibrils from fibripositors .", "In WT mouse tendons , fibripositors disappear and collagen fibrils are released to the ECM soon after birth ( marking the end of stage 1 ) , and the fibrils grow in diameter and length ( marking the start of stage 2 ) ( Kalson et al . , 2015 ) .", "In the absence of MMP14 , the fibrils are retained within fibripositors and the number of collagen fibrils formed during stage 1 is reduced .", "As a result , tendons in Mmp14-deficient mice are thinner compared to WT .", "Although MMP14 is capable of cleaving type I collagen at the ¾-¼ helical site , we show that cleavage at this site is not required for tendon development .", "Interestingly , FN accumulates in Mmp14-deficient tendons .", "Thus , we propose that the ability of MMP14 to cleave macromolecules other than type I collagen is essential for releasing collagen fibrils from fibripositors .", "Collagen fibrils are assembled on the surface of embryonic tenocytes and pulled into fibripositors by a mechanism powered by non-muscle myosin II ( Kalson et al . , 2013 ) .", "The absence of fibripositors in stage 2 shows that fibrils are ‘released’ from the plasma membrane for delivery to the ECM ( Kalson et al . , 2015 ) .", "We propose that collagen fibrillogenesis in embryonic tendon occurs via an ‘APR’ mechanism of collagen ‘attachment’ to the cell surface , non-muscle myosin II-powered ‘pulling’ on fibrils into fibripositors , and MMP14-mediated ‘release’ of the fibrils to the ECM .", "Mmp14-null mice had thinner tendons than WT mice .", "SBF-SEM analyses showed similar cell numbers in embryonic WT and null mouse tendons; therefore , we excluded the possibility that delayed development was the cause .", "However , Mmp14-null tendons had fewer collagen fibrils .", "As shown previously , the lateral size of the tendon is established during embryonic development when embryonic tenocytes assemble a finite number of collagen fibrils at fibripositors ( Kalson et al . , 2015 ) .", "The fibrils are released after birth and subsequently grow in length and diameter in a process of matrix expansion .", "Therefore , the fewer fibrils in Mmp14-deficient tendons appear to be a direct result of the inability of the cells to release collagen fibrils to the ECM before a new cycle of fibril assembly can begin .", "In the presence of continued collagen synthesis ( albeit at a reduced rate [Figure 1—figure supplement 1D] ) , the existing fibrils continue to grow in length and diameter at the expense of nucleation of new fibrils .", "At P0 and soon after birth , the fibrils in WT tendon grow in length to equal the length of fibrils in Mmp14 KO tendons .", "Tendon size was normal in Col-r/r embryos; therefore , the absence of type I collagen cleavage was not the cause of reduced fibril number in Mmp14-deficient tendons .", "However , tenocytes in embryonic Col-r/r tendons contained electron-dense vacuoles , which were morphologically similar to those previously observed ( Beertsen et al . , 1978; Everts et al . , 1996 ) .", "Therefore , although type I collagen is cleaved at the ¾-¼ site during embryonic development , cleavage is not essential for tendon development .", "While the Col-r/r mutation renders the triple helix resistant to cleavage by MMPs , rodent MMP13 recognizes an additional cleavage site C-terminal to the N-telopeptide crosslink ( Krane et al . , 1996 ) .", "Liu et al . ( 1995 ) reported that the MMP13 cleavage site in type I collagen permits normal remodeling during development and early postnatal life , but that cleavage at the ¾-¼ site is needed for subsequent remodeling and accounts for the observed progressive marked skin fibrosis in the Col-r/r .", "Our data agree with these conclusions and show that MMP13 ( and MMP2 ) is not essential for tendon development .", "The fact that degradative vacuoles are rare in embryonic Mmp14 KO tenocytes and that fibril numbers were reduced and fibril lengths are greater in Mmp14 KO cells , suggests that the vacuoles might be part of a mechanism to regulate fibril number and/or length .", "Two proteins , FN and periostin , stood out in the LC-MS/MS comparison of WT and Mmp14-null tendons as proteins that could help to explain the Mmp14-null tendon phenotype ( Supplementary file 1 shows number of peptides from periostin and FN were over-represented in Mmp14 KO tendon ) .", "Periostin is a member of the matricellular family of secreted proteins that modulate cell–ECM interactions ( Sage and Bornstein , 1991; Murphy-Ullrich and Sage , 2014 ) .", "Periostin is highly expressed by epithelial cells , is bound by αvβ3 and αvβ5 integrins , is upregulated in epithelial tumors to support adhesion and migration ( Gillan et al . , 2002; Yuyama et al . , 2002; Liu and Du , 2015 ) , and is a prognostic marker for TH2-driven asthma ( Parulekar et al . , 2014 ) and lung fibrosis ( Amara et al . , 2015 ) .", "Additional studies have shown that periostin supports tendon formation in an ectopic mouse model of the development of tenogenic tissue ( Noack et al . , 2014 ) .", "Evidence also suggests that periostin interacts with type I collagen to regulate collagen fibrillogenesis ( Noack et al . , 2014 ) .", "It has also been reported that periostin deficiency might cause collagen fibril disorganization and affect the distribution of FN ( Tabata et al . , 2014 ) .", "We showed that periostin was increased in the tendon epithelium ( Taylor et al . , 2011 ) that surrounds the body of the tendon , in P10 Scx-Cre::Mmp14 lox/lox mice compared to WT mice ( Figure 6—figure supplement 1A ) .", "Periostin can be cleaved by MMP14 in vitro ( Stegemann et al . , 2013 ) and therefore its accumulation in the epithelium could be a direct result of substrate accumulation .", "It is also possible that the elevated levels seen in the epithelium are an indirect result of MMP14 deficiency in the fibrous core of the tendon .", "We observed elevated levels of FN in Mmp14 KO tendon , as shown by LC-MS/MS and by immunofluorescence .", "MMP14 has been shown to cleave several macromolecules in vitro including FN ( d'Ortho et al . , 1997; Ohuchi et al . , 1997; Tam et al . , 2004; Butler and Overall , 2007 ) ; therefore , the accumulation of FN in Mmp14-deficient tendon might be a direct result of the absence of MMP14 .", "We observed a potential cleavage site between Ala ( 1078 ) and Thr ( 1079 ) in FN that occurred in WT but not in Scx-Cre::Mmp14 lox/lox tendon .", "FN is a core component of extracellular matrices ( Mao and Schwarzbauer , 2005 ) and has an important role in development ( George et al . , 1993 ) and wound healing ( Sakai et al . , 2001 ) .", "Mice lacking FN are embryonic lethal with defects in mesoderm formation ( George et al . , 1993 ) .", "Furthermore , FN co-distributes with type I and III collagen ( Fleischmajer and Timpl , 1984 ) .", "To understand if the presence of elevated levels of FN could explain the fibripositor phenotype , we incubated tendon-like constructs with exogenous FN .", "This resulted in a profound increase in appearance of fibripositors .", "Taking the EM , LC-MS/MS , and tendon-construct data together , we propose that FN forms a ‘molecular bridge’ between the cell and the collagen fibril .", "Thus , cleavage of the bridge and removal of fibripositors triggers the onset of stage 2 of tendon development and subsequent expansion of the matrix .", "An unexpected observation was the effect on skeletal size of deleting MMP14 from tendon .", "The shortened long bones in Scx-Cre::Mmp14 lox/lox mice suggests important consequences of tendon development on skeletal growth .", "LC-MS/MS analyses showed under-representation of decorin , COMP , PCOLCE , and TNXB in Mmp14-null tendons .", "These proteins are directly involved in regulating collagen fibril size and shape; mice lacking decorin have fibrils with irregular outlines ( Danielson et al . , 1997 ) , COMP can act as a catalyst for collagen fibril formation ( Halasz et al . , 2007 ) , PCOLCE enhances the cleavage of procollagen to collagen ( Takahara et al . , 1994 ) and mice lacking TNXB have reduced numbers of collagen fibrils ( Mao et al . , 2002 ) .", "In contrast , several proteins were over-represented in Mmp14-deficient tendons .", "These included filamin A , which has functions in cell–ECM adhesion ( Nakamura et al . , 2011 ) and mechanotransduction ( Jahed et al . , 2014 ) .", "As tendons are predominately ECM , changes in ECM composition and cell–ECM interactions are likely to have profound effects on cell signaling ( e . g . , ECM growth factor presentation ) as well as the growth and mechanical properties of tendon leading to changes in musculoskeletal development .", "Finally , the tendons , cartilage , muscle , and bone are peripheral circadian clocks , each with their unique circadian transcriptome ( see [Yeung et al . , 2014a] and reviewed by Dudek and Meng ( 2014 ) ) .", "Therefore , changes in the organization and mechanical properties of the tendon might affect its circadian entrainment and that of adjacent musculoskeletal tissues ." ], [ "The care and use of all mice in this study was carried out in accordance with UK Home Office regulations , UK Animals ( Scientific Procedures ) Act of 1986 under the UK Home Office licence ( PPL 40/3485 ) .", "All animals were sacrificed by a Schedule 1 procedure by trained personnel .", "Mmp14 KO mice were as described previously ( Zhou et al . , 2000 ) .", "To generate mice in which Mmp14 is ablated in tendon-lineage cells , we crossed mice-expressing Cre recombinase under the control of Scleraxis ( Scx-Cre; C57BL/6 ) ( Blitz et al . , 2013 ) with mice carrying the floxed exons ( exons 2 to 4 ) of the Mmp14 gene ( Mmp14 lox/lox; C57BL/6 ) ( Zigrino et al . , 2012 ) .", "Mmp13 KO embryos were a generous gift from Zena Werb ( Stickens et al . , 2004 ) .", "Mmp2 heterozygous mice were imported from RIKEN BioResource Center ( GelAKO/RBRC00398; C57 ) ( Itoh et al . , 1997 ) and bred to homozygosity .", "Col-r/r mice were imported from Jackson Laboratory ( B6;129S4-Col1a1tm1Jae/J ) ( Liu et al . , 1995 ) .", "X-ray analyses were performed as described previously ( Yeung et al . , 2014b ) .", "The methods used were as described previously ( Kalson et al . , 2010 ) .", "Tendon ( from tail ) diameters were measured from digital photographs .", "The diameter , d , was then used to calculate transverse area according to the formula πd2/4 .", "This assumed a circular transverse shape as used in mechanical testing of tissue engineered ligament ( Hairfield-Stein et al . , 2007 ) .", "An average of three diameter measurements was recorded for each tendon .", "The original contour length of tendons was measured from a digital photograph of the mounted construct .", "A tare load of 10 mN was applied at the start of the tensile test to fully straighten the tendon .", "The length at failure was determined from the Instron test ( giving change in length LΔ ) .", "The tendons were tested to failure with a strain rate of 5 mm per minute ( equivalent to approximately 1% strain per second ) .", "A minimum of 3 tail tendons was examined for each experiment .", "The tendons were prepared for TEM and SBF-SEM as described ( Starborg et al . , 2013 ) , with care being taken to maintain the length and tension during fixation .", "Sections ( 70-nm thick ) were examined for TEM using an FEI Tecnai 12 instrument fitted with a 2k × 2k-cooled CCD camera ( F214A , Tietz Video and Image Processing Systems , Gauting , Germany ) .", "Serial section electron tomography was completed as described using semi-thick ( 300 nm ) serial sections were collected on formvar-coated copper slot grids .", "Orthogonal tilt series were then acquired on a FEI Tecnai Polara TEM operated at 300 kV ( Kalson et al . , 2013 ) .", "Tomograms were generated and contours modeled in IMOD ( Kremer et al . , 1996 ) .", "The methods used for SBF-SEM were as described ( Starborg et al . , 2013 ) using a Gatan 3View microtome within an FEI Quanta 250 scanning microscope .", "Cell number measurements were made on 3 separate SBF-SEM samples for WT and Mmp14 KO tail tendons at P0 .", "The volume of the tendon tissue in each SBF-SEM 3D reconstruction was calculated and all the cells in the volume were reconstructed using IMOD .", "Each cell nucleus contained within the reconstruction was identified and counted .", "Cells per 1000 μm3 of tissue were calculated to allow comparison between samples .", "ImmunoEM was performed as previously described using high-pressure freezing and freeze substitution into LR White resin ( Canty et al . , 2004 ) .", "A rabbit anti-Collagen-I antibody ( T40777R; Meridian Life Science , Inc . ) and a mouse anti-MMP14 antibody ( MAB3328; Merk Millipore ) were used .", "Cleanly dissected neonatal ( P7-10 ) mouse Achilles tendons were snap frozen in liquid nitrogen and disrupted in 0 . 1 M Tris , pH 7 . 5 using a B Braun Mikro-dismembrator S ( 2 × 90 s , 2000 rpm ) .", "Tissue samples were digested with trypsin ( 12 . 5 ng/µl; Sigma ) overnight at 37°C in 25 mM ammonium bicarbonate ( pH 7 . 5 ) .", "Human rhFurin ( 2 ng/ml ) in 100 µl activation buffer ( 50 mM Tris-HCl , 1 mM CaCl2 , 0 . 5% Brij-35 , pH 9 ) was added to 4 μl human rhProMMP14 ( 0 . 37 μg/μl ) and incubated for 1 . 5 hr at 37°C .", "Recombinant human FN ( 1 µg/ml ) was incubated with activated rhMMP14 ( 1 ng/ml ) at 37°C for 1 hr .", "For gel-top analysis , MMP14-treated FN was briefly separated by electrophoresis under reducing conditions .", "A gel top band was excised from the stained gel and processed using in-gel tryptic digestion .", "For in-gel tryptic digestion , proteins were excised from SDS-PAGE gels and dehydrated using acetonitrile followed by vacuum centrifugation .", "Dried gel pieces were reduced with 10 mM dithiothreitol and alkylated with 55 mM iodoacetamide .", "Gel pieces were then washed alternately with 25 mM ammonium bicarbonate followed by acetonitrile and dried by vacuum centrifugation .", "Samples were digested with trypsin , as above .", "Digested samples were analyzed by LC-MS/MS using an UltiMate 3000 Rapid Separation LC ( RSLC , Dionex Corporation , Sunnyvale , CA ) coupled to an Orbitrap Elite ( Thermo Fisher Scientific , Waltham , MA ) mass spectrometer .", "Peptide mixtures were separated using a gradient from 92% A ( 0 . 1% FA in water ) and 8% B ( 0 . 1% FA in acetonitrile ) to 33% B , in 44 min at 300 nl/min , using a 250 mm × 75 μm i . d . 1 . 7 μM BEH C18 , analytical column ( Waters ) .", "Peptides were selected for fragmentation automatically by data-dependent analysis .", "Data produced were searched using Mascot ( Matrix Science UK ) , against uniprot with taxonomy of Mus musculus selected .", "Cysteine carbamidomethylation was selected as a fixed modification , and lysine and proline oxidation included as variable modifications , for all enzymes .", "For trypsin , methionine oxidation was additionally included as a variable modification and the enzyme selected as ‘semi-trypsin’ .", "Data were validated using Scaffold ( Proteome Software , Portland , OR ) .", "Mouse Achilles tendons ( at P7 ) were dissected clean of contaminating the muscle , snap frozen in liquid nitrogen and disrupted using a B Braun Mikro-dismembrator S ( 2 × 90 s , 2000 rpm ) .", "Proteins were extracted directly into RIPA buffer ( 50 mM Tris , pH 7 . 6 , 150 mM NaCl , 0 . 1% SDS , 1 mM EDTA and 1% NP-40 ) containing EDTA-free protease inhibitor cocktail ( Roche ) and quantified using a BCA assay .", "Samples ( 100 µg ) were reduced and analyzed by western blotting and densitometry .", "Western blots were stripped with 2% SDS , 62 . 5 mM Tris HCl pH 6 . 8 and 100 mM 2-mercaptoethanol for 50 min at 50°C .", "Fibronectin ( FN ) was detected using a rabbit polyclonal antibody ( ab2413; Abcam ) , and periostin was detected using a goat polyclonal antibody ( AF2955; R&D Systems ) .", "From WT and Scx-Cre::Mmp14 lox/lox mice , whole hind limbs from E15 . 5 embryos , lower hind limbs without the skin from P0 pups and dissected Achilles tendons from P10 pups were cryo-preserved in OCT-embedding matrix ( Thermo Scientific ) .", "Longitudinal sections of 8-µm thickness were fixed with 4% paraformaldehyde in PBS for 15 min , permeabilized with 0 . 2% Triton X-100 in PBS for 10 min and then blocked with 2% BSA in PBS for 1 hr .", "FN was detected using a rabbit polyclonal antibody ( ab23750; Abcam; diluted 1:500 ) , and periostin was detected using a goat polyclonal antibody ( ab14041; Abcam; diluted 1:500 ) .", "Cy3-conjugated secondary antibodies ( Invitrogen ) were used and sections were mounted using Vector Shield containing DAPI ( Vector Laboratories ) .", "Fluorescent images were taken using a digital camera attached to an Olympus BX51 and captured using MetaVue imaging software ( Molecular Devices ) .", "P7 tail tendons were labeled with 14C-proline for 1 hr and separate extracellular and intracellular extracts prepared as described ( Canty et al . , 2004 ) .", "The intracellular extract was analyzed by electrophoresis using 4–12% pre-cast Bis-Tris gels and MES running buffer .", "The gels were divided and the top half of the gel ( >70 kDa ) fixed , dried , and analyzed by autoradiography to detect 14C-collagen .", "The bottom half of the gel ( <70 kDa ) was analyzed by Western blotting with an antibody to β-actin; signal was detected using a CCD camera system .", "The gels were analyzed by densitometry with the relative band intensities determined by comparison to serial dilutions of an independent sample ." ] ]

[ "Type I collagen-containing fibrils are major structural components of the extracellular matrix of vertebrate tissues , especially tendon , but how they are formed is not fully understood .", "MMP14 is a potent pericellular collagenase that can cleave type I collagen in vitro .", "In this study , we show that tendon development is arrested in Scleraxis-Cre::Mmp14 lox/lox mice that are unable to release collagen fibrils from plasma membrane fibripositors .", "In contrast to its role in collagen turnover in adult tissue , MMP14 promotes embryonic tissue formation by releasing collagen fibrils from the cell surface .", "Notably , the tendons grow to normal size and collagen fibril release from fibripositors occurs in Col-r/r mice that have a mutated collagen-I that is uncleavable by MMPs .", "Furthermore , fibronectin ( not collagen-I ) accumulates in the tendons of Mmp14-null mice .", "We propose a model for cell-regulated collagen fibril assembly during tendon development in which MMP14 cleaves a molecular bridge tethering collagen fibrils to the plasma membrane of fibripositors ." ]

[ "A scaffold of proteins called the extracellular matrix surrounds each of the cells that make up our organs and tissues .", "This matrix , which contains fibres made of proteins called collagens , provides the physical support needed to hold organs and tissues together .", "This support is especially important in the tendons—a tough tissue that connects the muscle to bone—and other ‘connective’ tissues .", "An enzyme called MMP14 is able to cut through chains of collagen proteins .", "It belongs to a family of proteins that are involved in breaking down the extracellular matrix to enable cells to divide and for other important processes in cells .", "Some cancer cells exploit MMP14 to enable them to leave their tissue of origin and spread around the body .", "Therefore , when researchers bred mutant mice that lacked MMP14 , they expected to see excessive growth of collagen fibres in the connective tissues of the mice .", "However , these mice actually have extremely thin , fragile connective tissue and die soon after birth .", "Earlier in 2015 , a group of researchers demonstrated that the first stage of tendon development in mice involves the formation of collagen fibres , which are attached to structures that project from tendon cells called fibripositors .", "Then , soon after the mice are born , the fibripositors disappear and the collagen fibres are released into the extracellular matrix where they grow longer and become thicker .", "Now , Taylor , Yeung , Kalson et al . —including some of the researchers from the earlier work—have used electron microscopy to investigate how a lack of MMP14 leads to fragile tendons in young mice .", "The experiments show that MMP14 plays a crucial role in the first stage of tendon development by detaching the collagen fibres from the fibripositors .", "MMP14 also promotes the formation of new collagen fibres; the tendons of mutant mice that lack MMP14 have fewer collagen fibres than normal mice .", "Further experiments revealed that the release of collagen fibres from fibripositors does not require MMP14 to cleave the chains of collagen proteins themselves .", "Instead , it appears that MMP14 cleaves another protein that is associated with the fibres , called fibronectin .", "Taylor , Yeung , Kalson et al . 's findings show that MMP14 plays an important role in the development of tendons by releasing collagen fibres from fibripositors and promoting the formation of new fibres .", "The next challenge is to find out how MMP14 regulates the number of collagen fibres in mature tendons and other tissues , and how defects in this enzyme can lead to cancer and other diseases ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods", "Ethics statement" ]

[ "immunology and inflammation", "genetics and genomics" ]

[ [ "Lineage specification factors promote cellular differentiation by binding DNA regulatory elements to stably alter the local chromatin state and affect gene transcriptional outputs ( Visel et al . , 2009; Thurman et al . , 2012; Nord et al . , 2013; Ostuni et al . , 2013 ) .", "Since the majority of protein-coding genome sequence is under constraint , due to the cost of deleterious mutational perturbations in protein function , changes in non-coding regulatory portions of the genome are thought to serve as a major source of evolutionary innovation and substrates for selection ( King and Wilson , 1975; Schmidt et al . , 2010; Goncalves et al . , 2012; Ward and Kellis , 2012 ) .", "Histone modifications and chromatin accessibility , which serve as proxies for epigenetic transcriptional control , can exhibit conservation shared with the underlying genetic elements in a given cell lineage across species and within the genetically diverse human population ( Kasowski et al . , 2013a; Kilpinen et al . , 2013a; Long et al . , 2013; McVicker et al . , 2013a; Stergachis et al . , 2014; Vierstra et al . , 2014 ) .", "However , it remains unknown if changes in the chromatin state of regulatory elements associated with cell lineage specification are conserved in their lineage-specificity ( Odom et al . , 2007 ) .", "That is , it is unknown to what extent regulatory elements are cell lineage-specific across multiple organisms and individuals .", "Strong conservation would imply that the differentiation process of a specialized cell population is highly constrained at a genomic level , whereas extensive variation would suggest that lineage specification is dependent on only a few immutable genetic regulatory elements .", "We examined conservation of chromatin states and gene expression in human and mouse regulatory T ( Treg ) cells , a key cell population required for maintenance of tolerance to ‘self’ and restrained inflammatory responses during infection .", "Treg cell differentiation and function is controlled by a late-acting lineage specification factor Foxp3 , which is induced upon signaling through T cell ( TCR ) and cytokine receptors ( Hill et al . , 2007; Ohkura et al . , 2012; Samstein et al . , 2012 ) .", "Treg cells generated in the thymus exhibit a ‘naive’ phenotype and display low , if any suppressor activity in contrast to activated Treg cells , which acquire highly potent suppressor function as the result of TCR signaling-dependent activation and division ( Battaglia and Roncarolo , 2009; Levine et al . , 2014; Vahl et al . , 2014 ) .", "Genetic Foxp3 deficiency or elimination of Treg cells in mice results in lymphoproliferation and myeloproliferation leading to widespread fatal autoimmune lesions largely driven by activation of ‘self’- , commensal microbiota- , and food-derived antigen-reactive CD4 T cells ( Brunkow et al . , 2001; Fontenot et al . , 2003; Khattri et al . , 2003; Kim et al . , 2007 ) .", "Human IPEX patients with loss-of-function Foxp3 mutations and congenital Treg cell deficiency present with neonatal diabetes , thyroiditis , autoimmune anemia and neutropenia , autoimmune hepatitis , exudative dermatitis , enteropathies , and hyper-IgE syndrome ( Bennett et al . , 2001; Torgerson and Ochs , 2007; Verbsky and Chatila , 2013 ) .", "To characterize evolutionary conservation and human-to-human genetic variation that underlies the lineage specification of Treg cells , we investigated the active regulatory DNA elements and gene expression in murine and human resting and activated Treg cells and compared them to their counterpart resting naive CD4+ T cells ( Tn ) and activated and effector CD4+ T cells ( Teff ) .", "The analysis of orthologous lineage-specific epigenetic features revealed that while the vast majority of regulatory loci were genetically conserved , the lineage-specific epigenome was markedly different in mouse and human , supporting a model whereby few conserved elements contribute to the specification of the Treg cell lineage .", "A subset of the conserved lineage-specific elements contained nucleotide polymorphisms associated with altered epigenetic activity in human Treg cells isolated from a small cohort of healthy donors .", "Finally , we analyzed the polymorphic conserved elements using high-resolution epigenetic and genome-wide genetic disease association data and were able to localize disease-risk linked polymorphisms to regulatory loci that are exclusively epigenetically active in Treg cells .", "Our results support a role for Treg dysfunction in common polygenic diseases ." ], [ "To characterize the epigenetic state of human Treg and CD4+ T-cell populations , we isolated CD4+ T cells from the peripheral blood of individual anonymous human donors using negative selection ( see ‘Materials and methods’ ) .", "Previously characterized subsets of Treg cells and naive and effector CD4+ T cells were purified using FACS sorting based on expression of CD25 and CD45RO ( >97–99% purity ) ( Figure 1A–C , Figure 1—figure supplement 1A , B ) .", "Mouse resting Treg cells and CD4+ T cells were isolated from phosphate-buffered saline ( PBS ) -treated Foxp3DTR-GFP mice using FACS sorting based on the expression of GFP-DTR ( diphtheria toxin ( DT ) receptor ) fusion protein or lack thereof , respectively .", "In these mice , DNA sequence encoding IRES-driven GFP-DTR fusion protein was inserted in frame into the 3′ UTR of the endogenous Foxp3 gene .", "These knock-in mice enabled isolation of Treg cells based on GFP expression and their depletion upon DT injection ( Kim et al . , 2007 ) .", "The corresponding populations of activated CD4+ Teff and Treg cells were isolated from Foxp3DTR-GFP mice subjected to transient ablation of Treg cells followed by their recovery and activation in response to inflammation on day 11 after administration of a single dose of DT as described ( see ‘Materials and methods’ ) .", "We desired to compare aTreg vs Teff in addition to Treg vs Tn cell populations , since they have comparable antigen experience; however , human and mouse T-cell subsets isolated ex vivo may have experienced different in vivo activation conditions .", "Therefore , we compared activated Treg lineage-specific transcriptional and epigenetic features to those of conventional T effector populations for each organism to account for the species-specific activation associated changes .", "In total , we analyzed 16 human cell samples ( 7 donors: 7 aTreg , 4 rTreg , 2 Teff , 2 Tmem , and 1 Tn samples ) and 10 murine samples ( 2 aTreg , 4 rTreg , 2 Teff , and 4 Tn biological replicates independently isolated from different mice ) . 10 . 7554/eLife . 07571 . 003Figure 1 . Analysis of genetic and epigenetic conservation in mouse and human Treg and CD4+ T cell subsets .", "( A ) Schematic representation of profiled CD4+ T-cell subsets .", "Abbreviations: naive T cell ( Tn ) ; effector T cell ( Teff ) ; resting regulatory T cell ( rTreg ) ; activated regulatory T cells ( aTreg ) .", "( B ) The indicated human CD4+ T-cell subpopulations were FACS sorted based on CD3 , CD4 , CD45RO , and CD25 expression from preparations of peripheral blood mononuclear cells ( PBMCs ) from healthy human donors .", "Highly purified Treg cell subpopulations were obtained using a FACS Aria II fluorescent cell sorter ( Figure 1—figure supplement 1A ) .", "Epigenetic profiling was performed using the following 16 cell samples isolated from 7 healthy donors: including 7 aTreg , 4 rTreg , 2 Teff , 2 Tmem , and 1 Tn independently isolated cell populations .", "See also Figure 1—figure supplement 1A , B .", "( C ) Resting and activated murine CD4+ T-cell subpopulations were FACS sorted from Foxp3DTR-GFP mice injected with PBS or diphtheria toxin ( DT ) , respectively .", "In Foxp3DTR-GFP mice , Treg cells express diphtheria toxin receptor ( DTR ) .", "Mice injected with DT underwent punctual Treg cell depletion and consequent transient systemic inflammation , which resulted in activation of rebounding Treg and conventional T cells .", "A total of 10 mouse cell samples isolated using FACS sorting from DT-treated and DT-untreated Foxp3DTR mice were analyzed: 2 aTreg , 4 rTreg , 2 Teff , and 4 Tn biological replicates .", "( D , E )", "Genetic and epigenetic conservation at select loci .", "Multiple regulatory elements near LRRC32 and YY1 are genetically and epigenetically conserved: YY1 has two epigenetic elements that are not conserved in human; LRRC32 has a regulatory element that is genetically , but not epigenetically conserved .", "( D ) Acetylation at the LRRC32 locus shows multiple conserved genetic elements that illustrate concordant and discordant epigenetic states across species ( highlighted regions ) .", "The human LRRC32 locus ( top ) and murine Lrrc32 locus ( bottom ) feature extensive genetically orthologous elements ( lines connecting human and murine genomic coordinates ) containing species-specific insertions/deletions ( white space ) .", "H3K27ac ChIP-seq reads per million ( RPM ) are shown on y-axis for the indicated species and cell lineages .", "Orthologous regions with regulatory elements of interest are shown by blue background highlighting and red connecting lines .", "A genetically conserved element near LRRC32 is epigenetically active in mouse , but not in human ( leftmost highlighted region ) .", "( E ) Two regulatory elements near YY1 are epigenetically active in human but are not genetically conserved in mouse ( leftmost and rightmost highlighted regions ) .", "( F ) Genome-wide fractions of genetically conserved acetylated loci .", "Loci with high read counts are more frequently genetically conserved ( shown ) as are regulatory elements more proximal to gene body ( Figure 1—figure supplement 1H ) .", "( G ) Genome-wide quantification of epigenetic conservation .", "Axes show H3K27ac quantification ( reads per million: RPM ) of murine ( x-axis ) and human ( y-axis ) acetylated loci .", "Qualitatively , the vast majority of regulatory elements are epigenetically conserved in mouse and human Treg cells , with genome-wide quantitative correlation of r = 0 . 48 ( ‘†’ indicates that correlation is computed only for genetically conserved loci; non-conserved loci are shown on axes and by definition cannot be epigenetically conserved ) .", "Correlation across mouse biological replicates was r > 0 . 99 and between human donors r > 0 . 94 , indicating that the observed conservation and lack thereof are reflective of biology and not technical/replicate reproducibility ( Figure 1—figure supplement 1K ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 07571 . 00310 . 7554/eLife . 07571 . 004Figure 1—figure supplement 1 . Quality analysis of epigenetic datasets .", "( A ) High post-sort purity of human activated and resting Treg cell populations .", "( B ) Isolated cell counts for each of nine donors are shown .", "Donors D204 , D304 , and D563 had lower buffy coat sample volumes ( ∼25 ml ) than other donors ( ∼50 ml ) .", "( C , D )", "Examples of genetic conservation of inversion ( C ) and chromosome break ( D ) .", "( E ) Conservation analysis of the Foxp3 locus finds that downstream and proximal upstream elements are conserved , whereas distal upstream genomic elements encoding the GAGE transcripts are not conserved .", "( F ) Phylogenetic analysis of the Foxp3 locus across a broad set of organisms indicates evolutionary constraint of distal genetic elements .", "The proximal genetic elements controlling Foxp3 expression have been previously characterized ( Zheng et al . , 2010; Samstein et al . , 2012 ) .", "( G ) DNase-seq analysis confirms that the highlighted regulatory element is genetically conserved and even weakly accessible , but not histone H3K27 acetylated in human .", "( H ) Genome-wide fractions of genetically conserved DNase-accessible loci .", "Loci with ‘high’ read counts are more frequently genetically conserved .", "( I ) Genetic conservation of H3K27Ac sites is highest at promoter-proximal regulatory elements .", "Genetic conservation ( pie charts ) is shown for sites at given distances ( D ) from the most proximal gene promoter .", "Conservation at intronic loci is also shown .", "( J ) Conservation statistics for DNase hypersensitive sites ( DHSs ) ( similar to Figure 1F ) .", "Genome-wide quantitative correlation of mouse and human DHSs is r = 0 . 56 ( ‘†’ indicates that correlation is computed only for genetically conserved loci; non-conserved loci are shown on axes and by definition cannot be epigenetically conserved ) .", "( K ) ChIP-seq reproducibility in mouse and human .", "Representative donor-to-donor and mouse biological replicates are shown .", "Slightly increased variation in human donors is a combination of biological and technical variation .", "Lower chromatin ( cell ) input into the chromatin immunoprecipitation ( ChIP ) assay likely results in higher technical variation; however , we also identify donor-specific usage of regulatory elements such as ENTPD1 , which we confirmed as a high-probability eQTL ( Figure 4 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 07571 . 004 To identify active regulatory elements of the CD4+ T-cell epigenome , we performed chromatin immunoprecipitation ( ChIP ) of histone H3 acetylated at lysine 27 ( H3K27ac ) followed by high-throughput sequencing ( ChIP-seq ) .", "This histone modification serves as a reliable marker for active regulatory elements ( Creyghton et al . , 2010; Arvey et al . , 2012 ) .", "We observed ∼31 , 000 H3K27 acetylation peaks across the genome in CD4+ T-cell subsets , which we stratified by reads aligned per million ( RPM ) in each cell population .", "In addition to histone acetylation , we incorporated previously generated DNase-seq hypersensitive site ( DHS ) data sets to enable higher resolution positioning of active acetylated regulatory elements ( Arvey et al . , 2012; Samstein et al . , 2012; Thurman et al . , 2012; Epigenome Roadmap Consortium , 2015 ) .", "We found that the overall epigenetic features of chromatin in human- and mouse-activated Treg cells were highly conserved based on qualitative and quantitative analyses .", "Human and murine loci with sufficient read counts of H3K27ac were ‘lifted over’ and merged to form a single set of peaks that exist in either organism ( see ‘Materials and methods’ and Supplementary files 1-6 for details ) .", "This analysis captures micro- and macro-genetic differences , including sequence homology , insertions/deletions , inversions , and chromosome breaks ( Figure 1—figure supplement 1C–F ) .", "We identified loci that were genetically and epigenetically conserved ( e . g . , LRRC32 [encoding GARP]; Figure 1D ) , epigenetically active only in a single organism due to unique genetic elements ( e . g . , YY1; Figure 1E ) , and with loss or gain of histone modifications at genetically conserved sites ( e . g . , LRRC32 upstream gene C11ORF30 , Figure 1D , Figure 1—figure supplement 1G ) .", "Genome-wide comparison between human and mouse identified ∼27 , 000 orthologous regulatory elements ( Figure 1F , G , Figure 1—figure supplement 1H , I ) .", "The level of H3K27ac at conserved genetic elements was highly correlated ( r = 0 . 48 ) , with a plurality of shared elements being weakly active in both organisms ( Figure 1G ) .", "A similar correlation was revealed by analyses of chromatin accessibility at DHSs across organisms ( Figure 1—figure supplement 1J ) .", "We reasoned that the most critical genetic components of the Treg lineage identity would be genetically and epigenetically conserved in mouse and human and additionally be Treg lineage-specific in both species .", "We thus characterized regulatory elements with conserved lineage-specific activity , which we defined as increased or decreased H3K27ac amounts in Treg cells in comparison to non-Treg cells in both mouse and human .", "This was an extension of the above analyses in which we characterized genetic conservation of regulatory elements and the epigenetic activity of these elements in mouse and human Treg cells .", "Previous studies of multiple cell lines and specialized tissues from different organisms have shown that non-coding regulatory elements are more likely to be genetically conserved and active based on their epigenetic features ( Bernstein et al . , 2005; Schmidt et al . , 2010; Woo and Li , 2012 ) .", "However , it remained unclear whether cell lineage-specific regulatory elements , which define identity and function of a given cell type , are conserved at genetic , epigenetic , and lineage-specific functional levels ( Cheng et al . , 2014; Vierstra et al . , 2014; Yue et al . , 2014 ) .", "We identified a handful of regulatory elements with cell lineage-specific acetylation in both human and mouse .", "For instance , the LRRC32 locus , which encodes the GARP protein that acts as a functionally important marker of activated Treg cells in humans ( Wang et al . , 2009 ) , has multiple regulatory elements that are genetically conserved , epigenetically conserved , and only active in the Treg cell lineage ( Figure 2A ) .", "Intriguingly , while the genome-wide epigenetically active landscape was similar in human and mouse , quantification of cell lineage specificity revealed that very few of the elements are Treg cell-specific in both species ( Figure 2B and Figure 2—figure supplement 1A–C ) .", "Specifically , we were able to identify conserved epigenetic modifications at a small set of regulatory elements , which could be assigned to a handful of genes , including FOXP3 , IL2RA ( CD25 ) , CTLA4 , IKZF2 ( HELIOS ) , IL2RB , DUSP4 , BLIMP1 , TNFRSF8 , TNFRSF9 , CCR8 , and LRRC32 ( Figure 2—figure supplement 1D , E ) .", "Interestingly , there was greater statistical enrichment for conserved loci downregulated in a Treg-specific fashion , which included IL7R ( CD127 ) , IL2 , PDE3B , LEF1 , IL21 , PDE7A , TNFSF8 , and THEMIS . 10 . 7554/eLife . 07571 . 005Figure 2 . Conservation of the lineage-specific epigenome identifies the core Treg cell transcriptional control program .", "( A ) The LRRC32 locus contains multiple epigenetically active regulatory elements that are conserved and Treg lineage-specific in mouse and human .", "The layout is same as in Figure 1D , E , with H3K27ac and DNase-seq RPM quantification shown on the y-axis for multiple mouse and human CD4+ T-cell subpopulations .", "The DNase-seq track provides high-resolution localization of protein-bound DNA in acetylated loci .", "( B ) Lineage-specific conservation of epigenetic activity occurs at only a handful of Treg regulatory elements .", "Changes in histone acetylation ( H3K27ac ChIP RPM ) in aTreg compared to Teff cells are shown for mouse ( x-axis ) and human ( y-axis ) cells for genetically conserved loci .", "( C ) Treg cell lineage-specific gene expression is similarly conserved as lineage-specific chromatin features in mouse and man .", "Changes in gene expression in aTreg compared to Teff cells are shown for mouse ( x-axis ) and human ( y-axis ) cells .", "( D ) Conserved gene expression changes are associated with conserved lineage-specificity of histone acetylation .", "Expression changes are shown for genes most proximal to regulatory elements that were lineage-specific in both species .", "See also Figure 2—figure supplement 2 .", "( E ) Species-specific gene expression changes are associated with species-specific changes in histone acetylation .", "Acetylated loci were classified as upregulated or downregulated in a cell type-specific manner in mouse , human , or both organisms .", "These loci were mapped to their most proximal gene expressed in either mouse or human ( lines ) and cumulative fraction is shown ( y-axis ) .", "The differential expression in aTreg vs Teff cells was plotted for these genes in human ( left ) and mouse ( right ) .", "Regulatory elements that are lineage-specific in both species ( dashed lines ) have been associated with genes with the most conserved differential expression patterns .", "Regulatory elements that are lineage-specific in only mouse ( red/gray for up/downregulated loci ) are near genes that are more differentially expressed in mouse cell subsets .", "Similarly , elements that are lineage-specific in only human ( purple/green for up/downregulated loci ) are associated with differential expression in human .", "Statistical significance was assessed by the Kolmogorov–Smirnov test; all relevant distribution shifts are statistically significant; p-values are shown for relevant comparisons in Figure 2—figure supplement 1F . DOI: http://dx . doi . org/10 . 7554/eLife . 07571 . 00510 . 7554/eLife . 07571 . 006Figure 2—figure supplement 1 . The core Treg lineage specification program is robust across multiple donors and is enriched for significant changes in gene expression .", "( A ) Treg cell lineage-specific acetylation at the CTLA4 locus ( highlighted region ) .", "The layout and axes are same as in Figure 2A .", "( B ) The Treg lineage-specific H3K27ac landscape is reproducible in both mouse and human .", "Representative donor-to-donor and mouse biological replicates are shown as Treg vs Teff cell ChIP-seq log2 RPM fold-change on the x- and y-axes; actual transform is log2[ ( RPMtreg + 0 . 5 ) / ( RPMteff + 0 . 5 ) ] , which preferentially decreases fold changes of lower read count peaks and thus reduces noise in the plot .", "Sources of variance are likely to be similar to those in Figure 1—figure supplement 1K .", "An example of a genotype-associated inconsistent lineage-specificity is ENTPD1 , which is further characterized in Figure 4 .", "( C ) Lineage-specific changes in accessibility at DHSs are globally similar to H3K27ac , showing lineage-specific conservation at a subset of loci in mouse and human .", "( D ) Treg lineage-specific downregulation and upregulation of histone acetylation is significantly conserved ( p < 10−7 , 10−3 , respectively ) .", "Venn diagram shows overlap counts of loci in mouse and human with differential lineage acetylation .", "Statistical significance was tested by the one-sided hypergeometric test .", "( E ) Conservation of the Treg cell lineage-specific program is statistically significant ( y-axis ) irrespective of fold-change cutoff ( x-axis ) .", "All H3K27ac peaks were ranked by ChIP-seq RPM fold change in aTreg vs Teff cells ( x-axis; rank of upregulated and downregulated peaks is shown as red and blue , respectively ) .", "Genome-wide , downregulation of histone acetylation is more statistically conserved than upregulation .", "The rank of differentially acetylated loci in each species was tested for statistical significance ( y-axis , left ) of overlap enrichment between species ( y-axis , right ) .", "( F ) Statistical analysis of Figure 2E .", "Kolmogorov–Smirnov test p-values for the indicated comparisons ( shown in black arrows ) are shown on plots , proceeding left-to-right .", "For instance , regulatory elements that are downregulated in both mouse and human Treg cells ( dashed magenta line ) are near genes that are more downregulated in human Treg cells vs Teff cells ( left panel ) than regulatory elements downregulated only in human ( green line; p < 0 . 005 ) .", "Similarly , human-only downregulated elements ( green line ) are associated with much more pronounced gene repression than murine-only downregulated elements ( gray line ) in human cells ( left panel; p < 10−8 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 07571 . 00610 . 7554/eLife . 07571 . 007Figure 2—figure supplement 2 . Genes with lineage-specific elements in each organism at different genetic locations exhibit limited lineage-specific expression changes .", "( A ) Examples of lineage-specific elements in human and mouse that are partially or non-epigenetically and/or genetically conserved in the other organism .", "At the PRDM1 locus , the locus is comparably acetylated in both organisms ( labeled 1 ) , whereas a downstream enhancer shows lineage-specificity only in mouse; this element is epigenetically inactive in human ( 2 ) .", "Another enhancer is acetylated in a lineage-specific manner in human and epigenetically active , but not lineage-specific in mouse ( 3 ) .", "( B ) Overlap of genes with conserved or organism-specific differentially acetylated loci in aTreg vs Teff cells .", "Regulatory elements were assigned to genes within 100 kbp or nearest gene .", "The overlap of genes with human-specific and mouse-specific consistently differentially regulated elements ( n = 13 and n = 5 for downregulated and upregulated elements , respectively ) is not statistically significant ( see ‘Materials and methods’ for nominal and empirical statistical tests ) .", "Note the comparable values for overlap of discordant up/downregulated loci ( e . g . , n = 31 down in human , up in mouse; n = 12 up in human , down in mouse ) .", "( C–F )", "Regulatory elements that are lineage-specific either at the conserved orthologous genetic elements or nearby conserved or non-conserved regions have varying impact on gene expression .", "Heatmaps of differential gene expression in aTreg vs Teff cells; non-expressed genes are excluded .", "Panel F also shows maximum lineage-specific differential histone acetylation , since in that panel every gene can be assigned a maximally lineage-specific element .", "( C ) Loci that are lineage-specific elements in both human and mouse have concordant differential gene expression .", "Statistical analysis of this set is shown in Figure 2E , Figure 2—figure supplement 1F .", "Foxp3 differential expression is not detected in mouse due to knock in of IRES-DTR-GFP allele .", "( D ) Genes with regulatory elements that are epigenetically active in both organisms , but lineage-specific in only one .", "( E ) Genes with regulatory elements that are lineage-specific in only mouse or human and not genetically conserved in the other organism .", "( F ) Genes with at least one lineage-specific regulatory element in mouse or human .", "Genes with differential expression ( fold change >2 ) are shown above ( n = 22 and n = 26 ) and remaining genes are shown below .", "The additional two columns show human and mouse differential histone acetylation . DOI: http://dx . doi . org/10 . 7554/eLife . 07571 . 007 To assess potential functional relevance of species-specific features of H3K27 acetylation , we analyzed genome-wide RNA expression levels in the human and mouse Treg and CD4+ T-cell populations ( Figure 2C; Supplementary files 10-12 ) .", "Comparison of lineage-specific gene expression revealed similar conservation of gene expression patterns .", "We also observed that organism-specific cell lineage-associated variation in H3K27 acetylation correlated with organism-specific gene expression changes ( Figure 2D , E and Figure 2—figure supplement 1F ) .", "The latter result is consistent with organism-specific regulatory elements maintaining functional impact on Treg cell lineage-specific epigenetic activity .", "We next explored conservation of the regulatory network architecture by considering a broader definition of locus epigenetic ‘conservation’ .", "Namely , previous studies have identified broad conservation of the regulatory networks even if the exact regulatory elements and transcription factor-binding sites are not conserved ( Stergachis et al . , 2014; Vierstra et al . , 2014 ) .", "Thus , we examined all regulatory elements associated with a given gene ( most proximal gene body ) to see if a lineage-specific regulatory element in mouse could ‘re-emerge’ in a different location as a non-homologous lineage-specific regulatory element in human .", "This definition could be considered ‘functional homology’ in contrast to our previous analyses that have focused on individually conserved regulatory elements .", "Interestingly , this and other functional definitions of lineage-specific conservation ( see ‘Materials and methods’ ) yielded very few additional genes ( statistically non-significant gene counts , see ‘Materials and methods’ ) , nearly all of which had non-conserved gene expression patterns in Treg vs conventional CD4+ T cells ( Figure 2—figure supplement 2 ) .", "This implies that lineage specification of Treg cells is governed by a specific evolutionarily constrained small set of regulatory elements , which is in contrast to the more plastic conservation of global genome-wide regulatory network architectures ( Stergachis et al . , 2014; Vierstra et al . , 2014 ) .", "Given the critical role of Foxp3 in establishment and maintenance of Treg cell identity and function , and Treg-specific chromatin repression ( Arvey et al . , 2014 ) , we wanted to know if conservation of the lineage-specific epigenome is associated with conserved binding sites of Foxp3 .", "We quantified Foxp3 binding in human and murine Treg cells and found that many Foxp3-bound loci were genetically conserved and bound in both organisms ( Figure 3A , Figure 3—figure supplement 1A , B; Supplementary files 7-9 ) .", "While there was substantial conservation of Foxp3 binding , there were also many sites that were species specific or had insufficient binding for robust quantification in the weaker Foxp3-bound sites in one of the species ( Figure 3B ) . 10 . 7554/eLife . 07571 . 008Figure 3 . Epigenetic and genetic conservation of Foxp3-binding elements and corresponding differential gene expression .", "( A ) The TCF7 locus provides examples of conserved ( first intron , promoter-proximal ) , mouse-specific ( upstream ) , and human-specific ( first intron , promoter-distal ) Foxp3 binding .", "Plot layout and axes are similar to Figure 2A .", "Orthologous regulatory elements ( light blue ) and their orthological mapping ( red ) are shown .", "( B ) Genome-wide , regulatory elements are bound by Foxp3 in both mouse and human ( red ) or in a human-specific ( green ) or mouse-specific ( blue ) manner .", "( C ) Foxp3 binds loci in human and mouse that have decreased acetylation in the Treg cells at the PDE3B locus .", "This locus contains species-specific Foxp3-binding sites that are associated with species-specific decrease in acetylation in Treg cells ( marked as 1 , 3 ) and conserved Foxp3 binding that is associated with conserved Treg lineage decreases in acetylation ( 2 ) .", "The decrease in acetylation at the PDE3B promoter is conserved in both species .", "( D ) Conserved and species-specific Foxp3 binding is associated with decreased histone H3K27 acetylation at regulatory elements .", "Changes in human or mouse H3K27 acetylation ( y-axis ) at genetically conserved regulatory elements are shown to be associated with human- or mouse-specific , or conserved Foxp3 binding ( x-axis ) .", "Statistical significance was calculated by two-sample t-test using all acetylated loci as the background distribution; p-values are shown as *: p < 0 . 05; **: p < 0 . 01; ***: p < 0 . 001 .", "( E ) Species-specific Foxp3 binding in mouse and human Treg cells is associated with non-conserved forkhead box DNA motif .", "Enrichment of the forkhead motif ( odds ratio , y-axis ) is shown for those Foxp3-binding sites that are genetically conserved and bound only in human ( left ) or mouse ( center ) Treg cells or both ( right ) .", "Odds ratio and statistical significance were calculated using Fisher's exact test , where motif counts in Foxp3-binding sites were compared to flanking 200nt regions to estimate the empirical enrichment over chance .", "p-values are shown as *: p < 0 . 05; **: p < 0 . 01; ***: p < 0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 07571 . 00810 . 7554/eLife . 07571 . 009Figure 3—figure supplement 1 . Foxp3 binding sites in mouse and human are reproducible and associated with transcriptional repression .", "( A ) Foxp3 binding is consistent in multiple human donors across two laboratory studies .", "Each study consists of two replicates .", "The NCBI SRA accession SRP006674 ( Birzele et al . , 2011 ) had two high-quality technical replicates ( SRR192544 , SRR192545 ) of a single donor , which were merged into single average ( x-axis ) .", "The NCBI SRA accession SRP017669 ( Schmidl et al . , 2014 ) had high-quality Foxp3 ChIP-seq in two distinct donors ( SRR639420 , SRR639419 ) , which were averaged in the above comparison ( y-axis ) and analyzed individually in other analyses .", "( B ) Mouse Foxp3 ChIP-seq was performed on activated and resting Treg cell populations .", "Average ChIP signal is shown for 2 aTreg cell biological replicates on each axis ( left ) and single biological replicates in rTreg cells ( right ) .", "These panels are from Arvey et al . , 2014 and are reproduced here with copyright permission .", "( C ) Examples of Foxp3 binding that is conserved , species specific , and the association with Treg lineage-specific differences in acetylation .", "The examples include: a mouse-specific Foxp3-binding site is associated with a mouse-specific active regulatory element that is downregulated in the Treg lineage ( 1 ) ; a Treg lineage-specific decrease in acetylation at the PDE7A promoter in both mouse and human ( 2 ) ; and a human-specific Foxp3-binding site ( 3 ) .", "Figure design and axes similar to Figure 3C in main text .", "( D ) Foxp3-binding sites are associated with decreases in gene expression .", "Statistical significance is shown for comparison of conserved , mouse , and human-specific Foxp3-bound genes against all expressed genes in the respective organism ( p-values are one-sided Kolmogorov–Smirnov tests ) .", "Conserved and mouse-specific binding sites have statistically indistinguishable effect on gene expression in mouse ( p < 0 . 18 , two-sided test ) ; whereas conserved and mouse-specific sites are more repressed than human-specific sites ( p < 0 . 03 and p < 10−4 , respectively ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 07571 . 009 Recent reports have implicated Foxp3 in repression of chromatin ( Arvey et al . , 2014; DuPage et al . , 2015 ) ; therefore , we characterized the relationship between conservation of Foxp3 binding and conservation of Treg lineage-specific decreases in H3K27ac .", "We found that conserved Foxp3 binding was associated with decreased H3K27ac in both human and mouse ( Figure 3C , D , Figure 3—figure supplement 1C ) .", "Importantly , at human- or mouse species-specific Foxp3-bound sites , H3K27ac was decreased exclusively in the corresponding organism ( Figure 3D , Figure 3—figure supplement 1D ) .", "Species-specific Foxp3 occupancy at genetically conserved loci was also associated with presence of forkhead DNA-binding motifs at the corresponding Foxp3-bound sites ( Figure 3E ) .", "These results suggest that preservation of the forkhead motif likely enables Foxp3 protein recruitment , Foxp3-mediated chromatin repression at conserved loci , and that DNA sequence divergence can account for differential Foxp3 occupancy of otherwise genetically conserved orthologous regulatory elements .", "To determine if non-coding regulatory elements specific to resting or activated Treg cells are subject to genetic variation in human , we explored polymorphisms associated with these elements .", "We examined a narrow genomic window ( 150 bp ) at each of the ∼85 , 000 DHSs to find that ∼80 , 000 contained at least 1 polymorphism cataloged in the single nucleotide polymorphism database ( dbSNP ) and that ∼45 , 000 of these contained polymorphisms with significant ( >0 . 05 ) minor allele frequency ( MAF ) in at least one of the thousand genome project ( 1000G ) populations ( Figure 4A; ‘Materials and methods’; Supplementary file 13 ) .", "Consistent with evolutionary constraint , genetically conserved elements had decreased maximum MAF relative to expected frequency ( Figure 4B; ‘Materials and methods’ ) .", "This pattern held across regulatory elements regardless of their distance from protein-coding regions or binding of Foxp3 ( Figure 4—figure supplement 1A–C ) . 10 . 7554/eLife . 07571 . 010Figure 4 . Common human genetic polymorphisms in Treg lineage-specific regulatory elements can alter histone acetylation .", "( A ) The CD4+ T-cell epigenome is subject to human-to-human genetic variation .", "The number ( y-axis ) of DHSs with at least one polymorphism greater than a given minor allele frequency ( MAF , x-axis ) .", "MAFs are binned by <0 . 05 ( 0 ) , 0 . 05–0 . 15 ( 0 . 1 ) , 0 . 15–0 . 25 ( 0 . 2 ) , 0 . 25–0 . 35 ( 0 . 3 ) , 0 . 35–0 . 45 ( 0 . 4 ) , >0 . 45 ( 0 . 5 ) for each population in the 1000G project ( ‘Materials and methods’ ) .", "( B ) Human DHSs not genetically conserved in mouse ( red ) contain greater genetic variation than those DHS that are conserved ( black ) .", "Empirical p-value is computed by sampling maximum polymorphism MAF across conservation-permuted DHSs , fitting a linear regression , and testing for greater conserved vs shuffled DHS slopes: βconserved > βshuffle .", "( C ) Genetic polymorphisms are correlated with H3K27 acetylation at the ENTPD1 locus .", "The activated Treg cell-specific expression of ENTPD1 is associated with a haplotype represented by single nucleotide polymorphism ( SNP ) rs1342790 .", "Resting Treg ( rTreg ) and naive ( Tn ) and effector ( Teff ) CD4+ T-cell populations have limited acetylation of the ENTPD1 locus; in contrast , activated Treg cells have acetylation that is associated with the 'A' genotype of rs1342790 .", "( D ) Quantification of the ENTPD1 genetic association reveals allele-specific histone modification in heterozygous individuals .", "Meta-analysis of the ENTPD1 locus in B lymphoblastoid cell lines was performed to provide additional statistical power ( Kasowski et al . , 2013a ) .", "Genotypes ( x-axis ) and their corresponding reads crossing the polymorphism per million reads aligned ( RPM; y-axis ) were calculated for each individual with more than 5 reads at rs1342790 .", "Values across zygosity and cell type were made comparable by doubling heterozygous RPMs and mean normalizing .", "Statistical significance was estimated by independent assessment of heterozygous and homozygous allelic normalized read counts ( ‘Materials and methods’ ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 07571 . 01010 . 7554/eLife . 07571 . 011Figure 4—figure supplement 1 . Common human genetic polymorphisms exist in Treg lineage-specific regulatory elements and Foxp3 binding sites .", "These polymorphisms can be associated with variation in histone acetylation .", "( A ) Increased sequence polymorphism frequency in non-conserved DHSs stratified by localization relative to the most proximal gene .", "Non-conserved DHSs ( solid lines ) at promoters , exons , introns , and distal elements showed patterns of polymorphism consistent with decreased constraint in contrast to conserved DHSs ( dashed lines ) .", "( B ) Human Foxp3-bound regulatory elements are subject to human-to-human genetic variation .", "The number ( y-axis ) of DHSs with at least one polymorphism greater than a given MAF ( x-axis ) .", "( C ) Human Foxp3-bound regulatory elements not genetically conserved in mouse ( red ) contain greater genetic variation than the conserved ones ( black ) .", "Empirical p-value is computed by sampling maximum polymorphism MAF across conservation-permuted DHSs .", "( D ) Representative examples of lineage-to-lineage and donor-to-donor variation .", "ChIP-seq H3K27ac RPM intra-donor variation for D204 aTreg and rTreg cells is shown ( left ) as well as inter-donor variation between D204 and D304 aTreg cells ( right ) .", "Axes are log2 ( x + 0 . 5 ) transformed .", "( E ) QQ-plot quantification of allele specificity in 2227 enhancers with robustly detectable heterozygous polymorphisms reveals potentially significant associations .", "Axes show observed ( y-axis ) and expected ( x-axis ) −log10 p-values .", "Since the observed p-value distribution diverges from the expected ( null ) p-value distribution , the most significant p-values are likely not a result of multiple hypothesis testing .", "Observed −log10 p-values were capped at 5 for ease of visualization .", "( F ) A polymorphic allele at COMMD10 does not affect acetylation in B cells but is associated with differential acetylation in T-cell populations .", "A linear regression model with fixed intercept is fitted to the B and T-cell populations .", "Difference in slope parameters β1 ≠ β2 was tested for significance with two-tailed t-test with n1 + n2 −4° of freedom where n1 and n2 are the number of heterozygous alleles in B and Treg cells .", "As indicated in the legend , homozygous cell lines/donors are shown as black circles , while heterozygous sample cell type is represented by color and symbol . DOI: http://dx . doi . org/10 . 7554/eLife . 07571 . 011 To test if genetic variation in the Treg cell-specific regulatory elements could alter enhancer functionality , we characterized DNA sequence polymorphisms associated with inter-individual quantitative variability in H3K27ac modifications across multiple cell populations in a cohort of unrelated donors ( Figure 4—figure supplement 1D , E ) .", "For example , the Treg cell lineage-specific enhancer containing the single nucleotide polymorphism ( SNP ) rs2882971 at the ENTPD1 locus demonstrated genetic association with H3K27ac levels across individuals and in an allele-specific manner in a given individual ( Figure 4C ) .", "This is consistent with previous reports showing ENTPD1 RNA and protein expression association with a strong cis-eQTL ( Orrù et al . , 2013; Ferraro et al . , 2014 ) .", "We confirmed the genetic association with chromatin state through meta-analysis of lymphoblastoid cell lines , which enabled sufficient statistical power ( Figure 4D ) .", "We also identified potential associations where the enhancer decorated with H3K27ac was present in multiple CD4+ T-cell subsets , but exhibits only allele-specific variation in Treg cells , suggesting that Treg cell-specific chromatin modulators or transcription factors may preferentially act on a single allele of a polymorphic locus ( Figure 4—figure supplement 1F ) .", "Our observation that the inter-individual genetic variation present in the Treg cell-specific epigenome can potentially alter Treg cell function raised a question if this variation may contribute to polygenic human disease pathogenesis .", "In particular , while fatal monogenic IPEX disorder resulting from loss-of-function Foxp3 mutations ( Khattri et al . , 2003 ) highlights the critical role for Treg cells in preventing autoimmunity , the role of these cells in common polygenic human diseases has been notoriously difficult to assess with cellular functional analyses confounded by patients' disease state .", "As a result , assessments of Treg cell function in patients with complex autoimmune and inflammatory diseases frequently lead to conflicting conclusions open to alternative interpretations ( Miyara et al . , 2011 ) .", "Furthermore , recent findings from genome-wide association ( GWA ) studies have revealed that most disease-predisposing susceptibility loci reside in non-coding portions of the genome ( Welter et al . , 2014; Figure 5—figure supplement 1 ) , suggesting that the majority of polygenic disease is in fact due to variation in transcriptional control rather than protein structure and function .", "Recent studies have also found that active chromatin is enriched for variants that contribute to polygenic diseases , including autoimmunity ( Maurano et al . , 2012; Trynka et al . , 2013; Farh et al . , 2014 ) , and that these variants can alter transcriptional regulator protein-DNA interactions , resulting in differential gene expression ( Ferraro et al . , 2014; Lee et al . , 2014; Ye et al . , 2014 ) .", "Therefore , we reasoned that if disease-predisposing polymorphisms were embedded in chromatin with function exclusively in Treg cells , then Treg cell dysfunction could contribute to disease etiology .", "Through meta-analysis of GWA studies of SNPs , we identified hundreds of statistically significant disease-associated polymorphisms residing in the CD4+ T effector and Treg cell epigenome .", "One such strictly Treg cell-specific epigenetic element was found at the CTLA4 locus , which harbors a risk allele for multiple autoimmune disorders , including rheumatoid arthritis , type 1 diabetes ( T1D ) , Graves' disease , and systemic lupus erythematosus ( Scalapino and Daikh , 2008 ) .", "While early GWA genotyping approaches were too coarse to localize the causative polymorphism , fine mapping by the ImmunoChip across a large case–control T1D cohort demonstrated that the most disease-associated variants , represented by rs2882971 , lie within Treg cell-specific enhancers that are epigenetically conserved and lineage-specific in both human and mouse ( Figure 5A , Figure 2—figure supplement 1A ) ( Onengut-Gumuscu et al . , 2015 ) .", "Functional relevance of a non-coding CTLA4 polymorphism in linkage disequilibrium with rs2882971 ( rs3087243 ) has been experimentally validated as influencing the CTLA4 splice form dosage , with disease-predisposing variants decreasing the soluble isoform that is expressed in Treg cells ( Ueda et al . , 2003; Atabani et al . , 2005; Gerold et al . , 2011 ) .", "The linkage disequilibrium across the locus suggests that the causative polymorphism is either in ( 1 ) upstream Treg lineage-specific epigenetically active enhancers near rs2882971 or ( 2 ) downstream epigenetically and transcriptionally inactive DNA near rs3087243 .", "Pairwise conditioning of rs2882971 and rs3087243 resulted in a non-statistically significant association for both polymorphisms ( logistic regression for an additive model yielded SNP coefficient p-values that were greater than 0 . 05 ) .", "This indicates that genotype–phenotype statistics alone is unable to resolve the causative SNP .", "In contrast , our epigenetic approach provides orthogonal data that implicate upstream enhancers . 10 . 7554/eLife . 07571 . 012Figure 5 . Disease-associated SNPs are enriched in Treg lineage-specific regulatory elements .", "( A ) Fine mapping of genetic disease association identifies Treg-specific upstream enhancers ( highlighted in blue ) at the CTLA4 locus as being associated with predisposition to type 1 diabetes ( T1D ) .", "The high-resolution ImmunoChip SNP array analysis suggests that the functional polymorphism resides in the Treg-specific enhancer .", "The x-axis shows genomic position and y-axis shows RPM for chromatin tracks and −log10 ( p-value ) for association study data .", "Dashed horizontal lines show genome-wide statistical significance thresholds for T1D disease association studies .", "The highlighted polymorphism rs2882971 is representative of multiple SNPs in linkage disequilibrium .", "( B ) Autoimmune disease-risk polymorphisms have extensive overlap with the pan-CD4+ T-cell epigenome .", "Disease-risk polymorphisms was obtained from the NHGRI GWAS catalog and statistical significance of overlap was determined by a one-tailed hypergeometric test using all known disease-associated polymorphisms to model the null distribution ( ‘Materials and methods’ ) .", "( C ) Treg and Teff cell-specific epigenetic elements contain a significant number of autoimmune disease-risk polymorphisms .", "Metabolic diseases are shown as a control .", "T1D and type 2 diabetes ( T2D ) are shown as representative autoimmune and metabolic diseases .", "Aggregated disease sets are provided in Supplementary file 14 .", "( D ) Polymorphisms in conserved Treg lineage-specific epigenetic elements are enriched for autoimmune-associated genetic variation .", "Genes near epigenetic elements containing risk polymorphisms are divided into categories: genetically non-conserved , genetically conserved , lineage-specific elements epigenetically active in human , and lineage-specific epigenetic modification conserved in both human and mouse .", "Autoimmune ( AI ) , T1D , metabolic ( Met ) , T2D , and psychiatric ( Psych ) disease sets are shown . DOI: http://dx . doi . org/10 . 7554/eLife . 07571 . 01210 . 7554/eLife . 07571 . 013Figure 5—figure supplement 1 . Non-coding disease-associated polymorphisms are assayed by genotyping arrays .", "( A ) Polymorphisms associated with common polygenic diseases reside predominantly in the non-coding portion of the genome .", "Annotations of statistically significant SNP-phenotype associations are taken from the NHGRI GWAS catalog .", "( B ) Only a subset of DHSs contain variants that exhibit high LD with polymorphisms assayed using the Illumina genotyping bead array .", "The number of DHSs ( y-axis ) satisfying certain critera ( x-axis ) are shown .", "Of the ∼85 K DHSs , ∼45 K have at least one variant with significant MAF ( >0 . 05 across 1000G ) .", "The majority of these loci have limited LD ( <0 . 8 ) with any exonic polymorphism with 100 kbp .", "However , only ∼7 K DHSs have a polymorphism that is in strong LD ( >0 . 8 ) with an assayed polymorphism on the Illumina Human 660 K genotyping chip .", "( C ) Chromatin state at the INS locus supports the unique role of insulin in beta islet cells in the etiology of T1D ( similar axes to Figure 5A ) .", "This is consistent with an extensive literature on beta islet cells and supports the relevance of our approach to characterizing cell type- and tissue-specific disease relevance using chromatin state . DOI: http://dx . doi . org/10 . 7554/eLife . 07571 . 013 Next , we sought to identify specific diseases influenced by polymorphisms that may cause Treg cell dysfunction .", "We analyzed GWA case–control cohorts for autoimmune and autoinflammatory diseases in addition to metabolic and psychiatric disorders ( ‘Materials and methods’; Supplementary file 14 ) .", "We identified dozens of Treg cell lineage-specific epigenetic elements that harbor genetic variation associated with and potentially contributing to polygenic autoimmune disease such as T1D ( Figure 5B , C; ‘Materials and methods’ ) .", "Analysis of these risk alleles revealed that many were in LD or were proximal to genes with epigenetically conserved Treg lineage-specific elements ( Figure 5D ) , suggesting that these elements have important conserved function .", "In contrast , an aggregate GWAS data set of metabolic ( e . g . , type 2 diabetes ) and psychiatric disorders lacked disease associations in conserved Treg lineage-specific elements .", "Furthermore , psychiatric disorders risk-alleles were generally localized in non-conserved genetic elements , which is consistent with recent reports finding substantial human-specific genetic diversity in cognition-associated genes in comparison to recent evolutionary ancestors ( Ogawa and Vallender , 2014 ) ." ], [ "In the past few years , many studies have characterized genome-wide epigenetic activity in progenitor cells or related differentiated cell lineages to identify hundreds of lineage-specific regulatory elements ( Epigenome Roadmap Consortium , 2015 ) .", "To isolate biologically important elements , conservation of genetic DNA sequence and epigenetic activity has been used as a proxy for functional significance ( Odom et al . , 2007; Schmidt et al . , 2010; Long et al . , 2013; Vierstra et al . , 2014 ) .", "We compared genome-wide features of regulatory elements and their activity in two closely related cell lineages , Treg cells , and effector CD4+ T cells , which represent alternative cell fate choices during T-cell differentiation and fulfill opposing anti-inflammatory and pro-inflammatory immune functions .", "In our comparison of Treg and effector T-cell lineages , we found that ∼85% of regulatory elements were genetically conserved and epigenetically active in both human and mouse , ∼1–2% were lineage-specific in human or mouse , and <0 . 1% were lineage-specific in both human and mouse .", "This indicates that of those elements that are specific to the Treg lineage , less than 10% were lineage-specific in both mouse and human .", "Thus , the bulk of lineage-specific epigenetic activity was unique to each organism with only a small set of conserved Treg lineage defining regulatory elements , including enhancers near FOXP3 , IL2RA , LRRC32 , IKZF2 , IL2 , IL7R , and PDE3B .", "Interestingly , our analysis also indicates the potential importance of CCR8 , DUSP4 , TNFRSF19 , TNFRSF1B , THEMIS , PDE7A , and SWAP70 to Treg cellular function , as lineage-specific epigenetic activity was conserved in both mouse and human .", "These findings suggest that lineage specification programs may be dependent on but a few immutable genetic regulatory elements , while tolerating considerable epigenetic variation at numerous additional organism-specific lineage-specific elements .", "Given previous findings regarding the extensive turnover of DNA elements in genome-wide regulatory networks ( Stergachis et al . , 2014; Vierstra et al . , 2014 ) , it is noteworthy that we found limited turnover of regulatory elements that are lineage-specific in both mouse and human .", "This confirms the unique importance of these individual regulatory elements .", "However , our observation that many lineage-specific regulatory elements are lineage-specific in only a single organism highlights the plasticity of genome-wide regulatory networks .", "Furthermore , we found that the Foxp3-mediated transcriptional control program was subject to forkhead-binding motif ‘turnover’ that was associated with functional consequences .", "While these organism-specific elements contributed to gene regulation , the regulatory elements with conserved lineage-specific epigenetic activity were associated with far more pronounced regulation of Treg cell lineage-specific gene expression .", "Although our studies support a role for Treg cell dysfunction in disease pathogenesis , other factors are almost certainly required , such as environmental contributors and dysfunction of multiple other cell lineages and physiological processes .", "For instance , it is reasonable to consider that autoimmune diabetes development is impacted most significantly by HLA risk alleles ( Wong et al . , 2004; Stadinski et al . , 2010 ) , and to a lesser degree by other susceptibility determinants including potentially compromised Treg cells .", "Though , we observed statistical enrichment of risk alleles near Treg cell enhancers , it is worth noting that the majority of polymorphisms exist as broad haplotype blocks that cannot be perfectly resolved to a single or several lineage-specific enhancers .", "Although methods for parsing risk alleles into causative and specific cell lineages have been proposed ( Maurano et al . , 2012; Trynka et al . , 2013; Farh et al . , 2014; Seumois et al . , 2014; Onengut-Gumuscu et al . , 2015 ) , these approaches , similar to our study , rely on aggregate proximity statistics and imperfect modeling of genetic linkage .", "As whole genome sequencing and mappings of epigenetic activity improve and expand to more cell populations ( Epigenome Roadmap Consortium , 2015 ) , we expect a significant fraction of loci will have clear epigenetic partitioning in many diseases .", "We propose that for those risk alleles without clear causative polymorphism due to broad linkage disequilibrium , hypothesis-driven lineage-focused epigenomic QTL studies may be an alternative for gaining increased resolution of disease causing polymorphisms .", "Our study suggests that the vast majority of Treg cell lineage-specific elements are not conserved between mice and humans suggesting that during cellular differentiation , only a handful of epigenetic elements implement the core transcriptional program that underlies establishment of a differentiated Treg cell identity and function .", "In support of this model , disease-associated polymorphisms clustered at genic loci containing core lineage-specific epigenetic elements .", "Nevertheless , most Treg lineage-specific regulatory elements were lineage-specific only in human and could harbor extensive natural genetic variation that could influence transcription , alter cellular function , and contribute to polygenic disease .", "This orthogonal high-resolution epigenetic and genetic analysis enabled characterization of disease-predisposing genetic variation associated with Treg-specific enhancers , and therefore , implicated Treg cells in complex autoimmune disease ." ], [ "Buffy coat preparations from normal human peripheral blood donors were obtained from the New York Blood Center ( NYBC ) on same day as donation .", "CD4+ T cells were enriched through negative selection with RosetteSep antibody cocktails ( Stem Cell Technologies #15062 , Vancouver , BC Canada ) .", "Isolated CD4+ T cells were stained for CD3 ( PE-TexasRed , Invitrogen #MHCD0317 , Waltham , MA , United States ) , CD4 ( APC-eFluor 780 , eBioscience #47-0049-42 , San Diego , CA , United States ) , CD45RA ( APC , BioLegend #304112 ) , CD45RO ( PE-Cy7 , BioLegend #304230 ) , and CD25 ( PE , BioLegend #302606 ) , and resting and activated Treg and effector CD4+ T-cell subsets were purified on a BD Biosciences Aria2 fluorescent cell sorter .", "Murine CD3+CD4+ cells were isolated as previously described ( Kim et al . , 2007; Arvey et al . , 2014 ) .", "Briefly , naive CD4+ T cells ( GFP− ) and resting Treg cells ( GFP+ ) were isolated from untreated Foxp3DTR mice , whose Treg cells express GFP-DTR ( DT Receptor ) fusion protein driven cells by an IRES-DTR-GFP coding DNA sequence knocked into 3′-UTR of the Foxp3 gene .", "Activated T effector cells ( GFP− ) and activated Treg cells ( GFP+ ) were FACS sorted from Foxp3DTR mice 11 days after DT treatment ( day 0 and 1; 20 μg/kg ) .", "DT treatment resulted in transient ablation of Treg cells and systemic inflammation .", "Cells harvested from lymph node and spleen were enriched by positive selection ( Dynabeads , Invitrogen ) and sorted on a FACS Aria2 fluorescent sorter .", "ChIP-seq analysis of H3K27Ac in mouse and human cells was performed as previously described ( Arvey et al . , 2014 ) .", "Briefly , ChIP was performed using the H3K27Ac-specific antibody ( Abcam ab4729 , Cambridge , United Kingdom ) raised against the conserved acetylated epitope ‘LATKAARKSAPA’ .", "Sequencing was performed using an Illumina Hi-Seq 2000 instrument and standard library preparation and adaptors .", "Reads were aligned to hg19 using BWA using the ‘mem’ algorithm with parameters ‘-k 22 -L’ .", "Only nuclear chromosomes were included in our analysis , which excluded all contigs of unknown physical mapping , episomal DNA , and mitochondrial DNA .", "Peaks were called using MACSv2 using default parameters and a permissive threshold for total peak count ( p < 0 . 0001 ) .", "The peaks for all experiments were then combined and reads from all experiments were remapped onto the combined set of peaks .", "We removed peaks with low reads per million ( <1 RPM in all samples ) or high-input signal ( >0 . 5 RPM ) ; or peaks that were blacklisted by the ENCODE consortium analysis of artifactual signals in mouse or human cells ( Kundaje , 2013 ) .", "ChIP tracks were plotted by taking strand-shifted reads ( covering 200 nt from original read start ) and computing overlaps for the center 100 nt of each read , as previously described ( Arvey et al . , 2014 ) .", "This simple transformation is superior to simple raw read overlap , as it enhances signal at actual protein–DNA-binding sites .", "Human and murine T effector and Treg cell DHSs were assayed as previously described ( Bernstein et al . , 2012; Samstein et al . , 2012 ) .", "To confirm specificity of the most Treg-specific regulatory elements , we compared active regulatory elements across a number of tissues and immune cell populations identified by ChIP-seq and DNase-seq by the Epigenome Roadmap and ENCODE consortia ( Bernstein et al . , 2012 ) .", "The pancreatic islet active genomic element data set was obtained from recent studies ( Pasquali et al . , 2014; Epigenome Roadmap Consortium , 2015 ) .", "Additional human Treg cell epigenetic data were obtained from recent studies ( Andersson et al . , 2014; Schmidl et al . , 2014 ) .", "Murine Foxp3 ChIP-seq data sets were generated in our previous studies ( Samstein et al . , 2012; Arvey et al . , 2014 ) and human Foxp3 ChIP-seq data sets were obtained from SRP006674 ( technical replicates SRR192544 , SRR192545 ) and SRP017669 ( SRR639419 , SRR639420 ) ( Birzele et al . , 2011; Schmidl et al . , 2014 ) .", "Reproducibility of mouse and human ChIP-seq studies was confirmed ( Figure 3—figure supplement 1 ) .", "Murine gene expression microarray data sets were generated in our previous studies ( Samstein et al . , 2012; Arvey et al . , 2014 ) .", "Mouse Foxp3 gene expression is derived from RNA-seq ( data not shown ) since the Affymetrix 430 2 . 0 array probe assays the 3′-UTR region of the gene that contains the knocked in IRES-DTR-GFP construct .", "Human gene expression was obtained from a previously published study ( Miyara et al . , 2009 ) .", "Acetylated , DNase hypersensitive , and Foxp3-bound loci were identified as described above .", "To identify orthologous loci in human and mouse , the UCSC liftOver program ( Hinrichs et al . , 2006 ) was used with the hg19 to mm9 chains downloaded from: human: http://hgdownload-test . cse . ucsc . edu/goldenPath/hg19/liftOver/ , mouse: http://hgdownload-test . cse . ucsc . edu/goldenPath/mm9/liftOver/ using default parameters .", "If an orthologous locus was not identified , iterations over smaller regions of the locus were tested for orthology .", "This was particularly important for acetylation events , which typically spanned 2–5 kbp , with only a fraction requiring conservation to obtain conserved function .", "We used the following iteration scheme , starting at the center and then scanning outward in increments depending on assay: histone acetylation ChIP-seq: 3–5 kbp loci were searched in 300 bp increments , DNase-seq: 200 bp loci were searched in 50-bp increments , Foxp3 ChIP-seq: 300 bp loci were searched in 50-bp increments .", "Our high-level results were robust to diverse parameter settings and code is provided online .", "Conserved epigenetic activity was quantified as follows .", "After matching human-to-mouse loci , we then mapped all mouse-to-human elements and took the union of all elements in each species , respectively .", "Raw read counts were mapped to the union set of all genetically conserved regulatory elements and non-genetically conserved loci were set to zero .", "For analysis of gene-level conservation ( e . g . , gene expression ) , human HUGO gene names were mapped to murine gene names by Mouse ENCODE mapping ( Yue et al . , 2014 ) .", "To test for mobility ( since individual regulatory elements may be active in both mouse and human , but be lineage-specific only in a single organism , we determined if lineage-specific regulatory elements appear as an independent regulatory elements near the same gene ) of lineage-specific epigenetic elements within a genic locus ( defined as the closest gene transcription start site ( TSS ) and genes within 100 kbp ) , we addressed the following questions: ( 1 ) Is there mobility of lineage-specific epigenetic elements within a locus ?", "( 2 ) Do lineage-specific epigenetic elements become epigenetically active , but lineage non-specific , conserved genetic elements ?", "( 3 ) Would loosening of cutoffs for lineage-specificity and taking the maximally lineage-specific element across a large locus reveal conservation of the lineage specification program across non-conserved regulatory elements ?", "For ( 1 ) , we identified genic loci that contained movement of lineage-specific epigenetic elements in mouse and human ( Figure 2—figure supplement 2D , example shown in Figure 2—figure supplement 2A ) .", "Gene expression was concordant for 3/45 elements examined ( not statistically significant ) .", "For ( 2 ) , a handful of lineage-specific elements became epigenetically inactive in the other organism ( <15 , not statistically significant ) and had limited lineage-specific gene expression ( Figure 2—figure supplement 2E ) .", "For ( 3 ) , loosened rank cutoffs identified weakly lineage-specific elements in human ( n = 632/496 , up/down ) and mouse ( n = 759/625 , up/down ) at proximal but not necessarily orthogonal gene loci .", "Nearby genes were largely unaffected or exhibited discordant lineage-specific gene expression , indicating that this set may have limited functional relevance .", "Nearby genes with weak levels of differential expression ( >0 . 5-fold or q < 0 . 01 , where q is false discovery rate ( FDR ) value ) are shown in Figure 2—figure supplement 2F ( n = 26 up; n = 22 down ) , which includes many genes with regulatory elements that are lineage specific in both mouse and human as characterized in Figure 2—figure supplement 2C .", "Genotype calling was performed according to best practices described by the Broad Institute Genome Analysis Tool Kit v3 . 1 . 1 ( GATK ) documentation ( DePristo et al . , 2011 ) .", "We used Picard ( v0 . 92 ) to designate read groups by donor and cell type .", "Potentially monoclonal reads were removed by selecting a single read at random for each position in the genome with multiple read start sites ( custom Python script ) .", "This strategy avoids reference alignment bias of other tools ( e . g . , samtools rmdup ) that select reads with maximum alignment score .", "The standard GATK pipeline was used to perform local realignment around potential indels and recalibrate quality scores across experiments .", "Final calling and estimation of allelic read depth were performed using the Unified Genotyper algorithm on variants compiled in dbSNP ( v138 ) .", "Variant calling quality was assessed by the Variant Recalibrator algorithm using HapMap 3 . 3 , 1000G Project , and dbSNP v138 as calibrating resources for estimating sensitivity and specificity of calls .", "Low-quality tranches were discarded and not considered in downstream analyses .", "Heterozygous SNPs were identified using GATK ( see above ) .", "This set of SNPs was then filtered to remove low quality or unreliable genotype calls by discarding SNPs that:failed QC after GATK quality recalibration ( as described above ) ;were heterozygous in less than 2 donors;were homozygous in less than 2 donors;showed allelic imbalance in input ( ‘non-ChIPed’ ) DNA at nominal p-values <0 . 05 .", "Allelic read depth was determined from GATK estimates from ‘rmdup’ data output .", "Two-tailed binomial tests for allelic imbalance was used to filter all heterozygous polymorphisms with p-values > 0 . 2 , as these provide little evidence for allele-specific enhancer usage .", "Remaining polymorphisms were aggregated into H3K27ac regulatory elements and tested for LD r2 > 0 . 99 in the five 1000G populations AFR , EUR , CEU , CHB , and JPT .", "These polymorphisms were then aggregated for each regulatory element , under the assumption of allelic read depth independence .", "This statistic was then analyzed for divergence from an empirical null distribution constructed by random uniform p-value aggregation under identical assumptions .", "This is shown in the QQ-plot of Figure 4—figure supplement 1E .", "Since B lymphoblastoid cell lines ( B-LCL ) share many regulatory elements with CD4+ T cells , we performed meta-analysis with the inclusion of the B-LCL data sets ( Kasowski et al . , 2013a; McVicker et al . , 2013a ) to increase confidence in shared allele-specific chromatin expression alleles ( e . g . , ENTPD1 as shown in Figure 4 ) .", "Additionally , a small set of loci presented allele-specific modifications in only CD4+ T cells ( e . g . , Figure 4—figure supplement 1F ) ; however , our analysis was underpowered to detect such events genome-wide .", "A test of allelic preference across both homozygous and heterozygous individuals was performed on normalized estimates of read count coverage of a given genotype .", "That is , reads with a given genotype were quantified by RPM .", "Heterozygous RPM values were doubled to make them comparable to homozygous RPM values .", "These were then mean-normalized by data set ( LCL and primary T cells ) .", "Statistical test of allele specificity incorporating homozygous individuals was performed using two approaches: ( 1 ) independent test of heterozygous and homozygous individuals and ( 2 ) linear regression across all genotypes .", "For ( 1 ) , test of heterozygous allele specificity is described above and quantitative differences in H3K27ac across homozygous individuals was estimated by t-test .", "As homozygous and heterozygous individuals represent independent observations , the product of p-values was used as an estimate of statistical significance of allele specificity .", "For ( 2 ) , linear regression of normalized RPM values to major allele counts was modified to include values for the two heterozygous read overlap counts , shifting the regression domain from {0 , 1 , 2} to {0 , 1 , 2 , 3} , where 0 , 3 are homozygous and 1 , 2 are the respective heterozygous regressor values .", "Polymorphisms identified in prior GWA studies were obtained from the NHGRI GWAS Catalog ( Welter et al . , 2014 ) ( downloaded 12 April , 2014 ) .", "At this time , the catalog was in hg18 coordinates , which were converted to hg19 coordinates using the liftOver program and UCSC whole genome lift over chains .", "These polymorphisms were then analyzed for overlap with regions surrounding regulatory elements using the hypergeometric overlap test .", "Our analysis crudely corrected for linkage disequilibrium , which has confounded significance statistics in previous studies ( Maurano et al . , 2012 ) , by taking broad 100 kbp regions ( or most proximal gene body ) surrounding regulatory elements to include disease-associated polymorphisms across the haplotype block .", "This empirically eliminated over-counting events arising from multiple epigenetic elements overlapping a single genetic haplotype .", "We include GWA studies if they identified >5 independent SNPs associated with the disease .", "The phenotypic traits and diseases analyzed are presented in Supplementary file 14 .", "All source code is open source and available for download at https://bitbucket . org/aarvey/treg_epigen ." ], [ "All mice were bred and housed in the animal facility at the Memorial Sloan Kettering Cancer Center , in accordance with institutional guidelines .", "Human buffy coat samples were obtained from NYBC .", "Human subject informed consent was obtained by the NYBC , under guidance of the NYBC committee for protection of human subjects .", "The informed consent allowed for research use and publication .", "Donor identities were anonymous and subject IDs are different from those provided by NYBC .", "The Memorial Sloan Kettering Cancer Center IRB determined analysis of human data to be exempt research per 45 CFR 46 . 101 . b ( 4 ) , 45 CFR 164 . 512", "( i ) ( 2 )", "( ii ) , and 45 CFR 46 . 116 ( d ) ." ] ]

[ "Regulatory T ( Treg ) cells , which suppress autoimmunity and other inflammatory states , are characterized by a distinct set of genetic elements controlling their gene expression .", "However , the extent of genetic and associated epigenetic variation in the Treg cell lineage and its possible relation to disease states in humans remain unknown .", "We explored evolutionary conservation of regulatory elements and natural human inter-individual epigenetic variation in Treg cells to identify the core transcriptional control program of lineage specification .", "Analysis of single nucleotide polymorphisms in core lineage-specific enhancers revealed disease associations , which were further corroborated by high-resolution genotyping to fine map causal polymorphisms in lineage-specific enhancers .", "Our findings suggest that a small set of regulatory elements specify the Treg lineage and that genetic variation in Treg cell-specific enhancers may alter Treg cell function contributing to polygenic disease ." ]

[ "The immune system protects the body from infection .", "Key to this protection is the ability to mount an immune response that is sufficient to deal with the threat , but is not so large that the damage it causes to the body exceeds its immediate benefit .", "Immune cells called regulatory T cells ( or Treg cells for short ) help to shut down the immune response after a threat has been successfully destroyed .", "They also prevent the immune system from attacking the body's own cells , a phenomenon known as autoimmunity .", "All cells in the body carry the same set of genes , but the activity of these genes varies between cell types to enable the cells to perform their different jobs .", "This is possible because our DNA contains regions called regulatory elements that can control the expression of particular genes .", "These regions can be activated in specific types of cells , which results in specific chemical modifications to DNA that only affect gene activity in those cells .", "The DNA sequences of these regulatory elements vary between individuals .", "This ‘genetic variation’ can lead to differences in the chemical modifications that occur to DNA , which is known as epigenetic variation .", "This means that Treg cells in one person may work in a different way to those in another individual , which could make some individuals more susceptible to autoimmune diseases than others .", "However , it was not clear how much genetic and epigenetic variation exists in Treg cells .", "Here , Arvey et al . examined Treg and other immune cells from human and mouse donors .", "The experiments show that some of the epigenetic modifications present in many individuals only in Treg cells , which indicates that they may be important for the activity of the Treg cells .", "Unexpectedly , most of the epigenetic modifications were specific to either mice or humans , but Arvey et al . identified a core set of genes that had been modified only in Treg cells in both species .", "In the human cells , Arvey et al . also identified genetic differences in regulatory elements that are associated with autoimmune diseases .", "Arvey et al . 's findings suggest that a small set of regulatory elements are crucial to the role of Treg cells in the immune system .", "Furthermore , genetic variation in these elements can lead to epigenetic changes in Treg cells that contribute to autoimmune diseases in humans .", "Further study may lead to the development of new treatments for these diseases ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "evolutionary biology" ]

[ [ "Adaptive evolution is a process in which selective events result in the replacement of less-fit genotypes with a more fit ones .", "Intuitively , a series of selective events , each improving fitness relative to the immediate predecessor , should translate into a cumulative increase in fitness relative to the ancestral state .", "However , whether or not this is borne out over long evolutionary time scales has long been the subject of debate ( Ruse , 1993; Dawkins , 1997; Gould , 1997; Shanahan , 2000 ) .", "The failure to identify broad patterns of progress over evolutionary time scales—despite clear evidence of selection acting over successive short time intervals—is what Gould , 1985 referred to as ‘the paradox of the first tier' . This paradox implies that evolution exhibits nontransitivity , a property that is best illustrated by the Penrose staircase and the Rock–Paper–Scissors game . The Penrose staircase is a visual illusion of ascending sets of stairs that form a continuous loop such that—although each step appears higher than the last—no upward movement is realized . In the Rock–Paper–Scissors game each two-way interaction has a clear winner ( paper beats rock , scissors beats paper , and rock beats scissors ) , yet due to the nontransitivity of these two-way interactions , no clear hierarchy exists among the three . In ecology , nontransitive interactions among extant species are well documented as contributors to biological diversity and community structure ( Kerr et al . , 2002; Károlyi et al . , 2005; Laird and Schamp , 2006; Reichenbach et al . , 2007; Menezes et al . , 2019 ) and arise by way of resource ( Sinervo and Lively , 1996; Precoda et al . , 2017 ) or interference competition ( Kirkup and Riley , 2004 ) . First put forward in the 1970s ( Gilpin , 1975; Jackson and Buss , 1975; May and Leonard , 1975; Petraitis , 1979 ) , the importance of nontransitivity in ecology has garnered extensive theoretical and experimental consideration over the last half century ( e . g . Sinervo and Lively , 1996; Kerr et al . , 2002; Allesina and Levine , 2011; Rojas-Echenique and Allesina , 2011; Soliveres et al . , 2015; Liao et al . , 2019 ) . What is unknown is whether nontransitive interactions arise for direct descendants along a line of genealogical succession . This is the crux of Gould’s paradox and has broad implications for our understanding of evolutionary processes . For instance , if an evolved genotype is found to be less fit in comparison to a distant ancestor , the adaptive landscape upon which the population is evolving may not contain true fitness maxima ( Barrick and Lenski , 2013; Van den Bergh et al . , 2018 ) and , more broadly , directionality and progress may be illusory ( Gould , 1996 ) . Testing the hypothesis that nontransitive interactions arise along lines of genealogical descent , however , is not possible in natural populations because it requires our ability to directly compete an organism against its immediate predecessor as well as against its extinct genealogical ancestors . Fortunately , laboratory experimental evolution , in which populations are preserved as a ‘frozen fossil record’ , affords us with the unique opportunity to test for nontransitivity along a genealogical lineage by directly competing a given genotype against the extant as well as the extinct .", "An early study of laboratory evolution of yeast in glucose-limited chemostats appeared to demonstrate that nontransitive interactions arise along a line of genealogical descent ( Paquin and Adams , 1983 ) .", "However , the specific events that led to nontransitivity in this case are unknown , and it is likely the case that the authors were measuring interactions between contemporaneous lineages in a population , rather than individuals along a direct line of genealogical descent , as they report ( see Discussion ) .", "Indeed , adaptive diversification is common in experimental evolution due to spatial structuring ( Rainey and Travisano , 1998; Frenkel et al . , 2015 ) and metabolic diversification ( Paquin and Adams , 1983; Helling et al . , 1987; Turner et al . , 1996; Spencer et al . , 2008; Kinnersley et al . , 2014 ) , and is typically maintained by negative frequency-dependent selection , in which rare genotypes are favored .", "Collectively this work reinforces theory and observational evidence on the power of ecological nontransitivity as a driver and maintainer of diversity but is silent as to whether genealogical succession can also be nontransitive .", "Here we determine the sequence of events leading to the evolution of nontransitivity in a single yeast population during a 1000-generation evolution experiment .", "We show that nontransitivity arises through multilevel selection involving both the yeast nuclear genome and the population of a vertically transmitted virus .", "Many fungi , including the yeast Saccharomyces cerevisiae , are host to non-infectious , double-stranded RNA ‘killer’ viruses ( Wickner , 1976; Schmitt and Breinig , 2002; Schmitt and Breinig , 2006; Rowley , 2017 ) .", "Killer viruses produce a toxin that kills non-killer containing yeasts .", "The K1 toxin gene contains four subunits ( δ , α , γ , and β ) , which are post-translationally processed and glycosylated to produce an active two-subunit ( α , β ) secreted toxin ( Bostian et al . , 1983 ) .", "Immunity to the toxin is conferred by the pre-processed version of the toxin , thus requiring cells to maintain the virus for protection .", "We show that nontransitivity arises due to multilevel selection: adaptation in the yeast nuclear genome and the simultaneous stepwise deterioration of the killer virus .", "By expanding our study of host-virus genome evolution to over 100 additional yeast populations , we find that multilevel selection , and thus the potential for the evolution of nontransitive interactions , is a common occurrence given the conditions of our evolution experiment ." ], [ "Previously we evolved ~600 haploid populations of yeast asexually for 1000 generations in rich glucose medium ( Lang et al . , 2011 ) .", "We characterized extensively the nuclear basis of adaptation for a subset of these populations through whole-genome whole-population time-course sequencing ( Lang et al . , 2013 ) and/or fitness quantification of individual mutations ( Buskirk et al . , 2017 ) .", "For one population ( BYS1-D08 ) we were surprised to observe that a 1000-generation clone lost in direct competition with a fluorescently labeled version of the ancestor .", "To test the hypothesis that a nontransitive interaction arose during the adaptive evolution of this population , we isolated individual clones from three time points: Generation 0 ( Early ) , Generation 335 ( Intermediate ) , and Generation 1000 ( Late ) ( Figure 1A ) .", "These time points were chosen , in part , to coincide with the completion of selective sweeps in the population ( Lang et al . , 2013 ) .", "The Intermediate clone was isolated following a selective sweep that fixes three nuclear mutations including a beneficial mutation in YUR1 .", "The Late clone was isolated following three more selective sweeps that fix an additional 10 nuclear mutations including a beneficial mutation in STE4 .", "We performed pairwise competition experiments between the Early , Intermediate , and Late clones at multiple starting frequencies .", "We find that the Intermediate clone is 3 . 8% more fit relative to the Early clone and that the Late clone is 1 . 2% more fit relative to the Intermediate clone ( Figure 1B , left panel ) .", "The yur1 mutation in the Intermediate clone and the ste4 mutation in the late clone were previously estimated to provide a 4 . 6 ± 0 . 5% and 2 . 6 ± 0 . 4% fitness advantage , respectively ( Buskirk et al . , 2017 ) , consistent with the fitness differences between the Intermediate and Early clones and the Late and Intermediate clones .", "The expectation , assuming additivity , is that the Late clone will be more fit than the Early clone , by roughly 5 . 0% .", "Surprisingly , we find that the Late clone is less fit than expected , to the extent that it often loses in pairwise competition with the Early clone ( Figure 1B , left panel ) .", "Furthermore , the interaction between the Early and Late clones exhibits positive frequency-dependent selection , thus creating a bi-stable system where the fitness disadvantage of the Late clone can be overcome if it starts above a certain frequency relative to the Early clone ( Figure 1—figure supplement 1 ) .", "Positive frequency-dependent selection is rare in experimental evolution and can only arise through a few known mechanisms .", "It has been observed previously in yeast that harbor killer viruses ( Greig and Travisano , 2008 ) , which are dsRNA viruses that encode toxin/immunity systems .", "Using a well-described halo assay ( Woods and Bevan , 1968 ) , we find that the ancestral strain of our evolved populations exhibits the phenotype expected of yeast that harbor the killer virus: it inhibits growth of a nearby sensitive strain and resists killing by a known killer strain ( Figure 1—figure supplement 2 ) .", "We asked if the observed nontransitivity in the BYS1-D08 lineage could be explained by evolution of the killer phenotype .", "Phenotyping of the isolated clones revealed that the Intermediate clone no longer exhibits killing ability ( K-I+ ) and that the Late clone possesses neither killing ability nor immunity ( K-I- , Figure 1A , Figure 1—figure supplement 3 ) .", "Killer toxin has been shown to impart frequency-dependent selection in structured environments due to a high local concentration of secreted toxin ( Greig and Travisano , 2008 ) .", "We hypothesized that a stepwise loss of killing ability followed by loss of immunity , along with the acquisition of beneficial yur1 and ste4 nuclear mutations , were responsible for the frequency-dependent and nontransitive interaction between Early and Late clones .", "To determine if killer toxin production by the Early clone is necessary for it to outcompete the toxin-susceptible Late clone , we repeated the competition between the Early and Late clones using a virus-cured version of the Early clone .", "We find that removing the virus from the Early clone abolishes the frequency-dependent fitness advantage of the Early clone; the Late clone is 4 . 3% more fit than the cured Early clone at all frequencies ( Figure 1B , right panel ) due to the presence of adaptive mutations in the nuclear genome of the Late clone .", "Therefore the presence of killer virus in the Early clone and the subsequent loss of killer virus-associated phenotypes in the Late clone were necessary for the evolution of frequency dependence and nontransitivity .", "To determine if viral evolution alone is sufficient to account for the observed fitness gains in nontransitive interactions , we focused on the first step in the evolutionary sequence: the transition from the Early clone to Intermediate clone .", "We transferred the killer virus from the Intermediate clone to the cured Early clone and assayed fitness relative to the Early clone .", "Because the virus from the Intermediate clone no longer produces toxin , we suspected that it may provide a fitness benefit to the host .", "However , we find that the evolved killer virus from the Intermediate clone confers no significant effect on host fitness compared to the killer virus from the Early clone ( Figure 1B , right panel ) .", "This shows that the fitness benefit of the Intermediate clone relative to the Early clone is due to adaptation in the nuclear genome .", "Taken together these experiments show that the sequence of events leading to the evolution of nontransitivity involves changes to both the host and viral genomes .", "To determine the extent of killer phenotype evolution across all populations , we assayed the killer phenotype of 142 populations that were founded by a single ancestor and propagated at the same bottleneck size as BYS1-D08 ( Lang et al . , 2011 ) .", "We find that approximately half of all populations exhibit a loss or weakening of killing ability by Generation 1000 , with ~10% of populations exhibiting neither killing ability nor immunity ( Figure 2 ) .", "Of note , we did not observe loss of immunity without loss of killing ability , an increase in killing ability or immunity , or reappearance of killing ability or immunity once it was lost from a population ( Figure 2—figure supplement 1 ) , apart from the noise associated with scoring of population-level phenotypes .", "Several populations ( i . e . BYS2-B09 and BYS2-B12 ) lost both killing ability and immunity simultaneously , suggesting that a single event can cause the loss of both the killer phenotypes .", "Mutations in nuclear genes can affect killer-associated phenotypes .", "The primary receptors of the K1 killer toxin are β-glucans in the yeast cell wall ( Pieczynska et al . , 2013 ) .", "We observe a statistical enrichment of mutations in genes involved in β-glucan biosynthesis ( sixfold Gene Ontology [GO] Biological Process enrichment , p<0 . 0001 ) .", "Furthermore , of the 714 protein-coding mutations dispersed across 548 genes , 40 occur within 11 of the 36 genes ( identified by Pagé et al . , 2003 ) that , when deleted , confer a high level of resistance to the K1 toxin ( χ2 = 18 . 4 , df = 1 , p=1 . 8 × 10−5 ) .", "Nevertheless , the presence of mutations in nuclear genes that have been associated with high levels of resistance is not sufficient to account for the loss of killing ability ( χ2 = 1 . 037 , df = 1 , p=0 . 309 ) or immunity ( χ2 = 0 . 103 , df = 1 , p=0 . 748 ) .", "We sequenced viral genomes from our ancestral strain and a subset of yeast populations ( n = 67 ) at Generation 1000 ( Figure 3 ) .", "We find that our ancestral strain , which was derived from the common lab strain W303-1a , contains the M1-type killer virus ( encoding the K1-type killer toxin ) with only minor differences from previously sequenced strains ( Figure 3—figure supplement 1 ) .", "Our ancestral strain also possesses the L-A helper virus , which supplies the RNA-dependent RNA polymerase and capsid protein necessary for the killer virus , a satellite virus , to complete its life cycle ( Ribas and Wickner , 1992 ) .", "We sequenced viral genomes from 57 populations that changed killer phenotype and 10 control populations that retained the ancestral killer phenotypes .", "Viral genomes isolated from populations that lost killing ability possess 1–3 mutations in the M1 coding sequence – most being missense variants ( Figure 3A ) .", "In contrast , only a single mutation , synonymous nonetheless , was detected in M1 across the 10 control populations that retained the killer phenotype .", "The correlation between the presence of mutations in the viral genome and the loss of killing ability ( χ2 = 59 . 3 , df = 1 , p=1 . 4 × 10−13 ) is strong evidence that viral mutations are responsible for the changes in killer phenotypes .", "We estimate that by Generation 1000 half of all populations have fixed viral variants that alter killer phenotypes ( for comparison , IRA1 , the most common nuclear target , fixed in ~25% of populations over the same time period ) .", "Of the 57 populations that lost killing ability , 42 fixed one of three single nucleotide polymorphisms , resulting in amino acid substitutions D106G , D253N , and I292M and observed 13 , 14 , and 15 times , respectively ( Supplementary file 1 ) .", "Given their prevalence , these polymorphisms likely existed at low frequency in the shared ancestral culture ( indeed , we can detect one of the common polymorphisms , D106G , in individual clones at the Early time point , indicating that this mutation was heteroplasmic in cells of the founding population ) .", "Killer phenotypes are consistent across populations that fixed a particular ancestral polymorphism ( Supplementary file 1 ) .", "In addition to the three ancestral polymorphisms , we detect 34 putative de novo point mutations that arose during the evolution of individual populations ( Supplementary file 1 ) .", "Mutations are localized to the K1 coding sequence , scattered across the four encoded subunits , and skewed toward missense mutations relative to nonsense or frameshift ( Figure 3B ) .", "Of the 78 identified mutations , 14 are predicted to fall at or near sites of protease cleavage or post-translational modification necessary for toxin maturation .", "Overall , however , the K1 coding sequence appears to be under balancing selection ( dN/dS = 0 . 90 ) , indicating that certain amino acid substitutions ( e . g . those that eliminate immunity but retain killing ability ) are not tolerated .", "In addition , substitutions are extremely biased toward transitions over transversions ( Supplementary file 2 , R = 6 . 4 , χ2 = 44 . 2 , df = 1 , p<0 . 0001 ) , a bias that is also present in other laboratory-derived M1 variants ( R = 4 . 1 ) ( Suzuki et al . , 2015 ) and natural variation of the helper L-A virus ( R = 3 . 0 ) ( Diamond et al . , 1989; Icho and Wickner , 1989 ) .", "The transition:transversion bias appears specific to viral genomes as the ratio is much lower within evolved nuclear genomes ( R = 0 . 8 ) , especially in genes inferred to be under selection ( R = 0 . 5 ) , suggesting a mutational bias of the viral RNA-dependent RNA polymerase ( Lang et al . , 2013; Fisher et al . , 2018; Marad et al . , 2018 ) .", "Though point mutations are the most common form of evolved variation , we also detected two viral genomes in which large portions of the K1 ORF are deleted ( Figure 3B ) .", "Despite the loss of the majority of the K1 coding sequence , the deletion mutants maintain cis signals for replication and packaging ( Ribas and Wickner , 1992; Ribas et al . , 1994 ) .", "Notably , the two populations that possess these deletion mutants also possess full-length viral variants .", "The deletion mutants we observe are similar to the ScV-S defective interfering particles that have been shown to outcompete full-length virus presumably due to their decreased replication time ( Kane et al . , 1979; Ridley and Wickner , 1983; Esteban and Wickner , 1988 ) .", "To compare the dynamics of viral genome evolution , nuclear genome evolution , and phenotypic evolution we performed time-course sequencing of viral genomes from three yeast populations that lost killing ability and for which we have whole-population , whole-genome , and time-course sequencing data for the nuclear genome ( Lang et al . , 2013 ) .", "As with the evolutionary dynamics of the host genome , the dynamics of viral genome evolution feature clonal interference ( competition between mutant genotypes ) , genetic hitchhiking ( an increase in frequency of an allele due to genetic linkage to a beneficial mutation ) , and sequential sweeps ( Figure 4 , Figure 4—figure supplement 1 ) .", "Interestingly , viral sweeps often coincide with nuclear sweeps .", "Since the coinciding nuclear sweeps often contain known driver mutations , it is possible that the viral variants themselves are not driving adaptation but instead hitchhiking on the back of beneficial nuclear mutations .", "This is consistent with the observation that the introduction of the viral variant from the Intermediate clone did not affect the fitness of the Early clone ( Figure 1B ) .", "To determine if the loss of killer phenotype is caused solely by mutations in the killer virus , we transferred the ancestral virus ( K+I+ ) and five evolved viral variants into the virus-cured Early clone via cytoduction ( Figure 5A ) .", "The five viral variants were selected to span the range of evolved killer phenotypes: one exhibited weak killing ability and full immunity ( KwI+: D253N ) , three exhibited no killing ability and full immunity ( K-I+: P47S , D106G , I292M ) , and one exhibited neither killing ability nor immunity ( K-I-: −1 frameshift ) .", "Following cytoduction , we observed that the killer phenotype of each cytoductant matched the killer phenotype of the population of origin , which demonstrates that viral mutations are sufficient to explain changes in killer phenotypes ( Figure 5—figure supplement 1 ) .", "To determine if any viral variants affect host fitness , we competed all five cytoductants against the killer-containing Early clone ( K+I+ ) and the virus-cured Early clone ( K-I- ) in pairwise fashion .", "Frequency-dependent selection was observed in all cases in which one competitor exhibited killing ability and the other competitor lacked immunity ( Figure 5A ) .", "For example , cytoductants containing either the ancestral virus or the weak-killing D253N variant exhibited a frequency dependent advantage over the virus-cured Early clone .", "However , in all competitions where the killer-associated phenotypes were compatible , host fitness was not impacted by the specific viral variant , or even by the presence of the virus itself .", "These data suggest that production of active toxin and maintenance of the virus have no detectable fitness costs to the host .", "These findings support previous theoretical and empirical studies ( Pieczynska et al . , 2016; Pieczynska et al . , 2017 ) that claim that mycoviruses and their hosts have co-evolved to minimize cost .", "Based on the lack of a measurable effect of viral mutations on host fitness when killing-mediated interactions are absent , we hypothesized that the evolved viral variants may have a selective advantage within the viral population of individual yeast cells .", "A within-cell advantage has been invoked to explain the invasion of internal deletion variants ( e . g . ScV-S [Kane et al . , 1979] ) but has not been extended to point mutations .", "To test evolved viral variants for a within-cell fitness advantage , we generated a heteroplasmic diploid strain by mating the ancestor ( with wild-type virus ) with a haploid cytoductant containing either the I292M ( K-I+ ) or −1 frameshift ( K-I- ) viral variant .", "The heteroplasmic diploids were propagated for seven single-cell bottlenecks every 48 hr to minimize among-cell selection .", "At each bottleneck , we assayed the yeast cells for killer phenotypes and we quantified the ratio of the intracellular viral variants by RT-PCR and sequencing .", "We find that killing ability was lost from all lines , suggesting that the evolved viral variants outcompeted the ancestral variant ( Figure 5B ) .", "Sequencing confirmed that the derived viral variant fixed in most lines ( Figure 5C ) .", "In some lines , however , the derived viral variant increased initially before decreasing late .", "Further investigation into one of these lines revealed that the decrease in frequency of the viral variant corresponded to the sweep of a de novo G131D variant ( Figure 5C , inset ) .", "Viral variants therefore appear to constantly arise , and the evolutionary success of the observed variants results from their selective advantage over viral competitors within the context of an individual cell .", "We speculate that an intracellular competition between newly arising viral variants also explains the loss of immunity from populations that previously lost killing ability ( Figure 2—figure supplement 1 ) , given the relaxed selection for the maintenance of functional immunity in those populations ." ], [ "We examined phenotypic and sequence co-evolution of an intracellular double-stranded RNA virus and the host nuclear genome over the course of 1000 generations of experimental evolution .", "We observe complex dynamics including genetic hitchhiking and clonal interference in the host populations as well as the intracellular viral populations .", "Phenotypic and genotypic changes including the loss of killing ability , mutations in the host-encoded cell-wall biosynthesis genes , and the virally encoded toxin genes occur repeatedly across replicate populations .", "The loss of killer-associated phenotypes—killing ability and immunity to the killer toxin—leads to three phenomena with implications for adaptive evolution: positive frequency-dependent selection , multilevel selection , and nontransitivity .", "Frequency-dependent selection can be either negative , where rare genotypes are favored , or positive , where rare genotypes are disfavored .", "Of the two , negative frequency-dependent selection is more commonly observed in experimental evolution , arising , for example , from nutrient cross-feeding ( Helling et al . , 1987; Turner et al . , 1996; Spencer et al . , 2008; Kinnersley et al . , 2014; Plucain et al . , 2014; Green et al . , 2020 ) and spatial structuring ( Rainey and Travisano , 1998; Frenkel et al . , 2015 ) .", "Positive frequency-dependent selection , in contrast , is not typically observed in experimental evolution .", "By definition , a new positive frequency-dependent mutation must invade an established population at a time when its fitness is at its minimum .", "Even in situations in which positive frequency-dependent selection is likely to occur , such as the evolution of cooperative group behaviors and interference competition ( Chao and Levin , 1981 ) , a mutation may be unfavorable at the time it arises .", "A crowded , structured environment provides an opportunity for allelopathies to offer a local advantage .", "Here we describe an alternative mechanism for the success of positive frequency-dependent mutations through multilevel selection of the host genome and a toxin-encoding intracellular virus .", "The likelihood of such a scenario occurring is aided by the large population size of the extrachromosomal element: each of the ~105 cells that comprise each yeast population contains ~102 viral particles ( Bostian et al . , 1983; Ridley and Wickner , 1983 ) .", "Nontransitivity in our experimental system is due , in part , to interference competition .", "The production of a killer toxin by the Early clone kills the toxin-susceptible Late clone in a frequency-dependent manner: higher starting frequencies of the Early clone result in higher concentrations of toxin in the environment .", "Interference competition can drive ecological nontransitivity ( Kerr et al . , 2002; Kirkup and Riley , 2004 ) , suggesting that similar mechanisms may underlie both ecological and genealogical nontransitivity .", "The adaptive evolution of genealogical nontransitivity in our system does not follow the canonical model of a co-evolutionary arms race where the host evolves mechanisms to prevent the selfish replication of the virus and the virus evolves to circumvent the host’s defenses ( Daugherty and Malik , 2012; Rowley , 2017 ) .", "Rather , mutations that fix in the viral and yeast populations do so because they provide a direct fitness advantage in their respective populations .", "Nontransitivity arises through the combined effect of beneficial mutations in the host genome ( which improves the relative fitness within the yeast population , regardless of the presence or absence of the killer virus ) and the adaptive loss of killing ability and degeneration of the intracellular virus ( which provides an intracellular fitness advantage to the virus ) .", "The end result is a high-fitness yeast genotype ( relative to the ancestral yeast genotype ) that contains degenerate viruses , rendering their hosts susceptible to the virally encoded toxin .", "Though we did not find an impact of nuclear mutations on killer-associated phenotypes , we do observe a statistical enrichment of mutations in genes involved in β-glucan biosynthesis and in genes that when deleted confer a high level of resistance to the killer toxin .", "Nearly all mutations in these toxin-resistance genes are nonsynonymous ( 18 nonsense/frameshift , 21 missense , one synonymous ) , indicating a strong signature of positive selection .", "This suggests that the nuclear genome adapts in response to the presence of the killer toxin; however , the effect of these mutations may be beyond the resolution of our fitness assay .", "Among the viral variants we identified were two unique ~1 kb deletions; remnants of the killer virus that retain little more than the cis-acting elements necessary for viral replication and packaging .", "These defective interfering particles are thought to outcompete full-length virus due to their decreased replication time ( Kane et al . , 1979; Ridley and Wickner , 1983; Esteban and Wickner , 1988 ) .", "Defective interfering particles are common to RNA viruses ( Holland et al . , 1982 ) .", "Though there are several different killer viruses in yeast ( e . g . K1 , K2 , K28 , and Klus ) , each arose independently and has a distinct mechanism of action ( Rodríguez-Cousiño et al . , 2017 ) .", "Nontransitive interactions may therefore arise frequently through cycles of gains and losses of toxin production and toxin immunity in lineages that contain RNA viruses .", "Reports of nontransitivity arising along evolutionary lines of descent are rare ( de Visser and Lenski , 2002; Beaumont et al . , 2009 ) .", "The first ( and most widely cited ) report of nontransitivity along a direct line of descent occurred during yeast adaptation in glucose-limited chemostats ( Paquin and Adams , 1983 ) .", "This experiment was correctly interpreted under the assumption—generally accepted at the time—that large asexual populations evolved by clonal replacement , where new beneficial mutations arise and quickly sweep to fixation .", "This strong selection/weak mutation model , however , is now known to be an oversimplification for large asexual populations , where multiple beneficial mutations arise and spread simultaneously through the population leading to extensive clonal interference ( Gerrish and Lenski , 1998; Kvitek and Sherlock , 2013; Lang et al . , 2013 ) .", "In addition , the duration of the Paquin and Adams experiment was too short for the number of reported selective sweeps to have occurred ( 4 in 245 generations and 6 in 305 generations , for haploids and diploids , respectively ) .", "The strongest known beneficial mutations in glucose-limited chemostats , hexose transporter amplifications , provide a fitness advantage of ~30% ( Gresham et al . , 2008; Kvitek and Sherlock , 2011 ) and would require a minimum of ~150 generations to fix in a population size of 4 × 109 ( Otto and Whitlock , 1997 ) .", "We contend that Paquin and Adams observed nontransitive interactions among contemporaneous lineages—ecological nontransitivity—rather than nontransitivity among genealogical descendants .", "Apart from the present study , there are no other examples of nontransitivity arising along a line descent , but numerous examples of nontransitive interactions among contemporaneous lineages ( Sinervo and Lively , 1996; Kerr et al . , 2002; Kirkup and Riley , 2004; Károlyi et al . , 2005; Laird and Schamp , 2006; Reichenbach et al . , 2007; Precoda et al . , 2017; Menezes et al . , 2019 ) .", "Here we present a mechanistic case study on the evolution of nontransitivity along a direct line of genealogical descent .", "We determine the specific nuclear and viral changes that lead to nontransitivity in our focal population ( Figure 6 ) .", "Our results show that the continuous action of selection can give rise to genotypes that are less fit compared to a distant ancestor .", "We show that nontransitive interactions can arise quickly due to multilevel selection in a host/virus system .", "In the context of this experiment multi-level selection is common—most yeast populations fix nuclear and viral variants by Generation 1000 .", "Overall , our results demonstrate that adaptive evolution is capable of giving rise to nontransitive fitness interactions along an evolutionary lineage , even under simple laboratory conditions ." ], [ "Details of the evolution experiment have been described previously ( Lang et al . , 2011 ) .", "Briefly , population BYS1-D08 is one of ~600 populations that were evolved for 1000 generations at 30°C in YPD + A and T ( yeast extract , peptone , dextrose plus 100 μg/ml ampicillin and 25 μg/ml tetracycline to prevent bacterial contamination ) .", "Each day populations were diluted 1:210 into 128 μl of YPD + A and T in round-bottom 96-well plates using a BiomekFX liquid handler .", "The dilution scheme equates to 10 generations of growth per day at an effective population size of ~105 .", "Unless otherwise specified , yeast strains were propagated at 30°C in YPD + A and T . The ancestor and evolved populations were described previously ( Lang et al . , 2011 ) .", "Early , Intermediate , and Late clones were isolated by resurrecting population BYS1-D08 at the Generation 0 , 335 , and 1000 , respectively .", "These specific time points were selected to coincide with the completion of a selective sweep ( Lang et al . , 2013 ) , when the population is expected to be near clonal .", "For each time point we isolated multiple clones from a YPD plate and assayed each one to verify that the killer phenotype was uniform .", "The ancestral strain was cured of the M1 and LA viruses by streaking to single colonies on YPD agar and confirmed by halo assay , PCR , and sequencing .", "We integrated a constitutively expressed fluorescent reporter ( pACT1-ymCitrine ) at the CAN1 locus in the cured ancestral strain as well as the Intermediate ( Generation 335 ) and Late ( Generation 1000 ) clones .", "Karyogamy mutants were constructed by introducing the kar1Δ15 allele by two-step gene replacement in the cured a MATα version of the ancestor ( Georgieva and Rothstein , 2002 ) .", "The kar1Δ15-containing plasmid pMR1593 ( Mark Rose , Georgetown University ) was linearized with BglII prior to transformation and selection on -Ura .", "Mitotic excision of the integrated plasmid was selected for plating on 5-fluorotic acid ( 5-FOA ) .", "Then we replaced NatMX with KanMX to enable selection for recipients during viral transfer .", "Competitive fitness assays were performed as described previously ( Lang et al . , 2011; Lang et al . , 2013 ) .", "To investigate frequency dependence , competitors were mixed at various ratios at the initiation of the experiment .", "Competitions were performed for 50 generations under conditions identical to the evolution experiment ( Lang et al . , 2011 ) .", "Every 10 generations , competitions were diluted 1:1000 in fresh media and an aliquot was sampled by BD FACS Canto II flow cytometer .", "Flow cytometry data was analyzed using FlowJo 10 . 3 .", "Relative fitness was calculated as the slope of the change in the natural log ratio between the experimental and reference strain .", "To detect frequency-dependent selection , each 10-generation interval was analyzed independently to calculate starting frequency and fitness .", "Killer phenotype was measured using a high-throughput version of the standard halo assay ( Crabtree et al . , 2019 ) and a liquid handler ( Biomek FX ) .", "Assays were performed using YPD agar that had been buffered to pH 4 . 5 ( citrate-phosphate buffer ) , dyed with methylene blue ( 0 . 003% ) , and poured into a 1-well rectangular cell culture plate .", "Killing ability was assayed against a sensitive tester strain ( yGIL1097 ) that was isolated from a separate evolution experiment initiated from the same ancestor .", "The sensitive tester was grown to saturation , diluted 1:10 , and spread ( 150 µl ) evenly on the buffered agar .", "Query strains were grown to saturation , concentrated 5× , and spotted ( 2 µl ) on top of the absorbed lawn ( Figure 1—figure supplement 2 , left ) .", "Immunity was assayed against the ancestral strain ( yGIL432 ) .", "Query strains were grown to saturation , diluted 1:32 , and spotted ( 10 µl ) on the buffered agar .", "The killer tester was grown to saturation , concentrated 5× , and spotted ( 2 µl ) on top of the absorbed query strain ( Figure 1—figure supplement 2 , right ) .", "Plates were incubated at room temperature for 2–3 days before assessment .", "Killer phenotype was scored according to the scale in shown in Figure 2 .", "Nucleic acids were isolated by phenol–chloroform extraction and precipitated in ethanol .", "Isolated RNA was reverse-transcribed into cDNA using ProtoScript II First Strand cDNA Synthesis Kit ( NEB ) with either the enclosed Random Primer Mix or the M1-specific oligo M1_R3 .", "PCR was performed on cDNA using Q5 High-Fidelity Polymerase ( NEB ) .", "The K1 ORF was amplified using primers M1_F1 or M1_F5 and M1_R6 .", "The M1 region downstream of the polyA stretch was amplified using M1_F7 and M1_R3 .", "The LA virus was amplified using LA_F2 and LA_R2 , LA_F2 and LA_R3 , or LA_F3 and LA_R6 .", "PCR products were Sanger sequenced by Genscript .", "Mutations were identified and peak height quantified using 4Peaks ( nucleobytes ) .", "For intracellular competitions , mutation frequency was quantified by both Sanger and Illumina sequencing ( see below ) , with both methods producing nearly identical results ( Figure 5—figure supplement 2 ) .", "The Sanger sequencing data was aligned to publicly available M1 and LA references ( GenBank Accession Numbers U78817 and J04692 , respectively ) using ApE ( A plasmid Editor ) .", "The ancestral M1 and LA viruses differed from the references at 7 sites ( including 3 K1 missense mutations ) and 19 sites , respectively ( Figure 3—figure supplement 1 ) .", "Viruses were transferred to MATa strains using the MATα karyogamy mutant as an intermediate .", "Viral donors ( MATa , ura3 , NatMX ) were first transformed with the pRS426 ( URA3 , 2µ ORI ) for future indication of viral transfer .", "Cytoduction was performed by mixing a viral donor with the karyogamy mutant recipient ( MATα , ura3 , KanMX ) at a 5:1 ratio on solid media .", "After a 6 hr incubation at 30°C , the cells were plated on media containing G418 to select for cells with the recipient nuclei .", "Recipients that grew on -Ura ( indicator of cytoplasmic mixing ) and failed to grow on ClonNat ( absence of donor nuclei ) then served as donors for the next cytoduction .", "These karyogamy mutant donors ( MATα , URA3 , KanMX ) were mixed with the selected recipient ( MATa , ura3 , NatMX ) at a 5:1 ratio on solid media .", "After a 6 hr incubation at 30°C , the cells were plated on media containing ClonNat to select for cells with recipient nuclei .", "Recipients that grew on -Ura ( indicator of cytoplasmic mixing ) and failed to grow on G418 ( absence of the donor nucleus ) were then cured of the indicator plasmid by selection on 5-FOA .", "Killer phenotype was confirmed by halo assays and the presence of the viral variants in the recipient was verified by Sanger sequencing .", "Multiplexed libraries were prepared using a two-step PCR .", "First , cDNA was amplified by Q5 High-Fidelity Polymerase ( NEB ) for 10 cycles using primers I292M_read1 and I292M_read2 or frameshift_read1 and frameshift_read2 to incorporate a random 8 bp barcode and sequencing primer binding sites .", "The resulting amplicons were further amplified by Q5 PCR for 15 cycles using primers i5_adapter and i7_adapter to incorporate the sequencing adaptors and indices .", "Libraries were sequenced on a NovaSeq 6000 ( Illumina ) at the Genomics Core Facility at Princeton University .", "Raw FASTQ files were demultiplexed using a dual-index barcode splitter ( https://bitbucket . org/princeton_genomics/barcode_splitter ) and trimmed using Trimmomatic ( Bolger et al . , 2014 ) with default settings for paired-end reads .", "Mutation frequencies were determined by counting the number of reads that contain the ancestral or evolved allele ( mutation flanked by five nucleotides ) .", "Within-cell viral competitions were performed by propagating a heteroplasmic diploid and monitoring killer phenotype and viral variant frequency .", "Diploids were generated by crossing the ancestor with a cytoductant harboring either the I292M or −1 frameshift viral variant .", "For each viral variant , three diploid lines ( each initiated by a unique mating event ) were passaged every other day on buffered YPD media for a total of seven single-cell bottlenecks to minimize among-cell selection .", "A portion of each transferred colony was cryopreserved in 15% glycerol .", "Cryosamples were revived , assayed for killer phenotype , and harvested for RNA .", "Following RT-PCR , samples were sent for Sanger sequencing and Illumina sequencing .", "Variant frequency deviated from the expected frequency of 0 . 5 at the initial time point , presumably due to an unavoidable delay between the formation of the heteroplasmic diploid and initiation of the intracellular competition from a single colony .", "Alternatively , viral copy number may vary between donor and recipient cells ." ] ]

[ "A common misconception is that evolution is a linear ‘march of progress’ , where each organism along a line of descent is more fit than all those that came before it .", "Rejecting this misconception implies that evolution is nontransitive: a series of adaptive events will , on occasion , produce organisms that are less fit compared to a distant ancestor .", "Here we identify a nontransitive evolutionary sequence in a 1000-generation yeast evolution experiment .", "We show that nontransitivity arises due to adaptation in the yeast nuclear genome combined with the stepwise deterioration of an intracellular virus , which provides an advantage over viral competitors within host cells .", "Extending our analysis , we find that nearly half of our ~140 populations experience multilevel selection , fixing adaptive mutations in both the nuclear and viral genomes .", "Our results provide a mechanistic case-study for the adaptive evolution of nontransitivity due to multilevel selection in a 1000-generation host/virus evolution experiment ." ]

[ "It is widely accepted in biology that all life on Earth gradually evolved over billions of years from a single ancestor .", "Yet , there is still much about this process that is not fully understood .", "Evolution is often thought of as progressing in a linear fashion , with each new generation being better adapted to its environment than the last .", "But it has been proposed that evolution is also nontransitive: this means even if each generation is ‘fitter’ than its immediate predecessor , these series of adaptive changes will occasionally result in organisms that are less fit than their distant ancestors .", "Laboratory experiments of evolution are a good way to test evolutionary theories because they allow researchers to create scenarios that are impossible to observe in natural populations , such as an organism competing against its extinct ancestors .", "Buskirk et al . set up such an experiment using yeast to determine whether nontransitive effects can be observed in the direct descendants of an organism .", "At the start of the experiment , the yeast cells were host to a non-infectious ‘killer’ virus that is common among yeast .", "Cells containing the virus produce a toxin that destroys other yeast that lack the virus .", "The populations of yeast were given a nutrient-rich broth in which to grow and subjected to a simple evolutionary pressure: to grow fast , which limits the amount of resources available .", "As the yeast evolved , they gained beneficial genetic mutations that allowed them to outcompete their neighbors , and they passed these traits down to their descendants .", "Some of these mutations occurred not in the yeast genome , but in the genome of the killer virus , and this stopped the yeast infected with the virus from producing the killer toxin .", "Over time , other mutations resulted in the infected yeast no longer being immune to the toxin .", "Thus , when Buskirk et al . pitted these yeast against their distant ancestors , the new generation were destroyed by the toxins the older generation produced .", "These findings provide the first experimental evidence for nontransitivity along a line of descent .", "The results have broad implications for our understanding of how evolution works , casting doubts over the idea that evolution always involves a direct progression towards new , improved traits ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "chromosomes and gene expression", "tools and resources" ]

[ [ "The genome is packaged by histone proteins that are decorated with a wide variety of modifications .", "This provides a versatile marking mechanism that integrates cellular signals and plays a key role in processes such as transcription and DNA repair .", "Regulating the proper composition and modifications of chromatin is critical , as exemplified by the many cancer-promoting chromatin alterations and the epigenetic drugs in clinical trials ( Brien et al . , 2016 ) .", "At a first level , chromatin modification is under the control of modifying and demodifying enzymes , many of which have been identified .", "However , it is becoming increasingly clear that there is a second level of control that can involve a plethora of mechanisms , including targeting of enzymes to their site of action , cofactor availability , histone modification cross-talk , and regulation by protein-protein interactions .", "Understanding the establishment of epigenetic states and designing strategies to perturb them in treatment of disease will require a thorough and comprehensive understanding of the network of signals that feeds into the histone modification systems .", "A specific histone modification of great clinical importance that is still poorly understood is methylation of histone H3 on lysine 79 ( H3K79 ) ( Nguyen and Zhang , 2011; Vlaming and van Leeuwen , 2016 ) .", "H3K79 is methylated by Dot1/DOT1L .", "Human DOT1L is important in a subset of leukemias that express an MLL fusion protein caused by rearrangements of the MLL gene ( MLL-r ) ( Wang et al . , 2016 ) .", "DOT1L inhibitors have been developed and are currently in clinical trials for the treatment of MLL-r leukemia ( Stein and Tallman , 2015 ) .", "DOT1L has also been implicated in other cancers , and it has a role in normal development and cellular reprogramming ( reviewed in McLean et al . ( 2014 ) and Nguyen and Zhang ( 2011 ) ) .", "Furthermore , Dot1 plays a role in meiotic checkpoint activation and the DNA damage response ( Nguyen and Zhang , 2011 ) .", "Interestingly , the exact mechanism of action of H3K79 methylation remains poorly understood ( Vlaming and van Leeuwen , 2016 ) .", "The discovery of the important roles of H3K79 methylation has spiked an interest in the regulation of this modification .", "Both H3K79 methylation and the Dot1 ( L ) enzyme are conserved from budding yeast to humans .", "Although some regulators , such as MLL fusion partners AF9 and AF10 , are specific to DOT1L , other regulators are conserved throughout evolution ( Vlaming and van Leeuwen , 2016 ) .", "The best-characterized regulatory pathway is a trans-histone cross-talk: ubiquitination of H2B by Bre1 in yeast or RNF20/40 in mammals promotes H3K79 methylation ( Weake and Workman , 2008 ) .", "Other H3K79 methylation regulators may yet be discovered and could be potential new drug targets in diseases in which DOT1L has been implicated .", "To search for regulators of H3K79 methylation we took advantage of the yeast knock-out collection .", "Thus far , systematic analysis of chromatin regulatory mechanisms has been challenging; the available techniques are either laborious or indirect ( see discussion ) .", "Here we describe a screening strategy to identify chromatin regulators in a quantitative , systematic , and high-throughput manner using a direct read-out of the chromatin status in mutant libraries .", "In this method , which we call Epi-ID , chromatin status on DNA barcodes in the yeast genome is directly interrogated by chromatin immunoprecipitation ( ChIP ) on a pool of cells and read out by parallel deep sequencing .", "With the Epi-ID protocol , it is possible to screen thousands of strains for effects on a chromatin feature of interest .", "We used Epi-ID to screen the yeast knock-out collection for factors that influence H3K79 methylation ( H3K79me ) .", "This screen identified all known regulators of H3K79me , as well as several new regulators that could be validated .", "We found that histone deposition and homologous recombination negatively regulate H3K79me and that adenosine kinase promotes H3 methylation through its effect on the methionine cycle .", "Finally , the histone acetyltransferase module in the SAGA complex was identified as an H3K79me regulator and subsequently shown to regulate H2B ubiquitination and other downstream methylation marks , probably through the stability of the deubiquitinating enzyme Ubp8 , which is also a member of the SAGA complex .", "In summary , Epi-ID is a direct , efficient and widely applicable screening technology .", "The Epi-ID screen presented here yielded a comprehensive picture of the H3K79 methylation regulome , and the technique can be readily applied to other chromatin modifications or chromatin-binding proteins ." ], [ "DNA barcodes , unique sequences that can serve as identifiers , enable experiments on pools of cells ( e . g . Yan et al . , 2008 ) .", "When counted by high-throughput sequencing , they yield quantitative information .", "Classically , collections of barcoded yeast knock-out strains have been used for competitive growth assays , e . g . to find genes that mediate drug toxicity or resistance ( Giaever and Nislow , 2014 ) .", "Here we take advantage of a yeast collection with barcodes in the genomic DNA , and thus packaged into chromatin , and use the barcodes to report on chromatin modification status or binding events .", "By performing ChIP on pools of barcoded cells and counting the abundance of the barcodes by high-throughput sequencing , the relative enrichment of each barcode can be determined .", "Since each barcode corresponds to a gene deletion , the enrichment of the barcode reports on the effect of the gene deletion on the abundance of the chromatin feature assessed by ChIP ( Figure 1A ) .", "We have successfully tested this concept previously in combination with the recombination-induced tag exchange ( RITE ) assay to find regulators of histone turnover in a small set of candidates ( Verzijlbergen et al . , 2011 ) .", "Now , we modified the technique such that it is applicable to screening the complete set of non-essential knock outs and applied Epi-ID to find regulators of H3K79 methylation by Dot1 . 10 . 7554/eLife . 18919 . 003Figure 1 . Outline and proof-of-concept of Epi-ID .", "( A ) The barcoded knock-out library used for Epi-ID was created by crossing the NatMX knock-out library ( Tong and Boone , 2006 ) with the Barcoder collection ( Yan et al . , 2008 ) .", "Two 20-base-pair barcodes ( UpTag ( U ) and DownTag ( D ) ) flank a KanMX selection marker replacing the HO gene .", "For an Epi-ID experiment , barcoded mutant strains are pooled and ChIP experiments are performed on the pool .", "The barcodes of the different mutant strains can be counted by high-throughput sequencing and serve as a read-out for the amount of epitope present at the barcode .", "( B ) Average data of two Epi-ID screens on approximately 4100 yeast deletion strains .", "Each dot represents a deletion strain , with the exception of the Dot1 overexpression strain ( Dot1 OE ) .", "Control strains and some known regulators are highlighted .", "( C ) Schematic depiction of the H2Bub pathway regulating H3K79 methylation . DOI: http://dx . doi . org/10 . 7554/eLife . 18919 . 00310 . 7554/eLife . 18919 . 004Figure 1—source data 1 . Epi-ID data . DOI: http://dx . doi . org/10 . 7554/eLife . 18919 . 00410 . 7554/eLife . 18919 . 005Figure 1—figure supplement 1 . Technical details on Epi-ID .", "( A ) PCR set-up , introducing sequencing adapters and an index in a single PCR .", "Two custom sequencing primers were designed to include the U1/D1 sequence just upstream of the UpTag and DownTag .", "Starting the reads with the barcode yields maximum complexity in the first base pairs , which is good for clustering efficiency .", "These sequencing primers are compatible with standard Illumina sequencing conditions and can be mixed together to sequence UpTag and DownTag in one lane .", "A 6-base-pair index is introduced in the reverse primer , to allow for multiplexing .", "( B ) ChIP-qPCR data of the different methylation states , normalized to H3 , at several loci .", "HO promoter and HO terminator are near the UpTag and DownTag , respectively .", "Error bars show the range of two biological replicates .", "( C ) Dependence of H3K79me levels on Dot1 activity , modified from De Vos et al . ( 2011 ) .", "Although this is a model based on global H3K79 methylation , qualitatively similar changes in methylation can be expected on lowly versus highly methylated loci .", "Dashed lines indicate estimated levels of H3K79 methylation at the loci tested in the qPCR .", "HML , a silent mating type locus , has very low levels of methylation , the two promoters have intermediate levels and the two ORFs have high levels of methylation .", "HO promoter and HO terminator have a low to intermediate level of H3K79 methylation , both an increase and a decrease of methylation are possible , making these loci suitable for a regulator screen .", "( D ) It is important to start the PCR with sufficient material to minimize jackpot effects .", "Scatter plots showing the correlation between barcode counts after a PCR starting with approximately 2500 copies per barcode ( on the x axis ) , with barcode counts of PCRs on diluted sample .", "The correlation decreases with dilution , 250 copies per barcode seems to be sufficient to have minimal jackpot effect . DOI: http://dx . doi . org/10 . 7554/eLife . 18919 . 005 First , we generated a new barcoded yeast knock-out library by crossing a knock-out library that does not contain barcodes ( Tong and Boone , 2006 ) , with a Barcoder library ( Yan et al . , 2008 ) ( Figure 1A ) .", "Barcoder strains ( ~1100 ) harbor two unique 20-base-pair barcodes at a common location .", "The barcodes are upstream ( UpTag ) and downstream ( DownTag ) of a selection marker ( KanMX ) integrated at a safe-harbor locus , the well-studied HO gene .", "We obtained a set of approximately 4300 strains , divided over five subsets with unique barcodes .", "With this new library , chromatin structure or modification status can be measured at a common locus in many knock-out strains , avoiding possible position effects of the integrated barcoded cassette .", "However , the UpTag and DownTag differ in their genomic contexts , being surrounded by promoters or terminators , respectively ( Figure 1A ) .", "Thus , the two barcode positions will give information on Dot1 regulation in different functional contexts .", "Second , we optimized the barcode-seq library construction ( Figure 1—figure supplement 1A ) .", "The sequencing library was generated in a single round of amplification .", "UpTag and DownTag were amplified separately , using primers that annealed to common sequences immediately flanking the barcodes .", "An index introduced in this PCR allowed for extensive multiplexing , of up to at least 150 samples in one Illumina HiSeq lane .", "Before performing the H3K79me Epi-ID , we confirmed the presence of H3K79 methylation around the barcodes by ChIP-qPCR in a wild-type strain .", "Both barcode loci showed an intermediate level of H3K79 methylation in a wild-type strain ( Figure 1—figure supplement 1B ) .", "Here it is important to consider Dot1’s distributive mechanism of methylation ( Frederiks et al . , 2008 ) .", "This distributive mode of action leads to a characteristic shift in methylation states with changing Dot1 activity ( Figure 1—figure supplement 1C ) .", "With increasing Dot1 activity , H3K79me1 will first increase and then decrease , as it is being converted into higher methylation states .", "Around the barcodes , H3K79me1 was high and H3K79me3 was low compared to coding sequences; this intermediate level of H3K79 methylation was consistent with the intergenic location of the barcodes and indicated that they should be able to report increased as well as decreased Dot1 activity ( Figure 1—figure supplement 1C ) .", "We applied the optimized Epi-ID protocol to screen for regulators of H3K79 methylation .", "H3K79me1 and H3K79me3 were used for measuring changes in Dot1 activity since the combination of these methylation states provides the most informative and robust readout of Dot1 activity .", "Input DNA and total H3 ChIP were used for normalization purposes .", "All barcode count data can be found in Figure 1—source data 1 .", "The average data of two Epi-ID experiments was plotted as H3K79me1 versus H3K79me3 , each normalized to H3 ( Figure 1B ) .", "Data was median-normalized within each library subset , based on the assumption that most mutant strains have a wild-type level of H3K79 methylation .", "Uniquely-barcoded dot1Δ , bre1Δ and Dot1 over-expression control strains were added to each library subset as internal controls .", "The E3 ligase Bre1 ubiquitinates histone H2B on lysine 123 , thereby promoting Dot1 activity , and in a bre1Δ strain H3K79 methylation is reduced ( Weake and Workman , 2008 ) .", "A Dot1 over-expression strain has high levels of methylation .", "The spiked-in controls were clear outliers: dot1Δ strains showed low H3K79me1 and H3K79me3 at both the UpTag and DownTag , bre1Δ strains showed low H3K79me3 and high H3K79me1 , and Dot1 over-expression strains showed high H3K79me3 and low H3K79me1 .", "The independent dot1Δ and bre1Δ strains present in the original library behave the same as their added counterparts .", "The results of the spiked-in control strains confirmed that Epi-ID can be used to identify strains with lower and higher levels of H3K79 methylation in pools of mutants .", "Several other strong outliers could readily be explained , since they were known to affect H2B ubiquitination and H3K79 methylation ( Figure 1C ) .", "Positive regulators of H3K79 methylation were Rad6 and Lge1 , which form the H2B ubiquitination complex together with Bre1 ( Weake and Workman , 2008 ) , and Rtf1 , which is part of the PAF transcription-elongation complex and recruits Bre1/Rad6 to chromatin of transcribed regions ( Piro et al . , 2012 ) .", "Ubp8 and its partners in the deubiquitinase ( DUB ) module of the SAGA complex ( Sgf73 , Sgf11 and Sus1 ) together deubiquitinate H2B and predominantly act at the 5’ ends of transcribed regions ( Bonnet et al . , 2014; Morgan et al . , 2016; Schulze et al . , 2011 ) .", "In the Epi-ID screen , deletion of the genes encoding these proteins led to increased methylation on the UpTag , but not on the DownTag , as expected given their respective promoter and terminator context .", "Notably , deletion of the other H2B DUB , UBP10 , did not increase H3K79me on the barcodes , consistent with the observation that Ubp10 preferentially acts on telomeres ( Gardner et al . , 2005 ) .", "In summary , Epi-ID identified all established H3K79 methylation regulators acting via H2Bub in euchromatin .", "H3K79 methylation is very stable and accumulates on old histones ( De Vos et al . , 2011 ) .", "No H3K79 demethylases are known ( Sweet et al . , 2010; Zee et al . , 2010 ) and the Epi-ID screen provided no evidence of an H3K79 demethylase either .", "None of the genes with a known demethylase domain was found among the negative regulators ( Figure 2—figure supplement 2 ) .", "Given the likely absence of a demethylase , the main mechanism to counteract Dot1 activity may be dilution of methylated histones .", "This can occur by replication-independent turnover of histones ( Zentner and Henikoff , 2013 ) , or by dilution during S phase when the biggest influx of new histones occurs .", "A negative correlation between histone turnover and H3K79me3 level has been observed in genome-wide maps ( Radman-Livaja et al . , 2011; Weiner et al . , 2015 ) .", "Based on these observations and computational modeling , it has been proposed that slow-growing cells accumulate more H3K79 methylation ( De Vos et al . , 2011 ) .", "To test this hypothesis without having to change temperature or carbon source , we used barcode-seq to derive growth rates for each of the knock-out clones in the pool of cells ( Figure 2—source data 1 and Figure 2—figure supplement", "1 ) and determined the relation between growth rate and H3K79me level .", "As a single score for H3K79 methylation level , we used the H3K79me3/H3K79me1 ratio .", "This score was very robust , with a Pearson correlation of 0 . 89 between two screens .", "Figure 2A shows a negative correlation between the growth rate and the H3K79 methylation score on both UpTag and DownTag ( r = −0 . 4 ) and methylation was significantly higher for the slow growers than for the remainder of the yeast strains ( Figure 2A ) .", "It can also be appreciated from this plot that the known regulators identified in Figure 1 remain outliers after taking into account the growth defects that some of them have . 10 . 7554/eLife . 18919 . 006Figure 2 . H3K79 methylation regulation by growth and acetyltransferases .", "( A ) Scatter plots of growth rate and H3K79 methylation ( me3/me1 ) for UpTag and DownTag , each dot representing a mutant strain .", "The Pearson correlation coefficient is shown in the plot .", "The red line is the linear model fitting the data best and was used for correcting the H3K79 methylation score .", "Highlighted strains were ignored in the analysis , because they lacked validated H3K79me regulators .", "Alongside the scatter plot is a Tukey box plot to compare the median and spread of H3K79 methylation for the bottom-10% slowest growers , compared to the other 90% .", "These populations are highly significantly different , as determined by a T test .", "( B ) Bar charts of growth-corrected H3K79 methylation scores of deletion strains , showing the strongest positive and negative outliers on UpTag and DownTag .", "Because individual outliers are shown , a cutoff was applied on variation between the two biological replicates ( c . o . v . <0 . 35 ) to increase confidence .", "Mean and individual data points of two experiments are shown .", "Strain bre1_C is the control bre1Δ strain that was taken along multiple times in each experiment , whereas the other bre1Δ strain was part of the library .", "Strain rtt109Δ and NatA complex mutants have been highlighted , as well as mutants of the SAGA HAT module that will be discussed later on .", "( C ) ChIP-qPCR analysis at the HO promoter , near the UpTag .", "Plotted is the ratio between H3K79me3 and H3K9me1 IP values .", "Four wild-type ( mean with SD ) and two rtt109Δ strains ( individual data points shown ) were compared by an unpaired T test . DOI: http://dx . doi . org/10 . 7554/eLife . 18919 . 00610 . 7554/eLife . 18919 . 007Figure 2—source data 1 . Growth rates calculated for all deletion strains . DOI: http://dx . doi . org/10 . 7554/eLife . 18919 . 00710 . 7554/eLife . 18919 . 008Figure 2—source data 2 . Growth-corrected H3K79me scores . DOI: http://dx . doi . org/10 . 7554/eLife . 18919 . 00810 . 7554/eLife . 18919 . 009Figure 2—source data 3 . ChIP-qPCR data at the HO promoter , WT vs rtt109Δ .", "DOI: http://dx . doi . org/10 . 7554/eLife . 18919 . 00910 . 7554/eLife . 18919 . 010Figure 2—figure supplement 1 . Growth rate determination .", "( A ) Approximate cell counts at different time points , for each of the pools of cells .", "t = 0 is after scraping the cells off an agar plate on which they had grown for approximately 16 hr .", "The coloured dots indicate the cell counts estimated by optical density , the dashed line starts from the average cell count at t = 0 ( disregarding 4B ) and increases with the standard logarithmic growth rate of 0 . 42 h−1 ( Di Talia et al . , 2007 ) .", "Since the measurements were close to the dashed line throughout the experiment , it was fair to assume logarithmic growth .", "When calculating individual growth rates , the assumption of a median growth rate of 0 . 42 h−1 in each pool was used rather than the cell counts , since it was deemed more robust .", "( B ) Comparison of the growth rates obtained from two replicate experiments; each dot is a deletion strain .", "The growth rates per strain can be found in Figure 2—source data 1 .", "( C ) Distribution of calculated growth rates for over 4000 knock-out strains . DOI: http://dx . doi . org/10 . 7554/eLife . 18919 . 01010 . 7554/eLife . 18919 . 011Figure 2—figure supplement 2 . Growth-corrected H3K79me scores of genes that contain potential demethylase domains . Data taken from Figure 2—source data 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 18919 . 011 Having determined the relationship between growth and H3K79 methylation , we calculated a growth-corrected methylation score for each strain , by dividing the H3K79me3/H3K79me1 ratio by the methylation shift expected for the fitness of the strain ( Figure 2—source data 2 ) .", "The expected shift was based on the linear model fitting the data ( the red line in Figure 2A ) .", "The robust top outliers based on this growth-corrected methylation score are shown in Figure 2B .", "Rtt109 was one of the strongest negative regulators of H3K79 methylation on the UpTag ( Figure 2B ) , and high H3K79 methylation upon deletion of RTT109 could be validated by ChIP-qPCR ( Figure 2C ) .", "Rtt109 is a histone acetyltransferase that acetylates newly synthesized histone H3 on lysine 56 ( Driscoll et al . , 2007; Han et al . , 2007 ) .", "Through this activity , Rtt109 promotes histone transport and nucleosome assembly ( Dahlin et al . , 2015 ) .", "RTT109 deletion directly leads to decreased turnover at ‘hot’ nucleosomes , mostly found in promoters ( Dion et al . , 2007; Kaplan et al . , 2008 ) .", "The fact that Rtt109 was one of the strongest negative regulators of H3K79me at the UpTag , i . e . in a promoter region , indicates that histone turnover is an important determinant of the H3K79me level .", "Altogether , these data support the idea that no H3K79 demethylase is active in yeast and show that the deposition of new histones ( replication-coupled or -independent ) is an important mechanism to counteract H3K79 methylation .", "Among the strongest positive regulators of H3K79me on both the UpTag and DownTag were Nat1 and Ard1 , the two components of the NatA N-acetyltransferase complex .", "The DownTag score of the nat1Δ strain was filtered out in Figure 2B based on its variation between replicates , but it was a positive regulator as well .", "Ard1 has been reported to promote H2Bub and specifically H3K79me3 , but the role of Nat1 remained uncertain ( Takahashi et al . , 2011 ) .", "We confirmed the effect of Ard1 on H2B ubiquitination and H3K79 methylation , and found an identical effect for Nat1 ( Figure 3A ) .", "Also H3K4me3 and H3K36me3 were decreased in nat1Δ and ard1Δ strains , and again the effect was partial compared to the bre1Δ strain ( Figure 3A ) .", "H3K4me3 is known to depend on H2B ubiquitination ( Dover et al . , 2002 ) , but the decrease in H3K36me3 we observed in the bre1Δ strain was not reported before .", "We confirmed the decrease in H3K36me3 in the absence of H2B ubiquitination ( Figure 3—figure supplement 1C ) and observed that H3K36me2 was not affected .", "We conclude that the NatA complex is required for a normal H2Bub level and thereby promotes all downstream methylation events .", "Notably , that NatA acts upstream of Dot1 is in agreement with our previous observation that NatA and Dot1 act in the same silencing pathway ( van Welsem et al . , 2008 ) . 10 . 7554/eLife . 18919 . 012Figure 3 . Positive regulators of H3 methylation .", "( A ) Immunoblots of H3 methylation and H2B in the indicated strains .", "Biological replicates are shown in Figure 3—figure supplement 1A .", "( B ) Representative image of immunoblots of H3 methylation and H2B in wild-type and ado1Δ strains .", "Alongside is the quantification of the three biological replicates run on these gels .", "Uncropped blots are shown in Figure 3—figure supplement 2A , more replicates in 2B .", "( C ) Schematic depiction of the methionine cycle in budding yeast .", "Dot1 and Ado1 have been highlighted .", "In addition to Dot1 , SAM is utilized by many other methyltransferases .", "( D ) Levels of SAM and SAH on a logarithmic scale , and their ratio on a linear scale .", "n = 7–8 , comparison by multiple T test . DOI: http://dx . doi . org/10 . 7554/eLife . 18919 . 01210 . 7554/eLife . 18919 . 013Figure 3—source data 1 . Raw SAM and SAH measurements and calculations . DOI: http://dx . doi . org/10 . 7554/eLife . 18919 . 01310 . 7554/eLife . 18919 . 014Figure 3—figure supplement 1 . NatA immunoblots .", "( A ) Immunoblots of H3 methylation and H2B in the indicated deletion strains .", "Biological replicates of Figure 3A .", "( B ) Model of the dependence of H3K79 methylation on Dot1 activity ( modified from De Vos et al . , 2011 ) , with dashed lines indicating the estimated Dot1 activities in the indicated strains .", "The decrease in H3K79me3 and increase in K79me1 fit with lower overall Dot1 activity , but the effect of deletion of the NatA complex is not as strong as BRE1 deletion .", "( C ) Immunoblots showing the effect of an H2B-K123R mutation and BRE1 deletion on H3K36me2 ( no effect ) and H3K36me3 ( decrease ) .", "Bands shifted by the extra weight of the FLAG tagged have been marked with an F . ( D ) Dot1 expression levels are unaltered in nat1Δ and ard1Δ strains .", "( E ) Because a C-terminal tag on Bre1 disrupts its function ( Wood et al . , 2003 ) and an N-terminal tag interferes with potential N acetylation of the native N terminus of Bre1 , we performed an epistasis experiment to address whether the role of NatA in H2B ubiquitination was mediated by N-acetylation of Bre1 .", "FLAG-Bre1 has a normal activity that is still NatA dependent , which demonstrates that the NatA complex does not act though N-acetylation of Bre1 .", "( F ) TAP blots were used to measure the expression levels of C-terminally TAP-tagged versions of the indicated proteins .", "Representative blot .", "( G ) Quantification of the blot shown in panel F , as well as blots from two independent experiments .", "Only Rtf1 expression is significantly altered , but an increase in Rtf1 is not consistent with a decrease in H2Bub . DOI: http://dx . doi . org/10 . 7554/eLife . 18919 . 01410 . 7554/eLife . 18919 . 015Figure 3—figure supplement 2 . Ado1 immunoblots .", "( A ) Uncropped blots of Figure 3B , showing H3 methylation and H2B ubiquitination in wild-type and ado1Δ strains , as well as some controls .", "Alongside is the quantification of these blots .", "Replicates are independent samples of the same strains .", "Methylation of H3K79 , H3K4 and H3K36 all decrease on average , but the decreases seem to be ( partially ) compensated by an increase in H2Bub , especially in the first sample .", "( B ) Immunoblots like in A , from independent samples of one wild-type and one knock-out strain , with the quantification alongside .", "( C ) Dot1 blots show that ADO1 deletion does not alter Dot1 expression . DOI: http://dx . doi . org/10 . 7554/eLife . 18919 . 015 NatA acetylates about one third of the proteins encoded in the yeast genome and may affect their stability or interactions to other proteins ( Aksnes et al . , 2016 ) .", "We checked whether altered protein levels of Dot1 and H2Bub regulators , all of which are putative NatA targets based on sequence ( Aksnes et al . , 2016 ) , could explain the reduced H3K79me and H2Bub abundance in nat1Δ strains .", "However , no significant decrease in Dot1 and H2B ubiquitination factors or increase in H2Bub deubiquitinating factors was observed ( Figure 3—figure supplement 1D–G ) .", "Fully understanding which of the many N-acetylated proteins are responsible for the role of NatA in control of H2B ubiquitination and the downstream methylation events will require a comprehensive mutational analysis of the yeast N-terminal proteome .", "Another strong positive H3K79me regulator was ADO1 .", "At both the UpTag and DownTag the ado1Δ strain showed very low H3K79 methylation .", "Immunoblot analysis confirmed ADO1 as a positive regulator of H3K79 methylation , showing that also on a global level there is a shift from H3K79me3 to H3K79me1 in the ado1Δ strain ( Figure 3B ) .", "In addition , H3K4me3 and H3K36me3 were decreased in ado1Δ cells ( Figure 3B ) .", "To rule out that the methylation decreases were caused by low H2B ubiquitination , H2Bub levels were determined by immunoblot .", "H2Bub was not decreased in ado1Δ cells ( Figure 3B ) , if anything it was somewhat increased , which could mask a stronger methylation defect ( Figure 3—figure supplement 2A , B ) .", "Dot1 protein expression was not altered in the ado1Δ strain ( Figure 3—figure supplement 2C ) .", "The ADO1 gene encodes yeast adenosine kinase , responsible for phosphorylating adenosine to generate AMP .", "Clearance of adenosine by Ado1 may indirectly affect the methylation cycle and thereby the methylation potential in the cell ( Boison , 2013; Figure 3C ) .", "Disruption of the methionine cycle by genetic perturbation or nutrient supply is known to affect histone methylation in yeast and other organisms ( Janke et al . , 2015; Sadhu et al . , 2013 ) .", "To investigate the role of adenosine kinase in the SAM cycle , we determined SAM and SAH levels in ado1Δ and wild-type cells .", "As shown in Figure 3D , SAH levels increased 19-fold in the absence of adenosine kinase .", "This was expected based on the reaction scheme shown in Figure 3C , and is in agreement with the lower histone H3 methylation levels ( Figure 3B ) , since SAH is a known inhibitor of methyltransferases ( Etchegaray and Mostoslavsky , 2016; Richon et al . , 2011 ) .", "However , SAM levels increased by as much as 75-fold , causing an unexpected 4-fold increase of the SAM/SAH ratio .", "The increase in SAM levels may be caused by a global inhibition of methyltransferases by excess SAH and therefore reduced SAM usage , or by compensatory mechanisms such as altered expression of methionine biosynthesis genes ( Kanai et al . , 2013 ) .", "Although the SAM/SAH ratio is often considered to determine the activity of methyltransferases ( Etchegaray and Mostoslavsky , 2016 ) , our findings suggest that the absolute levels of SAH and SAM are also important .", "To find the more subtle second-level regulators of H3K79me in a systematic and unbiased manner , we first used Cutoff Linked to Interaction Knowledge ( CLIK ) to determine the threshold above which outliers were likely to be genuine regulators ( Dittmar et al . , 2013 ) .", "CLIK is based on the notion that the rate of interactions among true positives in a screen is higher than for a random set of genes .", "The algorithm indeed identified many genetic and/or physical interactions between genes at both the low methylation and high methylation ends of the data , for both UpTag and DownTag ( Figure 4—figure supplement 1 ) , and determined cutoffs based on the points in the rank list where interaction density dropped .", "The groups defined in this way were considered candidate regulators .", "Rather than focusing on individual genes in these groups , we took two bioinformatics approaches to identify complexes and processes that regulate H3K79 methylation .", "First , the built-in complex enrichment analysis in the CLIK tool identified enriched complexes in all groups of candidate regulators ( Table 1 and Table 1—source data 1 ) .", "Few new complexes were identified among the positive regulators , so we focused our attention on the negative regulators .", "Strikingly , of the enriched complexes on the UpTag , Mms22-Rtt101-Mms1 , Slx5-Slx8 , and Rad51-Rad57 were all involved in DNA repair , specifically at replication forks ( see below ) ( Branzei et al . , 2006; Costes and Lambert , 2012; Duro et al . , 2008; Su et al . , 2015 ) .", "Second , we used PANTHER ( Mi et al . , 2016 ) to identify processes enriched among the candidate negative regulators at the UpTag ( Table 2 ) .", "With the exception of two very general terms , all enriched GO processes fall into the DNA repair category .", "The highest enrichment scores were found for double-strand break ( DSB ) repair via homologous recombination ( HR ) and its parent recombinational repair ( Table 2 , Figure 4A ) . 10 . 7554/eLife . 18919 . 016Table 1 . Enriched complexes in the candidate regulator groups ( thresholds determined by the CLIK tool ) , on UpTag and DownTag .", "Complex enrichment was determined by the built-in complex enrichment tool on the CLIK website .", "Within each group , enriched complexes are ranked by p value , only complexes with a p value below 0 . 01 are shown .", "For complexes with an asterisk , all components present in the data were found in the CLIK group . DOI: http://dx . doi . org/10 . 7554/eLife . 18919 . 01610 . 7554/eLife . 18919 . 017Table 1—source data 1 . CLIK groups and complexes enriched in each group . DOI: http://dx . doi . org/10 . 7554/eLife . 18919 . 017Complex Candidate regulators Not in candidate regulator group Not in dataset P value Low methylation ( positive regulators ) on UpTag ( top-49 of 4231 ) Lge1/Bre1 complex *2001 . 2E-04Low methylation ( positive regulators ) on DownTag ( top-64 of 4238 ) Lge1/Bre1 complex *2002 . 1E-04Proteasome complex2830 . 009H+-transporting ATPase2830 . 009High methylation ( negative regulators ) on UpTag ( top-247 of 4231 ) Rad51-Rad57 *5004 . 9E-07SAGA complex45139 . 2E-04Slx5/Slx8 complex *2000 . 003Mms22/Rtt101/Mms1 complex *2010 . 003Mdm12/Mmm1/Mdm10 complex *2010 . 003ER V-ATPase assembly complex *2000 . 003DNA-directed RNA polymerase II , holoenzyme3480 . 005SWI/SNF complex3450 . 005High methylation ( negative regulators ) on DownTag ( top-274 of 4238 ) Mediator complex64141 . 3E-05Kornberg's mediator ( SRB ) complex65142 . 7E-05Rpd3L complex5431 . 2E-04Slx5/Slx8 complex *2000 . 004Mms22/Rtt101/Mms1 complex *2010 . 004Actin cytoskeleton-regulatory complex *2010 . 004Bub1/Bub3 complex *2000 . 004Kinetochore34110 . 00810 . 7554/eLife . 18919 . 018Table 2 . Enriched processes in the group of candidate negative regulators on the UpTag .", "Enrichment determined by PANTHER ( Mi et al . , 2016 ) .", "Terms are organized by hierarchy; daughter terms are indented .", "P values were Bonferroni-corrected . DOI: http://dx . doi . org/10 . 7554/eLife . 18919 . 018GO biological process completeEnrichmentp valueDNA metabolic process ( GO:0006259 ) 2 . 35 . 2E-03cellular response to DNA damage stimulus ( GO:0006974 ) 2 . 91 . 8E-04> DNA repair ( GO:0006281 ) 3 . 24 . 6E-05> > double-strand break repair ( GO:0006302 ) 4 . 48 . 8E-04> > recombinational repair ( GO:0000725 ) 5 . 91 . 2E-04> > > double-strand break repair via homologous recombination ( GO:0000724 ) 5 . 55 . 2E-03chromosome organization ( GO:0051276 ) 2 . 36 . 3E-04macromolecular complex subunit organization ( GO:0043933 ) 1 . 87 . 7E-0310 . 7554/eLife . 18919 . 019Figure 4 . DNA repair genes are negative regulators of H3K79 methylation .", "( A ) Genes from the GO process of double-strand break repair by homologous recombination , which are present in the dataset .", "Genes from the CLIK group of negative regulators are on the left ( in red ) , positive regulators on the right ( in green ) .", "( B ) ChIP-qPCR analysis of H3K79 methylation on the HO promoter , close to the UpTag .", "K79me IPs were normalized to H3; mutants were normalized to wild-type .", "4–6 replicates per strain , compared to one by one-sample T test .", "( C ) MS analysis of H3K79 methylation levels in wild type and mutants .", "Mean and individual data points are shown . DOI: http://dx . doi . org/10 . 7554/eLife . 18919 . 01910 . 7554/eLife . 18919 . 020Figure 4—source data 1 . Normalized ChIP-qPCR data on HO promoter . DOI: http://dx . doi . org/10 . 7554/eLife . 18919 . 02010 . 7554/eLife . 18919 . 021Figure 4—source data 2 . Raw mass spectrometry data . DOI: http://dx . doi . org/10 . 7554/eLife . 18919 . 02110 . 7554/eLife . 18919 . 022Figure 4—figure supplement 1 . CLIK plots . This figure is related to the data displayed in Table 1 .", "Plots were generated by the CLIK web tool ( http://www . rothsteinlab . com/tools/clik; Dittmar et al . , 2013 ) using lists of ORFs ranked on growth-corrected H3K79me score as input .", "The main clusters shown in the magnifications were analysed for enriched complexes , and the results of this analysis can be found in Table 1 and Table 1—source data 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 18919 . 02210 . 7554/eLife . 18919 . 023Figure 4—figure supplement 2 . Excluding mechanisms of H3K79me regulation by Slx5 , Slx8 and Mms21 . ( A ) Growth assay after serial dilution on solid media of strains before and after removal of the ( endogenous ) 2µ plasmid .", "PCR shows that the plasmid was indeed lost after curing , and that in this case the plasmid had already spontaneously been lost in the slx5Δ strain .", "Removal of the over-replicated 2µ plasmid relieves the growth defect in slx5Δ and slx8Δ strains , as described previously ( Burgess et al . , 2007 ) .", "( B ) Mini Epi-ID experiment for ubiquitinated H2B on the indicated strains .", "H2Bub/H3 was normalized to wild-type .", "3–9 replicates each .", "Comparison with wild-type was done using one-way ANOVA , ubp8Δ is shown for comparison .", "WT and ubp8Δ data is the same as in Figure 2G .", "H2Bub level at the UpTag was unaltered in the SUMO-ubiquitin pathway mutants , suggesting that H3K79me regulation occurs through another mechanism .", "( C ) Immunoblot analysis of Dot1 levels in the indicated strains , Pgk1 serves as a loading control .", "Slx5 and Slx8 do not regulate Dot1 expression level , suggesting that H3K79me regulation occurs through another mechanism . DOI: http://dx . doi . org/10 . 7554/eLife . 18919 . 023 To investigate this further , we performed validation experiments for the strongest individual outliers .", "Slx5 and Slx8 form a heterodimeric SUMO-targeted Ubiquitin Ligase complex ( StUbL ) that is required to maintain genomic stability ( Nagai et al . , 2008; Xie et al . , 2007 ) .", "Strains lacking Slx5 or Slx8 grow very slowly due to the amplification of endogenous 2µ circles ( Burgess et al . , 2007 ) , so we first relieved the growth defect of these strains by curing them ( and a wild-type control ) of the 2µ plasmid ( Figure 4—figure supplement 2A ) .", "Because the substrates of the Slx5/Slx8 StUbL complex largely overlap with those of the SUMO ligase Mms21 ( Albuquerque et al . , 2013 ) , we also generated a strain in which the SUMO ligase activity of Mms21 was compromised ( Zhao and Blobel , 2005 ) .", "As shown in Figure 4B , ChIP-qPCR showed an increase in methylation at the HO promoter , near the UpTag , in both slx8Δ and mms21ΔC , implicating the SUMO-ubiquitin pathway in regulating H3K79 methylation .", "A small global increase in methylation was observed in these strains by mass spectrometry ( Figure 4C ) , suggesting that the effect was not limited to the barcode regions .", "No DNA-damaging agents were present in the Epi-ID experiment .", "However , also in untreated cells homologous recombination is needed to restart replication forks that have stalled or collapsed , for instance at structure-forming sequences or replication-fork barriers ( Costes and Lambert , 2012 ) .", "Mms22 , Rtt101 and Mms1 ( of which Rtt101 was not in the dataset ) form a Cul4-like E3 ubiquitin ligase that sits at replication forks and promotes restart of stalled forks , presumably through homologous recombination ( Buser et al . , 2016; Vaisica et al . , 2011 ) .", "Rad52 and its partners are central players in homologous recombination , also at replication forks ( Costes and Lambert , 2012 ) .", "The Slx5 and Slx8 StUbL complex and Mms21 promote translocation of DNA breaks and collapsed replication forks to nuclear pores ( Burgess et al . , 2007; Horigome et al . , 2016; Su et al . , 2015 ) .", "Interestingly , also the nuclear pore component required for this translocation , Nup84 ( Su et al . , 2015 ) , was identified as a negative regulator of H3K79me in the Epi-ID screen .", "At the pore , Slx5/8 modulate homologous recombination by targeting protein substrates for degradation , including Rad52 itself ( Su et al . , 2015 ) .", "Other factors from the process of HR-mediated DSB repair that were among the negative regulators ( Figure 4A ) include Sgs1 , which also facilitates re-initiation of stalled replication forks ( Ashton and Hickson , 2010 ) , and Ctf4 , which tethers Mms22 to the replisome ( Buser et al . , 2016 ) .", "Taken together , among the second-level regulators of H3K79me are many factors required to allow recovery of stalled replication forks .", "This suggests that this process regulates H3K79me , either because the repair itself counteracts methylation or inhibits Dot1 , or because unrepaired collapsed replication forks lead to accumulation of H3K79 methylation .", "Since H2B ubiquitination has been described to facilitate fork recovery ( Lin et al . , 2014 ) , we wondered if the regulation of H3K79 methylation could be through H2B ubiquitination .", "However , H2Bub levels were not increased ( Figure 4—figure supplement 2B ) , and neither was Dot1 expression ( Figure 4—figure supplement 2C ) .", "Unraveling the mechanism of this regulation will require further studies .", "Another interesting point is the function of this regulation .", "Dot1 has been shown to function in several DNA repair pathways; it mediates checkpoint activation and recombinational repair after UV damage , and represses translesion synthesis ( Conde and San-Segundo , 2008; Rossodivita et al . , 2014 ) .", "Dot1 has not yet been studied in the context of replication fork stalling , but our finding that H3K79 methylation is regulated in this context warrants further investigations .", "Finally , since homologues of all involved proteins can be found in mammals , it will be interesting to see if also the described H3K79me regulation is conserved .", "SAGA complex subunits were highly enriched among the negative H3K79me regulators on the UpTag ( Table 1 ) .", "These not only included subunits of the deubiquitinase ( DUB ) module , but also subunits of the histone acetyltransferase ( HAT ) module .", "Indeed , Gcn5 and its partners in the HAT module , Ada2 and Ngg1 ( Ada3 ) , were among the strongest negative regulators ( Figure 2B ) .", "No data was obtained on Sgf29 , the last SAGA HAT module subunit .", "The specificity these factors showed for the UpTag is not surprising given the activity of Gcn5 at promoters ( Bonnet et al . , 2014 ) .", "Mass spectrometry measurements demonstrated that the effect of Gcn5 was not limited to the UpTag , since a gcn5Δ strain also had more H3K79me3 and less H3K79me1 globally ( Figure 5A ) .", "To investigate the role of Gcn5 in more detail , we analyzed the behavior of a specific point mutant , Gcn5-F221A .", "This mutant protein has previously been shown to be catalytically inactive ( Kuo et al . , 1998; Lanza et al . , 2012 ) .", "To allow for a direct , quantitative , and sensitive comparison , we performed a custom-designed Epi-ID experiment with a set of gcn5Δ strains with single-copy plasmids .", "In contrast to wild-type Gcn5 , the Gcn5-F221A mutant was not able to rescue the knock-out phenotype ( Figure 5B; Figure 5—figure supplement 1A , C ) .", "This finding suggests that H3K79me regulation by Gcn5 depends on its catalytic activity .", "However , it cannot be excluded that the F221A mutation acts by other mechanisms such as disrupting nucleosome binding or altering the stability of Gcn5 . 10 . 7554/eLife . 18919 . 024Figure 5 . Gcn5 regulates H2B ubiquitination and H3 methylation .", "( A ) MS analysis of H3K79 methylation levels in wild-type and gcn5Δ strains .", "Mean and individual data points of two biological replicates .", "( B ) Custom Epi-ID experiment on strains harboring empty or GCN5-encoding CEN plasmids , grown in YC-LEU .", "Gcn5* contains the F221A mutation , abrogating catalytic activity .", "H3K79me1/H3 and H3K79me3/H3 are normalized to the mean of ten wild-types , five or six replicates per mutant .", "( C ) Custom Epi-ID results for H2Bub , H3K4me3 and H3K36me3 , from the same experiment as shown in panel B . ( D ) ChIP-qPCR analysis of H2Bub and H3K9ac , normalized to H2B , at the HO promoter near the UpTag .", "n = 3 each , the wild-type average was set to 1 .", "( E ) Immunoblots of a GCN5+ and gcn5Δ strain , showing the levels of ( ubiquitinated ) H2B , Dot1 and Pgk1 as a loading control .", "The FLAG blot shows expression levels of Bre1 , which was N-terminally FLAG-tagged in these strains .", "Replicate blots can be found in Figure 5—figure supplement 2A .", "( F ) TAP blots were used to measure the expression levels of C-terminally TAP-tagged versions of the indicated proteins .", "Representative blot; note that the Rad6-TAP band is only just above the non-specific band indicated with an asterisk .", "( G ) Quantification of the FLAG and TAP blots shown in panels E and F , as well as two TAP blots from independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 18919 . 02410 . 7554/eLife . 18919 . 025Figure 5—source data 1 . Raw mass spectrometry data . DOI: http://dx . doi . org/10 . 7554/eLife . 18919 . 02510 . 7554/eLife . 18919 . 026Figure 5—source data 2 . Data from custom Epi-ID experiment . DOI: http://dx . doi . org/10 . 7554/eLife . 18919 . 02610 . 7554/eLife . 18919 . 027Figure 5—source data 3 . Data from H2Bub and H3K9ac ChIP-qPCR experiment . DOI: http://dx . doi . org/10 . 7554/eLife . 18919 . 02710 . 7554/eLife . 18919 . 028Figure 5—figure supplement 1 . Custom Epi-ID experiments with controls .", "( A ) Custom Epi-ID experiment shown in Figure 5B , with the control strains included in the experiment .", "All strains harbored a LEU2 CEN plasmid ( empty , or containing GCN5 , as indicated ) and were grown up in a pool in YC-LEU .", "Gcn5* bears the F221A mutation , which abrogates catalytic activity .", "One replicate for set1Δ and set2Δ , three replicates for dot1Δ , bre1Δ and ubp8Δ , 5 or six replicates per gcn5Δ mutant and ten replicates of wild type .", "( B ) Custom Epi-ID results for H2Bub , H3K4me3 and H3K36me3 , from the same experiment as shown in panel A . Gcn5 data as shown in Figure 5C .", "H2Bub and H3K36me3 are expected to be low at this intergenic ( promoter-like ) locus , explaining why the controls for these marks show smaller decreases than for H3K4me3 , which is enriched around the transcription start site .", "( C ) Independent custom Epi-ID experiment .", "Like in panel A , but with five wild types and three replicates per GCN5 mutant . DOI: http://dx . doi . org/10 . 7554/eLife . 18919 . 02810 . 7554/eLife . 18919 . 029Figure 5—figure supplement 2 . Immunoblots confirming the effect of Gcn5 on global H2Bub and Ubp8 levels .", "( A ) Replicates of Figure 5E .", "Immunoblots showing that Gcn5 does not alter Dot1 expression , and causes a global increase in H2Bub .", "BRE1 harbors an N-terminal FLAG tag in these strains .", "( B ) Immunoblot showing H2B ubiquitination in the indicated strains .", "All strains were on the same blot , but the blot was cropped to remove irrelevant samples .", "The asterisk indicates an aspecific band .", "( C ) Immunoblot quantification of H2Bub/H2B levels in mutant strains , normalized to wild-type ( 1 , indicated with a dashed line ) .", "( D ) Immunoblot showing the effects of GCN5 or NAT1 deletion on Ubp8-TAP abundance in chromatin .", "Sir2 was used as a loading control .", "( E ) Quantification of the blot shown in D . DOI: http://dx . doi . org/10 . 7554/eLife . 18919 . 02910 . 7554/eLife . 18919 . 030Figure 5—figure supplement 3 . Gcn5 regulates H2Bub and H3K9ac at several loci . ChIP-qPCR analysis of H2Bub and H3K9ac , normalized to H2B , at several loci .", "n = 3 each , the wild-type average was set to 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 18919 . 030 Dot1 protein expression was unaffected by deletion of GCN5 ( Figure 5E ) .", "Since Gcn5 is found in the SAGA complex , just like the H2Bub DUB Ubp8 , we wondered if Gcn5 could function through H2B ubiquitination .", "Indeed , gcn5Δ strains were found to have significantly more H2Bub on the UpTag than wild-type strains , and this was also the case for cells expressing the inactive Gcn5-F221A protein ( Figure 5C ) .", "The effect of Gcn5 on the H2Bub level at this locus was comparable to the effect of Ubp8 ( Figure 5—figure supplement 1B ) .", "Since H2Bub levels were affected , we also tested the effect on H3K4me3 and H3K36me3 in the custom Epi-ID experiment and found both to be increased in the absence of Gcn5 acetyltransferase activity ( Figure 5C ) .", "Thus , at the UpTag we found the activity of Gcn5 to negatively regulate H2B ubiquitination and , probably through its ubiquitination effect , methylation of H3K4 , H3K36 and H3K79 .", "To validate the H2Bub increase caused by GCN5 deletion by an independent method and to test whether the effect was not limited to the barcode region , we took two approaches .", "First , using ChIP-qPCR , both an increase in H2Bub and a decrease in H3K9ac could be observed at the HO promoter , near the UpTag in gcn5Δ strains ( Figure 5D ) .", "These changes were also observed at several other loci , but not at all of the loci examined ( Figure 5—figure supplement 3 ) .", "In the future , it will be interesting to determine where H2Bub regulation by Gcn5 occurs and how the specificity for certain regions is established .", "Second , immunoblotting was used to determine the global change in H2Bub level .", "Both gcn5Δ and ada2Δ strains showed increased H2Bub -levels ( Figure 5E , Figure 5—figure supplement A–C ) .", "Gcn5 may modulate H2Bub by acetylating histones or by interactions between the HAT and DUB modules .", "Indeed , acetylation on one of the DUB module components has been reported ( Henriksen et al . , 2012 ) .", "Furthermore , interactions between the HAT module and the DUB module have also been observed by independent approaches in yeast and human ( Atanassov et al . , 2009; Durand et al . , 2014; Han et al . , 2014 ) .", "To investigate this further , we examined the expression of H2Bub regulators and observed that Ubp8 protein expression was significantly decreased in the absence of Gcn5 ( Figure 5E–G ) .", "A reduction in Ubp8 protein level was also observed in chromatin fractions ( Figure 5—figure supplement 2D–E ) .", "Thus , the lower Ubp8 levels can at least in part explain the observed change in H2Bub in gcn5Δ cells .", "These findings suggest that Gcn5 is required for Ubp8 stability , potentially by regulating the association with its partners in the SAGA DUB module .", "Taken together , we identified the SAGA HAT as a negative regulator of H3K79 methylation , and then discovered that it regulates H2B ubiquitination and subsequent methylation events on three lysine residues on histone H3 , at least in part by maintaining Ubp8 protein expression .", "The sensitivity of the Epi-ID technique allowed for identification of this regulatory mechanism , even though the global effects are small and previously went unnoticed ( Lee et al . , 2005 ) .", "Our finding that the SAGA HAT module regulates the DUB , together with the finding that the DUB module regulates HAT activity ( Han et al . , 2014 ) , suggests that the two activities in the SAGA complex are highly interactive , despite being organized in distinct modules ." ], [ "Chromatin regulatory mechanisms are important for genome function and are often deregulated in cancer and other diseases ( Brien et al . , 2016 ) .", "To enable efficient and direct screening for chromatin regulators , we developed Epi-ID , in which chromatin modification or binding-events at DNA barcodes are directly interrogated by ChIP followed by deep sequencing .", "The Epi-ID technique has several advantages over the methods that are currently available .", "First , in contrast to other techniques used to screen for chromatin regulators ( Dover et al . , 2002; Peng et al . , 2008; Schulze et al . , 2009 ) , Epi-ID can be performed on pools of cells , making it easily applicable to large collections of mutants .", "Second , Epi-ID is a direct method , in contrast to reporter gene assays that have been used to read out chromatin changes ( Rossmann et al . , 2011 ) .", "Third , it is a very sensitive method that is able to pick up differences that cannot be detected by immunoblot .", "Finally , by comparing all knock-outs directly in a competitive manner , no complex normalization methods are required .", "As a proof of concept we applied Epi-ID to delineate the regulome of H3K79 methylation in budding yeast .", "All known H3K79 methylation regulators were identified , as well as several new regulators .", "All tested candidate regulators could be validated on a global level and/or locally , by ChIP-qPCR .", "It is clear from our data that the well-studied H2Bub-H3K79me trans-histone cross-talk is the main Dot1 regulatory pathway .", "Not only the factors directly involved in H2B ( de ) ubiquitination were identified as regulators of H3K79 methylation , also new H2Bub modulators were discovered .", "One of these was Gcn5 , which is responsible for the HAT activity in SAGA and was found to negatively regulate H3 methylation and H2Bub , at least in part by maintaining Ubp8 protein abundance .", "No candidate H3K79 demethylase was identified and histone deposition was found to negatively regulate H3K79 methylation , supporting the hypothesis that in the absence of a demethylase histone dilution is the main mechanism to counteract Dot1 activity .", "Several proteins involved in DNA repair through homologous recombination were scored as negative regulators of Dot1 activity , particularly proteins required for the recovery of stalled replication forks through recombination .", "Finally , there are several candidate regulators that we have not yet studied further .", "These factors may regulate Dot1 indirectly , through one of the mechanisms described above , or through a yet to be discovered mechanism .", "Especially for these uncharacterized candidates , indirect effects of gene knock-outs on neighboring genes should also be considered ( Ben-Shitrit et al . , 2012 ) .", "Future studies will be required to clarify the mechanisms of some of these regulators .", "The resource on Dot1 regulation presented here can be used as a starting point for such studies .", "The Epi-ID screen reported here was performed using the knock-out collection of non-essential genes and offers a valuable resource for future studies on the regulation of H3K79 methylation in yeast and other organisms .", "However , Epi-ID is not limited to the use of knock-out collections but is highly flexible with regard to the library being screened .", "Combining barcode libraries with libraries of temperature sensitive alleles , histone mutants , or hypomorphic ( DamP ) alleles ( Ben-Aroya et al . , 2008; Dai et al . , 2008; Yan et al . , 2008 ) may lead to new insights into the contribution of essential genes in regulating chromatin structure and function .", "Dedicated , smaller custom libraries can also be made , as illustrated by the experiment testing the rescue potential of Gcn5-containing plasmids ( Figure 5B ) .", "In this case one indexed Epi-ID experiment was an efficient and robust alternative to performing multiple replicate ChIPs and qPCR analyses .", "The technology can easily be applied to chromatin features other than H3K79 methylation .", "As an example , we showed a small Epi-ID experiment for H2B ubiquitination ( Figure 5C ) .", "Furthermore , a previous version of the technique has been used to identify histone turnover factors , albeit on a smaller scale ( Verzijlbergen et al . , 2011 ) , and Epi-ID for H2A . Z levels has also been performed successfully ( Korthout , van Leeuwen et al . , submitted ) .", "The only requirement for an Epi-ID experiment is that a ChIP against the chromatin feature of interest can pull down one or both of the barcoded regions at the HO locus .", "We envision that it will be feasible to generate a library of clones with barcodes at a locus of interest in the near future , which would eliminate this limitation .", "Such strategies will allow for determining the regulome of many other chromatin features and investigating the cross-talk between the different networks .", "Yeast offers the unique possibility to use genetic crosses and SGA technology to combine gene inactivations with chromatinized DNA barcodes .", "However , the concept of Epi-ID , i . e . barcode-ChIP-seq in mutant backgrounds , is not restricted to yeast and is in principle transferable to other organisms .", "The basic requirement is that mutants can be uniquely identified by a chromatinized DNA sequence , which is the case in shRNA , CRISPR and CRISPRi libraries when the genetic elements to inactivate genes are integrated in the genome ( e . g . Evers et al . , 2016 ) .", "With the recent developments in genome targeting and editing , tools are becoming available to integrate barcoded libraries at a common locus to avoid position effects .", "Taken together , Epi-ID is a highly adaptable technique that enables the identification of new chromatin regulators ." ], [ "Yeast strains and plasmids used in this study are listed in Supplementary file 1 and Supplementary file 2 , respectively .", "Yeast media were described previously ( Tong and Boone , 2006; Van Leeuwen and Gottschling , 2002 ) .", "Library manipulations were done using the RoToR from Singer Instruments ( Watchet , UK ) and the synthetic genetic array ( SGA ) technology ( Tong and Boone , 2006 ) .", "The collection of barcoded knockouts was made by crossing a set of 1140 Barcoder strains ( Yan et al . , 2008 ) to each of five plates making up the MATα NatMX knock-out collection ( Tong and Boone , 2006 ) .", "After mating , diploids were selected with G418 and CloNat double selection on rich media .", "After sporulation , the proper MATa mutant strains were selected in several pinning steps: first on haploid MATa selection ( YC-His+Can+SAEC ) , then twice on MATa double resistance selection ( YC-His+Can+SAEC+MSG+G418+CloNat ) .", "The barcodes and deletions were checked for a few mutants and were found to be correct .", "Three extra Barcoder strains were generated by amplifying the barcoded KanMX locus from strains from the MATa haploid gene knockout library ( Open Biosystems , Huntsville , AL ) and integrating it at the HO locus in strain BY4741 .", "The dot1Δ and bre1Δ strains were taken from the NatMX library and a Dot1 over-expression strain was generated by integrating the TDH3 promoter amplified from pYM-N15 ( Janke et al . , 2004 ) in front of the DOT1 gene in strain Y7092 .", "These strains were crossed to the extra Barcoder strains to generate barcoded controls that have the same background as the library ( NKI4557-4559 ) .", "NKI4560 was generated by selecting a G418-resistant , CloNat-sensitive haploid after the library cross .", "A custom set of barcoded deletion strains was made by re-arraying MATα NatMX deletion strains using the Stinger extension to the RoToR system ( Singer Instruments ) , crossing these to Barcoders and selecting double-resistant MATa haploids .", "Uniquely barcoded CloNat-sensitive MATa haploids were used as wild-type strains .", "Strains were transformed with single-copy CEN-LEU2 plasmids using an adaptation of the microtiter plate LiAc protocol ( Gietz , 2014 ) .", "The Gcn5-containing plasmids were generated by replacing the TRP1 marker in pRS414-GCN5 and pRS414-gcn5-F221A ( van Oevelen et al . , 2006 ) by the LEU2 marker using homologous recombination in yeast .", "NKI4657 was generated by integrating a barcoded KanMX at the HO locus of BY4733 .", "In this background , several deletions were made for validation experiments .", "The endogenous 2µ plasmid was cured from some strains by transforming the pBIS-GALkFLP ( URA3 ) plasmid into the cells ( Tsalik and Gartenberg , 1998 ) , inducing mutant FLP recombinase on galactose-containing medium and then selecting for the loss of the plasmid on media containing 5-fluoroorotic acid ( FOA ) .", "Loss of the 2µ plasmid was confirmed by PCR .", "To test the role of the SUMO ligase activity of the essential Mms21 , we truncated this essential gene at amino acid 183 to eliminate the catalytic domain , yet still support viability ( Zhao and Blobel , 2005 ) .", "This truncation was made by integrating a stop codon , CYC1 terminator and NatMX cassette in the place of the C-terminal MMS21 sequence .", "Strains expressing FLAG-tagged ( mutant ) H2B , NKI4609 and NKI4610 , were made by transforming pRG422 and pRG423 , respectively , into NKI4602 and selecting cells that had lost the original plasmid .", "To determine effects on the expression levels of H2Bub regulators , strains with TAP-tagged alleles were derived from the TAP-Fusion ORF collection ( Dharmacon; Ghaemmaghami et al . , 2003 ) .", "GCN5 and NAT1 were replaced by NatMX in these strains .", "Because we could not retrieve a correct RAD6-TAP clone from the library , RAD6 was TAP-tagged in BY4741 and derivates thereof , with the TAP-KanMX cassette amplified from pFvL29 .", "Finally , because Bre1-TAP loses its interaction with Rad6 ( Wood et al . , 2003 ) , we used KY2513 , in which Bre1 carries a FLAG tag on the N terminus and deleted GCN5 and NAT1 in this strain .", "Briefly , the Epi-ID screens consisted of five steps: ( 1 ) preparing chromatin from pools of cells , ( 2 ) Chromatin Immunoprecipitation experiments , ( 3 ) PCR reactions on purified DNA , ( 4 ) Sequencing , ( 5 ) Data analysis .", "The five plates of the collection were treated separately until after the PCR .", "The five plates were grown on rich media for one night and cells were scraped off and pooled in liquid media in the morning .", "The cultures were grown for 4–5 hr until in log phase and cells were cross-linked with formaldehyde , washed and harvested .", "Chromatin was prepared essentially as in Vlaming et al . ( 2014 ) , in the presence of SDS for H3C and methyl ChIPs , but sonication of chromatin from ~5E9 cells in 1 . 5 mL was done in 15 mL tubes .", "The ChIP was also performed as in Vlaming et al . ( 2014 ) , using polyclonal antibodies against H3K79me1 , H3K79me3 and the H3 C terminus ( Frederiks et al . , 2008 ) .", "In the small-scale Epi-ID experiments , also antibodies against H3K4me3 ( RRID:AB_306649 , lot GR273043-1 ) and H3K36me3 ( RRID:AB_306966 , lot GF260274-1 ) were used , as well as an H2BK123ub antibody that will be described elsewhere ( manuscript in preparation ) .", "The UpTag and DownTag were amplified separately from the purified DNA ( Figure 1—figure supplement 1A ) .", "The scale of each Epi-ID experiment was chosen such that on average an estimated number of 250 copies of each barcode was present in each PCR reaction to minimize jackpot effects ( Figure 1—figure supplement 1D ) .", "The forward primer ( AATGATACGGCGACCACCGAGATCTCGCTCTTCCGATCTAGATGTCCACGAGGTCTCT/AATGATACGGCGACCACCGAGATCTACACTCTTCCGATCTACGGTGTCGGTCTCGTAG for UpTag/DownTag ) introduced the Illumina P5 sequence and extra nucleotides for annealing of the 5’ end of the custom sequencing primers .", "With the reverse primer ( CAAGCAGAAGACGGCATACGANNNNNNGTCGACCTGCAGCGTACG/CAAGCAGAAGACGGCATACGANNNNNNAACGAGCTCGAATTCATCGA for UpTag/DownTag ) the Illumina P7 sequence was introduced , as well as a 6-base-pair index .", "The uniquely barcoded amplicons were then mixed equimolarly and purified from an agarose gel with a size selection of 100–150 bp .", "The purified DNA was sequenced ( single read , >50 bp ) on a HiSeq2500 platform ( Illumina , San Diego , CA ) with High Output Run Mode , using a mix of custom sequencing primers for the UpTag and DownTag ( CGCTCTTCCGATCTAGATGTCCACGAGGTCTCT/ ACACTCTTCCGATCTACGGTGTCGGTCTCGTAG ) .", "Since the above-mentioned primer sequences were not compatible with paired-end flow cells , the oligonucleotide sequences were slightly altered ( see Supplementary file", "3 ) to sequence small-scale Epi-ID experiments on a MiSeq ( Illumina ) .", "A Perl script , eXtracting Counting And LInking to Barcode References ( xcalibr ) , was written to transform raw sequencing reads to tables with counts for each index-barcode combination .", "In short , the U2/D2 sequences were used to assign a read to UpTag or DownTag and the index sequence behind the U2/D2 sequence and the barcode sequence in the beginning of the read were identified .", "The xcalibr source code is available at https://github . com/NKI-GCF/xcalibr .", "In the counts table , counts below ten were removed .", "After that , any barcode below 10% of the median in an input sample of a plate was considered not present on that plate and counts for all indices belonging to this plate were removed .", "The tables were median-normalized for each index and converted to a table with ORFs and experiments based on the barcode-index combinations .", "In this table IP columns could be divided over each other or over input .", "H3K79me data of strains with H3/input of <0 . 5 was discarded .", "Analysis for the small-scale experiments was different in the normalization step , where the average wild-type count was set to 1 .", "Cells were grown up for an Epi-ID experiment , but cells were collected for gDNA isolation at two time points prior to harvesting the cross-linked cells .", "Input material from chromatin made for the Epi-ID experiment ( not described in this study ) was used for the third time point .", "gDNA was isolated as described by Hoffman and Winston ( 1987 ) .", "The relative abundance of the barcodes at each time was used to estimate growth rate for all the deletion strains , assuming a wild-type median growth rate of 0 . 42 h−1 ( Di Talia et al . , 2007 ) and using the formula N ( t ) =N0*eμ*t , where N ( t ) is the number of cells at time t , N0 is the number of cells at t0 and µ is the growth rate .", "Growth rates that gave a goodness of fit of <0 . 95 were discarded .", "Growth rates were calculated using data of the UpTag and DownTag separately , and these values were averaged .", "A few cases were removed , where different rates were calculated for the UpTag and DownTag ( c . o . v . >0 . 2 ) .", "Finally , the growth rates calculated for two independent experiments were averaged .", "To create a growth-corrected H3K79 methylation score , the log2-transformed me3/me1 value expected based on the fitness of the strain was subtracted from the original log2-transformed me3/me1 value .", "The expected value was calculated using the fits shown in Figure 2A .", "Quantitative immunoblotting was performed as described previously ( Vlaming et al . , 2014 ) .", "Protein extracts were made using NaOH or SUMEB lysis buffer .", "Chromatin samples were prepared as described before ( Vlaming et al . , 2014 ) , but without SDS , and incubated with Laemmli buffer at 95°C for 1 hr .", "The antibodies used in the Epi-ID experiment were also used on blots , as well as antibodies against H3K79me2 ( RRID:AB_1587126 ) , Dot1 ( van Leeuwen et al . , 2002 ) , H2B ( 39238 , Active Motif , Carlsbad , CA ) , Pgk1 ( RRID:AB_221541 ) , FLAG ( RRID:AB_259529 ) , TAP ( RRID:AB_10709700 ) and Sir2 ( RRID:AB_656455 ) .", "Analysis of H3K79 methylation levels in the gcn5Δ strain was performed as described before ( De Vos et al . , 2011 ) , using multiple reaction monitoring ( nanoLC-MRM ) using a 4000 Q TRAP mass spectrometer ( AB SCIEX , Framingham , MA ) .", "To measure H3K79 methylation levels in the DNA repair mutants , samples were prepared in the same way as described before ( De Vos et al . , 2011 ) .", "Samples were dissolved in 10% formic acid prior to reverse phase nano-flow liquid chromatography on an EASY nLC 1000 system ( Thermo Scientific , San Jose , CA ) coupled to a Thermo Orbitrap Fusion hybrid mass spectrometer ( Thermo Scientific . San Jose , CA ) .", "Briefly , peptides were separated on a ReproSil‐Pur 120 C18‐AQ 2 . 4 μm ( Dr . Maisch GmbH , Ammerbuch , Germany ) 75 μm × 500 mm analytical column ( packed in house ) in a 30 min . linear gradient from 10% to 40% solvent B ( 0 . 1% formic acid in 8:2 ( v/v ) acetonitrile:water ) followed by a 15‐min wash at 100% solvent B at ~250 nl/min .", "Nanospray was achieved using the Proxeon nanoflex source and fused silica gold coated emitters ( pulled and coated in-house ) at 1 . 55 kV .", "The mass spectrometer was configured in targeted mode to select precursor ions by quadrupole isolation at 1 . 6 Th , followed by HCD fragmentation with a normalized collision energy of 25 and Orbitrap MS2 fragment detection .", "The instrument was run in top speed mode with 3 s cycles .", "PRM parameters were optimized using a set of four purified synthetic peptides with the sequence EIAQDFK*TDLR ( K*: K-me0 , -me1 , -me2 and -me3 ) .", "Briefly , doubly and triply charged precursors were fragmented in the Orbitrap Fusion and their four most abundant fragment ions were selected and validated using Skyline software ( RRID:SCR_014080; MacLean et al . , 2010 ) .", "Label-free quantification was achieved by comparing the area of each methylation state to the sum of the areas of all methylation states .", "As the four different peptides have slightly different physicochemical properties and thus might have slightly different ionization efficiencies in the mass spectrometer , final peptide intensities were corrected using a relative response factor to obtain more accurate results .", "The relative response factor was obtained by measuring the peak areas of the four differently methylated peptide standards from an equimolar mixture and dividing peptide areas by the area of the unmethylated peptide .", "ChIP-qPCR experiments were performed as in Vlaming et al . ( 2014 ) , in the presence of SDS for IPs against H3 or H3K79 methylation .", "A DC protein assay ( Bio-Rad , Hercules , CA ) was used to quantify protein content of chromatin samples , with the purpose of equalizing the amount of chromatin going into each ChIP .", "Antibodies used were the same as for Epi-ID or immunoblot experiments , as well as an antibody against H3K9ac ( RRID:AB_2118292 , lot GR243602-1 ) .", "Oligos used for qPCR can be found in Supplementary file", "3 . Each sample was measured in two technical duplicates in the qPCR and the average value of these two was taken as one value when combining biological replicates .", "40–80M logarithmically-growing cells were pelleted and washed with cold TBS .", "Pellets were resuspended in 500 µL 5% perchloric acid and spun down after 30 min on ice , after which the supernatant was collected .", "SAM and SAH measurements were performed as described before ( Struys et al . , 2000 ) .", "Briefly , SAM and SAH were extracted from yeast lysates using solid phase extraction ( Oasis HLB , Waters , Milford , MA ) .", "Subsequently , the concentrations were determined using positive electrospray liquid chromatography tandem mass spectrometry ( API5000 , Applied Biosystems , Foster City , CA ) .", "The intra- and inter-assay CVs for SAM were 6 . 8% and 4 . 2% , respectively .", "The intra- and inter-assay CVs for SAH were 6 . 9% and 5 . 5% , respectively .", "The online tool Cutoff Linked to Interaction Knowledge ( CLIK ) from the Rothstein laboratory was used to determine outlier groups and calculate enriched protein complexes ( http://www . rothsteinlab . com/tools/clik; RRID:SCR_014690; Dittmar et al . , 2013 ) .", "The CLIK analysis relied on BioGRID version 3 . 4 . 130 ( RRID:SCR_007393 ) for interaction data , and the curated list of protein complexes from Baryshnikova et al . ( 2010 ) .", "PANTHER ( RRID:SCR_004869; Mi et al . , 2016 ) was used to find enriched GO processes in a CLIK group .", "Comparisons between wild-type and mutant strains for different methylation states were done using two-way ANOVA and corrected for multiple comparisons using the Šídák method , unless otherwise indicated .", "Sample sizes are reported in the figure legends and are always biological replicates , meaning that the cells were grown independently .", "All performed statistical tests can be found in Supplementary file", "4 . The significance thresholds used were p<0 . 05 ( * ) , p<0 . 01 ( ** ) and p<0 . 001 ( *** ) .", "The asterisks indicate significant differences compared to wild type .", "R ( RRID:SCR_001905; R Core Team , 2016 ) and GraphPad Prism 6 ( RRID:SCR_002798 ) were used for data analysis and plotting .", "Error bars represent standard deviation ." ] ]

[ "Given the frequent misregulation of chromatin in cancer , it is important to understand the cellular mechanisms that regulate chromatin structure .", "However , systematic screening for epigenetic regulators is challenging and often relies on laborious assays or indirect reporter read-outs .", "Here we describe a strategy , Epi-ID , to directly assess chromatin status in thousands of mutants .", "In Epi-ID , chromatin status on DNA barcodes is interrogated by chromatin immunoprecipitation followed by deep sequencing , allowing for quantitative comparison of many mutants in parallel .", "Screening of a barcoded yeast knock-out collection for regulators of histone H3K79 methylation by Dot1 identified all known regulators as well as novel players and processes .", "These include histone deposition , homologous recombination , and adenosine kinase , which influences the methionine cycle .", "Gcn5 , the acetyltransferase within the SAGA complex , was found to regulate histone methylation and H2B ubiquitination .", "The concept of Epi-ID is widely applicable and can be readily applied to other chromatin features ." ]

[ "To fit into the nucleus of eukaryotic cells ( which include plant , animal and yeast cells ) , DNA wraps around histone proteins to form a structure called chromatin .", "Histones can be modified by a variety of chemical tags , which affect how easily nearby DNA can be accessed by other molecules in the cell .", "These modifications therefore help to control the activity of the genes encoded in the DNA and other key processes such as DNA repair .", "If histone modifications are not regulated correctly , diseases such as cancer may result .", "Enzymes generally perform the actual modification , but there is another layer of regulation that controls the activity of these enzymes that not much is known about .", "The activity of an enzyme that performs a histone modification known as H3K79 methylation ( which involves a methyl chemical group being added to a particular region of a particular histone protein ) has been linked to some forms of leukemia .", "Collections of mutant yeast cells can be used to identify the factors that regulate histone modifications in both yeast and human cells .", "However , current methods that screen for these regulators are time consuming .", "To make the search for histone modification regulators more efficient , Vlaming et al . developed a new screening procedure called Epi-ID that can measure the amount of a specific histone modification in thousands of budding yeast mutants at the same time .", "In Epi-ID , each mutant yeast cell has a unique DNA sequence , or “barcode” .", "The mutant cells are mixed together and the barcodes that are modified by a particular histone modification – such as H3K79 methylation – are isolated and then counted using a DNA sequencing technique .", "A high barcode count of a certain mutant indicates that more of the histone modification occurs in that mutant .", "Using Epi-ID to survey H3K79 methylation enabled Vlaming et al . to successfully identify all previously known H3K79 methylation regulators , as well several new ones .", "These new regulators included enzymes that deposit histones on DNA , that carry out DNA repair , and that modify or de-modify histone proteins .", "To move forward with the newly identified regulators , it will be important to analyze how they control H3K79 methylation in yeast cells and to determine whether the regulators also control H3K79 methylation in human cells .", "Finally , Epi-ID can be used to identify regulators of other types of histone modifications .", "A better understanding of chromatin regulation – and H3K79 methylation regulation in particular – can increase our understanding of diseases in which chromatin is deregulated , and may yield new strategies for the treatment of such diseases ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "cell biology" ]

[ [ "Endocytosis is the process by which cells internalize various molecules , such as proteins and lipids , from the plasma membrane and outside the cell .", "Recent live-cell imaging studies of yeast and mammalian cells have revealed that the actin cytoskeleton plays important roles in the formation and internalization of clathrin-coated vesicles ( CCVs ) and post-internalization events in the endocytic pathway , including vesicle transport and endosome motility ( Engqvist-Goldstein and Drubin , 2003; Girao et al . , 2008; Kaksonen et al . , 2006 ) .", "In the early stages of the endocytic pathway , transient actin polymerization at the endocytic site is required for the formation and internalization of a CCV ( Kaksonen et al . , 2003; Merrifield et al . , 2002 ) .", "This process is regulated by several actin nucleation promoting factors ( NPFs ) , including the type I myosins Myo3/5p , the actin binding protein Abp1p , the yeast WASP homologue Las17p , and the Eps15-like protein Pan1p ( Goode et al . , 2015; Weinberg and Drubin , 2012 ) .", "Compared to Las17p and Myo3/5p , Pan1p has a lower NPF activity , and mutation in the Arp2/3 complex binding region of Pan1p causes only a minor defect in actin polymerization at endocytic sites because of the functional redundancy with Las17p ( Sun et al . , 2006; Toshima et al . , 2005 ) .", "However , a form of Pan1p mutated to prevent phosphorylation at 15 threonines , Pan1-15TA , directly binds to F-actin with high affinity and its expression causes abnormal cytoplasmic actin concentrations , also called actin clumps ( Toshima et al . , 2005 ) .", "Thus , Pan1p could be acting after F-actin is assembled by other NPFs as well as or instead of during the initiation of actin polymerization during endocytosis .", "Pan1p’s abilities to bind F-actin and promote actin polymerization are regulated by the Prk1 family protein kinases Prk1p and Ark1p , which are related to the mammalian proteins GAK and AAK1 ( Smythe and Ayscough , 2003 ) .", "The Prk1 family kinases are important regulators of endocytosis and the actin cytoskeleton in both yeast and mammalian cells ( Smythe and Ayscough , 2003 ) .", "In budding yeast , Ark1p and Prk1p are recruited to endocytic sites 1–2 s after commencement of actin assembly and CCV internalization , and phosphorylate several endocytic proteins , including Sla1p , Ent1/2p , Yap1801/2p , Scd5p , and Pan1p , to disassemble endocytic coat proteins and actin ( Cope et al . , 1999; Henry et al . , 2003; Toret et al . , 2008; Watson et al . , 2001; Zeng and Cai , 1999; Zeng et al . , 2001 ) .", "Pan1p is one of the key targets of Ark1/Prk1 kinases , and phosphorylation of Pan1p by Ark1/Prk1 kinases is believed to be important for disassembly of the Pan1p complex , composed of several endocytic proteins ( Toshima et al . , 2005; Wendland and Emr , 1998; Zeng and Cai , 1999; Zeng et al . , 2001 ) .", "Interestingly , disruption of the normal phosphorylation cycle by deletion or chemical inhibition of Ark1/Prk1 kinases leads to the concentration of actin in association with endocytic vesicles ( Sekiya-Kawasaki et al . , 2003; Toshima et al . , 2005 ) , suggesting a role for Pan1p and other substrates in regulating interaction between endocytic vesicles and the actin cytoskeleton .", "After being internalized , endocytic vesicles move away from the plasma membrane in an association with actin cables that is still mechanistically unexplained ( Huckaba et al . , 2004; Toshima et al . , 2006 ) .", "Yeast actin cables , which are bundles of actin filaments that align along the long axis of budding yeast , are crucial for the establishment of cell polarity ( Yang and Pon , 2002 ) .", "Actin cables are also used as tracks for polarized transport during the secretion of exocytic vesicles and the segregation of organelles from mother to daughter cells ( Bretscher , 2003 ) .", "Many of these types of transport along actin cables are known to depend on the type V myosins , Myo2/4p , which mediate the movement of cargo from the minus to plus ends of actin filaments ( Bretscher , 2003 ) .", "However , transport of endocytic vesicles along actin cables is not likely to depend on these myosins , because a temperature sensitive mutant of MYO2 ( myo2-66 ) or a deletion of MYO4 gene did not exhibit any defect in endocytosis ( Govindan et al . , 1995; Haarer et al . , 1994 ) .", "Other myosins , such as type II myosin ( Myo1p ) and type I myosin ( Myo3/5p ) also do not seem to mediate this transport .", "Myo1p has an important role in controlling actin cable dynamics at the bud sites or neck , but it is not localized to endocytic vesicles ( Huckaba et al . , 2006 ) .", "Myo3/5p are necessary for promoting actin assembly and endocytosis at cortical patches , but they stay at the cell cortex when endocytic vesicles are internalized along actin cables ( Sun et al . , 2006 ) .", "Interestingly , a previous study demonstrated that endocytic vesicle movement occurs at the same velocity and in the same direction as the movement of actin cables ( Huckaba et al . , 2004 ) .", "They also reported that an endocytic vesicle stays at the same position on the cable and moves together with the actin cable , suggesting that endocytic vesicles are fixed on the actin cables and move as a result of actin cable flow ( Huckaba et al . , 2004 ) .", "In addition to endocytic vesicles , early endosomes also associate with the actin cytoskeleton , and the motility of endosomes is significantly inhibited by treatment of latrunculin A ( LatA ) , a drug that sequesters actin monomers ( Chang et al . , 2003; Fernandez-Borja et al . , 2005; Toshima et al . , 2006; Voigt et al . , 2005 ) .", "Similarly to endocytic vesicles , early endosome motility also does not depend on Myo2/4p ( Toshima et al . , 2006 ) .", "These results suggest that unknown molecular mechanisms exist that bind endocytic vesicles and endosomes to actin cables .", "We sought to understand the role of Pan1 phosphorylation during endocytosis using a form of Pan1 that mimics the ark1Δ prk1Δ phenotype .", "We examined cells expressing Pan1-18TA , which is mutated to prevent phosphorylation at all 18 threonines; this mutant showed almost the same endocytic defect as ark1Δ prk1Δ cells , resulting in stable association between endocytic vesicles and actin cables .", "Interestingly , the pan1-18TA mutant also leads to accumulation of early endosomes in actin clumps .", "Thus , phosphorylation of Pan1p seems to regulate the interaction between endocytic compartments and the actin cytoskeleton ." ], [ "Our group had previously demonstrated that expression of a form of Pan1 containing a mutation of 15 Ark1p/Prk1p consensus sequences ( LxxQxTG ) to alanine causes an endocytic defect and abnormal clumping of actin in the cytosol .", "However , the defect in the pan1-15TA mutant was not as pronounced as that in the ark1△ prk1△ mutant ( Toshima et al . , 2005 ) .", "We first sought to determine if the presence of other functionally important phosphorylation sites in Pan1p was responsible for the difference in phenotypes .", "In a previous intensive investigation , Cai and colleagues identified the [L/I/V/M]xx[Q/N/T/S]xTG motif as a further potential site of phosphorylation by Ark1/Prk1 kinases ( Huang et al . , 2003 ) .", "Pan1p contains three more such Ark1p/Prk1p consensus sequences ( MQPNIT464G , MMPQTT480G , and MMPQTT487G ) all located in the second LR region ( Figure 1A ) ( Huang et al . , 2003 ) .", "When we additionally mutated these sites to create pan1-18TA , we observed a more severe growth retardation phenotype than in pan1-15TA ( Figure 1B ) .", "The Pan1-18TA protein was expressed normally , but its phosphorylation was mostly inhibited ( Figure 1C ) .", "pan1-18TA mutant cells displayed prominent actin concentrations and a more severe defect in endocytic internalization ( Figure 1D , E ) .", "Pan1-18TA-GFP also showed defects in localization , with 95% colocalizing with actin clumps or smaller , peripheral actin patches , similar to Pan1-15TA ( Figure 1F ) ( Toshima et al . , 2005 ) .", "This is in contrast to wild-type cells , in which Pan1p is recruited to cortical patches early , arriving ~20 s before actin is detected , and associates with actin for ~10–15 s ( Kaksonen , 2003 ) , resulting in ~30% of Pan1p colocalizing with Abp1p ( Figure 1F ) . 10 . 7554/eLife . 10276 . 003Figure 1 . Construction and characterization of a Pan1p phosphorylation-site mutant .", "( A ) Structure of a Pan1p phosphorylation mutant .", "The two amino-terminal Eps15 homology ( EH ) domains , long repeat ( LR ) regions , predicted coiled-coil ( CC ) , acidic region ( A ) , and carboxy-terminal prolin-rich domain ( PR ) domain , are indicated .", "The fifteen consensus phosphorylation sites previously mutated in Pan1-15TA are indicated below the protein in black .", "The three additional sites mutated in Pan1-18TA are in red .", "( B ) Plate showing the growth phenotype of pan1-15TA and pan1-18TA mutants .", "A dilution series of cells was plated on YPD plates and incubated for 2–3 days at 25 or 37°C , respectively .", "( C ) Analysis of the phosphorylation state of the Pan1-18TA mutant .", "Protein expression was analyzed by immunoblotting 20 μg of total cell lysate ( TCL ) with an anti-GFP antibody ( left panel ) .", "Phosphorylated proteins were purified from TCL with Phos-tag agarose , run on SDS-PAGE and immunoblotted with the anti-GFP antibody ( right panel ) as described in Materials and methods .", "Lane 1 , JJTY369; lane 2 , JJTY509; lane 3 , JJTY5486 .", "( D ) Alexa Fluor 488-phalloidin staining of fixed wild-type and pan1-18TA cells to visualize actin .", "( E ) The effect of Pan1p phosphorylation-site mutations on endocytic internalization .", "Radiolabeled α-factor internalization assays performed on wild-type ( blue ) , pan1-15TA ( yellow ) , pan1-18TA ( magenta ) , pan1-18TA sla1-10TA ( green ) , or ark1△ prk1△ ( black ) cells at 25°C .", "Each curve represents the average of three independent experiments , and error bars indicate the SD at each time point .", "( F ) The localization of Pan1-GFP in wild-type and pan1-18TA cells .", "Cells expressing Pan1-GFP and Abp1-mCherry were grown to early to mid-logarithmic phase in YPD medium at 25°C and observed by fluorescence microscopy .", "Merged images of GFP and mCherry channels are shown in the right panels .", "( G ) Endocytic cargo is transported to the vacuole through the actin clumps in pan1-18TA .", "pan1-18TA cells were labeled with A594-α-factor as described in the Methods .", "The images were acquired simultaneously at 1 , 5 , 15 , and 30 min after washing out unbound A594-α-factor and warming the cells to 25°C .", "Scale bars , 2 . 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 10276 . 00310 . 7554/eLife . 10276 . 004Figure 1—figure supplement 1 . Construction and characterization of a Sla1p phosphorylation-site mutant .", "( A ) Structure of the Sla1p phosphorylation mutant .", "The three src homology 3 ( SH3 ) domains , two Sla1 homology domains ( SHD1/2 ) , clathrin-binding motif ( CBM ) and carboxyl-terminal Sla1 repeats ( SR ) are indicated .", "The threonine in the 10 consensus Prk1 phosphorylation sites indicated below the protein was mutated to an alanine to make Sla1-10TA .", "( B ) Phosphorylation state of Sla1-10TA mutant .", "Expression of proteins were analyzed by immunoblotting 20 μg total cell lysate ( TCL ) with anti-GFP antibody ( left panel ) .", "Phosphorylated proteins were purified from TCL by Phos-tag agarose , run on SDS-PAGE and immunoblotted with anti-GFP antibody ( right panel ) .", "Lane 1 , JJTY369; lane 2 , JJTY130; lane 3 , JTY4139 .", "( C ) Plate showing the growth phenotype of the sla1-10TA mutant .", "A dilution series of cells was plated on YPD plates and incubated for 2–3 days at 25 or 37°C , respectively .", "( D ) The in vivo effect of the Sla1p phosphorylation-site mutant .", "Wild-type , sla1-10TA , and pan1-18TA sla1-10TA cells were fixed and stained with Alexa Fluor 488-phalloidin to visualize actin .", "( E ) Upper panels are single frames from a two-color movie showing Sla1-GFP ( green ) and Abp1-mCherry ( red ) in wild-type ( top ) or sla1-10TA ( bottom ) cells .", "Lower panels are time series of patches marked by arrowheads in upper panels .", "The time to acquire one image pair was 1 s .", "( F ) Average lifetime of Sla1-GFP and Abp1-mCherry ± SD in wild-type and sla1-10TA cells .", "Data were taken from 60–90 s movies with a 1 sec frame interval .", "n = 50 patches for each strain .", "( G ) Effect of Sla1p phosphorylation-site mutation on endocytic internalization .", "Radiolabeled α-factor internalization assays performed on wild-type ( blue ) or sla1-10TA ( magenta ) cells at 25°C .", "Each curve represents the average of three independent experiments , and error bars indicate the SD at each time point .", "Scale bars , 2 . 5 μm .", "( H ) The growth phenotype of sla1-10TA , pan1-18TA , sla1-10TA pan1-18TA double mutants , and ark1△ prk1△ .", "A dilution series of cells was plated on YPD plates and incubated for 2–3 days at 25 or 37°C , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 10276 . 004 We next entertained the hypothesis that Prk1p phosphorylation of some of its other known targets such as Sla1p , Ent1/2p , Yap1801/2p and Scd5p ( Watson et al . , 2001; Zeng et al . , 2001; 2007 ) , might also play a role in regulating actin organization and endocytosis .", "These target proteins were shown to be phosphorylated by Prk1p in vitro , but significant phenotypes caused by mutations of their phosphorylation-sites have not been observed ( Henry et al . , 2003; Huang et al . , 2003; Watson et al . , 2001; Zeng et al . , 2001 ) .", "Sla1p contains the most potential Prk1 phosphorylation sites among these proteins ( Zeng et al . , 2001 ) .", "We therefore mutated the threonines in all 10 of these [L/I/V/M]xx[Q/N/T/S]xTG sites ( Huang et al . , 2003 ) to alanine ( sla1-10TA ) , integrated this mutant into the endogenous SLA1 locus , and analyzed the phenotypes ( Figure 1—figure supplement 1A ) .", "We first confirmed that the Sla1-10TA mutant was expressed at similar levels to the wild-type protein , and that its phosphorylation was severely inhibited ( Figure 1—figure supplement 1B ) .", "While cells lacking the SLA1 gene were temperature-sensitive for growth at 37°C , the sla1-10TA mutant exhibited almost the same growth as wild-type cells ( Figure 1—figure supplement 1C ) .", "Alexa Fluor 488-phalloidin staining of F-actin in fixed sla1-10TA mutants closely resembled that of wild-type cells , with brightly stained actin patches and weakly stained actin cables ( Figure 1—figure supplement 1D ) .", "We next examined the dynamics of the clathrin-coat module and the actin patches in the sla1-10TA mutants , using GFP-tagged Sla1-10TA and Abp1-mCherry as markers respectively ( Kaksonen et al . , 2003 ) .", "Consistent with previous reports , Sla1p and Abp1p patches formed in the cell cortex with lifetimes of 36 ± 7 s and 13 ± 3 s , respectively , culminating in inward movement ( Figure 1—figure supplement 1E , F ) ( Kaksonen et al . , 2003 ) .", "Sla1-GFP localization was immediately followed by a burst of Abp1-mCherry recruitment in wild-type cells ( Figure 1—figure supplement 1E ) .", "In the sla1-10TA mutants , Sla1p and Abp1p patches formed and disappeared with the typical inward movement , and their lifetimes were slightly prolonged to 43 ± 11 s and 15 ± 3 s , respectively ( Figure 1—figure supplement 1F ) .", "We also examined the effect of sla1-10TA mutants on endocytic internalization by assessing the ingression of 35S-labeled α-factor , and found that it was only slightly affected ( Figure 1—figure supplement 1G ) .", "Furthermore , the pan1-18TA sla1-10TA double mutant exhibited only a negligible additive effect , when compared to the pan1-18TA single mutant ( Figure 1E , and Figure 1—figure supplement 1D , H ) .", "These findings indicate that Pan1p is the major in vivosubstrate of Ark1/Prk1 kinases during their regulation of endocytosis .", "We next visualized sequential steps in the endocytic pathway in the pan1-18TA mutant using Alexa Fluor 594-labeled yeast mating pheromone α-factor ( A594-α-factor ) , a marker of endocytosis ( Toshima et al . , 2006 ) .", "Interestingly , internalized A594-α-factor moved to actin clumps in pan1-18TA before being transported to the vacuole , while the actin remained in clumps ( Figure 1G ) .", "This result suggests that the endocytic cargo can transit through the actin clumps before arriving at the vacuole .", "We next sought to determine if other organelles along the endocytic route also accumulate in these actin clumps .", "To this end , we employed Vps21p and Sec4p as markers of Rab proteins that function in the endocytic or exocytic pathway ( Hutagalung and Novick , 2011 ) , Vps8p and Vps11p as markers of the CORVET and HOPS complexes ( Balderhaar and Ungermann , 2013 ) , a set of proteins from the ESCRT complex ( Hse1p , Mvb12p , Vps36p , and Vps24p ) ( Bilodeau et al . , 2002; Hurley , 2008 ) , Vps4p , Ear1p , and Vps15p as markers of MVBs ( Ear1p , and Vps15p ) ( Burda et al . , 2002; Leon et al . , 2008 ) , Vps26p as a marker of the retromer complex ( Seaman , 2004 ) , Vps52p as a marker of the GARP ( Golgi-associated retrograde protein ) complex ( Bonifacino and Hierro , 2011 ) , and Sec7p as a marker of the trans-Golgi network ( Franzusoff et al . , 1991 ) .", "None of these proteins show clump-like localization in wild-type cells ( Figure 2—figure supplement 1 ) .", "Among the 14 proteins examined , three – Hse1p , Mvb12p , and Vps36p – showed a clear change in localization to Abp1-mCherry-labeled actin clumps in the pan1-18TA mutant ( Figure 2A , B ) .", "Hse1p , Mvb12p , and Vps36p , which function at an early stage of the ESCRT pathway on the way to the MVB , were contained in 60–65% of the actin clumps , whereas Vps24p and Vps4p , which function at a later stage of the ESCRT pathway , were contained in 25–30% of the actin clumps ( Figure 2A , B ) .", "Vps8p and Vps11p , which mediate early to late transitions of endosomes , exhibited levels of actin clump localization ( ~30% ) similar to that of Vps24p ( Figure 2B ) .", "In contrast , Vps26p or Vps52p , both of which are required for retrograde transport from late endosomes to the Golgi , showed lower localization ( ~15% and ~7% , respectively ) , and Sec7p and Sec4p , which reside on the Golgi or secretory pathway , were rarely contained in actin clumps in the pan1-18TA mutant ( <10% ) ( Figure 2A , B ) .", "These results indicate that earlier stage endosomes are highly localized to actin clumps in pan1-18TA mutant cells . 10 . 7554/eLife . 10276 . 005Figure 2 . The localization of endosomal proteins in pan1-18TA cells .", "( A ) Localization of GFP-tagged endosomal proteins in pan1-18TA .", "pan1-18TA cells expressing Abp1-mCherry and GFP-tagged endosomal proteins were grown to early to mid-logarithmic phase in YPD medium at 25°C and observed by fluorescence microscopy .", "Merged images of GFP and mCherry channels are shown in the lower panel .", "Arrowheads indicate examples of colocalization .", "Scale bar , 2 . 5 μm .", "( B ) Quantification of actin clumps including GFP-tagged endosomal proteins .", "The percentages were calculated as the ratio of actin clumps ( n = 100 ) colocalizing with each protein in each experiment .", "Error bars indicate the standard deviation ( SD ) from at least three independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 10276 . 00510 . 7554/eLife . 10276 . 006Figure 2—figure supplement 1 . Localization of endosomal proteins in wild-type cells . Cells expressing each GFP-tagged endosomal protein were grown to early to mid-logarithmic phase in YPD medium at 25°C and observed by fluorescence microscopy . Scale bars , 2 . 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 10276 . 006 We wished to confirm that early endosomes accumulate at actin clumps in pan1-18TA mutant cells .", "As we had seen the ESCRT-0 component Hse1p localize to actin clumps in the mutant , we wished to investigate its organellar localization in wild-type cells more precisely; therefore we tagged Hse1p with three tandem repeats of GFP ( 3GFP ) or mCherry and confirmed their functionality ( Figure 3—figure supplement 1A ) .", "Hse1-3GFP was clearly detected as numerous small puncta throughout the cytoplasm and prevacuolar compartments ( PVCs ) ( Figure 3—figure supplement 1B ) .", "Examining Hse1-3GFP in the overlay of 30 consecutive time-lapse frames made it easy to distinguish the Hse1p localizing at endosomes in the cytoplasm ( Figure 3—figure supplement 1B , right cell ) and at the PVCs ( Figure 3—figure supplement 1B , left cell ) .", "By comparing the localization of Hse1-mCherry with GFP-tagged markers , we found that Hse1p exhibited high colocalization with Mvb12p , partial colocalization with Ear1p and Vps26p , and little colocalization with Sec7p ( Figure 3—figure supplement 1C , D ) .", "We next utilized A594-α-factor to compare the spatiotemporal localization of Hse1p with Vps26p in the endocytic pathway ( Figure 3—figure supplement 1E , F ) ( Toshima et al . , 2006; 2009 ) .", "Hse1-3GFP was highly colocalized with A594-α-factor-labeled endosomes at 5–10 min after α-factor internalization , whereas Vps26-GFP was mainly colocalized with A594-α-factor-labeled endosomes at 10 min ( Figure 3—figure supplement 1G ) .", "In pan1-18TA cells , similar levels of Hse1p also colocalized with Vps26p ( Figure 3—figure supplement 1D , H ) .", "These data indicate that Hse1p is widely localized from early to late endosomes and partially colocalized with Vps26p at late endosomes both in wild-type and pan1-18TA cells .", "Unexpectedly , Vps21p , yeast Rab5 , which is known to be localized at early-to-late endosomes ( Cabrera et al . , 2013; Puchner et al . , 2013; Toshima et al . , 2014 ) , exhibited lower localization at the actin clumps ( ~33% ) , compared to Hse1p ( Figure 2A , B ) .", "To determine the relative localization of Hse1p and Vps21p at early stage of endocytosis , we slowed endocytic transport by removing glucose from culture medium ( Aoh et al . , 2011 ) , and compared their localization with internalized A594-α-factor .", "Consistent with a recent study by Arlt et al . reporting that an ESCRT-I subunit , Vps23 , is recruited to endosomes earlier than Vps21p ( Arlt et al . , 2015 ) , we found that Hse1p colocalizes with A594-α-factor slightly more than Vps21p at 10 min after α-factor internalization ( Figure 3—figure supplement 2A ) .", "These results , therefore , suggest that endosomes at the early stage of endocytosis are highly localized to actin clumps .", "The localization of early endosomal proteins at actin clumps in the pan1-18TA mutant suggests that early endosomes might associate with the actin cytoskeleton in wild-type cells .", "Previous studies have also indicated that actin cables mediate the directed movements of early endosomes ( Chang et al . , 2003; Toshima et al . , 2006 ) , but which endosomes , and how they associate with actin cables , has not yet been clarified .", "We utilized Hse1p as a marker to address these questions .", "However because Hse1p is found at early to late endosomes , we classified these endosomes into two categories using Vps26-mCherry: endosomes not labeled with Vps26-mCherry ( early stage endosomes ) and endosomes labeled with Vps26-mCherry ( late stage endosome ) ( Figure 3A , C ) .", "Vps26-mCherry mostly colocalized with Hse1p at the vacuolar membrane ( Figure 3A , C ) .", "Quantification of endosome velocity revealed that early-stage endosomes moved with an average speed of 125 ± 119 nm/s ( n = 100 ) , whereas late-stage endosomes moved with an average speed of 156 ± 130 nm/s ( n = 100 ) in wild-type cells ( Figure 3A , B ) .", "We then investigated the effects of the actin-sequestering drug , LatrunculinA ( LatA ) , on the movement of these endosomes .", "Concomitantly , LatA treatment led to a significant decrease in the velocity of early-stage endosomes ( ~23 ± 47 nm/s ) ( Figure 3C , D ) .", "In contrast , the velocity of late-stage endosomes was not significantly affected by LatA treatment ( ~119 ± 112 nm/s ) ( Figure 3C , D ) .", "Similar results were obtained by analyzing Vps21p-containing endosomes ( Figure 3—figure supplement 2B ) .", "The velocity of Vps21p-containing endosomes not labeled with Vps26-mCherry ( ~146 ± 128 nm/s ) was decreased by LatA treatment ( ~55 ± 42 nm/s ) , whereas that of ones labeled with Vps26-mCherry ( ~138 ± 128 nm/s ) was not significantly affected ( ~99 ± 91 nm/s ) .", "To further confirm the association between Hse1p-labeled endosomes and the actin cytoskeleton , we labeled actin cables with tdTomato-tagged Abp140p ( Yang and Pon , 2002 ) in wild-type cells .", "Simultaneous imaging revealed that Hse1p-labeled endosomes localized along , and moved on , actin cables ( Figure 3E , F , and Video 1 ) .", "Since two distinct sets of actin assembly-promoting machinery have been identified in yeast , the Arp2/3 complex and formins ( Goode et al . , 2015 ) , we next utilized specific inhibitors toward these regulators .", "The Arp2/3 complex inhibitor CK-666 specifically disassembled Arp2/3 complex-dependent actin patches , whereas SMIFH2 disassembled formin-dependent actin cables ( Nolen et al . , 2009; Rizvi et al . , 2009 ) .", "As expected , SMIFH2 treatment led to actin cable disassembly , followed by a decrease in the velocity of endosomes ( ~60 ± 53 nm/s ) .", "In contrast , CK-666 inhibited vesicle internalization , but endosome motilities were little affected ( ~126 ± 123 nm/s ) ( Figure 3G , H , and Video 2 ) .", "These results indicate that the movement of early-stage endosomes is dependent on formin-dependent actin polymerization . 10 . 7554/eLife . 10276 . 007Figure 3 . Interaction of endosomes expressing Hse1-3GFP with the actin cytoskeleton .", "( A and C )", "Movement of Hse1-3GFP-containing endosomes in living cells .", "Wild-type cells expressing Vps26-3mCherry and Hse1-3GFP were grown to log phase at 25°C , treated with DMSO ( LatA- ) ( A ) or 250 μM LatA ( C ) for 30 min at 25°C , and subsequently imaged at 1 s intervals .", "In the upper row the left two panels show the individual channels and the right most image shows a merged overlay of the signal from both channels in the first 19 s .", "The lower row shows merged images of both channels at indicated time .", "Yellow or red arrowheads indicate examples of Hse1p-containing endosomes that do or do not co-label with Vps26p , respectively .", "Scale bars , 2 . 5 μm .", "The schematic panels on the right show tracking of the endosome indicated by yellow or red arrowheads in the lower microscope images .", "Scale bars , 0 . 5 μm .", "( B and D )", "Quantification of the velocity of Hse1p-containing endosomes .", "Endosome velocities were acquired at 1 s intervals and categorized according to a velocity range .", "( E ) Hse1p-containing endosomes move along actin cables .", "Wild-type cells expressing Hse1-3GFP and Abp140-tdTomato were grown to early to mid-logarithmic phase and each image pair was acquired simultaneously at 1 s intervals .", "Scale bar , 2 . 5 μm .", "( F ) Higher magnification view of the boxed area in ( E ) at successively later time points specified in s above .", "Arrowheads indicate an endosome moving along an actin cable .", "( G ) The effect of SMIFH2 or CK-666 on movement of Hse1-3GFP-containing endosomes .", "Wild-type cells expressing Abp140-tdTomato and Hse1-3GFP were grown to log phase at 25°C , treated with 25 μM SMIFH2 ( the upper row ) or 100 μM CK-666 ( the lower row ) for 30 min at 25°C , and subsequently imaged at 1 s intervals .", "The left three panels show the individual channels and their merged image for a particular time point , and the right most image shows a merged overlay of Hse1-GFP signals in 30 sec .", "Red arrowheads indicate examples of Hse1p-containing endosomes .", "Scale bars , 2 . 5 μm .", "( H ) Quantification of the velocity of Hse1p-containing endosomes .", "Endosome velocities were acquired at 1 s intervals and categorized according to a velocity range . DOI: http://dx . doi . org/10 . 7554/eLife . 10276 . 00710 . 7554/eLife . 10276 . 008Figure 3—figure supplement 1 . Localization of 3GFP- or mCherry-tagged Hse1p in wild-type cells .", "( A ) The functionality of 3GFP- and mCherry-tagged Hse1p was confirmed by testing their ability to complement the growth phenotype of hse1△ cells in a dilution series of each plated on YPD containing 15 mM caffeine , and incubated at 25°C .", "( B ) Hse1p is localized at endosomes and PVCs in wild-type cells .", "The left panel is a single frame from a single-color movie showing Hse1-3GFP in wild-type cells .", "The middle panel is an image of the overlay of the Hse1-GFP localization seen in each 1 s frame interval of a 30 s movie .", "Examining Hse1-3GFP in the overlay of 30 consecutive time-lapse frames made it easy to distinguish the Hse1p localizing at endosomes in the cytoplasm and at the PVCs .", "The merged image of the Hse1-3GFP overlay and the differential interference contrast ( DIC ) images are shown in the right panel .", "( C ) Localization of Hse1-mCherry and GFP-tagged proteins in living cells .", "Merged images of GFP and mCherry channels are shown in the lower panels .", "Wild-type cells expressing Hse1-mCherry and GFP-tagged proteins were grown to early to mid-logarithmic phase at 25°C in YPD medium and observed by fluorescence microscopy .", "Each image pair was acquired simultaneously .", "( D ) The histogram represents the percentage of Hse1-mCherry labeled compartments colocalizing with the indicated organelle markers .", "In each experiment ( n = 100 ) Hse1-mCherry labeled compartments were counted for each marker protein .", "Error bars indicate the SEM from at least three independent experiments .", "( E and F )", "Localization of 3GFP-tagged Hse1p ( E ) or Vps26p ( F ) in endocytic compartments .", "Cells were labeled with A594-α-factor as described in the Methods .", "The images were acquired simultaneously at 0 , 5 , and 15 min after washing out unbound A594-α-factor and warming the cells to 25°C .", "Arrowheads indicate examples of colocalization .", "( G ) Quantification of the colocalization of Hse1-3GFP or Vps26-GFP with A594-α-factor at each time point .", "The percentages of colocalization were calculated as the ratio of A594-α-factor localized in the respective GFP positive compartments ( n = 50 ) in each experiment .", "Error bars indicate the SEM from at least three independent experiments .", "( H ) Hse1p resides both in actin clump and late endosomes .", "Each image pair was acquired using fluorescence microscopy equipped with high-speed filter changer .", "Time to acquire one image pair is 3 . 5 s .", "Arrowheads indicate example of colocalization of Hse1-3GFP and Vps26-mCherry ( yellow ) or Abp1-CFP ( red ) .", "Scale bars , 2 . 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 10276 . 00810 . 7554/eLife . 10276 . 009Figure 3—figure supplement 2 . Localization and movement of GFP-Vps21-containing endosomes in living cells .", "( A ) Localization of Hse1-3GFP or GFP-Vps21 in endocytic compartments .", "Cells were labeled with A594-α-factor as described in the Materials and methods .", "The images were acquired simultaneously at 10 min after washing out unbound A594-α-factor and warming the cells to 25°C .", "Arrowheads indicate examples of colocalization .", "The bar graphs represent the colocalization of Hse1-3GFP or GFP-Vps21 with A594-α-factor at 10 or 20 min after A594-α-factor internalization .", "The percentages of colocalization were calculated as the ratio of GFP signals in A594-α-factor positive compartments ( n = 50 ) in each experiment .", "Error bars indicate the SEM from at least three independent experiments .", "( B ) Movement of GFP-Vps21-containing endosomes in living cells .", "Wild-type cells expressing Vps26-3mCherry and GFP-Vps21 were grown to log phase at 25°C , treated with DMSO ( LatA- ) or 250 μM LatA for 30 min at 25°C , and subsequently imaged at 1 s intervals .", "In the upper row the left two panels show the individual channels and the right most image shows a merged images .", "The lower left panel shows merged overlay of the signal from both channels in the first 30 s .", "The bar graphs represent the average velocity of Vps21p-containing endosomes ( n = 100 ) .", "Scale bars , 2 . 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 10276 . 00910 . 7554/eLife . 10276 . 010Video 1 . Localization of Hse1-3GFP ( left; green in merge ) and Abp140-tdTomato ( center; red in merge ) in wild-type cells . The interval between frames is 1 s . DOI: http://dx . doi . org/10 . 7554/eLife . 10276 . 01010 . 7554/eLife . 10276 . 011Video 2 . Localization of Hse1-3GFP ( left; green in merge ) and Abp140-Tomato ( center; red in merge ) in wild-type cells treated with 25 μM SMIFH2 or 100 μM CK-666 . Arrowheads indicate examples of Hse1p-labeled endosome .", "The interval between frames is 1 s . DOI: http://dx . doi . org/10 . 7554/eLife . 10276 . 011 In addition to endosomes , endocytic vesicles are also known to associate with actin cables ( Huckaba et al . , 2004; Toshima et al . , 2006 ) , but the molecules that regulate this association remain unclear .", "Therefore , we examined the effect of the pan1-18TA mutation on the localization and dynamics of actin cables and endocytic vesicles .", "In wild-type cells , actin cables are highly dynamic polarized structures ( Figure 4—figure supplement 1A and Video 3 ) ( Yang and Pon , 2002 ) .", "In contrast , the pan1-18TA mutant exhibited less polarized and more aggregated actin cable structures , some of which associated with the large actin concentrations ( Figure 4—figure supplement 1A and Video 3 ) .", "The movement of this Pan1p/actin aggregate in pan1-18TA was sensitive to SMIFH2 but not CK-666 , suggesting that the aberrant structure associates with actin cables ( Figure 4—figure supplement 1B–E ) .", "In a recent study , we showed that ~85% of endocytic vesicles were internalized along actin cables at the internalization step of endocytosis ( Toshima et al . , 2015 ) .", "Here we show that in this step , Pan1-mCherry-labeled vesicles in wild-type cells associated with actin cables for ~4 . 6 s and moved on cables about 0 . 4 μm after internalization ( Figure 4A , B , C ) .", "This association and these movements were significantly decreased by treating with 25 μM SMIFH2 ( Figure 4B , C , D ) .", "Over 80% of Pan1-mCherry-labeled endocytic vesicles , even in single focal plane images , also associated with and internalized along actin cables in the pan1-18TA mutant ( Figure 4E ) .", "In contrast to wild-type cells , Pan1-18TA-mCherry structures became stably associated with peripheral patches , as well as actin clumps , labeled by Abp140-3GFP ( Figure 4E ) .", "Interestingly , live-cell imaging revealed that many of internalized patches labeled by Pan1-18TA-mCherry stably associated with actin cables over 30 s , and move on actin cables more than 3 . 0 μm ( Figure 4B , C , E , and Video 4 ) .", "We also wished to determine the relationship of endocytic vesicles and endosomes in the absence of Pan1 phosphorylation .", "In the pan1-18TA mutant , we often observed that peripheral Pan1-18TA-mCherry patches colocalized and moved together with Hse1p-labeled endosomes ( Figure 4F , G , and Video 5 ) , whereas such colocalization was rarely observed in wild-type cells ( Figure 4F ) .", "These findings suggest that in the pan1-18TA mutant , endocytic vesicles and endosomes interact before fusion while they are both associated with actin cables , potentially tethering them together .", "Our data was collected with a wide field microscope; high-speed confocal microscopy could improve the quality of our results as it would permit 3-D analysis ( Kurokawa et al . , 2013 ) .", "However , since some vesicles and endosomes move in a single focal plane , the simultaneous double color live cell imaging used in this study permits analysis leading to substantive biological insights . 10 . 7554/eLife . 10276 . 012Video 3 . Localization of Abp140-3GFP in wild-type and pan1-18TA cells . The interval between frames is 1 s . DOI: http://dx . doi . org/10 . 7554/eLife . 10276 . 01210 . 7554/eLife . 10276 . 013Figure 4 . Interaction of Pan1p-containing compartments with actin cables and endosomes .", "( A ) Localization of Abp140-3GFP and Pan1-mCherry in a wild-type cell .", "The lower panels correspond to a time series of a higher magnification view of the boxed area in the upper right image .", "( B ) The residence time of Pan1-mCherry-labeled vesicles on an actin cable .", "The residence time was determined from 60 sequential two-dimensional images .", "n = 52 vesicles for each strain .", "Vesicles residing on cable over 30 s are indicated as >30 in the graph .", "( C ) Moving distance of Pan1-mCherry-labeled vesicles on actin cables .", "To determine each moving distance , the distance that the center of the Pan1-mCherry fluorescence moves on an actin cable was calculated based on pixel coordinates ( 1 pixel = 64 . 5 nm ) .", "n = 42 vesicles for each strain .", "( D ) Effect of the formin inhibitor SMIFH2 on the movement of Pan1-mCherry patches .", "Wild-type cells expressing Pan1-mCherry and Abp140-3GFP were grown to log phase at 25°C , treated with 25 μM SMIFH2 for 30 min at 25°C , and subsequently imaged at 1 s intervals .", "( E ) The localization of Abp140-3GFP and Pan1-18TA-mCherry in a pan1-18TA cell .", "The lower panels are single focal plane images corresponding to a time series of a higher magnification view of the boxed area in the upper right image .", "Arrowheads indicate examples of Pan1p-containing compartments moving along an actin cable .", "Yellow or red arrowheads indicates different vesicles .", "Upper and middle panels show GFP and mCherry channels , respectively , and lower panel shows their merged images .", "( F ) The localization of Hse1-3GFP and Pan1-mCherry in wild-type and pan1-18TA cells .", "( G ) Time series of single patches in the boxed area in ( F ) .", "Cells expressing Hse1-3GFP and Pan1-18TA-mCherry were grown to early to mid-logarithmic phase at 25°C in YPD medium and imaged at 1 s intervals .", "Scale bars , 2 . 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 10276 . 01310 . 7554/eLife . 10276 . 014Figure 4—figure supplement 1 . Actin cable dynamics in wild-type and pan1-18TA cells .", "( A ) Cells expressing Abp140-3GFP were grown to early to mid-logarithmic phase at 25°C in YPD medium and imaged at 1 s intervals .", "Right panels indicate a high magnification view of the boxed area in the left panels .", "( B and C )", "Effect of SMIFH2 or CK-666 on movement of actin clump .", "pan1-18TA cells expressing Abp140-3GFP and Pan1-mCherry were grown to log phase at 25°C , treated with 100 μM CK-666 ( B ) or 25 μM SMIFH2 ( C ) for 30 min at 25°C , and subsequently imaged at 1 s intervals .", "The upper images show the localization of Abp140-3GFP and Pan1-18TA-mCherry in a pan1-18TA cell .", "The lower panels correspond to a time series of a higher magnification view of the boxed area in the upper right image .", "( D ) The schematic panels show tracking of the actin clumps in the boxed areas in ( B ) or ( C ) .", "Positions of actin clumps were determined by calculating the center of fluorescence intensity .", "( E ) The bar graphs represent the average velocity of actin clumps ( n = 50 ) .", "Scale bars , 0 . 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 10276 . 01410 . 7554/eLife . 10276 . 015Video 4 . Localization of Abp140-3GFP ( left; green in merge ) and Pan1-18TA-mCherry ( center; red in merge ) in pan1-18TA cells . The interval between frames is 1 s . DOI: http://dx . doi . org/10 . 7554/eLife . 10276 . 01510 . 7554/eLife . 10276 . 016Video 5 . Localization of Hse1-3GFP ( left; green in merge ) and Pan1-18TA-mCherry ( center; red in merge ) in pan1-18TA cells . Arrowheads indicate examples of colocalization .", "The interval between frames is 1 s . DOI: http://dx . doi . org/10 . 7554/eLife . 10276 . 016 To further investigate the phosphorylation-dependent association between endocytic vesicle and actin cable , we next used an analogue-sensitive mutant of Prk1p in cells lacking Ark1p ( ark1△ prk1-as3 ) ( Sekiya-Kawasaki et al . , 2003 ) .", "This mutant shows specific sensitivity to 1NA-PP1 , an ATP analogue , and enabled us to investigate the direct and immediate consequence of Prk1p inactivation for the association between endocytic vesicles and actin cables .", "In the mutant untreated with 1NA-PP1 , Pan1-mCherry-labeled vesicles only transiently associated with actin cables , similar to wild-type cells ( Figure 5A and Video 6 ) .", "However , at 1 min after treatment of the mutant with 100 μM 1NA-PP1 , endocytic vesicles stably associated with , and moved on actin cables .", "By 3 min small aggregates containing Pan1p that associated with actin cables were formed ( Figure 5A and Video 6 ) .", "At 10 min after 1NA-PP1 treatment , a large actin clump that stably associated with actin cables was formed in the mutant , similar to the pan1-18TA mutant ( Figure 5A and Video 6; also see Figure 4C ) .", "These observations support the idea that Pan1p phosphorylation by Ark1/Prk1 kinases is necessary for the rapid dissociation of endocytic vesicles from actin cables . 10 . 7554/eLife . 10276 . 017Figure 5 . Interaction between endocytic vesicles and actin cables upon inhibition of Prk1p .", "( A ) The left images represent single frames from movies of ark1△ prk1-as3 mutant cells showing merged images of the GFP ( Abp140p ) and the mCherry ( Pan1p ) channel .", "The ark1△ prk1-as3 mutant cells expressing Abp140-3GFP and Pan1-mCherry were grown to log phase at 25°C , treated with 100 μM 1NA-PP1 for the indicated time at 25°C , and subsequently imaged at 1 s intervals .", "A time series of single patches in the boxed area for each strain are shown in the right panels .", "Blue arrowheads indicate Pan1-mCherry-labeled vesicles associating with actin cables .", "Scale bar , 2 . 5 μm .", "( B ) Localization of Abp140-3GFP and Pan1-mCherry in pan1-18TA and pan1-18TA△855 cells .", "Scale bar , 2 . 5 μm .", "( C ) Quantification of cells containing actin clumps .", "Cells expressing Abp140-3GFP were grown to log phase at 25°C and imaged .", "Data show mean ± SD from at least three experiments , with 50 cells counted for each strain per experiment . DOI: http://dx . doi . org/10 . 7554/eLife . 10276 . 01710 . 7554/eLife . 10276 . 018Video 6 . Localization of Abp140-3GFP ( left; green in merge ) and Pan1-mCherry ( center; red in merge ) in ark1△ prk1-as3 cells untreated or treated with 100 μM 1NA-PP1 . Arrowheads indicate examples of vesicles associated with actin cables .", "The interval between frames is 1 s . DOI: http://dx . doi . org/10 . 7554/eLife . 10276 . 018 Pan1p can bind directly to F-actin and its binding activity is regulated by phosphorylation through the Ark1/Prk1 kinases ( Toshima et al . , 2005 ) .", "Thus , we next examined whether Pan1p directly mediates the interaction between vesicles and actin cables via its actin binding .", "To completely destroy the actin binding activity of Pan1p , we used a Pan1 C-terminal deletion mutant ( pan1△855–1480 ) , which lacks actin binding and Arp2/3-activating regions ( Duncan et al . , 2001; Toshima et al . , 2005 ) .", "Interestingly , combining the pan1-18TA and pan1△855–1480 mutations ( pan1-18TA△855 ) caused accumulation of Pan1-mCherry vesicles similar to pan1-18TA mutant , but formation of actin clumps were significantly suppressed ( Figure 5B , C ) .", "The interaction between Pan1-mCherry-labeled vesicles and actin cables also decreased , but the vesicles still had an ability to bind actin cables ( Figure 4D ) , suggesting the existence of additional actin-binding coat protein ( s ) that stabilize the association of vesicles with actin cables .", "To be sure that this association is not merely a product of both being caught up in aberrant actin concentrations , we sought to isolate a mutant that could suppress actin clump formation in the pan1-18TA mutant .", "To this end , we deleted several actin-related genes involved in endocytic internalization in the pan1-18TA mutant and found three out of seven genes whose absence suppressed actin clump formation ( Figure 6A , B ) .", "Deletion of Sla2p , a yeast Hip1R-related protein , in the pan1-18TA mutant decreased the fraction of cells containing actin clumps by 95% as detected by Abp1-mCherry in maximum-intensity projections of Z stacks ( Figure 6—figure supplement 1A , B ) .", "These pan1-18TA sla2△ double mutants exhibited the elongated actin tails originating from non-motile endocytic sites seen in sla2△ ( Kaksonen et al . , 2003 ) ( Figure 6B ) .", "Deletion of both of the yeast type I myosins , Myo3p and Myo5p , led to an 85% reduction and deletion of Sac6p , the yeast homologue of the actin filament bundling protein fimbrin , almost completely suppressed actin clump formation in the pan1-18TA mutant ( Figure 6—figure supplement 1A , B ) .", "All three mutants showed a severe defect in the internalization of cortical actin patches ( Kaksonen et al . , 2003; 2005 ) , resulting in the loss of actin clumps in the pan1-18TA mutant .", "We then wished to examine the localization of early endosomes , using Hse1-GFP as a marker , in these double and triple mutants .", "Interestingly , Hse1p-labeled endosomes showed a change in localization to the cell periphery in all three suppressing mutants ( Figure 6B , C ) .", "We also observed that Pan1-18TA patches accumulate near Hse1p-labeled endosomes at the cell periphery in pan1-18TA sac6△ cells ( Figure 6D ) .", "As reported previously ( Gheorghe et al . , 2008 ) , deletion of the SAC6 gene increased the lifetime of actin patches , but around half of the endocytic vesicles were able to internalize ( Figure 6—figure supplement 1C , D ) , indicating that the formation of endocytic vesicles is delayed but eventually occurred .", "In contrast , in pan1-18TA sac6△ cells , the lifetime of actin patches were significantly increased and their internalizations were markedly decreased , but ~5 . 3% of patches were still internalized ( Figure 6—figure supplement 1D ) .", "Thus , it seems that endocytic vesicle formation is not completely blocked in pan1-18TA sac6∆ cells .", "To examine if endocytic vesicles are formed in this double mutant , we explored the ultrastructure of these mutants using electron microscopy .", "Interestingly , we observed many vesicle-like structures ( ~40–60 nm ) accumulating around the cell periphery in pan1-18TA sac6△ cells , whereas in wild-type cells such structures were rarely detected ( Figure 6—figure supplement 2A–C ) .", "We also observed that several endosome-like structures ( ~200–300 nm ) associate with these vesicles in this mutant ( Figure 6—figure supplement 2B ) .", "Quantification of these structures revealed that endosome-like structures associate with ~1–3 small vesicles in two-dimensional cross sections ( Figure 6—figure supplement 2D ) .", "Similarly to pan1-18TA sac6△ cells , in pan1-18TA sla2△ cells , Hse1p-labeled endosomes were observed at the cell periphery .", "sla2△ cells are reported to have a severe defect in endocytic internalization , but the internalization is not completely inhibited ( Raths et al . , 1993 ) and endocytic coats are assembled on the plasma membrane ( Kaksonen et al . , 2003; Skruzny et al . , 2012 ) .", "Thus , in pan1-18TA sla2△ cells , peripheral endosomes might interact with the occasionally formed endocytic vesicles at the cell periphery or with coat proteins accumulating on the plasma membrane .", "Examining Hse1-GFP in overlaid time-lapse images of pan1-18TA sac6△ cells demonstrated its localization exclusively at the cell periphery and vacuolar membrane , and that peripheral endosomes are less motile ( ~17 . 8 nm/s ) , compared to prevacuolar ones ( ~189 . 0 nm/s ) ( Figure 6E , F ) .", "Since the pan1-18TA sac6△ double mutant has apparently normal late endosomes at the vacuolar membrane , similar to wild-type cells ( Figure 6E ) , the peripheral immotile endosomes observed in the mutant are likely early endosomes .", "Such peripheral endosomes were rarely observed in the sac6△ mutant ( Figure 6E ) , suggesting that they are due to the mutation of the phosphorylation sites in Pan1p and not just due to the defects in the actin cytoskeleton .", "LatA treatment had no effect on endosome localization in pan1-18TA sac6△ cells ( Figure 6G ) , supporting the contention that endocytic vesicles and early endosomes stably associate , independently of actin . 10 . 7554/eLife . 10276 . 019Figure 6 . Interaction between endocytic vesicles and early endosomes in the pan1-18TA mutant .", "( A ) Localization of Hse1-3GFP-labeled endosomes and actin structures in pan1-18TA double mutant cells .", "The pan1-18TA and double mutant cells expressing Hse1-3GFP and Abp1-mCherry were grown to early to mid-logarithmic phase at 25°C in YPD medium and observed by fluorescence microscopy .", "( B ) Higher magnification view of the boxed areas in ( A ) .", "Arrowheads indicate examples of Hse1p-containing endosomes at the plasma membrane .", "( C ) Quantification of the number of Hse1p-containing endosomes localizing at the cell periphery in single focal plane images .", "Data show mean ± SD , with n = 50 cells counted for each strain .", "( D ) Localization of Pan1-18TA-mCherry and Hse1-3GFP in pan1-18TA sac6△ double mutant cells .", "( E ) Movement of Hse1p-containing endosomes in pan1-18TA , sac6△ , and the double mutant .", "Cells expressing Hse1-3GFP to visualize the endosomes were grown to log phase at 25°C , and imaged for 30 s at 1 s intervals .", "At time t0 = 0 s , Hse1-GFP is shown in green , at t1 = 30 s , Hse1-3GFP is shown in red .", "t0 , t1 overlay shows overlay image of t0 and t1 , and t0-t1 overlay shows overlay image of all 30 frames from t0 ( 0 s ) to t1 ( 30 s ) .", "Scale bars , 2 . 5 μm .", "( F ) Quantification of the velocity of Hse1p-containing endosomes at the cell periphery ( PM ) and the vacuolar membrane ( Vac ) .", "( G ) Localization of Hse1p-residing endosomes in the pan1-18TA sac6△ mutant treated with 250 μM LatA .", "Cells treated with 250 μM LatA for 30 min at 25°C were imaged for 30 s at 1 s intervals .", "At time t0 = 0 s , Hse1-GFP is shown in green , at t1 = 30 s , Hse1-3GFP is shown in red .", "t0 , t1 overlay shows overlay image of t0 and t1 .", "Scale bars , 2 . 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 10276 . 01910 . 7554/eLife . 10276 . 020Figure 6—figure supplement 1 . Actin structures and actin patch dynamics in pan1-18TA double mutant cells .", "( A ) Maximum intensity projections of Z stacks of pan1-18TA and double mutant cells labeled with Abp1-mCherry .", "The Z series was acquired through the entire cell at 0 . 2 μm intervals .", "( B ) Quantification of the fraction of cells containing actin clumps .", "The bar graphs represent the average percentage of cells containing actin clump ( s ) .", "The size of the actin clumps is not considered in this experiment .", "Data show mean ± SEM from at least three experiments , with 50 cells counted for each strain per experiment .", "( C ) Localization of Abp1-GFP in wild-type , sac6△ , and pan1-18TA sac6△ cells .", "Kymographs from the same movies are shown in the lower panels .", "( D ) The left bar graph represents average lifetimes of Abp1-GFP ± SD in indicated cells .", "Data were taken from 1- or 2-min movies with a 1- or 2-s frame interval .", "n = 50 patches for each strain .", "** , p value <0 . 001 , unpaired t-test .", "The right graph represents the percentage of patches internalized into the cytoplasm in indicated cells .", "Data show mean ± SEM from at least three experiments , with >50 patches counted for each strain per experiment .", "** , p value <0 . 001 , unpaired t-test .", "Scale bars , 0 . 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 10276 . 02010 . 7554/eLife . 10276 . 021Figure 6—figure supplement 2 . Ultrastructure of endocytic vesicles and endosomes observed in wild-type and pan1-18TA sac6△ cells .", "( A and B )", "Wild-type and pan1-18TA sac6△ cells were grown at 25°C , fixed using propane jet freezing method and processed for electron microscopic analysis .", "The lower panels show higher magnification views of the boxed areas in the upper panels .", "Arrowheads point to endocytic vesicle-like structures that accumulate in pan1-18TA sac6△ cells .", "E: endosome-like structures .", "Dotted lines represent the areas 100 nm outside from endosome-like structures .", "Scale bars: 2 μm ( upper panels ) , 0 . 5 μm ( lower panels ) .", "( C ) The bar graph represents the percentage of cell containing accumulation of ~50 nm vesicle ( n = 20 cells ) .", "( D ) Quantification of number of ~50 nm vesicles existing around endosome-like structures .", "Numbers of vesicles locating within 100 nm from outside of endosome-like structures were counted .", "Data show mean ± SD , with >50 endosomes counted for each strain . DOI: http://dx . doi . org/10 . 7554/eLife . 10276 . 021" ], [ "In yeast , endocytic vesicles require actin cables to mediate their transport to the early endosome .", "The vesicles move in a retrograde direction , from daughter toward mother cells ( Huckaba et al . , 2004; Toshima et al . , 2006 ) .", "This is distinct from most types of transport along actin cables , such as secretion and organellar division , that move from mother to daughter cells due to the action of myosin V motor proteins ( Myo2p and Myo4p ) .", "Findings that endocytic vesicle movement occurs at the same velocity and direction as that of actin cables have suggested that endocytic vesicles remain fixed on the actin cables and move as a result of actin cable flow ( Girao et al . , 2008; Huckaba et al . , 2004 ) .", "The molecular machinery that attaches endocytic vesicles to actin cables has not yet been elucidated , although a likely candidate would be an endocytic protein that binds F-actin .", "Several such proteins exist ( Engqvist-Goldstein and Drubin , 2003 ) .", "Considering that the association between endocytic vesicles and actin cables should be transient and controllable , Pan1p is an ideal candidate to mediate this interaction .", "Pan1p can bind directly to F-actin with high affinity ( KD<0 . 5 μM ) and its binding activity is regulated by phosphorylation through the Ark1/Prk1 kinases ( Toshima et al . , 2005 ) .", "In this study , we demonstrated that a Pan1-18TA △855 mutant lacking its C-terminal actin binding and Arp2/3-activating regions partially suppressed the formation of the actin clump and reduced the interaction between Pan1p-residing vesicles and actin cables .", "Thus , Pan1p seems to be one of the key regulators that fixes vesicles to the actin cable and then dissociate from the cable and the vesicle upon phosphorylation ( Figure 7 ) .", "However , the ability of vesicles to bind to actin cables was not completely lost in the mutant , implying the existence of additional actin-binding coat protein ( s ) that stabilize vesicle association with actin cables .", "Sla2p , the yeast HIP1R , and Ent1p , the yeast epsin , bind to both the plasma membrane and F-actin via their N-terminal lipid-binding domain and C-terminal actin-binding domain ( Skruzny et al . , 2012; Sun et al . , 2005; Yang et al . , 1999 ) .", "A recent study showed that Sla2p and Ent1p interact redundantly with F-actin , and strains carrying a deletion of both proteins’ actin-binding domains exhibit severe a defect in endocytosis ( Skruzny et al . , 2012 ) .", "Although the defects caused by these mutants are predominantly observed in vesicle formation , these proteins could be responsible for the residual association of endocytic vesicles with actin cables in the Pan1-18TA actin-binding mutant . 10 . 7554/eLife . 10276 . 022Figure 7 . Model of the actin cable-mediated endocytic pathway . Unphosphorylated Pan1p on an endocytic vesicle binds to actin to fix the vesicle to the actin cable .", "After being pinched off from the membrane , the endocytic vesicle moves into the cytosol as a result of actin cable flow , and then , interacts with the early endosome via potential tethering protein .", "Pan1p phosphorylation by Ark1/Prk1 kinases causes dissociation of coat proteins and the actin cable from the endocytic vesicle , making it possible for the vesicle to fuse to the endosome .", "This also results in the dissociation of the actin cable and the early endosome , which then moves to the vacuolar membrane , and matures into a late endosome . DOI: http://dx . doi . org/10 . 7554/eLife . 10276 . 022 Early endosomes also associate with actin cables ( Chang et al . , 2003; Toshima et al . , 2006 ) , but the mechanism is still unknown .", "In the pan1-18TA mutant , endocytic vesicles stably associate with and move together with endosomes .", "This finding suggests that endocytic vesicles are capable of tethering to early endosomes , but are inefficient at fusing with them due to the inhibition of endocytic vesicle uncoating caused by Pan1p-dephosphorylation .", "Many tethering proteins localized at target organelles have been shown to directly interact with coat proteins of transport vesicles ( Cai et al . , 2007 ) , supporting this idea .", "After phosphorylation of Pan1p by Ark1/Prk1 kinases , the endocytic vesicle is uncoated , making it possible to fuse to the early endosome .", "Thus the cycle of Pan1 phosphorylation could release endocytic vesicles from the actin cable precisely at the time of their fusion to the endosome , also allowing the endosome which is indirectly tethered to then move on and mature into a late endosome .", "Our experiments may also permit a clearer ordering of the mechanistic steps of endocytosis .", "Many lines of evidence indicate that cargo transport from early to late endosomes is achieved by endosome maturation , which is a successive and rapid process accompanied by Rab5-Rab7 conversion ( Balderhaar and Ungermann , 2013; Nordmann et al . , 2010; Poteryaev et al . , 2010; Rink et al . , 2005 ) .", "In yeast , attempts to visualize the conversion of Vps21p ( yeast Rab5 ) to Ypt7p ( yeast Rab7 ) on endosomes have so far been unsuccessful .", "Since Vps21p is localized not only at early , but also at late endosomes ( Toshima et al . , 2014 ) , it is difficult to determine the point at which the early endosome ends and the late endosome begins .", "In the pan1-18TA mutant , localization of endosomal proteins was clearly divided into several groups , and endosomes at the early stage were more highly localized to actin clumps .", "This observation may be informative when considering the timing of several events comprising the endocytic pathway .", "For instance , subunits of the ESCRT-0 , I , and II complex are highly localized but the subunit of ESCRT-III or Vps4p is localized only partially at actin clumps in the pan1-18TA mutant , suggesting that recruitment of early ESCRT components to the endosome occurs at a relatively early stage ( Figure 7 ) .", "Vps21p is a key regulator of early endocytic trafficking , being involved in fusion between early endosomes and the maturation of early to late endosomes ( Poteryaev et al . , 2010; Rink et al . , 2005; Russell et al . , 2012 ) .", "Deletion of the VPS21 gene results in accumulation of early endosomes in the cytosol ( Toshima et al . , 2014 ) .", "Yet Vps21p exhibited only partial localization at actin clumps , suggesting that Vps21p is recruited to the endosomal membrane after the start of ESCRT complex formation .", "Vps26p , a component of the retromer complex , is rarely localized at actin clumps and is predominantly localized at the vacuolar membrane , suggesting that the retromer complex mediates retrograde transport to the Golgi at the late endosome stage ( Figure 7 ) .", "Although Rab5-Rab7 conversion is important for endosome maturation what triggers initiation of early-to-late endosome transition is still unknown .", "We demonstrated that early endosomes associate with actin filaments and then change their localization to the vacuolar membrane .", "This suggests that dissociation of early endosomes from the actin filaments might be a trigger for the initiation of endosome maturation .", "In mammalian cells , before endosomes move from the plasma membrane to the lysosome along microtubules , endosomes associate with the cortical actin cytoskeleton underlying the plasma membrane ( Aschenbrenner et al . , 2004; Fernandez-Borja et al . , 2005 ) .", "The interaction between endosomes and actin is regulated by RhoB GTPase , an upstream recruiter and activator of mammalian Dia1 and PRK1/PKN ( Fernandez-Borja et al . , 2005; Mellor et al . , 1998 ) .", "Expression of activated RhoB facilitates the association of early endosomes with cortical actin filaments , which prevents the transfer of endosomes to microtubules and inhibits further transport ( Fernandez-Borja et al . , 2005 ) .", "The physiological importance of the interaction between endosomes and the actin cytoskeleton in mammalian cells has not been determined , but in analogy to our findings this might enable endosomes to fuse with endocytic vesicles more efficiently , at the same time preventing the progression of immature early endosomes to late endosomes .", "In conclusion , our results suggest that phosphorylation of Pan1p regulates the interaction between endocytic compartments and the actin cytoskeleton .", "Clarifying the molecular mechanisms regulating the interaction between endocytic vesicles and endosomes , and endosomes and the actin cytoskeleton is important for elucidating the whole picture of transport from the formation of an endocytic vesicle to its fusion to an early endosome ." ], [ "The yeast strains used in this study are listed in the strain list ( Supplementary file 1 ) .", "All strains were grown in standard rich medium ( YPD ) or synthetic medium ( SM ) supplemented with 2% glucose and appropriate amino acids .", "C-terminal GFP or mCherry tagging of proteins was performed as described previously ( Longtine et al . , 1998 ) .", "The pan1-18TA mutant was integrated as follows: First , to create a pan1 integration plasmid , the XmnI-DraI fragment of the PAN1 gene was cloned into pBluescript II SK , and the SalI fragment of the LEU2 gene was inserted into the SalI site 154-bp upstream of the PAN1 ORF ( Toshima et al . , 2005 ) .", "The mutated MscI-NheI pan1-18TA fragments were used to replace the PAN1 gene in the integration plasmid .", "To integrate pan1 mutants at the endogenous locus , the integration plasmids were digested with SacI and XbaI , and transformed into pan1△::HIS3/PAN1 diploid strains .", "Integrated pan1 mutants were selected on SC plates lacking leucine and sporulated to obtain pan1-18TA mutants .", "Phosphorylation site mutants were constructed by a PCR-based mutagenesis protocol ( Supplementary file 2 ) .", "Fluorescence microscopy was performed using an Olympus IX81 microscope equipped with a x100/NA 1 . 40 ( Olympus ) objective and Orca-AG cooled CCD camera ( Hamamatsu , JAPAN ) , using Metamorph software ( Universal Imaging ) .", "Simultaneous imaging of red and green fluorescence was performed using an Olympus IX81 microscope , described above , and an image splitter ( Dual-View; Optical Insights ) that divided the red and green components of the images with a 565-nm dichroic mirror and passed the red component through a 630/50 nm filter and the green component through a 530/30 nm filter .", "These split signals were taken simultaneously with one CCD camera , described above .", "Preparation of cell extracts was performed as described previously ( Toshima et al . , 2005 ) .", "In brief , the cells grown in 200 ml YPD to OD600 of 1 . 0 were harvested by centrifugation , washed with dH2O , resuspended to 1 ml of dH2O , drop-frozen in liquid N2 , and powdered with mortar and pestle .", "The cell extracts were prepared using lysis buffer ( 50 mM Tri-HCl , pH 8 . 0 , 150 mM NaCl , 8 M Urea , 1% Triton X-100 , phosphatase inhibitor cocktail ) .", "High molecular weight proteins over 100 K molecular weight were enriched using Amicon Ultra-0 . 5 100 K ( Millipore ) , and phosphorylated proteins included in the fraction were enriched using Phos-tag Agarose ( NARD Institute ) .", "The enrichment of phosphorylated proteins using Phos-tag Agarose was performed as previously ( Kinoshita-Kikuta et al . , 2006; 2009 ) .", "Immunoblot analysis was performed as described previously ( Toshima et al . , 2005 ) .", "The chicken polyclonal antibody to GFP ( GeneTex , GTX124117 ) was used at a dilution of 1:10000 and the HRP-conjugated rabbit polyclonal antibody to chicken IgY ( Promega , G135A ) at 1:10000 dilution was used as the secondary antibody .", "Immunoreactive proteins bands were visualized using the Western Lightning Plus ECL ( PerkinElmer ) .", "Endosome motility and velocity was analyzed using the ImageJ v1 . 32 software package .", "For quantification of endosome velocity , time-lapse images were acquired at 1 s intervals .", "To determine the velocity , the distance traveled by each endosome in 1 s was calculated based on pixel coordinates ( 1 pxl = 64 nm ) .", "Cells sandwiched between copper disks were frozen in liquid propane at -175°C and then freeze substituted with acetone containing 2% OsO4 and 2% distilled water at -80°C for 48 hr .", "The samples were kept at -20°C for 4 hr and then at 4°C for 1 hr , and dehydrated in anhydrous acetone two times and 100% ethanol three times .", "After being infiltrated with propylene oxide ( PO ) two times the samples were put into a 70:30 mixture of PO and resin ( Quetol-651 ) and then transferred to a fresh 100% resin , and polymerized at 60°C for 48 hr .", "The blocks were cut into 70-nm-thick sections , and the sections were mounted on copper grids .", "The specimens were stained with 2% uranyl acetate and Lead stain solution , and observed using a transmission electron microscope ( JEM-1400Plus; JEOL ) ." ] ]

[ "The actin cytoskeleton plays important roles in the formation and internalization of endocytic vesicles .", "In yeast , endocytic vesicles move towards early endosomes along actin cables , however , the molecular machinery regulating interaction between endocytic vesicles and actin cables is poorly understood .", "The Eps15-like protein Pan1p plays a key role in actin-mediated endocytosis and is negatively regulated by Ark1 and Prk1 kinases .", "Here we show that pan1 mutated to prevent phosphorylation at all 18 threonines , pan1-18TA , displayed almost the same endocytic defect as ark1Δ prk1Δ cells , and contained abnormal actin concentrations including several endocytic compartments .", "Early endosomes were highly localized in the actin concentrations and displayed movement along actin cables .", "The dephosphorylated form of Pan1p also caused stable associations between endocytic vesicles and actin cables , and between endocytic vesicles and endosomes .", "Thus Pan1 phosphorylation is part of a novel mechanism that regulates endocytic compartment interactions with each other and with actin cables ." ]

[ "The cells of all eukaryotes – including plants , animals and fungi – absorb many substances that they need from their surroundings by forming pockets around them , and then pinching off these pockets to create structures called vesicles .", "Clathrin is a protein that acts as a scaffold for these vesicles .", "Inside a eukaryotic cell , clathrin-coated vesicles first go to a structure known as an endosome , possibly by following a track made from filaments of a protein called actin .", "Researchers have shown previously that a yeast protein called Pan1 binds to actin filaments and helps the cells to create clathrin-coated vesicles .", "However it was unclear if the Pan1 protein is also important for transporting clathrin-coated vesicles to endosomes .", "Previous studies have also shown that adding phosphate groups on to the Pan1 protein prevents it from binding to clathrin-coated vesicles or actin filaments .", "Now , Toshima et al . show that a mutant version of the Pan1 protein , which cannot be modified in this way , can bind stably to both clathrin-coated vesicles and the actin filaments and connect them together .", "The experiments also showed that , in yeast cells that only produce the mutant version of Pan1 , clathrin-coated vesicles bind stably to endosomes without the need for actin .", "Thus , these findings show that the addition of phosphate groups onto Pan1 is part of a mechanism that regulates the interactions between clathrin-coated vesicles , endosomes and actin filaments .", "Following on from this work , one future challenge is to find which proteins directly connect clathrin-coated vesicles with endosomes .", "It will also be important to investigate if similar mechanisms are used in the cells of mammals ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "cell biology", "cancer biology" ]

[ [ "Molecular scaffolds affect the intensity and duration of signaling pathways by coordinating a discrete set of effectors at defined subcellular locations to regulate multiple cell fates ( Morrison and Davis , 2003; Pawson and Scott , 1997 ) .", "Kinase Suppressor of Ras 1 ( KSR1 ) serves as a scaffold for Raf , MEK , and ERK enabling the efficient transmission of signals within the mitogen activated protein kinase ( MAPK ) cascade ( Kortum and Lewis , 2004; Nguyen et al . , 2002 ) .", "Although KSR1 is dispensable for normal development , it is necessary for oncogenic Ras-induced tumorigenesis including colorectal cancer cells ( Kortum and Lewis , 2004; Nguyen et al . , 2002; Fisher et al . , 2011; Fisher et al . , 2015; Morrison et al . , 2009; Rao et al . , 2020 ) , suggesting that KSR1 may modulate aberrant signals that redirect the function of effectors typically involved in normal cellular homeostasis .", "Activating Ras mutations are present in over 40 % of colorectal cancers ( CRC ) , and associated with advanced disease and decreased overall survival ( Haigis , 2017; Serebriiskii et al . , 2019 ) .", "Activated Ras , a critical driver of both tumor growth and survival , is an alluring therapeutic target , yet targeting the majority of oncogenic Ras alleles is still a work in progress .", "Raf/MEK/ERK signaling can phenocopy Ras signaling essential for CRC growth and survival ( Schmitz et al . , 2007; Brandt et al . , 2019 ) .", "Therefore , understanding the effectors that transmit signals emanating from oncogenic Ras is a valuable step in detecting and targeting the pathways critical to tumor cell function and their adaptation to therapy .", "Oncogene-driven signaling pathways promote mRNA translation that enables expression of a subset of mRNAs to promote growth , invasion , and metastasis ( Chu et al . , 2016; Avdulov et al . , 2004; Truitt Morgan et al . , 2015; Pelletier et al . , 2015 ) .", "Tumor cells have an increased dependence on cap-dependent translation , unlike their normal complements ( Truitt Morgan et al . , 2015; Truitt and Ruggero , 2016 ) .", "Eukaryotic Translation Initiation Factor 4E ( eIF4E ) is a rate-limiting factor for oncogenic transformation , with reductions of as little as 40 % being sufficient to block tumorigenesis ( Truitt Morgan et al . , 2015 ) .", "eIF4E function is regulated by association of 4E-binding proteins ( 4EBPs ) .", "Importantly , disruption of KSR1 or ERK inhibition leads to dephosphorylation and activation of 4EBP1 , indicating that the function of KSR1 as an ERK scaffold is key to the aberrant regulation of mRNA translation ( McCall et al . , 2016 ) .", "This tumor-specific , KSR1-dependent regulation of mRNA translation of a subset of genes was predicted to selectively promote survival of CRC cells but not normal colon epithelia ( McCall et al . , 2016; Neilsen et al . , 2019 ) .", "Almost all CRC originates from epithelial cells lining the colon or rectum of the gastrointestinal tract , but in order to invade to the surrounding tissue , cancer cells lose cell adhesiveness to acquire motility and become invasive , characterized by the epithelial-to-mesenchymal transition ( EMT ) , which is central to tumor pathogenesis ( Ye and Weinberg , 2015; Nieto , 2013; Thiery et al . , 2009; Dongre and Weinberg , 2019 ) .", "EMT involves a complex cellular process during which epithelial cells lose polarity , cell-cell contacts and acquire mesenchymal characteristics .", "While EMT is crucial for cell plasticity during embryonic development , trans differentiation and wound healing , when aberrantly activated EMT has deleterious effects , which facilitate motility and invasion of cancer cells ( Nieto , 2013; Thiery et al . , 2009; Dongre and Weinberg , 2019; Nieto et al . , 2016 ) .", "EMT has been shown to be controlled by transcription-dependent mechanisms , especially through repression of genes that are hallmarks of epithelial phenotype such as E-cadherin .", "Loss of E-cadherin at the membrane has been associated with carcinoma progression and EMT ( Thiery et al . , 2009; Thiery , 2002; Oda et al . , 1994; Frixen et al . , 1991 ) .", "E-cadherin function is transcriptionally repressed through the action of EMT transcription factors ( TFs ) , including Snail-family proteins ( Snail1 , Slug ) , zinc finger E-box binding homeobox 1 and 2 ( ZEB1 and ZEB2 ) , and twist-related protein ( Twist ) ( Nieto et al . , 2016; Jolly et al . , 2017 ) .", "Transcriptional control of E-cadherin is unlikely to be sole determinant of EMT , invasion and metastasis .", "Inappropriate induction of non-epithelial cadherins , such as N-cadherin by epithelial cells are known to play a fundamental role during initiation of metastasis ( Nieman et al . , 1999; Liu et al . , 2017; Suyama et al . , 2002; Rosivatz et al . , 2004; Okubo et al . , 2017; Sadot et al . , 1998; Loh et al . , 2019 ) .", "N-cadherin disassembles adherent junction complexes , disrupting the intercellular cohesion and reorienting the migration of cells , away from the direction of cell-cell contact ( Nieman et al . , 1999; Scarpa et al . , 2015 ) .", "Upregulation of N-cadherin expression promotes motility and invasion ( Nieman et al . , 1999; Liu et al . , 2017; Suyama et al . , 2002; Hulit et al . , 2007 ) .", "Thus , central to the process of EMT is the coordinated loss of E-cadherin expression and the upregulation of N-cadherin gene expression , termed cadherin switching ( Loh et al . , 2019; Wheelock et al . , 2008; Tomita et al . , 2000; Maeda et al . , 2005; Araki et al . , 2011 ) .", "Previous studies have demonstrated transcriptional regulation of EMT through oncogenic Ras or its downstream effector signaling pathways via the activation of EMT-TFs ( Shin et al . , 2010; Shin et al . , 2019; Andreolas et al . , 2008; Liu et al . , 2014; Wong et al . , 2013; Wang et al . , 2010; Lemieux et al . , 2009 ) .", "Oncogenic Ras itself activates EMT-TF Slug to induce EMT in skin and colon cancer cells ( Wong et al . , 2013; Wang et al . , 2010 ) .", "Enhanced activity of ERK2 but not ERK1 , has been linked with Ras-dependent regulation of EMT ( Shin et al . , 2010; Shin et al . , 2019 ) .", "Several studies have also described an alternative program wherein cells lose their epithelial phenotype , via post-transcriptional modifications rather than transcriptional repression involving translational regulation or protein internalization ( Jechlinger et al . , 2003; Aiello et al . , 2018; Waerner et al . , 2006 ) .", "Expression profiling of polysome-bound mRNA to assess translational efficiency identified over 30 genes that were translationally regulated upon Ras and TGFβ inducing EMT ( Jechlinger et al . , 2003; Waerner et al . , 2006 ) .", "Functional characterization of the resultant proteins should reveal preferentially translated mRNAs essential to invasion and metastasis .", "EPSTI1 was identified as a stromal fibroblast-induced gene upon co-cultures of breast cancer cells with stomal fibroblasts ( Nielsen et al . , 2002 ) .", "EPSTI1 is expressed at low levels in normal breast and colon tissue but aberrantly expressed in breast tumor tissue ( Nielsen et al . , 2002 ) .", "EPSTI1 promotes cell invasion and malignant growth of primary breast tumor cells ( Li et al . , 2014; de Neergaard et al . , 2010 ) .", "We performed polysome profiling in CRC cells and found that KSR1- and ERK induces of EPSTI1 mRNA translation .", "EPSTI1 is both necessary and sufficient for coordinating the upregulation of N-cadherin with the downregulation of E-cadherin to stimulate cell motility and invasion in colon cancer cells .", "These data demonstrate that ERK-regulated regulation of mRNA translation is an essential contributor to EMT-like phenotype and reveal a novel effector of the cadherin switch whose characterization should yield novel insights into the mechanisms controlling the migratory and invasive behavior of cells ." ], [ "ERK signaling regulates global and selective mRNA translation through RSK1/2-dependent modification of cap-dependent translation ( McCall et al . , 2016; Roux et al . , 2007 ) .", "Phosphorylation of cap binding protein 4E-BP1 releases eIF4E to promote translation and the abundance of eIF4E is a rate-limiting factor for oncogenic Ras- and Myc-driven transformation ( Truitt Morgan et al . , 2015 ) .", "We showed previously that KSR1 maximizes ERK activation in the setting of oncogenic Ras ( Kortum et al . , 2006 ) , which is required for increased Myc translation via dephosphorylation of 4E-BP1 , supporting CRC cell growth ( McCall et al . , 2016 ) .", "These observations imply that the ERK scaffold function of KSR1 alters the translational landscape in CRC cells to support their survival .", "To determine the effect of KSR1 on translatomes in colon cancer cells , we performed genome-wide polysome profiling ( King and Gerber , 2016 ) .", "We stably expressed short hairpin RNA ( shRNA ) constructs targeting KSR1 ( KSR1 RNAi ) or a non-targeting control in two K-Ras mutant CRC cell lines , HCT116 and HCT15 ( Figure 1D , top panels ) .", "We isolated and quantified both total mRNA and efficiently translated mRNAs ( associated with ≥3 ribosomes ) using RNA sequencing ( Figure 1A , Figure 1—figure supplement 1 ) .", "We used Anota2seq ( Oertlin et al . , 2019 ) to calculate translation efficiency ( TE ) by comparing the differences in efficiently translated mRNAs to the total transcript of each mRNA and observed that a significant number of mRNAs ( [selDeltaTP ≥ log ( 1 . 2 ) and selDeltaPT ≥ log ( 1 . 2 ) ] and p-value < 0 . 05 ) showed either reduced TE or upregulated TE upon KSR1 disruption ( Figure 1B–C , Supplementary file 1 , Source data 1 ) in both HCT116 and HCT15 cells .", "Gene Set Enrichment Analysis ( GSEA ) ( Subramanian et al . , 2005 ) of significantly enriched genes in HCT116 and HCT15 ( Figure 1B , Figure 1—figure supplement 2A-B ) , identified 11 mRNAs in the gene set titled “Hallmark EMT signature” , “Jechlinger EMT Up” , and “Gotzmann EMT up” , that had significantly decreased translation upon KSR1 disruption ( Supplementary file 2 ) .", "Among the genes with decreased translation , EPSTI1 was one of the highly significant mRNAs .", "We sought to determine the functional relevance of KSR1-dependent induction of EPSTI1 to phenotypic plasticity in colon cancer cells .", "To confirm that EPSTI1 translation is KSR1-dependent , we observed that , EPSTI1 protein expression was decreased with the knockdown of KSR1 in HCT116 and HCT15 cells ( Figure 1D ) , while the total mRNA transcript was unchanged upon KSR1 disruption ( Figure 1E , left panel ) .", "EPSTI1 TE was markedly decreased upon KSR1 depletion ( Figure 1E , right ) .", "RT-qPCR analysis of sucrose-gradient fractions of monosome mRNA and polysome RNA distribution confirmed that EPSTI1 mRNA shifted from actively translating high-molecular-weight ( MW ) polysome fractions to low-MW fractions in KSR1 knockdown cells ( Figure 1F ) .", "In contrast , HPRT1 mRNA was insensitive to KSR1 knockdown in HCT116 and HCT15 cells , and qPCR analysis of HPRT1 mRNA isolated from sucrose gradient fractions of control and KSR1 knockdown cells showed no significant shift between the low-MW and the high-MW fractions ( Figure 1—figure supplement 2C ) .", "To determine if KSR1 promotes EPSTI1 degradation , we first assessed EPSTI1 turnover in HCT116 cells following treatment with a protein-synthesis inhibitor , cycloheximide ( CHX ) and observed that EPSTI1 has a 6 hr half-life ( Figure 1—figure supplement 2D ) .", "We analyzed EPSTI1 turnover using a combination of proteasome inhibitor , MG132 and CHX in control and CRISPR-targeted KSR1 HCT116 cells ( Figure 1—figure supplement 2E ) .", "EPSTI1 turnover was not sensitive to MG132 treatment in HCT116 cells lacking KSR1 expression .", "Therefore , in HCT116 cells , KSR1 does not mediate ubiquitin proteosome system ( UPS ) -mediated degradation of EPSTI1 .", "These data support our conclusion that EPSTI1 translation is induced by KSR1 .", "To confirm our observations in KSR1 knockdown cells , we tested the effect of CRISPR/Cas9-mediated targeting of KSR1 on EPSTI1 in CRC cell lines .", "EPSTI1 protein expression was decreased upon KSR1 depletion in HCT116 and HCT15 cells and EPSTI1 expression was restored in knockout cells upon expression of a KSR1 transgene ( + KSR1 ) ( Figure 2A ) .", "Similar to inhibition of KSR1 , treatment with ERK inhibitor SCH772984 ( Morris et al . , 2013 ) suppressed EPSTI1 protein expression in both CRC cell line HCT116 and tumorigenic patient derived colon organoid engineered with deletion of APC , p53 , SMAD4 , and K-RasG12D mutation ( PDO-11 AKPS ) ( Figure 2B; Drost et al . , 2015 ) .", "To determine if EPSTI1 expression is also dependent on mTOR signaling , we tested the effect of mTOR inhibition on EPSTI1expression .", "Though mTOR inhibitor , AZD8055 ( Chresta et al . , 2010 ) robustly inhibited phosphorylation of mTOR substrate p70 S6 kinase , its ability to decrease EPSTI1 expression in HCT116 cells was weak relative to treatment with the ERK inhibitor ( Figure 2C ) .", "These observations suggest the ERK affects EPSTI1 expression via mechanisms distinct from mTOR .", "While the total protein was reduced upon ERK inhibition in HCT116 , the EPSTI1 transcript levels were not altered significantly by SCH772984 treatment ( Figure 2D ) .", "We performed polysome profiling in HCT116 cells , either treated with DMSO or ERK inhibitor , SCH772984 and we isolated mRNA from low-MW monosome ( fractions 3–5 ) and high-MW polysome ( fractions 6–8 ) fractions ( Figure 2E ) .", "RT-qPCR demonstrated that EPSTI1 mRNA shifted from high-MW fractions to the low-MW fractions upon ERK inhibition ( Figure 2F ) .", "The distribution of mRNA for HPRT1 within the same profile was not altered by SCH772984 treatment ( Figure 2F ) .", "These data indicate that KSR1-dependent ERK signaling is a critical regulator of EPSTI1 mRNA translation in colon cells and organoids .", "KSR1 disruption inhibits HCT116 cell anchorage-independent growth in vitro and tumor formation in vivo ( Fisher et al . , 2015 ) .", "Similarly , disruption of KSR1 by CRISPR/Cas9-mediated targeting decreased HCT116 and HCT15 cell viability under anchorage-independent conditions on simulated by poly- ( HEMA ) coating ( Figure 3A ) .", "KSR1 transgene expression restored cell viability in HCT116 and HCT15 cells lacking KSR1 ( KSR1 CRISPR+ KSR1 ) ( Figure 3A ) .", "We showed previously that KSR1 expression is upregulated in colon cancer cell lines when compared to the non-transformed human colon epithelial cells ( HCECs ) ( Fisher et al . , 2015 ) .", "We observed that EPSTI1 protein is aberrantly expressed in colon cancer cell lines HCT116 and HCT15 , while its expression is detected weakly in HCECs ( Figure 3B ) .", "EPSTI1 protein expression is also markedly higher in AKPS organoids than normal colon organoids ( Figure 3B ) .", "To determine the regulation of EPSTI1 in human colon tumor maintenance , we performed siRNA knockdown of EPSTI1 in HCT116 and HCT15 cells .", "EPSTI1 disruption suppressed viability on poly- ( HEMA ) coated by 40 % in HCT15 cells , and over 70% , in HCT116 cells ( Figure 3C ) .", "EPSTI1 knockdown reduced colony formation in soft agar by 63 % in HCT116 cells and 71 % in SW480 cells ( Figure 3D ) .", "These observations show that KSR1-dependent translation of ESPTI1 is required for anchorage-independent growth of colon tumor cell lines .", "Considering the suggested role of EPSTI1 in promoting EMT-like phenotypes ( Nielsen et al . , 2002; Li et al . , 2014 ) , we sought to evaluate the biological role of EPSTI1 in colon cancer cells .", "Time-lapse images of control and EPSTI1 knockdown in HCT116 cell motility in a scratch wound was analyzed by measuring the relative wound density ( Johnston et al . , 2015 ) over 72 hours ( Figure 4A , bottom ) .", "IncuCyte software was used to calculate relative wound density , that is , the percentage of spatial cell density inside the wound relative to the spatial density outside of the wound area at a given time point .", "The calculation of cell migration using this method , avoids false changes in cell density due to proliferation .", "Motility was also assessed in control , CRISPR-targeted ( KSR1 CRISPR ) , and CRIPSR-targeted HCT116 cells expressing KSR1 ( KSR1 CRISPR+ KSR1 ) ( Figure 4A , top ) .", "Cells lacking either EPSTI1 or KSR1 were approximately 20 % less motile compared to control cells .", "Reintroduction of KSR1 expression in CRISPR-targeted HCT116 cells restored motility comparable to the control cells ( Figure 4A , top ) .", "EPSTI1 knockdown HCT116 and SW480 cells were subjected to Transwell invasion assays .", "EPSTI1 RNAi suppresses cell invasion through Matrigel by 72 % in HCT116 and by 75 % in SW480 .", "( Figure 4B , top right and bottom ) .", "Since KSR1 is required for EPSTI1 translation , we determined the functional contribution of KSR1 in regulating cell invasion .", "KSR1 depletion suppressed invasion by 64 % in HCT116 and by 53 % SW480 cells ( Figure 4B , top left and bottom ) .", "Overall , these results suggest the KSR1-dependent EPSTI1 signaling contributes to cell migration and invasion in CRC cells .", "To understand the underlying mechanism by which KSR1 or EPSTI1 promote motility and invasion in CRC cells , we evaluated their contribution to the expression of critical determinants of EMT that modulate cell adhesion , E- and N-cadherins and EMT-TFs .", "Compared to the non-targeting control , KSR1 disruption in HCT116 , HCT15 and SW480 cells had elevated levels of E-cadherin , along with a coincident decrease in EMT-TF Slug ( Figure 5A ) .", "Expression of Vimentin , and Snail1 was not changed in HCT116 cells ( Figure 5—figure supplement 1A ) .", "Upon knockdown of EPSTI1 with either of two siRNA oligos , we observed a decrease in the expression of N-cadherin , ZEB1 and Slug .", "Coincident with the decrease in EMT-TFs , E-cadherin levels were elevated ( Figure 5B ) .", "While there was no significant change in the Slug and ZEB1 mRNA upon EPSTI1 knockdown ( Figure 5—figure supplement 1B ) , EPSTI1 disruption decreased N-cadherin mRNA expression over 50 % in HCT116 and SW480 cells ( Figure 5C ) .", "Following EPSTI1 knockdown , we subjected control HCT116 cells and HCT116 cells overexpressing N-cadherin to Transwell invasion assay through Matrigel .", "EPSTI1 knockdown suppressed cell invasion .", "The expression of N-cadherin in cells lacking EPSTI1 was sufficient to restore invasiveness to HCT116 cells ( Figure 5—figure supplement 1C-D ) .", "This is consistent with previous observations that upregulation of N-cadherin expression enhances motility in multiple cancer cell lines ( Nieman et al . , 1999; Hulit et al . , 2007; Mrozik et al . , 2018 ) .", "These results indicate that the switch of E-cadherin to N-cadherin expression promotes the progression of migratory and invasive behavior orchestrated by EPSTI1 signaling in CRC cells .", "To determine the extent to which KSR1- and ERK-dependent EPSTI1 translation is critical to colon tumor cell growth and invasion , we expressed a MSCV-FLAG-EPSTI1-GFP construct in KSR1-CRISPR knockout HCT116 , SW480 , and HCT15 cells .", "CRISPR/Cas9-mediated deletion of KSR1 disrupted EPSTI1 expression , downregulated Slug and N-cadherin expression and elevated E-cadherin expression ( Figure 6A ) .", "E-cadherin staining was absent in control CRC cells but evident at the cell membrane in KSR1 knockout cells ( Figure 6B ) .", "Exogenous expression of EPSTI1 in cells lacking KSR1 restored the cadherin switch , by decreasing the expression of E-cadherin ( Figure 6A and B ) and increasing N-cadherin levels comparable to control cells ( Figure 6A ) .", "Suppression of E-cadherin and restoration of N-cadherin expression by the EPSTI1 transgene reestablished the ability of KSR1 knockout cells to migrate in monolayer culture ( Figure 6C ) and invade through Matrigel .", "Forced expression of EPSTI1 in these cells , increased the number of invading cells by over threefold ( Figure 6D ) .", "To determine the effect of EPSTI1 on cell proliferation , we analyzed the cell growth kinetics in HCT116 and SW480 cells ( Figure 6—figure supplement 1 ) .", "Over 3 days , EPSTI1 knockdown had no effect on cell proliferation compared to control HCT116 and SW480 cells .", "While EPSTI1 expression in KSR1 knockout cells had no significant effect on cell proliferation for 24 hr in HCT116 and SW480 cells ( Figure 6—figure supplement 1A-B ) , EPSTI1 expression increased the number of invading cells in that period over 50 % in HCT116 and over 70 % in SW480 cells ( Figure 6D ) .", "Although , EPSTI1 expression has a significant effect on cell proliferation over 7 days compared to KSR1 knockout cells in HCT116 and SW480 cells , EPSTI1 expression rescued migratory potential by over 60 % in KSR1-depleted HCT116 and SW480 cells within 24 hr ( Figure 6C ) .", "These data reveal that disabling the cadherin switch and inhibition of cell invasion by KSR1 disruption interrupts EPSTI1 translation , highlighting the pivotal role of this pathway for the induction of EMT-like phenotype in CRC cells .", "Knockdown of EPSTI1 in HCT116 and SW480 , decreased N-cadherin mRNA expression 50 % ( Figure 5C ) .", "Upon KSR1 depletion , N-cadherin mRNA decreased 32 % in HCT116% and 89% in SW480 cells ( Figure 7A ) .", "Ectopic expression of EPSTI1 in these cells restored the N-cadherin mRNA expression to levels observed in control SW480 cells , while in HCT116 KSR1 KO , forced EPSTI1 expression increased N-cadherin mRNA levels threefold above that seen in control HCT116 cells ( Figure 7A ) .", "We tested the effect of ectopic expression of EPSTI1 on invasion in non-transformed HCECs .", "We stably expressed MSCV-IRES-GFP or MSCV-IRES-EPSTI1-GFP in HCECs and subjected the cells to Transwell invasion assay through Matrigel ( Figure 7B , top ) , and observed that EPSTI1 alone was sufficient to dramatically induce the expression of N-cadherin and double the invasive activity of HCECs ( Figure 7B ) .", "These data indicate that EPSTI1 mediates the expression of N-cadherin to promote invasive behavior in non-transformed colon epithelial cells and colon cancer cells .", "The E- to N-cadherin switch promotes cancer cell survival following the loss of cell adhesion to the extracellular matrix ( Derksen et al . , 2006; Onder et al . , 2008 ) .", "KSR1 also promotes CRC cell survival when detached from a solid substrate ( Fisher et al . , 2015; McCall et al . , 2016 ) .", "To determine the extent to which EPSTI1 expression was sufficient to restore CRC cell viability in the absence of KSR1 , we grew cells under anchorage-independent conditions either on Poly- ( HEMA ) ( Figure 7C ) or on soft agar ( Figure 7D ) following forced expression of EPSTI1 in HCT116 , HCT15 , and SW480 cells lacking KSR1 .", "Anchorage-independent viability was measured over three days on poly- ( HEMA ) coated plates .", "Compared to control HCT116 and HCT15 cells , viability decreased approximately 75 % in cells lacking KSR1 .", "Ectopic expression of EPSTI1 restored viability to approximately 50 % of control levels in both cell lines ( Figure 7C ) .", "Similar to our previous findings ( Fisher et al . , 2015; Kortum et al . , 2006 ) , KSR1 disruption hampered the ability of Ras transformed cells to form colonies on soft agar , the number of colonies formed in HCT116 and SW480 cells dramatically decreased by 75 % in the absence of KSR1 .", "Forced expression of EPSTI1 was sufficient to reverse the suppression of colony formation caused by KSR1 disruption to levels observed in control HCT116 and SW480 cells ( Figure 7D ) .", "These results show that despite the absence of KSR1 to maintain and support cell growth , ectopic EPSTI1 expression was able to maintain anchorage-independent viability in CRC cells ." ], [ "Persistent oncogenic reprogramming of transcription and translation during EMT grants migratory and invasive properties to tumor cells ( Dongre and Weinberg , 2019; Nieto et al . , 2016 ) .", "Multiple studies have established a relationship between oncogenic Ras-mediated ERK signaling and EMT , either through Ras or its downstream effector signaling pathways activating EMT-TFs ( Shin et al . , 2010; Andreolas et al . , 2008; Liu et al . , 2014; Wong et al . , 2013; Wang et al . , 2010; Lemieux et al . , 2009; Diesch et al . , 2014 ) .", "Silencing of Erbin , a tumor suppresser known to disrupt KSR1-RAF1 interaction , promoted cell migration and invasion of colon cancer cells , but did not identify the mechanism on how KSR1-dependent MAPK signaling affected EMT ( Stevens et al . , 2018 ) .", "Mediators of EMT- like phenotype activate cap-dependent translation initiation have been associated with increased aggressiveness and metastases of cancer cells , and we have shown that KSR1 can affect translation initiation ( McCall et al . , 2016; Jechlinger et al . , 2003; Waerner et al . , 2006; Prakash et al . , 2019 ) .", "Our observations establish the novel role of the scaffold protein KSR1 promoting the preferential translation of an EMT-related gene , EPSTI1 , and outline a mechanism for KSR1-dependent stimulation of phenotypic plasticity .", "Using gene-expression analysis of the polysome-bound mRNA , we discovered KSR1 and ERK increase the translational efficiency of EPSTI1 mRNA .", "EPSTI1 mediates KSR1-dependent motility , invasion , and anchorage-independent growth coincident with its suppression of EMT-TF , Slug , elevating E-cadherin expression .", "EPSTI1 knockdown also decreased the expression of N-cadherin mRNA and protein .", "In the absence of KSR1 , ectopic expression of EPSTI1 was sufficient to suppress E-cadherin expression , stimulate N-cadherin expression and enhance motility and invasive behavior , this invasive behavior was also induced in non-transformed colon cells .", "These data demonstrate that a KSR1- and ERK-regulated component is critical to the execution of the transcriptional program that drives interconversion between epithelial and mesenchymal phenotypes .", "These studies of post-transcriptional regulation and mRNA translation reveal the importance of expanding beyond gene expression analysis for detecting mechanisms underlying epithelial plasticity and tumorigenicity .", "The association of EPSTI1 with tumor metastatic potential is supported by observations that EPSTI1 is highly upregulated in invasive breast cancer tissues and suggested the role of EPSTI1 in promoting metastasis , tumorsphere formation , and stemness ( Nielsen et al . , 2002; Li et al . , 2014; de Neergaard et al . , 2010 ) .", "Although the aberrant expression of EPSTI1 in breast cancer cells is well-established , there is little indication in the literature on the role of EPSTI1 to induce EMT , cancer invasion , and metastasis .", "The association of EPSTI1 induction of invasion in breast cancer cells was attributed to the increased expression of Slug and Twist mRNA and increased expression of fibronectin and α2β1 integrins ( de Neergaard et al . , 2010 ) .", "Another study suggested the interaction of EPSTI1 with valosin-containing protein ( VCP ) and the subsequent activation of NF-κB signaling contributed to the increased tumor invasion and metastasis ( Li et al . , 2014 ) .", "Future studies should evaluate the potential of EPSTI1 to directly affect N-cadherin and EMT-TF expression , assess the role of NF-κB signaling in EPSTI1-dependent CRC cell EMT and evaluate the potential of EPSTI1 to contribute to invasion and metastasis in vivo .", "Determining how KSR1- and ERK-dependent signaling promotes EPSTI1 translation should yield novel mechanisms underlying tumor cell metastatic behavior .", "We show that EPSTI1 mRNA is unchanged upon KSR1 disruption or ERK inhibition ( Figures 1E and 2D ) and KSR1 does not contribute to UPS-mediated protein degradation ( Figure 1—figure supplement 2E ) , suggesting that KSR1 regulates EPSTI1 through post-transcriptional modifications enhancing its preferential loading onto the polysomes .", "Differential mRNA splicing is implicated in EMT-related processes and splicing regulatory factors have been implicated in the motility and invasive behavior of tumor cells ( Park et al . , 2019; Pradella et al . , 2017 ) .", "One possibility is that KSR1 signaling promotes the splicing of EPSTI1 that promotes it’s the preferential translational contributing to increased motility and invasion .", "Upon removal of KSR1 or EPSTI1 , the tumor cells switch back from highly migratory and invasive EMT-like state to the epithelial state .", "However , the invasive property is not completely lost in KSR1/EPSTI1 disruption ( Figure 4B ) , which could be attributed to other mesenchymal markers retained in the cells , such as vimentin ( Figure 5—figure supplement 1A ) .", "Investigating other EMT-related mRNAs that are preferentially translated in response to KSR1-scaffolded ERK signaling may reveal additional mRNAs that make previously unappreciated contributions to cell migration , invasion , and EMT .", "Constitutive KSR1 or EPSTI1 knockout yields developmentally normal mice ( Lozano et al . , 2003; Nguyen et al . , 2002; Kim et al . , 2018 ) .", "While KSR1 or EPSTI1 may not be essential to EMT during normal development , they may play a role in other EMT-dependent events such as wound healing where cells collectively migrate , differentiate , and re-epithelialize keratinocytes around and/or within the damaged site .", "If their role in EMT is exclusive to tumor cells it will reveal a key vulnerability for therapeutic evaluation .", "Further characterization of KSR1 , EPSTI1 and the additional effectors repurposed by dysregulated translation in CRC should reveal additional novel mechanisms critical to CRC tumor survival and progression ." ], [ "Colorectal cancer cell lines HCT116 , HCT15 , and SW480 were acquired from American Type Culture Collection ( ATCC ) .", "The cells were cultured in Dulbecco’s modified Eagle’s medium ( DMEM ) containing high glucose with 10 % fetal bovine serum ( FBS ) and grown at 37 °C with ambient O2 and 5 % CO2 .", "Cells were routinely tested for mycoplasma .", "No further authentication of cell lines was performed by the authors .", "Non-transformed immortalized human colon epithelial cell line ( HCEC ) was a gift from J . Shay ( University of Texas [UT] Southwestern ) and were grown and maintained as described previously ( Fisher et al . , 2015; Roig et al . , 2010 ) .", "HCECs were grown in a hypoxia chamber with 2% O2 and 5% CO2 at 37°C in four parts DMEM to 1 part medium 199 ( Sigma-Aldrich #M4530 ) with 2% cosmic calf serum ( GE Healthcare , #SH30087 . 03 ) , 25 ng/mL EGF ( R&D , Minneapolis , MN #236-EG ) , 1 µg/mL hydrocortisone ( Sigma-Aldrich , #H0888 ) , 10 µg/mL insulin ( Sigma-Aldrich , #I550 ) , 2 µg/mL transferrin ( Sigma-Aldrich , #T1428 ) , 5 nM sodium selenite ( Sigma-Aldrich #S5261 ) , and 50 µg/mL gentamicin sulfate ( Gibco #15750–060 ) as described previously ( Fisher et al . , 2015 ) .", "Normal and quadruple mutant AKPS ( APC KO/KRASG12D/P53 KO/SMAD4KO ) tumor colon organoids obtained from the Living Organoid Biobank housed by Dr . Hans Clevers and cultured as described previously ( Drost et al . , 2015; van de Wetering et al . , 2015 ) .", "The normal organoids were cultured in medium containing advanced DMEM/F12 ( Invitrogen #12634 ) with 50 % WNT conditioned media ( produced using stably transfected L cells ) , 20 % R-spondin1 , 10 % Noggin , 1 X B27 ( Invitrogen #17504–044 ) , 10 mM nicotinamide ( Sigma-Aldrich #N0636 ) , 1 . 25 mM N-acetylcysteine ( Sigma-Aldrich #A9165-5G ) , 50 ng/mL EGF ( Invitrogen #PMG8043 ) , 5000 nM TGF-b type I receptor inhibitor A83-01 ( Tocris #2939 ) , 10 nM Prostaglandin E2 ( Tocris #2296 ) , 3 µM p38 inhibitor SB202190 ( Sigma-Aldrich #S7067 ) , and 100 µg/mL Primocin ( Invivogen #ant-pm-1 ) .", "The quadruple mutant AKPS organoids were grown in media lacking WNT conditioned media , R-spondin 1 , noggin and EGF and containing 10 µM nutlin-3 ( Sigma #675576-98-4 ) .", "Approximately 500 , 000 cells were transfected using a final concentration of 20 nM EPSTI1 ( J-015094-09-0020 and J-015094-12-0020 ) or non-targeting ( D-001810-01-20 and D-001810-02-20 ) ON-TARGETplus siRNAs from GE Healthcare Dharmacon using 20 µL of Lipofectamine RNAiMAX ( ThermoFisher #13778–150 ) and 500 µL OptiMEM ( ThermoFisher #31985070 ) .", "Cells were incubated for 72 hr before further analysis .", "A lentiviral pLKO . 1-puro constructs targeting KSR1 , and non-targeting control were transfected into HEK-293T cells using trans-lentiviral packaging system ( ThermoFisher Scientific ) .", "The virus was collected , and the medium was replaced 48 hr post transfection .", "HCT116 and HCT15 cells were infected with virus with 8 µg/mL of Polybrene for several days .", "The population of cells with depleted KSR1 was selected with 10 µg/mL puromycin .", "The KSR1 knockdown was confirmed via western blotting .", "pCAG-SpCas9-GFP-U6-gRNA was a gift from Jizhong Zou ( Addgene plasmid #79144 ) , KSR1 sgRNA and non-targeting control sgRNA was cloned into the pCas9 vector .", "Both the non-targeting control and sgKSR1 were transfected into HCT116 , HCT15 , and SW480 cells using PEI transfection as described previously ( Longo et al . , 2013 ) .", "The GFP-positive cells were sorted 48 hr post transfection , and colonies were picked by placing sterile glass rings around individual colonies .", "MSCV-IRES-GFP , MSCV-IRES-KSR1-GFP , MSCV-IRES-FLAG-EPSTI1 , and N-cadherin mGFP constructs were transfected into Phoenix GP cells using trans-lentiviral packaging system ( ThermoFisher Scientific ) .", "The virus was collected , and the medium was replaced 48 hours post transfection .", "HCECs/ KSR1-CRISPR HCT116 , HCT15 , and SW480 cells were infected with virus with 8 µg/mL of Polybrene for 96 hours .", "The population of cells with KSR1 expression was selected following GFP sorting using fluorescence-activated cell sorting ( FACS ) .", "The KSR1/EPSTI1 expression was confirmed via western blotting .", "Whole cell lysate was extracted in radioimmunoprecipitation assay ( RIPA ) buffer containing 50 mM Tris-HCl , 1% NP-40 , 0 . 5 % Na deoxycholate , 0 . 1 % Na dodecyl sulfate , 150 mM NaCl , 2 mM EDTA , 2 mM EGTA , and 1 X protease and phosphatase inhibitor cocktail ( Halt , ThermoFisher Scientific #78440 ) .", "Cytoplasmic and nuclear fractionation was performed using NE-PER Nuclear and Cytoplasmic Extraction Reagents ( ThermoFisher Scientific #PI78835 ) .", "The estimation of protein concentration was done using BCA protein assay ( Promega #PI-23222 , PI-23224 ) .", "Samples were diluted using 1 X sample buffer ( 4 X stock , LI-COR #928–40004 ) with 100 mM dithiothreitol ( DTT ) ( 10 X stock , 1 mM , Sigma #D9779-5G ) .", "The protein was separated using 8–12% SDS-PAGE and transferred to nitrocellulose membrane .", "The membrane was blocked with Odyssey TBS blocking buffer ( LICOR-Biosciences #927–50003 ) for 45 min at room temperature , then incubated with primary antibodies ( Key Resources Table ) at least overnight at 4°C .", "IRDye 800CW and 680RD secondary antibodies ( LI-COR Biosciences # 926–32211 , # 926–68072 ) were diluted 1:10 , 000 in 0 . 1% TBS-Tween and imaged on the Odyssey Classic Scanner ( LI-COR Biosciences ) .", "Cells were treated with 100 µg/mL cycloheximide ( Sigma #C4859 ) on ice in PBS for 10 min .", "The cells were lysed with 10 mM HEPES , 100 mM KCL , 5 mM MgCl2 , 100 µg/mL cycloheximide , 2 mM DTT , 1 % Triton-X100 , 2 . 5 µl RNaseOUT ( ThermoFisher Scientific #10777019 ) .", "The lysates were cleared by centrifugation for 10 min at 13 , 200 rpm at 4°C .", "Approximately 200 µL of the total RNA was collected in a new RNAse-free microcentrifuge tube and the remaining supernatant was loaded onto a 15–45% sucrose gradient .", "The samples were spun at 37 , 500 rpm for 2 hr at 4°C in SW55Ti Beckman ultracentrifuge and separated on a gradient fractionation system to resolve the polysomes .", "Polysome profiles were identified at 260 nM using an absorbance detector .", "Gradient fractions were collected dropwise at 0 . 75 mL/min .", "For RNAseq , the total RNA and RNA pooled from the polysome fraction ( fractions 6–9 ) of three sets of independently isolated cells was isolated using RNAzol ( Molecular Research Centre #RN 190 ) according to the manufacture’s protocol .", "RNA purity was evaluated by the UNMC DNA Sequencing Core using a BioAnalyzer .", "RNA sequencing ( RNA seq ) was conducted by the UNMC DNA Sequencing Core .", "For RNA-seq , RNA was purified from three biological replicates of total and polysome-bound RNA from HCT116 and HCT15 , control and KSR1 knockdown cells as previously described .", "Stranded RNA sequencing libraries were prepared as per manufactures’ protocol using TrueSeq mRNA protocol kit ( Illumina ) and 500 ng of the total RNA was used for each of the samples .", "Purified libraries were pooled at a 0 . 9 pM concentration and sequenced on an Illumina NextSeq550 instrument , using a 75 SR High-output flow cell , to obtain approximately 45 million single-end reads per sample .", "NGS short reads from RNA-seq experiments was downloaded from the HiSeq2500 server in FASTQ format .", "FastQC ( http://www . bioinformatics . babraham . ac . uk/projects/fastqc/ ) was used to perform quality control checks on the fastq files that contain the raw short reads from sequencing .", "The reads were then mapped to the Homo sapiens ( human ) reference genome assembly GRCh38 ( hg38 ) using STAR v2 . 7 alignment .", "The --quantMode GeneCounts option in STAR 2 . 7 ( Dobin et al . , 2013 ) was used to obtain the HTSeq counts per gene .", "Gencode v32 Gene Transfer Format ( GTF ) was used for the transcript/gene annotations .", "The output files were combined into a matrix using R . The gene counts were further used as input for downstream analysis using Anota2seq .", "The high-throughput sequencing data have been deposited in the Gene Expression Omnibus ( GEO ) database , http://www . ncbi . nlm . nih . gov/geo ( accession no . GSE164492 ) .", "The altered levels of total mRNA can impact the changes in the pool of polysome-bound mRNA , leading to a spurious calculation translational efficiency ( TE ) .", "Anota2seq ( Oertlin et al . , 2019 ) allows the quantification of actual changes in TE .", "TE was calculated using the R Bioconductor anota2Seq package for the HTSeq counts by first removing genes that did not contain expression values in more than 10% of the samples .", "16 , 023 genes remained after this step .", "TMM normalization was further performed prior to log2 counts per million computation ( CPM ) using the voom function of the limma package using the anota2seqDataSetFromMatrix function ( with parameters datatype = “RNAseq” , normalize = TRUE , transformation = “TMM-log2” ) .", "TE was calculated using the 2 × 2 factorial design model for the two cell lines ( HCT116 and HCT15 ) .", "Genes were considered significantly regulated at Adjusted p-value < 0 . 05 when passing filtering criteria ( parameters for anota2seqSelSigGenes function ) using Random variance Model [useRVM = TRUE] , [selDeltaPT > log2 ( 1 . 2 ) ] , [minSlopeTranslation >−1] , [maxSlopeTranslation <2] , [selDeltaTP> log2 ( 1 . 2 ) ] , [minSlopeBuffering >−2] and [maxSlopeBuffering <1] , [selDeltaP> log2 ( 1 ) ] , [selDetaT > log2 ( 1 ) ] .", "The scatterplots were obtained using the anota2seqPlotFC function .", "The heatmaps were generated using the TE values for the two cell lines using the R Bioconductor ComplexHeatmap package .", "Poly- ( HEMA ) stock solution ( 10 mg/mL ) was prepared by dissolving poly- ( HEMA ) ( Sigma #3932–25 G ) in 95% ethanol at 37°C until fully dissolved ( overnight ) .", "Ninety-six-well optical bottom plates ( Thermo Scientific Nunc #165305 ) were coated in 200 µL of poly- ( HEMA ) solution and allowing it to evaporate .", "Cells were plated in complete growth medium of the poly- ( HEMA ) coated plates at a concentration of 10 , 000 cells/ 100 µL .", "Cell viability was measured at the indicated time points by the addition of CellTiter-Glo 2 . 0 reagent ( Promega #G9242 ) and luminescence was measured ( POLARstar Optima plate reader ) according to the manufacturer’s protocol .", "A total of 6000 cells were seeded in 1 . 6 % NuSieve Agarose ( Lonza #50081 ) to assess anchorage-independent growth according to the protocol of Fisher et al . , 2015 .", "Colonies greater than 100 µm in diameter from six replicates per sample were counted , representative photomicrographs were taken after 10–14 days of incubation at 37°C and 5 % CO2 .", "Cells were harvested using 1 mL TRIzol ( ThermoFisher Scientific #15596026 ) and RNA extraction was performed using RNeasy spin columns ( Qiagen #74104 ) .", "RNA was eluted with nuclease-free water .", "The RNA was quantified using a NanoDrop 2000 ( Thermo Scientific ) and Reverse Transcription ( RT ) was performed with 2 µg RNA per 40 µl reaction mixture using iScript Reverse Transcription Supermix ( Bio-Rad #170–8891 ) .", "RT-qPCR was performed using primers antibodies ( Key Resources Table ) , and all targets were amplified using SsoAdvanced Universal SYBR green Supermix ( Bio-Rad #1725271 ) with 40 cycles on a QuantStudio 3 ( ThermoFisher Scientific ) .", "The analysis was performed using 2-ΔΔCT method ( Schmittgen and Livak , 2008 ) .", "For polysome gradients , the RNA levels were quantified from the cDNA using the standard curve method , summed across all fractions ( Kortum and Lewis , 2004; Nguyen et al . , 2002; Fisher et al . , 2011; Fisher et al . , 2015; Morrison et al . , 2009; Rao et al . , 2020 ) and presented as a percentage of the total fractions .", "An in vitro scratch test was performed with the IncuCyte Zoom according to the manufacturer’s instructions .", "Approximately 35 , 000 cells were seeded onto a 96-well ImageLock plates ( Essen BioScience #4379 ) and grown to 90–95% confluency .", "The scratches were created using WoundMaker ( Essen BioScience #4563 ) in all the wells , after which the cells were washed with 1 x PBS , and media without containing serum was replaced .", "Images of the cells were obtained every 20 min for a total duration of 72 hr using IncuCyte Kinetic Live Cell Imaging System ( Essen BioScience ) and analyzed using the IncuCyte Zoom software ( Essen BioScience ) .", "IncuCyte Software was used to calculate the relative wound density metric to quantify the cell migration over time .", "The metric is designed to be zero at t = 0% and 100% when cell density inside the wound is the same as the cell density outside the initial wound , thus , allowing to experimentally quantify the effects of cell migration separate from changes that occurs as result of cell proliferation .", "Transwell inserts ( 24-well Millicell cell culture , #MCEP24H48 ) were coated with 50 µL of Matrigel ( Corning , # 356234 ) and allowed to solidify for 15–30 min .", "Approximately 20 , 000 stably generated knockout cells , or cells after 48 hr of transfection were plated in serum free media in the upper chamber of transwell insert .", "Cells were allowed to invade toward 10% serum containing media in the lower chamber for 24 hr , after which cells and gel in the upper chamber was gently removed with a sterile cotton applicator and the cells in the lower side of the insert was fixed with 3 . 7% formaldehyde for 2 min , permeabilized with 100% methanol for 20 min and stained with Giemsa for 15 min .", "The numbers of cells were counted using an inverted microscope at ×20 magnification .", "Cells were plated on glass coverslips to 70–80% confluence for 48 hr in growth media .", "Cells were fixed in 1 % formaldehyde diluted in PBS for 15 min .", "The cells were rinsed three times with PBS for 5 min and coverslips were blocked for 1 hr with 1 X PBS/ 5% goat serum/0 . 3% Triton X-100 and then incubated with E-cadherin antibody ( #4A2 ) overnight .", "Cells were washed three times for 5 min with PBS and incubated in anti-mouse IgG Alexa Fluor 555 Conjugate ( Cell signaling #4409 ) at a dilution of 1:500 for 1 hr .", "Coverslips were rinsed three times for 5 min in PBS and briefly rinsed in distilled water prior to mounting in Prolong Gold Antifade Reagent with DAPI ( Cell signaling #8961 ) .", "All Images were acquired using a Zeiss LSM-780 confocal microscope and processed using ZEISS ZEN 3 . 2 ( blue edition ) software .", "Cells were transfected with siRNA targeting EPSTI1 or a non-targeting control as previously described .", "The next day , control , KSR1-CRISPR , KSR1-CRISPR HCT116 and SW480 cells expressing KSR1 or EPSTI1 , siControl and siEPSTI1 HCT116 and SW480 cells were counted and approximately 1 × 104 cells were plated in all wells of a 12-well plate for each condition .", "The next day , four of the wells from each 12-well plate were harvested and stained with 0 . 4% trypan blue ( Sigma , # T6146-5G ) and were then counted and recorded using Countess II automated cell counter ( ThermoFisher , #A27977 ) .", "This procedure was repeated for indicated days and the cells from day 7 were harvested and a western blot analysis was performed to ensure the expression of the target protein was maintained .", "Cell counts were then graphed in GraphPad .", "The ERK inhibitor SCH772984 was purchased from SelleckChem ( S7101 ) , Z-Leu-Leu-Leu-al ( MG132 , S2619 ) were purchased from Fisher and mTOR inhibitor AZD8055 ( HY_10422 ) MedChem Express ." ] ]

[ "The epithelial-to-mesenchymal transition ( EMT ) is considered a transcriptional process that induces a switch in cells from a polarized state to a migratory phenotype .", "Here , we show that KSR1 and ERK promote EMT-like phenotype through the preferential translation of Epithelial-Stromal Interaction 1 ( EPSTI1 ) , which is required to induce the switch from E- to N-cadherin and coordinate migratory and invasive behavior .", "EPSTI1 is overexpressed in human colorectal cancer ( CRC ) cells .", "Disruption of KSR1 or EPSTI1 significantly impairs cell migration and invasion in vitro , and reverses EMT-like phenotype , in part , by decreasing the expression of N-cadherin and the transcriptional repressors of E-cadherin expression , ZEB1 and Slug .", "In CRC cells lacking KSR1 , ectopic EPSTI1 expression restored the E- to N-cadherin switch , migration , invasion , and anchorage-independent growth .", "KSR1-dependent induction of EMT-like phenotype via selective translation of mRNAs reveals its underappreciated role in remodeling the translational landscape of CRC cells to promote their migratory and invasive behavior ." ]

[ "The majority of cancer deaths result from tumor cells spreading to other parts of the body via a process known as metastasis .", "90% of all cancers originate in epithelial cells that line the inner and outer surface of organs in our bodies .", "Epithelial cells , however , are typically stationary and must undergo various chemical and physical changes to transform in to migratory cells that can invade other tissues .", "This transformation process alters the amount of protein cells use to interact with one another .", "For example , epithelial cells from the colon produce less of a protein called E-cadherin as they transition into migrating cancer cells and make another protein called N-cadherin instead .", "A protein called KSR1 is a key component of a signaling pathway that is responsible for generating the proteins colon cancer cells need to survive .", "But it is unknown which proteins KSR1 helps synthesize and whether it plays a role in the metastasis of colon cancer cells .", "To investigate this , Rao et al . studied the proteins generated by cancerous colon cells cultured in the laboratory , in the presence and absence of KSR1 .", "The experiment showed that KSR1 increases the levels of a protein called EPSTI1 , which colon cancer cells need to transform into migratory cells .", "Depleting KSR1 caused cancer cells to generate less EPSTI1 and to share more features with healthy cells , such as higher levels of E-cadherin on their surface and reduced mobility .", "Adding EPSTI1 to the cancer cells that lacked KSR1 restored the traits associated with metastasis , such as high levels of N-cadherin , and allowed the cells to move more easily .", "These findings suggest that KSR1 and EPSTI1 could be new drug targets for reducing , or potentially reversing , the invasive behavior of colon cancer cells .", "However , further investigation is needed to reveal how EPSTI1 is generated and how this protein helps colon cancer cells move and invade other tissues ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "genetics and genomics" ]

[ [ "New species arise when reproductive barriers form , preventing gene flow between populations ( Coyne and Orr , 2004 ) .", "Recently , two approaches have substantially advanced the understanding of the genetic mechanisms underlying reproductive isolation ( Reviewed in Noor and Feder ( 2006 ) ; Reviewed in Wolf et al . ( 2010 ) ) .", "Genetic crosses in the laboratory involving model organisms have identified loci and genes causing hybrid defects , a common type of reproductive barrier caused by genetic interactions between divergent alleles at two or more loci ( Bateson , 1909; Dobzhansky , 1937; Muller , 1942 ) .", "In nature , recent technological advances enable fine-scale characterization of genome-wide patterns of divergence between incipient species and variation in hybrid zones .", "For example , ‘islands of divergence’ have been reported in species pairs from taxonomically diverse groups ( Turner et al . , 2005; Nadeau et al . , 2011; Nosil et al . , 2012; Ellegren et al . , 2013; Hemmer-Hansen et al . , 2013; Renaut et al . , 2013; Carneiro et al . , 2014; Poelstra et al . , 2014; Schumer et al . , 2014 ) .", "These high-divergence genomic outlier regions are sometimes referred to as ‘islands of speciation’ , resistant to introgression because they harbor genes causing reproductive isolation .", "However , other forces can create similar genomic patterns , thus islands may not always represent targets of selection that contributed to speciation ( Noor and Bennett , 2009; Turner and Hahn , 2010; Renaut et al . , 2013; Cruickshank and Hahn , 2014 ) .", "An alternative approach to identify genomic regions contributing to reproductive isolation is to map known reproductive barrier traits in naturally hybridizing populations .", "The potential for mapping in hybrid zones is long-recognized ( Kocher and Sage , 1986; Harrison , 1990; Szymura and Barton , 1991; Briscoe et al . , 1994; Reviewed in Rieseberg and Buerkle ( 2002 ) ) .", "Hybrid zones are ‘natural laboratories for evolutionary studies’ ( Hewitt , 1988 ) , enabling investigation of speciation in progress .", "The Dobzhansky-Muller model predicts that hybrid incompatibilities between incipient species accumulate faster than linearly with time ( Orr , 1995 ) , thus investigating taxa in the early stages of speciation facilitates identification of incompatibilities that initially caused reproductive isolation vs incompatibilities that arose after isolation was complete .", "Despite these advantages , few studies have mapped barrier traits or other fitness-related traits in nature , due to the logistical challenges of collecting dense genome-wide genetic markers in species with admixed populations and well-characterized phenotypes .", "Examples include associations between pollen sterility and genomic regions showing reduced introgression in a sunflower hybrid zone ( Rieseberg et al . , 1999 ) and loci contributing to variation in male nuptial color and body shape mapped in a recently admixed stickleback population ( Malek et al . , 2012 ) .", "House mice ( Mus musculus ) are a promising model system for genetic mapping in natural populations ( Laurie et al . , 2007 ) and have an abundance of genetic tools available to ultimately isolate and characterize the causative genes underlying candidate loci .", "Three house mouse subspecies—M .", "m .", "musculus , M . m .", "domesticus , and M . m .", "castaneus–diverged ∼500 , 000 years ago from a common ancestor ( Reviewed in Boursot et al . ( 1993 ) ; Salcedo et al . ( 2007 ) ; Geraldes et al . ( 2008 ) ) .", "M . m .", "musculus and M . m .", "domesticus ( hereafter , musculus and domesticus ) colonized Europe through different geographic routes and meet in a narrow secondary contact zone running through central Europe from Bulgaria to Denmark ( Sage et al . , 1986; Boursot et al . , 1993 ) .", "Genome-wide analyses of patterns of gene flow in several geographically distinct transects across the hybrid zone have identified genomic regions showing reduced introgression , which may contribute to reproductive isolation ( Tucker et al . , 1992; Macholan et al . , 2007; Teeter et al . , 2008 , 2010; Janousek et al . , 2012 ) .", "Reduced male fertility is common in wild-caught hybrids ( Albrechtová et al . , 2012; Turner et al . , 2012 ) and in musculus–domesticus hybrids generated in the laboratory ( Britton-Davidian et al . , 2005; Reviewed in Good et al . ( 2008a ) ) , implying that hybrid sterility is an important barrier to gene flow in house mice .", "Mapping studies using F1 and F2 , hybrids generated from laboratory crosses between house mouse subspecies have identified many loci and genetic interactions contributing to sterility phenotypes ( Storchova et al . , 2004; Good et al . , 2008b; White et al . , 2011; Dzur-Gejdosova et al . , 2012; Turner et al . , 2014 ) .", "Prdm9 , a histone methyltransferase , was recently identified as a gene causing F1 hybrid sterility and is the first mammalian hybrid incompatibility gene identified ( Mihola et al . , 2009 ) .", "Comparisons between different F1 crosses show that hybrid sterility alleles are polymorphic within subspecies ( Britton-Davidian et al . , 2005; Good et al . , 2008a ) .", "Furthermore , reduced fertility phenotypes observed in nature vary in severity; complete sterility , as documented in some F1 crosses , appears to be rare or absent in the hybrid zone ( Albrechtová et al . , 2012; Turner et al . , 2012 ) .", "Taken together , studies of hybrid sterility in house mice indicate that , even in the early stages of speciation , the genetic basis of hybrid defects can be complex .", "Studies of gene flow in the hybrid zone and of hybrid sterility in the laboratory both have advantages and have shed light on the speciation process .", "Mapping sterility phenotypes in natural hybrids can potentially integrate insights from the two approaches by identifying associations between hybrid incompatibility loci and reduced gene flow across the hybrid zone .", "In this study , we map sterility-related phenotypes in hybrid zone mice to investigate the genetic architecture of reproductive isolation between incipient species .", "We performed a genome-wide association study ( GWAS ) to map testis weight and testis gene expression in 185 first generation lab-bred offspring of wild-caught hybrid mice ( Figure 1—figure supplement 1 ) .", "GWAS have been powerful in humans , loci contributing to hundreds of quantitative traits associated with disease and other phenotypic variation has been identified ( Reviewed in Stranger et al . ( 2011 ) ) .", "Examples of GWAS for fitness-related traits in non-humans are only beginning to emerge ( Johnston et al . , 2011; Filiault and Maloof , 2012; Magwire et al . , 2012 ) .", "Our hybrid zone GWAS identified genomic regions associated with variation in relative testis weight ( testis weight/body weight ) and genome-wide testis expression pattern , including regions previously implicated in hybrid sterility as well as novel loci .", "Motivated by the Dobzhansky–Muller genetic model of hybrid defects , we tested for genetic interactions ( Dobzhansky–Muller interactions—‘DMIs’ ) between loci affecting testis weight or expression pattern .", "All loci except one showed evidence for interaction with at least one partner locus and most interact with more than one partner .", "The deviations in phenotype associated with most interactions were large–affected individuals have phenotypes below the range observed in pure subspecies–suggesting that these interactions indeed are hybrid incompatibilities .", "To our knowledge , this is the first GWAS for a reproductive barrier trait .", "Using natural hybrids provided high mapping resolution that will facilitate future studies to identify causative genes; for example , a majority of GWAS regions contain 10 or fewer genes .", "Moreover , this study provides the first genome-scale description of a hybrid incompatibility network in nature ." ], [ "We investigated two phenotypes in males from the house mouse hybrid zone: relative testis weight ( testis weight/body weight ) and genome-wide testis gene expression pattern .", "Both of these phenotypes have previously been linked to hybrid male sterility in studies of offspring from crosses between musculus and domesticus and mice from the hybrid zone ( Britton-Davidian et al . , 2005; Rottscheidt and Harr , 2007; Reviewed in Good et al . ( 2008a ) , ( 2010 ) ; White et al . ( 2011 ) ; Turner et al . ( 2012 ) , ( 2014 ) ) .", "We refer to these as ‘sterility phenotypes’ , following conventional terminology in the field , however , it is important to note that the severity of defects observed in most hybrid zone mice is consistent with reduced fertility/partial sterility ( Turner et al . , 2012 ) .", "Testis expression PC1 ( explaining 14 . 6% of the variance ) is significantly correlated with relative testis weight ( cor = 0 . 67 , P = 2 × 10−16 ) , indicating that there is a strong association between those two sterility phenotypes ( Figure 1—figure supplement 2 ) .", "Principal component 2 ( PC2 , 8 . 1% variance ) is strongly correlated with hybrid index ( % musculus autosomal SNPs: cor = 0 . 75 , P = 2 × 10−16 ) , thus the effect of hybrid defects does not obscure subspecies differences in expression .", "In the mapping population , 19/185 ( 10 . 2% ) individuals had relative testis weight below the minimum observed in pure subspecies males and 21/179 ( 11 . 7% ) individuals had expression PC1 scores below ( PC1 = −46 . 97 ) the pure subspecies range .", "We identified four SNPs significantly associated with relative testis weight in three regions on the X chromosome using stringent thresholds determined by permutation ( Table 1; Figure 1A ) .", "An additional 51 SNPs were significant using a more permissive significance threshold ( false discovery rate ( FDR ) < 0 . 1 ) .", "Significant SNPs were clustered in 12 genomic regions ( of size 1 bp to 13 . 3 Mb; Table 1 ) .", "We report GWAS regions defined using the permissive FDR threshold because we plan to combine mapping results from multiple phenotypes to identify candidate sterility loci , based on the idea that spurious associations are unlikely to be shared among phenotypes .", "Significant regions were located on the X chromosome and 9 autosomes , suggesting a minimum of 10 loci contribute to variation in testis weight .", "It is difficult to estimate the precise number of genes involved , because the extent of linkage disequilibrium ( LD ) of significant SNPs around a causative mutation depends on the phenotypic effect size , recombination rate , allele frequency , and local population structure .", "Multiple significant regions might be linked to a single causative mutation , or conversely , a significant region might be linked to multiple causative mutations in the same gene or in multiple genes . 10 . 7554/eLife . 02504 . 003Table 1 . Genomic regions significantly associated with relative testis weightDOI: http://dx . doi . org/10 . 7554/eLife . 02504 . 00310 . 7554/eLife . 02504 . 004Table 1—source data 1 . Protein-coding genes in significant relative testis weight regions . DOI: http://dx . doi . org/10 . 7554/eLife . 02504 . 004Region*1ChrPosition ( Mb ) †Length ( kb ) Sig .", "SNPs ( 5% perm ) ‡No .", "sign SNPs expression§Interactions#Concordant PC1 region¶Concordant sterility loci**Sterile Allele††No .", "genes ( coding ) ‡‡Candidate Genes§§RTW011173 . 30–173 . 3440 . 7115PC03d3 ( 3 ) RTW02233 . 152 . 6104PC04BHZm*1 ( 1 ) RTW032129 . 59–129 . 6559 . 8112PC08TWAd2 ( 1 ) RTW046132 . 63–100–M0RTW05964 . 40–103–U1 ( 1 ) RTW061124 . 250 . 8112PC26BHZD0RTW071237 . 16–41 . 524364 . 2447PC29D20 ( 12 ) Arl4aEFGRTW081351 . 44–104–TWBd0RTW091756 . 68–58 . 441752 . 2428PC43SCbinA; TWA; BHZM42 ( 39 ) Acsbg2E; ClppG; SafbG; Tmem146EGRTW10X12 . 17–1 ( 1 ) 14PC46ASHD; eQTLHSC; HTA; SCAm*1 ( 1 ) RTW11X85 . 13–98 . 4313294 . 335 ( 2 ) 353PC49ASHD; DBTA; eQTLHSC; FERTB; HTA; PBTA; SCB; TASA; TWBD; BHZD191 ( 67 ) ArEFG; ArxG; Pcyt1bEFG; Tex11EFG; ZfxEFGRTW12X127 . 57–134 . 136555 . 54 ( 1 ) 42PC50ASHD; eQTLHSC; shPC1A; SCD; TWD; BHZD158 ( 71 ) Nxf2G; Taf7lEFG*Significant SNPs <10 Mb apart were combined into regions .", "†Significant intervals were defined by positions of the most proximal and distal SNPs with LD > 0 . 9 to a significant SNP .", "‡The number of SNPs significant at FDR < 0 . 1 is reported; number of significant SNPs significant with <0 . 05 P value in permutations is in parentheses .", "§Number of significant SNPs enriched for associations with transcripts expressed on another chromosome ( P < 0 . 05; FDR < 0 . 1; >30 transcripts ) .", "#Number of regions with significant interactions .", "¶Overlapping regions significant for expression PC1 ( see Table 2 ) .", "**Sterility QTL overlapping or within 10 Mb from A ( White et al . 2011 ) , B ( Dzur-Gejdosova et al . 2012 ) , C ( Turner et al . 2014 ) , D ( Good et al . 2008b ) .", "Abbreviations for phenotypes: ASH: abnormal sperm head morphology , TW: testis weight , SC: sperm count , shPC1: sperm head shape PC1 , eQTLHS: trans eQTL hotspot , FERT: fertility , PBT: proximal bent sperm tail , HT: headless/tailless sperm , DBT: distal bent sperm tail , TAS: total abnormal sperm .", "BHZ: overlapping candidate regions with evidence from epistasis in the Bavarian hybrid zone transect ( Janousek et al . 2012 ) .", "††Sterile allele inferred on the basis of frequency of a majority of significant SNPs in pure subspecies samples: D–domesticus; M–musculus; lower-case indicates FST< 0 . 7 between pure subspecies; * indicates overlapping PC1 region is D sterile; U–nondiagnostic SNP and/or no majority allele .", "‡‡Number of genes ( protein-coding ) overlapping region .", "§§Genes with roles in male reproduction on the basis of Emale reproduction gene ontology terms ( see ‘Materials and methods’ ) or phenotypes of knockout models reported in F ( Matzuk and Lamb 2008 ) or GMGI database . 10 . 7554/eLife . 02504 . 005Figure 1 . Manhattan plot of GWAS results . Single SNPs associated with ( A ) relative testis weight , ( B ) testis expression principal component 1 , and ( C ) expression of transcripts located on other chromosomes ( trans ) .", "Dashed lines indicate significance thresholds based on: permutations for autosomes ( labeled 5% perm A ) , permutations for X chromosome ( labeled 5% perm X ) , false discovery rate <0 . 1 ( labeled 10% FDR ) , and 95th percentile of significant transcript association counts across SNPs ( labeled 95% ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 02504 . 00510 . 7554/eLife . 02504 . 006Figure 1—source data 1 . SNPs significantly associated with relative testis weight and/or testis expression PC1 ( excel file ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 02504 . 00610 . 7554/eLife . 02504 . 007Figure 1—figure supplement 1 . Geographic location of and genetic makeup of mapping population .", "( A ) Location of sampling area ( black box ) in European house mouse hybrid zone .", "( B ) Sampling locations for parents of mice in the mapping population .", "( C ) Structure analysis of mapping population .", "Individuals ( vertical bands ) are arranged by geographic origin and average percentage alleles from Mus musculus musculus . DOI: http://dx . doi . org/10 . 7554/eLife . 02504 . 00710 . 7554/eLife . 02504 . 008Figure 1—figure supplement 2 . Principal components analysis of genome-wide gene expression in testis .", "( A ) Plot of principal component 1 ( PC1 ) vs PC2 scores .", "Individuals with relative testis weight and/or sperm count below the pure subspecies range are indicated in blue ( ‘low fertility’ ) .", "Individuals with relative testis weight and sperm count within one standard deviation of the mean in pure subspecies individuals are indicated in red ( ‘fertile range’ ) .", "( B ) Plot of relative testis weight vs PC1 score .", "Correlation coefficient ( Pearson's ) and p value are indicated .", "( C ) Plot of hybrid index ( % musculus alleles on autosomes ) vs PC2 score .", "Correlation coefficient ( Pearson's ) and p value are indicated . DOI: http://dx . doi . org/10 . 7554/eLife . 02504 . 008 We identified 104 SNPs on the X and chromosome 1 significantly associated with expression PC1 using stringent permutation-based thresholds ( Table 2; Figure 1B ) .", "An additional 349 SNPs were significant with the more permissive threshold of FDR < 0 . 1 .", "Significant SNPs clustered in 50 genomic regions located on 18 autosomes and the X . 10 . 7554/eLife . 02504 . 009Table 2 . Genomic regions significantly associated with testis expression PC1DOI: http://dx . doi . org/10 . 7554/eLife . 02504 . 00910 . 7554/eLife . 02504 . 010Table 2—source data 1 . Protein-coding genes in significant testis expression PC1 regions . DOI: http://dx . doi . org/10 . 7554/eLife . 02504 . 010Region*ChrPosition ( Mb ) †Length ( kb ) Sig .", "SNPs ( 5% perm ) ‡No .", "sign SNPs expression§Interactions#Concordant RTW region¶Concordant sterility loci**Sterile Allele††No .", "genes ( coding ) ‡‡Candidate Genes§§PC0118 . 01–12 . 724715 . 24446BHZU40 ( 18 ) Mybl1GHPC02199 . 53–1119BHZD0PC031166 . 84–185 . 8318988 . 228 ( 1 ) 2547RTW01BHZD297 ( 229 ) Adcy10FGH; Atp1a4H; Ddr2GH; DeddH; Exo1FGH; F11rG; H3f3aGH; LbrH; Lmx1aH; MaelFH; MpzH; Vangl2HPC04221 . 72–49 . 0127288 . 0303045RTW02TWA; BHZD604 ( 334 ) Acvr2aGH; BmycH; Grin1H; Il1rnI; Lhx3H; Notch1F; Nr5a1GH; Nr6a1F; Odf2FH; Pax8GH; Sh2d3cH; Sohlh1GH; StrbpFGH; Tsc1HPC05267 . 00–1138TWAd1 ( 0 ) PC06284 . 56–84 . 68125 . 61138eQTLHSC; TWAd8 ( 7 ) PC072114 . 21–116 . 792579 . 47741eQTLHSC; TWA; BHZD20 ( 4 ) PC082129 . 59–129 . 6559 . 81116RTW03TWAd2 ( 1 ) PC09363 . 61–63 . 625 . 52236DBTAM1 ( 1 ) PC10382 . 14–1122eQTLHSCd0PC1143 . 14–11 . 168023 . 38844D98 ( 31 ) Ccne2H; Chd7H; Plag1HPC12452 . 80–1133U0PC13537 . 81–1140m1 ( 1 ) PC1465 . 78–5 . 90121 . 61128BHZd1 ( 1 ) PC1577 . 09–7 . 109 . 01124shPC1Ad1 ( 1 ) PC16735 . 47–1121shPC1Ad1 ( 1 ) PC177140 . 36–140 . 98620 . 93343D8 ( 7 ) PC18837 . 56–1122STAAd1 ( 1 ) PC19874 . 15–74 . 1720 . 91133STAAd1 ( 1 ) PC20890 . 23–106 . 7716539 . 65345STAA; BHZD146 ( 101 ) Bbs2GH; Ccdc135F; Csnk2a2GH; Katnb1H; Nkd1FHPC218118 . 11–120 . 562451 . 02240STAAU23 ( 16 ) PC22932 . 44–1134BHZm0PC23957 . 23–60 . 593359 . 65441D69 ( 54 ) 2410076I21RikF; Bbs4GH; Cyp11a1GHPC24991 . 04–91 . 22180 . 02233D0PC251034 . 9–35 . 08185 . 21127PBTAd0PC261124 . 250 . 81129RTW06BHZD0PC271167 . 99–69 . 471479 . 71131shPC1AD67 ( 46 ) AurkbH; Odf4F; ShbgF; Trp53HPC28127 . 85–16 . 138278 . 4191947D54 ( 32 ) ApobFGH; Gdf7GH; Pum2HPC291228 . 99–54 . 2225238 . 3464447RTW07BHZD150 ( 93 ) AhrGH; Arl4aGH; Immp2lFGH; Slc26a4HPC3012116 . 53–1135m0PC31136 . 74–6 . 85113 . 32235TWAD0PC321429 . 53–32 . 212675 . 55443STAA; TWBD44 ( 35 ) ChdhH; Dnahc1G; TktHPC331466 . 74–75 . 018274 . 92241SCBU98 ( 71 ) Fndc3aFGH; Gnrh1GH; Npm2F; Piwil2FGH; Rb1HPC3414121 . 69–121 . 7783 . 21131d1 ( 1 ) PC351527 . 75–31 . 463701 . 35547HTA; TASAD19 ( 8 ) PC361545 . 67–1136HTA; TASAd0PC371573 . 00–1127eQTLHSCd1 ( 1 ) PC38168 . 18–18 . 5110329 . 1565641BHZD201 ( 132 ) Prm1FGH; Prm2FGH; Prm3F; Ranbp1H; Rimbp3H; Rpl39lF; Snai2H; Spag6FGH; Tnp2FGH; Top3bI; Tssk1FH; Tssk2FHPC391629 . 16–29 . 179 . 71134d0PC401666 . 52–66 . 5313 . 12239STAAU0PC411690 . 92–90 . 9311 . 61135STAAd1 ( 1 ) PC421711 . 05–11 . 18132 . 73343eQTLHSC; FERTB; SCAB; TWABDh1 ( 1 ) PC431742 . 08–63 . 2921217 . 1131145RTW09SCA; TWA; BHZMd272 ( 209 ) Acsbg2F; ClppH; DazlFGH; Klhdc3F; Mea1F; Pot1bH; SafbH; Sgol1F; Tcte1H; Tdrd6H; Tmem146F; Ubr2FGH; Zfp318HPC441777 . 34–83 . 596248 . 82233TWAD53 ( 36 ) PC451944 . 82–45 . 74918 . 110946BHZD23 ( 16 ) BtrcGH; DpcdHPC46X11 . 34–19 . 347995 . 319 ( 7 ) 1944RTW10ASHD; eQTLHSC; HTA; SCAD; TWD; BHZD82 ( 21 ) PC47X36 . 94–1128ASHD; eQTLHSC; FERTB; HTA; shPC1A; SCABD; TWBD; BHZd0PC48X68 . 03–70 . 772742 . 24 ( 1 ) 343ASHAE; DBTA; eQTLHSC; FERTB; HTA; OFFE; PBTA; SCBE; TASA; TWBE; BHZU62 ( 41 ) Cetn2F; Mtm1HPC49X83 . 62–108 . 5324911 . 7125 ( 84 ) 12545RTW11DBTA; eQTLHSC; FERTB; HTA; PBTA; shPC1A; SCB; TASA; TWBD; BHZD407 ( 142 ) ArGH; ArxH; Atp7aH; Pcyt1bFGH; Tex11FGH; TsxH; ZfxFGHPC50X127 . 01–137 . 3710365 . 121 ( 11 ) 2145RTW12ASHD; eQTLHSC; shPC1A; SCD; TWD; BHZD212 ( 92 ) Nxf2H; Taf7lFGH; Tsc22d3H*Significant SNPs <10 Mb apart were combined into regions .", "†Significant intervals were defined by positions of the most proximal and distal SNPs with LD > 0 . 9 to a significant SNP .", "‡The number of SNPs significant at FDR < 0 . 1 is reported; number of significant SNPs significant with <0 . 05 P value in permutations is in parentheses .", "§Number of significant SNPs enriched for associations with transcripts expressed on another chromosome ( P < 0 . 05; FDR < 0 . 1; >30 transcripts ) .", "#Number of regions with significant interactions .", "¶Overlapping regions significant for relative testis weight ( see Table 1 ) .", "**Sterility QTL overlapping or within 10 Mb from A ( White et al . 2011 ) , B ( Dzur-Gejdosova et al . 2012 ) , C ( Turner et al . 2014 ) , D ( Good et al . 2008b ) , E ( Storchova et al . 2004 ) .", "Abbreviations for phenotypes: ASH: abnormal sperm head morphology , TW: testis weight , SC: sperm count , shPC1: sperm head shape PC1 , eQTLHS: trans eQTL hotspot , STA: seminiferous tubule area , FERT: fertility , PBT: proximal bent sperm tail , HT: headless/tailless sperm , DBT: distal bent sperm tail , TAS: total abnormal sperm , OFF: number of offspring .", "BHZ: overlapping candidate regions with evidence from epistasis in the Bavarian hybrid zone transect ( Janousek et al . 2012 ) .", "††Sterile allele inferred on the basis of frequency of a majority of significant SNPs in pure subspecies samples: D–domesticus; M–musculus; lower-case indicates FST< 0 . 7 between pure subspecies; * indicates overlapping PC1 region is D sterile; U–nondiagnostic SNP and/or no majority allele; Dh–two SNPs with domesticus sterile alleles , one SNP heterozygous genotype shows sterile pattern; Md–majority musculus sterile alleles but some SNPs diagnostic domesticus sterile alleles .", "‡‡Number of genes ( protein-coding ) overlapping region .", "§§Genes with roles in male reproduction on the basis of Fmale reproduction gene ontology terms ( see ‘Materials and methods’ ) or phenotypes of knockout models reported in G ( Matzuk and Lamb 2008 ) or HMGI database .", "To gain further insight into associations between sterility and gene expression , we mapped expression levels of individual transcripts .", "A total of 18 , 992/36 , 323 probes showed significant associations with at least one SNP .", "We focused on trans associations ( SNP is located on different chromosome from transcript ) , based on evidence from a study in F2 hybrids that trans expression QTL ( eQTL ) are associated with sterility while cis eQTL are predominantly associated with subspecies differences ( Turner et al . , 2014 ) .", "To identify SNPs significantly enriched for trans associations with expression , we used a threshold set at the 95% percentile of significant probe association counts across all SNPs ( i . e . , SNPs that showed associations with at least 30 transcripts , Figure 1C ) .", "There was substantial overlap between mapping results for testis weight and expression PC1; 48/55 SNPs significant for relative testis weight ( 9 regions ) were also significant for expression PC1 .", "A permutation test , performed by randomly shuffling the positions of GWAS regions in the genome , provides strong evidence that this overlap is non-random ( p < 0 . 0001 , 10 , 000 permutations ) .", "Most SNPs significant for testis weight and/or expression PC1 were significantly enriched for trans associations with individual transcripts ( relative testis weight: 49/55 SNPs , 8/12 regions; PC1: 440/453 SNPs , 50/50 regions ) .", "The combined mapping results provide multiple lines of evidence for contributions of all 50 PC1 regions and 9/12 testis weight regions .", "The three testis-weight regions ( RTW04 , RTW05 , RTW08 ) not significantly associated with testis expression phenotypes are more likely to be spurious and are weaker candidates for future study .", "Power to identify pairwise epistasis in GWAS for quantitative traits is limited even with very large sample sizes , due to multiple testing issues ( e . g . Marchini et al . , 2005 ) .", "The Dobzhansky-Muller model predicts that the effect of each hybrid defect gene depends on interaction with at least one partner locus .", "Hence , for hybrid sterility traits , there is a hypothesis-driven framework in which to limit tests for epistasis to a small subset of possible interactions .", "We tested for genetic interactions between all pairs of significant SNPs ( FDR < 0 . 1 ) located on different chromosomes for testis weight and for expression PC1 .", "We identified 142 significant pairwise interactions for relative testis weight , representing 22 pairs of GWAS regions ( Figure 2A ) .", "These results provide evidence for a minimum of 13 autosomal–autosomal and five X–autosomal interactions affecting testis weight . 10 . 7554/eLife . 02504 . 011Figure 2 . Significant GWAS regions and interactions in hybrid zone mice . Results for ( A ) relative testis weight and ( B ) testis expression principal component 1 in hybrid zone mice .", "In ( A ) orange and yellow boxes in outer rings ( outside grey line ) indicate quantitative trait loci ( QTL ) identified for testis weight and other sterility phenotypes in previous studies ( see Table 1 for details ) .", "Green boxes indicate significant GWAS regions for relative testis weight .", "Green lines represent significant genetic interactions between regions; shade and line weight indicate the number of significant pairwise interactions between SNPs for each region pair .", "In ( B ) orange boxes in outer rings indicate QTL for testis-related phenotypes ( testis weight and seminiferous tubule area ) identified in previous studies , yellow boxes indicate QTL for other sterility phenotypes and red boxes indicate trans eQTL hotspots ( see Table 2 for details ) .", "Green boxes indicate significant GWAS regions for relative testis weight .", "Purple boxes indicate significant GWAS regions for testis expression PC1 .", "Lines represent significant genetic interactions between regions; color and line weight—as specified in legend—indicate the number of significant pairwise interactions between SNPs for each region pair .", "Plot generated using circos ( Krzywinski et al . , 2009 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 02504 . 01110 . 7554/eLife . 02504 . 012Figure 2—source data 1 . Significant genetic interactions ( SNP pairs ) for relative testis weight ( excel file ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 02504 . 01210 . 7554/eLife . 02504 . 013Figure 2—source data 2 . Significant genetic interactions ( SNP pairs ) for testis expression PC1 ( excel file ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 02504 . 01310 . 7554/eLife . 02504 . 014Figure 2—figure supplement 1 . Genetic interactions associated with hybrid sterility in hybrid zone mice and in F2 hybrids . Orange boxes in outer rings indicate QTL for testis-related phenotypes ( testis weight and seminiferous tubule area ) identified in previous studies , yellow boxes indicate QTL for other sterility phenotypes , and red boxes indicate trans eQTL hotspots ( see Table 2 for details ) .", "Green boxes indicate significant GWAS regions for relative testis weight .", "Purple boxes indicate significant GWAS regions for testis expression PC1 .", "Lines represent significant genetic interactions identified in hybrid zone mice for relative testis weight ( in green ) and expression PC1 ( in purple ) , which are concordant with genetic interactions identified by mapping expression traits in F2 hybrids ( Turner et al . , 2014 ) .", "Plot generated using circos ( Krzywinski et al . , 2009 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 02504 . 014 We identified 44 , 145 significant interactions between SNPs for expression PC1 .", "The 913 GWAS region pairs provide evidence that at least 144 autosomal–autosomal interactions and 18 X–autosomal interactions contribute to expression PC1 ( Figure 2B ) .", "We used deviations from population means for single SNPs and two-locus genotypes to estimate the phenotypic effects of GWAS regions and interactions ( Figure 3A , B ) . 10 . 7554/eLife . 02504 . 015Figure 3 . Phenotypic effects of testis-weight loci and interactions . Histograms showing maximum deviations from the population mean for ( A ) single SNPs and ( B ) two-locus interactions .", "Dashed vertical lines indicate minimum values observed in pure subspecies males .", "( C ) Examples of phenotypic means by two-locus genotype for autosomal–autosomal and X–autosomal interactions .", "Genotypes are indicated by one letter for each locus: D—homozygous for the domesticus allele , H—heterozygous , M—homozygous musculus . DOI: http://dx . doi . org/10 . 7554/eLife . 02504 . 015 As expected , interactions had greater effects , on average , than single loci for both phenotypes ( relative testis weight: single locus mean = −1 . 81 mg/g , interaction mean = −4 . 07 mg/g; expression PC1: single locus mean = −81 . 51 , interaction mean = −130 . 77 ) .", "We provide examples of autosomal–autosomal and X–autosomal SNP pairs with significant interactions for each phenotype in Figure 3C .", "It is important to note that mean deviations are rough estimates of effect sizes , which don’t account for family structure .", "It is possible that some of the GWAS regions we mapped contribute to quantitative variation within/between subspecies , rather than hybrid defects .", "The lowest genotypic means for most interactions fell below the range observed in pure subspecies ( relative testis weight: 19/22 ( 86 . 3% ) region pairs; expression PC1: 877/913 ( 96% ) region pairs; Figure 3A , B ) , consistent with the hypothesis that interactions represent Dobzhansky–Muller incompatibilities .", "We performed simulations to assess the performance of the mapping procedure for different genetic architectures by estimating the power to detect causative loci and the false positive rate ( Figure 4 ) .", "We simulated phenotypes based on two-locus genotypes from the SNP dataset using genetic models for nine genetic architecture classes ( i . e . autosomal vs X linked , varied dominance ) with parameters based on the observed distribution of relative testis weight ( Figure 4—figure supplement 1 , Figure 4—source data 1 ) . 10 . 7554/eLife . 02504 . 016Figure 4 . Mapping power in simulations . Each panel illustrates results from a single genetic architecture model for ( A ) 100 autosomal–autosomal SNP pairs and ( B ) 100 X—autosomal SNP pairs .", "Each point represents the percentage of data sets generated from a single SNP pair in which locus 1 ( domesticus sterile allele; green ) , locus 2 ( musculus sterile allele; purple ) , or both loci ( orange ) were identified by association mapping ( ≥1 SNP significant by permutation based threshold within 10 Mb of ‘causal’ SNP ) .", "The x axis indicates the percentage of individuals with partial or full sterility phenotypes .", "Curves were fit using second order polynomials .", "In ( A ) , locus 1 indicates the SNPs with musculus alleles sterile and locus 2 indicates the SNPs with domesticus alleles sterile .", "In ( B ) , locus 1 is the X-linked SNP and locus 2 is the autosomal SNP . DOI: http://dx . doi . org/10 . 7554/eLife . 02504 . 01610 . 7554/eLife . 02504 . 017Figure 4—source data 1 . Z scores for simulation models . DOI: http://dx . doi . org/10 . 7554/eLife . 02504 . 01710 . 7554/eLife . 02504 . 018Figure 4—figure supplement 1 . Mapping simulation methods . Schematics of ( A ) choice of ‘causal’ SNP pairs from the genotype data , ( B ) phenotype distributions for simulations , ( C ) generation of simulated phenotype data sets , ( D ) association mapping .", "In ( B ) histogram shows the empirical distribution of relative testis weight in the mapping population , in standard deviation units . DOI: http://dx . doi . org/10 . 7554/eLife . 02504 . 01810 . 7554/eLife . 02504 . 019Figure 4—figure supplement 2 . Distances of significant SNPs to causal SNP in simulations . Distributions are shown at two scales for autosomal and X-linked loci . DOI: http://dx . doi . org/10 . 7554/eLife . 02504 . 019 The distribution of distances to the causal SNP for all significant SNPs located on the same chromosome ( Figure 4—figure supplement", "2 ) shows that the majority of significant SNPs ( 62 . 7% ) are within 10 Mb of the causal SNP , however , a small proportion of significant SNPs are >50 Mb from the causal SNP .", "In most cases , causal SNPs detected at long distances also had significant SNPs nearby , for example 83 . 4% of loci with significant SNPs 1–10 Mb distant also have significant SNPs within 1 Mb .", "These results provide support for our choice to define significant GWAS regions by combining significant SNPs within 10 Mb , and suggest that these regions are likely to encompass the causative gene .", "As expected , the power to detect one or both causative loci depended on the location ( autosomal vs X-linked ) , dominance , and frequency of both ‘causative’ alleles ( Figure 4 , Table 3 ) .", "For example , the mean percentage of simulations for which both loci were detected ( SNP < 10 Mb significant by permutation-based threshold ) was six times higher ( 14 . 4% ) for the X chromosome × autosomal-dominant architecture compared to the autosomal-recessive × autosomal-recessive architecture ( 2 . 6% ) .", "The relationship between power and the proportion of affected individuals in the mapping population was complex .", "Interestingly , power was high for some simulations with very few affected individuals .", "In these cases , the few individuals carrying the lower frequency sterility allele by chance also carried the sterility allele from the second locus , thus the average single allele effects were not diminished by individuals carrying one but not both interacting sterility alleles . 10 . 7554/eLife . 02504 . 020Table 3 . Results of mapping simulationsDOI: http://dx . doi . org/10 . 7554/eLife . 02504 . 020Locus 1 detected† , ‡Locus 2 detected‡ , §Both loci detected‡Mean No .", "Sig .", "SNPsArchitecture*Med .", "Distance to Causal SNP ( Mb ) X chromosome/Autosome0 . 2 Mb1 Mb10 Mb0 . 2 Mb1 Mb10 Mb0 . 2 Mb1 Mb10 Mb10 Mb#50 Mb#Diff .", "Chr .", "¶Permutation P<0 . 05rec-rec5 . 97 . 28 . 412 . 39 . 011 . 715 . 80 . 30 . 72 . 61 . 11 . 55 . 5rec-add2 . 618 . 322 . 228 . 012 . 615 . 821 . 03 . 24 . 47 . 23 . 52 . 34 . 4rec-dom2 . 027 . 431 . 839 . 219 . 122 . 226 . 45 . 57 . 812 . 96 . 98 . 55 . 0add-add1 . 46 . 77 . 710 . 547 . 551 . 955 . 82 . 73 . 14 . 77 . 99 . 26 . 1add-dom1 . 714 . 215 . 919 . 051 . 655 . 759 . 26 . 07 . 510 . 311 . 113 . 35 . 4dom-dom1 . 87 . 89 . 814 . 363 . 866 . 970 . 62 . 43 . 77 . 314 . 717 . 66 . 2X-rec12 . 2/4 . 810 . 314 . 026 . 210 . 012 . 718 . 80 . 11 . 34 . 95 . 69 . 94 . 8X-add9 . 1/2 . 033 . 939 . 148 . 524 . 325 . 631 . 03 . 85 . 311 . 421 . 935 . 75 . 7X-dom9 . 8/2 . 046 . 551 . 359 . 726 . 928 . 532 . 65 . 98 . 614 . 431 . 052 . 83 . 8FDR <0 . 1rec-rec10 . 016 . 621 . 434 . 718 . 523 . 535 . 53 . 55 . 415 . 05 . 18 . 334 . 7rec-add5 . 532 . 739 . 752 . 727 . 232 . 645 . 211 . 415 . 527 . 913 . 218 . 932 . 9rec-dom4 . 142 . 249 . 762 . 933 . 537 . 248 . 416 . 521 . 333 . 822 . 230 . 128 . 7add-add3 . 614 . 417 . 630 . 663 . 369 . 377 . 68 . 411 . 323 . 321 . 628 . 836 . 8add-dom3 . 526 . 531 . 142 . 065 . 570 . 678 . 118 . 222 . 833 . 529 . 239 . 329 . 1dom-dom3 . 616 . 422 . 135 . 376 . 879 . 885 . 99 . 415 . 129 . 335 . 548 . 026 . 5X-rec12 . 2/7 . 810 . 314 . 026 . 220 . 025 . 240 . 50 . 73 . 111 . 010 . 317 . 534 . 6X-add9 . 1/4 . 733 . 939 . 148 . 533 . 236 . 648 . 36 . 39 . 420 . 928 . 746 . 130 . 0X-dom9 . 8/5 . 046 . 551 . 359 . 737 . 041 . 250 . 911 . 416 . 327 . 238 . 865 . 521 . 6*Architecture abbreviations: add–additive; dom–dominant; rec–recessive .", "†Locus 1 for autosomal pairs is musculus sterile allele; locus 1 for X-autosomal pairs is X-linked .", "‡3‘detected’–≥1 significant SNP within given distance criterion .", "§Locus 2 for autosomal pairs has a domesticus sterile allele; locus 2 for X-autosomal pairs is autosomal .", "#Mean number significant SNPs within distance criterion for either locus .", "¶Mean number significant SNPs on chromosomes not containing ‘causal’ SNPs .", "It is important to note that our empirical results suggest that the two-locus models used to simulate phenotypes are overly simplified .", "We predict that involvement of a sterility locus in multiple incompatibilities would reduce the influence of allele/genotype frequencies of any single partner locus on power .", "To estimate the false discovery rate from simulations , we classified significant SNPs not located on the same chromosome as one of the causative SNPs as false positives .", "Choosing an appropriate distance threshold for false vs true positives on the same chromosome was not obvious given the distribution of distances to causal SNPs ( Figure 4-figure supplement 2 ) .", "We classified significant SNPs <50 Mb from causative SNPs as true positives and excluded SNPs >50 Mb when calculating FDR .", "Using permutation-based significance thresholds , the median false positive rate was 0 . 014 ( calculated for simulations with ≥10 SNPs within 50 Mb of either causative locus ) .", "These results suggest that significant SNPs from the GWAS identified using this more stringent threshold are likely to be true positives .", "By contrast , the median false positive rate was 0 . 280 using the FDR < 0 . 1 threshold , indicating this threshold is more permissive than predicted .", "Thus , there is a substantial chance that SNP associations with relative testis weight and expression PC1 identified using this threshold are spurious and evidence is weak for GWAS regions comprising one SNP significantly associated with a single phenotype ." ], [ "The potential to leverage recombination events from generations of intercrossing in hybrid zones to achieve high-resolution genetic mapping of quantitative traits has been recognized for decades ( Reviewed in Rieseberg and Buerkle , ( 2002 ) ) .", "Until recently , collection of dense genotype datasets and large sample sizes has not been feasible in natural populations due to logistics and costs .", "This study demonstrates that loci and genetic interactions contributing to reproductive barrier traits can be identified in a GWAS with a modest sample size ( see also related study mapping craniofacial phenotypes in this mapping population Pallares et al . 2014 ) .", "Sample sizes approximating those used for human GWAS are not necessary if the prevalence and genetic architecture of the trait of interest are favorable .", "In general , epistasis makes genetic mapping more difficult .", "However , for hybrid defects , dependence of the phenotype on epistasis conversely may facilitate mapping .", "Despite substantial deleterious effects in hybrids , incompatibility alleles are not subject to negative selection within species and may be at high frequency or fixed within species .", "Hence , the prevalence of affected individuals in a hybrid zone for epistatic traits may be much higher than for deleterious traits in pure populations ( e . g . disease in humans ) .", "Combining mapping of multiple sterility-related phenotypes substantially improved power to identify sterility loci .", "We identified a few loci for each phenotype using stringent significance thresholds based on permutation .", "In addition , most loci identified using more permissive thresholds showed significant associations with more than one phenotype .", "Spurious associations are unlikely to be shared across phenotypes , thus evidence from multiple phenotypes provided confidence for contributions of nine genomic regions to testis weight ( on the X and 5 autosomes ) and 50 genomic regions to expression PC1 ( on the X and 18 autosomes ) .", "The high resolution of mapping in the hybrid zone provides an advantage over laboratory crosses .", "For example , significant regions identified here ( median = 2 . 1 Mb , regions with defined intervals ) are much smaller than sterility QTL identified in F2s ( 35 . 1 Mb; White et al . , 2011 ) .", "Many GWAS regions contain few enough genes that it will be possible to individually evaluate the potential role of each in future studies to identify causative genes .", "For example , 8/12 testis-weight regions and 28/50 expression PC1 regions contain 10 or fewer protein-coding genes .", "We identified candidate genes with known roles in reproduction in four testis-weight regions and 17 expression PC1 regions ( Tables 1–2 ) .", "However , for the majority of regions ( 8/12 relative testis weight , 33/50 expression PC1 ) , there are no overlapping/nearby genes previously linked to fertility .", "It is unlikely that these regions would be prioritized if contained in large QTL intervals .", "High resolution mapping is possible using mapping resources such as the collaborative cross ( Aylor et al . , 2011 ) and heterogeneous stocks ( Svenson et al . , 2012 ) , but these populations represent a small proportion of genetic diversity in house mice ( Yang et al . , 2011 ) and hybrid incompatibility alleles may have been lost during strain production .", "Comparisons of different F1 crosses between strains of domesticus and musculus have shown that hybrid sterility phenotypes and loci depend on the geographic origins of parental strains ( Britton-Davidian et al . , 2005; Good et al . , 2008a ) , suggesting that most hybrid sterility alleles are segregating as polymorphisms within subspecies .", "Several of the loci identified in this study of hybrid zone mice are novel , providing additional evidence that sterility alleles are polymorphic within subspecies .", "However , a majority of loci we identified in natural hybrids are concordant with previously identified sterility QTL ( Tables 1–2 , Figure 2 ) .", "This similarity suggests that there are common genetic factors underlying hybrid sterility in house mice , although there was no statistical support that genome-wide patterns of overlap with previous studies for testis weight or expression PC1 were non-random ( p > 0 . 05 , 10 , 000 permutations ) .", "Prdm9 , discovered by mapping F1 hybrid sterility , is the only characterized hybrid sterility gene in mice ( Mihola et al . , 2009 ) .", "None of the GWAS regions identified here overlap Prdm9 ( chromosome 17 , 15 . 7 Mb ) .", "However , one expression PC1 region ( PC42 ) is ∼4 Mb proximal to Prdm9 .", "Reductions in PC1 are observed in individuals that are heterozygous or homozygous for the domesticus allele at PC42 .", "This pattern is partially consistent with sterility caused by Prdm9 , which occurs in heterozygous individuals carrying sterile alleles from domesticus ( Dzur-Gejdosova et al . , 2012; Flachs et al . , 2012 ) .", "We did not find evidence for significant associations between SNPs near Prdm9 and testis weight; the nearest GWAS region ( RTW09 ) is ∼41 Mb distal and low testis-weight is associated with the musculus allele .", "There is concordance between some of the genetic interactions between loci identified here and interactions identified by mapping sterility phenotypes and testis expression traits in an F2 cross between musculus and domesticus ( White et al . , 2011; Turner et al . , 2014 ) ( Figure 2—figure supplement 1 ) .", "Precise overlap between some GWAS regions and interaction regions from F2s identifies strong candidates for future studies to identify the causative mechanisms and genes underlying sterility loci .", "For example , an interaction between chromosome 12 and the central X chromosome ( RTW11 , PC49 ) identified for testis weight and expression PC1 overlaps an interaction affecting testis expression in F2 hybrids ( Turner et al . , 2014 ) .", "The 4 . 3 Mb interval of overlap among chromosome 12 loci ( RTW07 , PC29 , 32 . 38–41 . 43 Mb F2s ) encompasses 12 protein-coding genes , including a gene with a knockout model showing low testis weight and sperm count ( Arl4a ) ( Schurmann et al . , 2002 ) , and two genes with roles in regulating gene expression ( Meox2 , Etv1 ) .", "We compared the positions of GWAS regions to 182 regions ( 163 autosomal , 19 X-linked ) with evidence for epistasis based on a genome-wide analysis of genomic clines in a transect across the house mouse hybrid zone in Bavaria ( Janousek et al . , 2012 ) , the same region where the progenitors of the mapping population were collected .", "Five testis-weight regions and 18 expression-PC1 regions overlap candidate regions from the hybrid zone genomic clines analysis ( Tables 1–2 ) , however , the patterns of overlap were not statistically significant ( p > 0 . 05 , 10 , 000 permutations ) .", "Future introgression analyses using high-density markers within and around GWAS regions may be useful in identifying causative genes and estimating the contributions of sterility alleles to reduced gene flow .", "Three GWAS regions associated with testis weight and five expression PC1 regions are located on the X chromosome .", "The X-chromosomal regions surpass the stringent permutation-based significance thresholds and thus have strong statistical support .", "These results are consistent with evidence for an important role for the X in hybrid sterility from laboratory crosses between subspecies strains geographically diverse in origin ( Guenet et al . , 1990; Elliott et al . , 2001; Oka et al . , 2004 , 2007; Storchova et al . , 2004; Good et al . , 2008a , 2008b; Mihola et al . , 2009; White et al . , 2012 ) and evidence for greatly reduced gene flow of X-linked loci across the European hybrid zone ( Tucker et al . , 1992; Payseur et al . , 2004; Macholan et al . , 2007; Teeter et al . , 2008 , 2010 ) .", "A disproportionately large contribution of the X chromosome is a common feature of reproductive isolation in many taxa , the so-called ‘large X effect’ ( Coyne and Orr , 1989 ) .", "The musculus derived X chromosome has been implicated repeatedly in genetic studies of sterility in F1 and F2 hybrids ( Reviewed in Good et al . ( 2008a ) ; White et al . ( 2011 ) ) .", "By contrast , domesticus alleles were associated with the sterile pattern for most loci we identified on the X in hybrid zone mice ( Tables 1–2 ) .", "A testis expression-QTL mapping study performed in F2s also showed that domesticus ancestry in the central/distal region of the X was associated with a sterile expression pattern ( Turner et al . , 2014 ) .", "Differences between studies might reflect geographic variation in sterility alleles , but identification of domesticus-sterile X alleles only in generations beyond the F1 suggests that interactions with recessive autosomal partner loci are essential .", "The importance of recessive sterility alleles was demonstrated previously by the discovery of multiple novel recessive loci in an F2 mapping study ( White et al . , 2011 ) .", "F1 hybrids are essentially absent in nature ( Teeter et al . , 2008; Turner et al . , 2012 ) because the hybrid zone is ≥30 km wide ( Boursot et al . , 1993 ) , thus pure subspecies individuals rarely encounter each other .", "Consequently , recessive autosomal loci acting in F2 and advanced generation hybrids contribute to the maintenance of reproductive isolation in the hybrid zone and may have played important roles in its establishment .", "Despite a growing list of sterility loci and genes identified in a variety of animal and plant taxa , there are few cases of Dobzhansky–Muller incompatibilities for which all partner loci are known ( Phadnis , 2011 ) .", "Hence , there remain many unanswered questions about the genetic architecture of hybrid defects .", "For example , how many incompatibilities contribute to reproductive barriers in the early stages of speciation ?", "How many partner loci are involved in incompatibilities ?", "Are these patterns consistent among taxa ?", "The interactions contributing to sterility phenotypes we mapped in hybrid zone mice reveal several general features of the genetic architecture of hybrid sterility .", "Most sterility loci interact with more than one partner locus .", "This pattern is consistent with evidence from studies mapping sterility in F1 musculus–domesticus hybrids ( Dzur-Gejdosova et al . , 2012 ) and mapping interactions affecting testis gene expression in F2 hybrids ( Turner et al . , 2014 ) .", "We did not have sufficient power to map interactions requiring three or more sterility alleles , but interactions between alleles from the same subspecies imply their existence .", "Loci causing male sterility in Drosophila pseudoobscura Bogota–USA hybrids also have multiple interaction partners; seven loci of varying effect size interact to cause sterility ( Phadnis , 2011 ) .", "In hybrids between Drosophila koepferae and Drosophila buzzatii , sterility is associated with many loci of small effect , consistent with a polygenic threshold model ( Moran and Fontdevila , 2014 ) .", "These studies suggest that biological pathways/networks are often affected by multiple Dobzhansky–Muller interactions; a single pairwise interaction between incompatible alleles disrupts pathway function enough to cause a hybrid defect phenotype , but when more incompatible alleles are present the effects of multiple pairwise interactions are synergistic .", "Variation in the effect sizes of sterility loci might then reflect variation in the number of networks in which the gene is involved and the connectedness/centrality of the gene within those networks .", "Characteristics of the incompatibility network are important for generating accurate models of the evolution of reproductive isolation .", "A ‘snowball effect’—faster-than-linear accumulation of incompatibilities caused by epistasis—is predicted on the basis of the Dobzhansky–Muller model ( Orr , 1995; Orr and Turelli , 2001 ) .", "Patterns of accumulation of hybrid incompatibilities in Drosophila and Solanum provide empirical support for the snowball hypothesis ( Matute et al . , 2010; Moyle and Nakazato , 2010 ) .", "Because most GWAS regions have many interaction partners , our results are not consistent with the assumption of the snowball model that incompatibilities are independent , suggesting that network models of incompatibilities ( Johnson and Porter , 2000; Porter and Johnson , 2002; Johnson and Porter , 2007; Palmer and Feldman , 2009 ) may be more accurate for understanding the evolution of reproductive barriers in house mice .", "Involvement of hybrid sterility loci in interactions with multiple partner loci also has important implications for understanding the maintenance of the hybrid zone .", "Because deleterious effects of a sterility allele are not dependent on a single partner allele , the marginal effect of each locus and thus visibility to selection are less sensitive to the allele frequencies at any single partner locus in the population .", "Identifying and functionally characterizing incompatibility genes is an important goal in understanding speciation but is unrealistic in most non-model organisms .", "By contrast , mapping reproductive isolation traits in natural populations to identify the number and location of loci and interactions is feasible .", "General features of the genetic architecture of hybrid sterility—the number of incompatibilities and number and effect size of interacting loci—are arguably more likely to be shared among organisms than specific hybrid sterility genes .", "Comparison of these features among taxa may reveal commonalities of the speciation process ." ], [ "The mapping population includes first-generation lab-bred male offspring of mice captured in the hybrid zone ( Bavaria ) in 2008 ( Turner et al . , 2012 ) ( Figure 1—figure supplement 1 ) .", "We included 185 mice generated from 63 mating pairs involving 37 unrelated females and 35 unrelated males .", "Many dams and sires were used in multiple mating pairs , thus our mapping population includes full siblings , half siblings , and unrelated individuals .", "Most mating pairs ( 53 pairs , 149 offspring ) were set up with parents originating from the same or nearby trapping locations .", "Eleven pairs ( 36 offspring ) include dams and sires originating from more distant trapping locations; phenotypes of these offspring were not reported in Turner et al . ( 2012 ) .", "Males were housed individually after weaning ( 28 days ) to prevent effects of dominance interactions on fertility .", "We measured combined testis weight and body weight immediately after mice were sacrificed at 9–12 weeks .", "We calculated relative testis weight ( testis weight/body weight ) to account for a significant association between testis weight and body weight ( Pearson's correlation = 0 . 29 , p = 4 . 9 × 10−5 ) .", "We classify individuals with relative testis weight below the range observed in pure subspecies as showing evidence for sterility ( Turner et al . , 2012 ) .", "To confirm that this is an appropriate threshold for inferring hybrid defects , we compared this value to relative testis weights reported previously for offspring from intraspecific and interspecific crosses ( Good et al . , 2008a ) .", "The pure subspecies minimum we observed is substantially lower ( >2 standard deviations ) than means for males from intraspecific crosses ( converted from single relative testis weight: musculusPWK × musculusCZECH − mean = 10 . 2 , standard deviation = 1 . 2; domesticusLEWES x domesticusWSB − mean = 11 . 0 , standard deviation = 1 . 0 ) and comparable to ( within 1 standard deviation ) values observed in F1 hybrids from 4/7 interspecific crosses that showed significant reductions ( mean plus one standard deviation 4 . 6–9 . 2 mg/g ) .", "We measured gene expression in testes of 179 out of the 185 males from the mapping population .", "Freshly dissected testes were stored in RNAlater ( Qiagen , Hilden , Germany ) at 4°C overnight , then transferred to −20°C until processed .", "We extracted RNA from 15–20 mg whole testis using Qiagen RNeasy kits and a Qiagen Tissue Lyser for the homogenization step .", "We verified quality of RNA samples ( RIN >8 ) using RNA 6000 Nano kits ( Agilent ) on a 2100 Bioanalyzer ( Agilent , Waldbronn , Germany ) .", "We used Whole Mouse Genome Microarrays ( Agilent ) to measure genome-wide expression .", "This array contains 43 , 379 probes surveying 22 , 210 transcripts from 21 , 326 genes .", "We labeled , amplified , and hybridized samples to arrays using single-color Quick-Amp Labeling Kits ( Agilent ) , according to manufacturer protocols .", "We verified the yield ( >2 μg ) and specific activity ( >9 . 0 pmol Cy3/μg cRNA ) of labeling reactions using a NanoDrop ND-1000 UV-VIS Spectrophotometer ( NanoDrop , Wilimington , DE , USA ) .", "We scanned arrays using a High Resolution Microarray Scanner ( Agilent ) and processed raw images using Feature Extraction Software ( Agilent ) .", "Quality control procedures for arrays included visual inspection of raw images and the distribution of non-uniformity outliers to identify large spatial artifacts ( e . g . caused by buffer leakage or dust particles ) and quality control metrics from Feature Extraction protocol GE1_QCMT_Dec08 .", "We mapped the 41 , 174 non-control probe sequences from the Whole Mouse Genome Microarray to the mouse reference genome ( NCBI37 , downloaded March 2011 ) using BLAT ( ( Kent , 2002 ) ; minScore = 55 , default settings for all other options ) .", "Probes with multiple perfect matches , more than nine imperfect matches , matches to non-coding/intergenic regions only , or matches to more than one gene were excluded .", "A total of 36 , 323 probes ( covering 19 , 742 Entrez Genes ) were retained .", "We preformed preprocessing of microarray data using the R package Agi4x44PreProcess ( Lopez-Romero , 2009 ) .", "We used the background signal computed in Feature Extraction , which incorporates a local background measurement and a spatial de-trending surface value .", "We used the ‘half’ setting in Agi4x44PreProcess , which sets intensities below 0 . 5 to 0 . 5 following background subtraction and adds an offset value of 50 .", "Flags from Feature Extraction were used to filter probes during preprocessing ( wellaboveBG = TRUE , isfound = TRUE , wellaboveNEG = TRUE ) .", "We retained probes with signal above background for at least 10% of samples .", "We used quantile normalization to normalize signal between arrays .", "Expression data were deposited in Gene Expression Omnibus as project GSE61417 .", "To identify major axes of variation in testis expression , we performed a principal components analysis using prcomp in R ( R Development Core Team 2010 ) with scaled variables .", "We extracted DNA from liver , spleen , or ear samples using salt extraction or DNeasy kits ( Qiagen ) .", "Males from the mapping population were genotyped using Mouse Diversity Genotyping Arrays ( Affymetrix , Santa Clara , CA ) by Atlas Biolabs ( Berlin , Germany ) .", "We called genotypes at 584 , 729 SNPs using apt-probeset-genotype ( Affymetrix ) and standard settings .", "We used the MouseDivGeno algorithm to identify variable intensity oligonucleotides ( VINOs ) ( Yang et al . , 2011 ) ; 53 , 148 VINOs were removed from the dataset .", "In addition , we removed 18 , 120 SNPs with heterozygosity >0 . 9 in any population because these SNPs likely represent additional VINOs .", "We performed additional filtering steps on SNPs included in the dataset used for mapping .", "We only included SNPs with a minor allele frequency >5% in the mapping population .", "SNPs without a genome position or with missing data for >15% of the individuals in the mapping population or pure subspecies reference panel were removed .", "We pruned the dataset based on linkage disequilibrium ( LD ) to reduce the number of tests performed .", "LD pruning was performed in PLINK ( Purcell et al . , 2007; Purcell", "n . d .", ") using a sliding window approach ( 30 SNPs window size , 5 SNPs step size ) and a VIF threshold of 1 x 10−6 ( VIF = 1/ ( 1−R2 ) , where R2 is the multiple correlation coefficient for a SNP regressed on all other SNPs simultaneously ) .", "This procedure essentially removed SNPs in perfect LD .", "These filtering steps yielded 156 , 204 SNPs .", "To identify ancestry-informative SNPs , we compared genotypes from 21 pure M .", "m . domesticus individuals ( 11 from Massif Central , France and 10 from Cologne/Bonn , Germany ) and 22 M .", "m . musculus individuals ( 11 from Námest nad Oslavou , Czech Republic and 11 from Almaty , Kazakhstan ) ( Staubach et al . , 2012 ) .", "We used Structure ( Pritchard et al . , 2000; Falush et al . , 2003 ) to graphically represent the genetic composition of our mapping population ( Figure 1—figure supplement 1 ) .", "We included one diagnostic SNP per 20 cM , 3–5 markers/chromosome totaling 60 SNPs genome wide .", "We used the ‘admix’ model in Structure and assumed two ancestral populations .", "To identify genomic regions significantly associated with relative testis weight and testis gene expression , we used a mixed model approach to test for single SNP associations .", "Admixture mapping—often applied in studies using samples with genetic ancestry from two distinct populations—was not appropriate for this study because it was not possible to account for relatedness among individuals in the mapping population ( Buerkle and Lexer , 2008; Winkler et al . , 2010 ) .", "We performed association mapping using GEMMA ( Zhou and Stephens , 2012 ) , which fits a univariate mixed model , incorporating an n x n relatedness ( identity-by-state ) matrix as a random effect to correct for genetic structure in the mapping population .", "We estimated relatedness among the individuals in the mapping population in GEMMA using all markers and the –gk 1 option , which generates a centered relatedness matrix .", "For each single SNP association test we recorded the Wald test p value .", "Phenotypes tested include relative testis weight ( testis weight/body weight , RTW ) , testis expression principal component 1 ( PC1 , 14 . 6% variance , associated with fertility , Figure 1—figure supplement 2 ) , and normal quantile ranks of gene expression values for individual transcripts .", "Neither RTW nor expression PC1 was significantly correlated with age at phenotyping ( RTW: cor = −0 . 02 , p = 0 . 72; PC1: cor = 0 . 01 , p = 0 . 90 ) , thus we did not include age in the model .", "SNP data , phenotypic data , and kinship matrix to run GEMMA area are available through Dryad at: doi:10 . 5061/dryad . 2br40 .", "To account for multiple testing , we first determined stringent significance thresholds by permutation .", "We randomized phenotypes among individuals 10 , 000 times , recording the lowest p value on the X and the lowest p value on any autosome for each permutation .", "Thresholds set to the fifth percentile across permutations for RTW were 5 . 73 × 10−7 ( autosomes ) and 5 . 83 × 10−5 ( X chromosome ) ; thresholds for expression PC1 were 1 . 66 × 10−8 ( autosomes ) and 1 . 01 × 10−5 ( X chromosome ) .", "Next , we identified regions using a more permissive significance threshold based on the 10% false discovery rate ( Benjamini and Hochberg , 1995 ) , equivalent to p = 3 . 49 × 10−5 for RTW and p = 2 . 86 × 10−4 for expression PC1 .", "To estimate the genomic interval represented by each significant LD-filtered SNP , we report significant regions defined by the most distant flanking SNPs in the full dataset showing r2 > 0 . 9 ( genotypic LD , measured in PLINK ) with each significant SNP .", "We combined significant regions <10 Mb apart into a single region .", "Identifying genetic interactions using GWAS is computationally and statistically challenging .", "To improve power , we reduced the number of tests performed by testing for interactions only among significant SNPs ( FDR < 0 . 1 ) identified using GEMMA .", "We tested all pairs of significant SNPs located on different chromosomes for each phenotype ( 692 pairs RTW , 82 , 428 pairs expression PC1 ) .", "To account for relatedness among individuals we used a mixed model approach , similar to the model implemented in GEMMA .", "We used the lmekin function from the coxme R package ( Therneau , 2012 ) to fit linear mixed models including the identity-by-state kinship matrix as a random covariate .", "We report interactions as significant for SNP pairs with p < 0 . 05 and FDR < 0 . 1 for interaction terms ( RTW: FDR < 0 . 1 ∼ p < 0 . 02; expression PC1: FDR < 0 . 09 ∼ p < 0 . 05 ) .", "We performed simulations to evaluate the performance of our mapping approach under varying genetic architectures and allele frequencies .", "We simulated phenotypes using several genetic models of two-locus epistasis and parameters based on the empirical distribution of relative testis weight .", "The simulation procedure is illustrated in Figure 4—figure supplement 1 .", "To preserve genetic structure , we simulated phenotypes using two-locus genotypes from the SNP dataset .", "We tested 100 autosomal–autosomal SNP pairs ( SNPs on different chromosomes ) and 100 X–autosomal pairs ( 50 with domesticus X-linked sterile alleles and 50 with musculus X-linked sterile alleles ) .", "The criteria used for choosing ‘causative’ SNPs were a minor allele frequency >0 . 05 in the mapping population and fixed in at least one subspecies .", "The ‘sterile’ allele could be polymorphic or fixed within subspecies but the alternate ‘non-sterile’ allele had to be fixed within the other subspecies—e . g . domesticus sterile alleles have frequencies 0 . 05–1 . 0 in the domesticus reference populations from France and Germany and the alternate allele at those SNPs are fixed in musculus samples from the Czech Republic and Kazakhstan .", "For each pair , the ‘causative’ SNPs were randomly selected from all SNPs meeting those criteria ( 144 , 506 possible domesticus sterile , 124 , 390 possible musculus sterile ) .", "For each SNP pair , we modeled all possible combinations of recessive , additive , and dominant sterile alleles .", "For each model type , we assigned mean Z scores for each possible two-locus genotype ( Figure 4—source data 1 ) .", "The magnitude of the most severe phenotype ( −2 . 3 standard deviations ) is based on observed relative testis weights in the most severely affected males .", "The mean Z score for heterozygotes in additive models was −1 . 15 .", "Mean Z scores for non-sterile genotypes in the models were randomly drawn from a uniform distribution between −0 . 5 and 0 . 5 .", "For each SNP pair/architecture , 100 data sets were generated by drawing phenotypes ( Z scores ) for each individual from a normal distribution with the appropriate two-locus mean and standard deviation = 0 . 75 .", "The standard deviation value , equivalent to 2 . 98 mg/g , was chosen on the basis of standard deviations in pure subspecies samples from the mapping population ( domesticus = 2 . 13 , musculus = 3 . 65; ( Turner et al . , 2012 ) ) .", "This value is higher than standard deviations in intraspecific F1 males ( domesticusLEWES × domesticusWSB = 1 . 2 , musculusPWK × musculusCZECH = 1 . 0; ( Good et al . , 2008a ) ) , suggesting estimates of mapping power may be conservative .", "In total , 90 , 000 simulations were performed , ( 9 architectures × 100 SNP pairs × 100 data sets ) .", "We identified significant SNPs for each data set using GEMMA , as described above for the empirical data .", "We used permutations to test for non-random co-localization of candidate sterility loci from this study and previous QTL and hybrid zone studies .", "The locations of significant GWAS regions for relative testis weight and expression PC1 were randomized 10 , 000 times using BEDTools ( Quinlan and Hall , 2010 ) .", "To assess overlap between significant regions for the two phenotypes , we counted the number of RTW regions overlapping PC1 regions ( and vice versa ) for each permutation .", "To test for overlap between GWAS identified regions and previously reported candidate regions for related phenotypes , we counted the number of permuted regions overlapping the positions of the published regions ( fixed ) for each replicate .", "GWAS regions for both phenotypes were compared to genomic regions with evidence for epistasis and reduced introgression in the Bavarian transect of the hybrid zone ( Janousek et al . , 2012 ) .", "In addition , RTW regions were compared to testis weight QTL from mapping studies in F2 hybrids from crosses between subspecies ( Storchova et al . , 2004; Good et al . , 2008b; White et al . , 2011; Dzur-Gejdosova et al . , 2012 ) and expression PC1 regions were compared to trans eQTL hotspots identified in F2 hybrids ( Turner et al . , 2014 ) .", "We used ENSEMBL ( version 66 , February 2012 ) Biomart to download gene annotations for genomic regions significantly associated with relative testis weight .", "We identified candidate genes in significant regions with roles in male reproduction using reviews of male fertility ( Matzuk and Lamb , 2008 ) , manual searches , MouseMine searches for terms related to male fertility ( http://www . mousemine . org/ ) , and gene ontology ( GO ) terms related to male reproduction or gene regulation ( plus children ) : meiosis GO:0007126; DNA methylation GO:0006306; regulation of gene expression GO:0010468; transcription GO:0006351; spermatogenesis GO:0007283; male gamete generation GO:0048232; gamete generation GO:0007276; meiotic cell cycle GO:0051321 .", "Many genes with roles in reproduction reported in publications were not annotated with related GO terms , highlighting the limitations of gene ontology .", "Moreover , genes causing sterility might not have functions obviously related to reproduction ." ] ]

[ "Mapping hybrid defects in contact zones between incipient species can identify genomic regions contributing to reproductive isolation and reveal genetic mechanisms of speciation .", "The house mouse features a rare combination of sophisticated genetic tools and natural hybrid zones between subspecies .", "Male hybrids often show reduced fertility , a common reproductive barrier between incipient species .", "Laboratory crosses have identified sterility loci , but each encompasses hundreds of genes .", "We map genetic determinants of testis weight and testis gene expression using offspring of mice captured in a hybrid zone between M . musculus musculus and M . m .", "domesticus .", "Many generations of admixture enables high-resolution mapping of loci contributing to these sterility-related phenotypes .", "We identify complex interactions among sterility loci , suggesting multiple , non-independent genetic incompatibilities contribute to barriers to gene flow in the hybrid zone ." ]

[ "Different species have often evolved from a common ancestor .", "In order to become distinct species , however , the different groups of descendants of that ancestor must have become isolated from one another at some point in their history so that they could no longer mate or reproduce .", "For example , a mountain or a river might create a physical barrier that keeps species apart , so that if the species meet up again they may struggle to mate or produce offspring .", "Furthermore , any ‘hybrid’ offspring that are produced may themselves struggle to survive or successfully reproduce .", "Examining the genes of the hybrid offspring that result when two recently separated species crossbreed could help us to understand how new species evolve .", "However , the challenges of finding enough suitable hybrids to compare means that few studies have so far investigated the genetic changes that occur to make reproduction between separate species difficult .", "Two subspecies of the house mouse—Mus musculus musculus and Mus musculus domesticus—live alongside each other in a region of central Europe and can mate and produce hybrid offspring .", "Male hybrid mice are commonly less fertile than non-hybrids; this acts as a barrier to reproductive success that helps to maintain the separation between the two subspecies .", "Turner and Harr captured wild hybrid mice , bred them in the laboratory , and studied their offspring .", "This strategy enabled them to measure fertility in mice very similar to wild-caught hybrids , but now all individuals can be measured at the same age and under the same environmental conditions .", "A method called a genome-wide association study can be used to survey the genes of individuals with a particular disease or physical characteristic in an effort to identify gene variants that are associated with that condition .", "In many species , the weight of a male's testes has been linked to their fertility—small testes mean the male is likely to be less fertile .", "Changes in how genes are ‘expressed’ in the testes can also reduce fertility .", "Turner and Harr used a genome-wide association study to investigate which genetic changes are linked to changes in testis weight or how genes in the testes are expressed in the offspring of hybrid mice .", "This revealed that many separate genetic regions are involved; including some that had not previously been identified .", "Turner and Harr then examined how these gene regions interact with each other .", "With the exception of one gene , all interacted with at least one of the other genetic regions that had been identified , forming a complex network of interactions .", "Although a genome-wide association study reveals which genes are altered in hybrid mice with small testes , it does not reveal which of these genes actually cause the changes in testis size and gene expression .", "However , the work of Turner and Harr greatly narrows down the candidates for further investigation ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "cell biology", "neuroscience" ]

[ [ "Proteins on the surface of cells are removed from the plasma membrane by endocytosis .", "Many cargo proteins are recruited to sites of endocytosis by the tetrameric adaptor complex AP2 ( Mahaffey et al . , 1990; Traub , 2003 ) .", "The adaptor complex in turn recruits the coat protein clathrin to the membrane .", "Clathrin converts the raft of cargo and adaptor proteins into a budding vesicle by forming a scaffold that shapes the membrane ( Mashl and Bruinsma , 1998 ) .", "Clathrin-mediated endocytosis probably functions in all tissues , but it is unclear whether this process is suited to the particularly high rates of endocytosis required at nerve terminals .", "Nonetheless , the predominant mechanism for synaptic vesicle endocytosis is thought to be mediated via AP2 and clathrin ( Dittman and Ryan , 2009 ) .", "Testing this model by disrupting clathrin is difficult to interpret because trafficking from the trans-Golgi relies in part on clathrin-coated vesicles .", "Thus , genetic analysis of AP2 mutants is more specific for endocytic trafficking of proteins from the cell surface .", "The AP2 adaptin complex has four subunits—two large subunits α and β2 , a medium subunit μ2 , and a small subunit σ2 ( Matsui and Kirchhausen , 1990 ) .", "It is generally thought that adaptor complexes act as obligate tetramers; loss of one subunit will destabilize the entire complex ( Dell'Angelica et al . , 1998; Kantheti et al . , 1998; Collins et al . , 2002; Motley et al . , 2003; Traub , 2003; Nakatsu et al . , 2004; Mitsunari et al . , 2005; Kim and Ryan , 2009 ) .", "AP2 functions at the plasma membrane as an interaction hub for transmembrane cargoes , accessory proteins , and clathrin ( Traub , 2003; Robinson , 2004 ) .", "Loss of single AP2 subunits is known to disrupt endocytosis at the plasma membrane .", "A null allele in α-adaptin is lethal and leads to an absence of synaptic vesicles at neuromuscular junctions in Drosophila embryos and thus appears to disrupt endocytosis ( Gonzalez-Gaitan and Jackle , 1997 ) .", "Similarly , in C . elegans loss of either of α- or β-adaptin by RNA interference perturbs the endocytosis of yolk protein from the plasma membrane ( Grant and Hirsh , 1999 ) .", "These data suggest that loss of either large subunit eliminates AP2 function .", "Recent data suggest that the medium subunit μ2 may not play an essential role for the endocytosis of synaptic vesicle components from the plasma membrane ( Gu et al . , 2008; Kim and Ryan , 2009 ) .", "Although μ2 is required in part for the localization of clathrin at synapses ( Gu et al . , 2008 ) , synaptic vesicles and constituent proteins are still recycled in the absence of μ2 ( Gu et al . , 2008; Kim and Ryan , 2009 ) .", "These contrasting results for α-adaptin vs μ2-adaptin mutants from different organisms suggest that α-adaptin is essential for synaptic vesicle endocytosis , whereas the μ2 subunit may not be essential .", "Here we evaluate the function of AP2 at synapses by studying mutations in α- and μ2-adaptins in C . elegans .", "Because null mutations for both of these genes are viable , we can compare the loss of these AP2 subunits in a single organism for the first time .", "Mutants lacking α-adaptin retain a partially functional AP2 hemicomplex consisting of μ2 and β-adaptin .", "Mutants lacking both α and μ2 subunits exhibit a more severe phenotype than the single mutants and are subviable .", "These results suggest that the single subunits retain some function , but that the double mutants lack all AP2 function .", "Nevertheless a moderate level of synaptic transmission remains in the double mutant and is able to sustain locomotory behavior , suggesting the presence of an AP2 independent mechanism capable of maintaining synaptic transmission at the synapse ." ], [ "In C . elegans , α-adaptin is encoded by the apa-2 gene ( Figure 1A ) .", "Two alleles of apa-2 have been isolated ( Figure 1B ) : b1044 is a 925 bp deletion that starts within the second intron , extends to the fourth exon and deletes a large fraction of the trunk domain .", "ox422 is premature stop mutation at Lys215 and would lead to a truncation of α-adaptin from the middle of the trunk domain to the carboxy terminus including the ear domain ( Figure 1C ) .", "We did not detect full-length APA-2 protein from either of these alleles ( Figure 1—figure supplement 1 ) , and they are likely to be null mutations . 10 . 7554/eLife . 00190 . 003Figure 1 . apa-2 cloning .", "( A ) Genetic map position of apa-2 on chromosome X . ( B ) Genomic structure of the apa-2 gene and the nature of mutant alleles .", "b1044 is a 925 bp deletion from the second intron to the fourth exon .", "ox422 is an A to T transversion .", "( C ) Protein domain structure of alpha adaptin .", "b1044 causes a deletion of aa93-318 in the trunk domain .", "ox422 changes Lys215 to a premature stop . DOI: http://dx . doi . org/10 . 7554/eLife . 00190 . 00310 . 7554/eLife . 00190 . 004Figure 1—figure supplement 1 . Western blot of α adaptin mutants apa-2 ( ox422 ) and apa-2 ( b1044 ) .", "Antibodies are rabbit polyclonal anti-α adaptin and mouse monoclonal anti-tubulin . DOI: http://dx . doi . org/10 . 7554/eLife . 00190 . 004 To determine the expression pattern of apa-2 , we inserted the coding sequence for GFP in frame at the 3′ end of the open reading frame ( Figure 2A ) .", "The GFP fusion construct rescued the mutant phenotype ( data not shown ) .", "The apa-2 gene appears to be expressed in most cells of the animal , and is highly expressed in the nervous system ( Figure 2B , C ) . 10 . 7554/eLife . 00190 . 005Figure 2 . α-adaptin is expressed ubiquitously .", "( A ) Schematic of apa-2::GFP translational reporter construct .", "The APA-2::GFP fusion construct is expressed under the control of the apa-2 promoter ( 1 . 9 kb upstream of ATG ) from an extrachromosomal array in a lin-15 rescued background .", "( B ) The expression pattern of the translational fusion protein APA-2::GFP in young adult hermaphrodite .", "The worm is oriented anterior left and dorsal up .", "GFP fluorescence is observed ubiquitously in transgenic worms .", "( C ) Detailed images of APA-2::GFP expression in three tissues: nervous system , intestine and hypodermis .", "The scale bar represents 50 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 00190 . 005 Unlike α-adaptin mutants in Drosophila ( Gonzalez-Gaitan and Jackle , 1997 ) , worms missing α-adaptin in C . elegans are viable; they grow to adulthood and are grossly similar to μ2-adaptin ( apm-2 ) mutants ( Figure 3A ) .", "About 30% of α mutants ( Figure 3A ) and about 5% of μ2 mutants ( Gu et al . , 2008 ) have cuticle protrusions on either side of the head called ‘jowls' .", "However , the variable dumpy phenotype of apa-2 is less severe than that of apm-2 ( Figure 3B ) .", "The α-adaptin mutants are egg-laying defective and mildly uncoordinated; they crawl forward well but are slightly jerky as they move backward .", "apa-2 mutants exhibit only mild defects in thrashing when placed in liquid ( Figure 3C ) . 10 . 7554/eLife . 00190 . 006Figure 3 . Tissue-specific rescue of α-adaptin mutant .", "( A ) Bright field images apm-2 ( e840 ) and tissue-specific rescue of apa-2 ( ox422 ) mutants .", "Worms are rescued by strains carrying single-copy transgenes .", "The jowls are indicated by the black arrowheads; most apm-2 animals lack jowls .", "The scale bar represents 100 μm .", "( B ) The dumpy phenotype of apa-2 mutants is rescued by neuronal expression .", "Body length of apm-2 ( e840 ) ( deletion allele of μ2 adaptin ) , apa-2 mutants and apa-2 tissue-specific rescued animals .", "Average body length at the L4 stage in μm ± SEM: wild type 763 ± 10 , apm-2 ( e840 ) 609 ± 12 ( p<0 . 0001 ) , apa-2 ( ox422 ) 711 ± 12 ( p=0 . 0037 ) , apa-2 ( b1044 ) 684 ± 15 ( p<0 . 0001 ) , skin-rescued apa-2 ( ox422 ) 727 ± 10 ( p=0 . 0203 ) , neuron-rescued apa-2 ( ox422 ) 820 ± 17 ( p=0 . 0098 ) , ubiquitous rescued apa-2 ( ox422 ) 773 ± 12 ( p=0 . 5301 ) .", "n = 10 L4 worms .", "( C ) Locomotion assay .", "Average body bends per minute ± SEM: wild type 94 . 4 ± 6 . 0 , apm-2 ( e840 ) 46 . 0 ± 17 . 4 ( p=0 . 0478 ) , apa-2 ( ox422 ) 82 . 4 ± 4 . 0 ( p=0 . 1347 ) , apa-2 ( b1044 ) 74 . 6 ± 2 . 7 ( p=0 . 0168 ) , ubiquitously-rescued apa-2 ( ox422 ) 93 . 4 ± 6 . 2 ( p=0 . 9106 ) , neuron-rescued apa-2 ( ox422 ) 98 . 2 ± 6 . 3 ( p=0 . 6738 ) , skin-rescued apa-2 ( ox422 ) 106 . 8 ± 5 . 2 ( p=0 . 1570 ) , skin-rescued apa-2 ( ox422 ) apm-2 ( e840 ) 83 . 2 ± 3 . 2 ( p=0 . 1382 ) .", "n = 5 adult hermaphrodites .", "n of apm-2 = 7 .", "* p<0 . 05 , ** p<0 . 01 , *** p<0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 00190 . 00610 . 7554/eLife . 00190 . 007Figure 3—figure supplement 1 . Tissue-specific expression of APA-2::GFP . The dpy-7 promoter drives expression ( skin ) .", "The rab-3 promoter drives neuronal expression in the neurons .", "The dpy-30 promoter drives ubiquitous expression .", "Worms are oriented anterior left and dorsal up .", "Images are confocal Z-stack projections of the head of the worm .", "All worms were imaged under identical conditions .", "The scale bar represents 20 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 00190 . 007 Expression of APA-2::GFP under a ubiquitous promoter can fully rescue the mutant phenotypes including the cuticle protrusions ( Figure 3A–C; Figure 3—figure supplement 1 ) .", "Expression of the α-adaptin specifically in the epidermis ( the equivalent of skin in C . elegans ) rescues the cuticle phenotype ( Figure 3A; Figure 3—figure supplement 1 ) , which is similar to rescue experiments in μ2-adaptin mutants ( Gu et al . , 2008 ) .", "However unlike μ2-adaptin mutants , the dumpy phenotype of apa-2 is rescued by neuron-specific but not skin-specific expression ( Figure 3B ) .", "In fact , the neuron-rescued worms are longer than the wild type ( Figure 3B ) .", "Thus , α- and μ2 mutants exhibit similar but distinct phenotypes suggesting α- and μ2-adaptins may not be required for identical functions of AP2 in worms .", "If the functions of α-adaptin and μ2-adaptin are different , then the double mutants will be synthetic , that is the phenotype of the double mutant will be much more severe than the single mutants .", "Indeed , when the apa-2 and apm-2 mutations are combined , only 4 . 3% of the double mutants coming from a heterozygote are viable and the brood size of these survivors is reduced to 1 . 4% compared to the wild type ( Figure 4A , B ) .", "The rare escapers grow twice as slowly as wild-type animals and are sick and dumpy ( Figure 4C and Figure 4—figure supplement 1 ) .", "RNAi of μ2 in α mutants and α in μ2 mutants produced similar results ( data not shown ) .", "These data suggest residual function of AP2 remains in both apa-2 and apm-2 single mutants . 10 . 7554/eLife . 00190 . 008Figure 4 . α- and μ2-adaptin double mutant is synthetic .", "( A ) Embryonic lethality ( % total embryos ) of AP2 mutants ± SEM: wild type 1 . 13 ± 0 . 25 n = 10 , apa-2 ( ox422 ) 2 . 42 ± 0 . 96 n = 9 ( p=0 . 1902 ) , apm-2 ( e840 ) 5 . 85 ± 2 . 56 n = 10 ( p=0 . 0831 ) , apa-2 ( ox422 ) apm-2 ( e840 ) 95 . 70 ± 4 . 30 n = 11 ( p<0 . 0001 ) .", "*** p<0 . 001 .", "( B ) The brood size of AP2 mutants±SEM: wild type 247 . 3 ± 8 . 8 n = 10 , apa-2 ( ox422 ) 104 . 8 ± 16 . 9 n = 9 ( p<0 . 0001 ) , apm-2 ( e840 ) 49 . 0 ± 14 . 2 n = 10 ( p<0 . 0001 ) , apa-2 ( ox422 ) apm-2 ( e840 ) 3 . 4 + 3 . 1 n = 11 ( p<0 . 0001 ) .", "( C ) Bright-field images of the wild type , apm-2 ( e840 ) , apa-2 ( ox422 ) and a surviving apa-2 ( ox422 ) apm-2 ( e840 ) adult .", "The scale bar represents 100 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 00190 . 00810 . 7554/eLife . 00190 . 009Figure 4—figure supplement 1 . AP2 mutants exhibit slowed postembryonic development . Mean days from L1 to L4 stage ± SEM: wild type 1 . 5 ± 0 n = 29 , apm-2 ( e840 ) 1 . 97 ± 0 . 17 n = 28 ( p=0 . 0068 ) , apa-2 ( ox422 ) 2 . 11 ± 0 . 09 n = 28 ( p<0 . 0001 ) , apm-2 ( e840 ) apa-2 ( ox422 ) 2 . 75 ± 0 . 17 n = 29 ( p<0 . 0001 ) .", "** p<0 . 01 , *** p<0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 00190 . 009 At a cellular level , α-adaptin mutants exhibit defects in endocytosis .", "Yolk is a lipoprotein particle composed of lipids and lipid-transport proteins called vitellogenins .", "Yolk particles are synthesized and secreted by the intestine and are then taken up from the extracellular space by maturing oocytes via receptor-mediated endocytosis .", "Yolk endocytosis is clathrin-dependent and can be assayed in animals expressing GFP-tagged vitellogenin-2 ( YP170::GFP ) ( Grant and Hirsh , 1999; Sato et al . , 2009 ) .", "In wild-type worms , YP170::GFP is enriched in the three most mature oocytes near the spermatheca .", "In α-adaptin mutants , the number of GFP-positive oocytes is decreased to one or two cells ( Figure 5A , B ) , which is similar to the defect in μ2 mutants ( Gu et al . , 2008 ) .", "By contrast , strong defects in YP170::GFP endocytosis are observed in mutants lacking the alternative clathrin adaptor Disabled ( Holmes et al . , 2007 ) .", "Thus , the AP2 complex appears to assist Disabled for yolk endocytosis , and α- and μ2-adaptin do not contribute differentially to this process .", "To determine if α- and μ2-adaptin contribute differentially to endocytosis , we needed to identify specific cargo . 10 . 7554/eLife . 00190 . 010Figure 5 . Endocytosis of α- and μ2-adaptin-dependent cargo . Fluorescence images have been inverted to aid visualization of signals .", "( A ) Yolk protein , YP170::GFP is endocytosed by maturing oocytes in the wild type and both apa-2 mutants .", "Black arrowheads point to maturing oocytes and black arrows point to fertilized embryos .", "( B ) The number of YP170::GFP positive oocytes ± SEM: wild type 2 . 5 ± 0 . 2 , apa-2 ( ox422 ) 1 . 6 ± 0 . 1 ( p=0 . 0003 ) , apa-2 ( b1044 ) 1 . 4 ± 0 . 1 ( p<0 . 0001 ) .", "n = 20 adult hermaphrodites , *** p<0 . 001 .", "Two-tailed Student's t-test .", "( C ) A diagram of eGFP-CD4 artificial cargo .", "eGFP was flanked by two 12 aa flexible linkers and inserted after the secretion signal peptide , the extracellular domain of CD4 was truncated to include one immunoglobulin domain , the cytoplasmic domain of CD4 was removed leaving a seven aa tail ( Feinberg et al . , 2008 ) , and the 11 aa Nef di-leucine motif was fused after CD4 ( Doray et al . , 2007 ) .", "Circles represent amino acids on the cytoplasmic face .", "( D ) eGFP-CD4-LL localization in intestine in the wild type and α adaptin mutant ( apa-2 ( ox422 ) X ) and μ2 adaptin mutant ( apm-2 ( e840 ) X ) mutants .", "Black arrowheads point to the intracellular organelle in the wild type ( see inset ) and to the lateral surface of the plasma membrane in the mutants .", "( E ) Quantification of fluorescence intensity of eGFP-CD4-LL in wild type and AP2 mutants .", "Total fluorescence was measured from regions of interest defined on the basolateral membrane and averaged .", "Fluorescence intensity arbitrary units mean ± SEM: wild type 1998 ± 275 n = 5 , apa-2 ( ox422 ) 14 , 907 ± 990 n = 6 ( p<0 . 0001 ) , apm-2 ( e840 ) 10 , 310 ± 1342 n = 6 ( p=0 . 0004 ) .", "The p value between apa-2 and apm-2 is <0 . 0203 .", "* p<0 . 05 .", "( F ) Endocytosis of MIG-14/wntless in the intestine in the wild type , μ2 adaptin mutant ( apm-2 ( e840 ) X ) and an α adaptin mutant ( apa-2 ( ox422 ) X ) .", "Fluorescence images are inverted to better view dim GFP fluorescence .", "( G ) Quantification of fluorescence intensity of MIG-14 in wild type and AP2 mutants .", "Total fluorescence was measured from regions of interest defined on the basolateral membrane and averaged .", "The data were captured on a Zeiss LSM 510 and the spectral fingerprinting feature was used to remove intestinal autofluorescence .", "Fluorescence intensity arbitrary units mean ± SD: wild type 7363 ± 3498 n = 18 , apm-2 ( e840 ) 92 , 648 ± 34 , 237 n = 18 ( p<0 . 0001 ) , apa-2 ( ox422 ) 25 , 110 ± 11 , 570 n = 18 ( p<0 . 0001 ) .", "The p value between apa-2 and apm-2 is <0 . 0001 .", "*** p<0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 00190 . 01010 . 7554/eLife . 00190 . 011Figure 5—figure supplement 1 . An AP2-independent cargo is not affected by AP2 subunits mutants . Human IL2 receptor α subunit Tac ( hTAC ) in the nematode intestine in the wild type , μ2 adaptin mutant ( apm-2 ( e840 ) X ) and an α adaptin mutant ( apa-2 ( ox422 ) X ) .", "Fluorescence images are inverted to better view GFP fluorescence . DOI: http://dx . doi . org/10 . 7554/eLife . 00190 . 01110 . 7554/eLife . 00190 . 012Figure 5—figure supplement 2 . Quantification of total fluorescence intensity of hTAC in the wild type and AP2 mutants . Total fluorescence was measured along the basolateral membrane and averaged .", "Fluorescence intensity mean ± SD: wild type 16 , 080 ± 7875 n = 18 , apm-2 ( e840 ) 22 , 948 ± 9488 n = 18 ( p=0 . 0240 ) , apa-2 ( ox422 ) 21 , 950 ± 12 , 953 n = 18 ( p=0 . 1096 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 00190 . 012 There is no known α-adaptin specific cargo in C . elegans .", "However , the α-adaptin subunit is involved in binding cargo with di-leucine motifs ( Kelly et al . , 2008 ) .", "We constructed an artificial cargo protein known to bind α-adaptin ( Figure 5C ) .", "We tagged the human CD4 protein with GFP , and appended the di-leucine motif ( ExxxLL ) from HIV Nef to the carboxy terminus ( Doray et al . , 2007 ) .", "We expressed the construct in the intestine .", "In wild-type worms CD4-dileucine is localized to intracellular compartments; however , in apa-2 mutants CD4-dileucine accumulates abnormally on basolateral membranes ( Figure 5D ) .", "By contrast in apm-2 mutants , the CD4-dileucine accumulation on the plasma membrane is milder ( 69% compared to α mutants; Figure 5D , E ) .", "These data suggest that recovery of di-leucine cargo depends on α-adaptin more than μ2-adaptin .", "MIG-14/wntless is a μ2-dependent cargo ( Pan et al . , 2008 ) .", "In the absence of the μ2 subunit , MIG-14::GFP is strongly mislocalized to the basal and lateral surfaces of intestine cells ( Figure 5F , G ) .", "In the absence of α-adaptin , MIG-14::GFP is only weakly mislocalized on the basolateral surface ( 27% compared to μ2 mutants; Figure 5F , G ) .", "MIG-14 contains a tyrosine in its carboxy terminus ( μ2 consensus target is Yxxϕ ) , but it is not known if this sequence is required for μ2 binding .", "As a control , the endocytosis of clathrin-independent cargo hTAC is unaffected in both adaptin mutants ( Figure 5—figure supplement 1 , 2 ) .", "Taken together , these data suggest that partially functional AP2 complexes might be present in mutations that eliminate single subunits .", "The open form of AP2 can be considered as two hemicomplexes: the α and the σ2 subunits are in close contact , and the μ2 and β subunits are in close contact in both open and closed forms of the complex ( Collins et al . , 2002; Jackson et al . , 2010 ) .", "However , these two hemicomplexes are only loosely associated in the open form of the AP2 complex ( Jackson et al . , 2010 ) .", "Here we demonstrate that in the absence of α-adaptin that a μ2-β hemicomplex remains , and that in the absence of μ2-adaptin that a α-σ2 complex remains in vivo .", "The β- and μ2-adaptins , which are not closely associated with α-adaptin , are stable in the absence of α−adaptin .", "Transgenes expressing GFP-tagged AP2 subunits were inserted as single copy transgenes and crossed into apa-2 mutants .", "Tagged β-adaptin and μ2-adaptin are localized to synaptic regions of the nerve ring ( the major neuropil of the worm , Figure 6B , C ) and at the plasma membrane in oocytes ( Figure 6—figure supplement 1 ) .", "The level of μ2-adaptin is reduced to 40% in apa-2 mutants as assayed by western blot ( Figure 6—figure supplement 1 ) or 20% as measured by fluorescence ( Figure 6 ) .", "On the other hand , the small σ2 subunit , which is normally tightly bound to α-adaptin , is unstable in apa-2 mutants .", "Tagged σ2 is no longer detectable in the nerve ring ( Figure 6 ) or in maturing oocytes ( Figure 6—figure supplement 2 ) , and the protein is reduced to about 10% of the wild-type level as assayed by fluorescence ( Figure 6 , Table 1 ) or western blot ( Figure 6—figure supplement 2 ) . 10 . 7554/eLife . 00190 . 013Figure 6 . AP2 hemicomplexes are partially stable in vivo . All images are inverted to better visualize GFP fluorescence .", "( A ) Synaptic localization of σ2 adaptin ( APS-2::GFP ) in α and μ mutants .", "The nerve ring is indicated by the black arrowhead .", "( B ) Synaptic localization of β adaptin ( APB-1::GFP ) in α and μ2 mutants .", "( C ) Synaptic localization of μ2 adaptin ( APM-2::GFP ) rescuing construct in μ2 mutants ( labeled as wild type* ) and an α μ2 double mutant ( apm-2 ( e840 ) apa-2 ( ox422 ) X , labeled as apa-2 ( ox422 ) * ) .", "The single copy APM-2::GFP transgene oxSi54 fully rescues the apm-2 ( e840 ) mutation .", "( D ) Synaptic localization of α adaptin ( APA-2::GFP ) in wild type and an μ2 mutant apm-2 ( e840 ) .", "The scale bar represents 20 μm .", "Please refer to Table 1 for detailed quantification . DOI: http://dx . doi . org/10 . 7554/eLife . 00190 . 01310 . 7554/eLife . 00190 . 014Figure 6—figure supplement 1 . μ2-adaptin is present in α-adaptin mutants . A rescuing construct of tagged μ2-adaptin was inserted into a μ2-adaptin null strain for all genotypes .", "Top: μ2-adaptin ( APM-2::GFP ) expression in the gonad of a μ2 mutant ( apm-2 ( e840 ) X , labeled as wild type* ) and an α μ2 double mutant ( apm-2 ( e840 ) apa-2 ( ox422 ) X , labeled as apa-2* ) .", "μ2-adaptin is enriched at the plasma membrane of oocytes ( black arrow heads ) .", "The contrast was increased to visualize the GFP signal at the plasma membrane .", "The scale bar represents 20 μm .", "Bottom: western blot for the expression level of μ2 adaptin-GFP in apa-2 ( ox422 ) .", "Antibodies are mouse monoclonal anti-GFP and anti-tubulin .", "μ2 adaptin is reduced to 42% but still present in an α adaptin mutant . DOI: http://dx . doi . org/10 . 7554/eLife . 00190 . 01410 . 7554/eLife . 00190 . 015Figure 6—figure supplement 2 . σ2-adaptin is more unstable in α-adaptin than in μ2-adaptin mutants . Top: σ2::GFP is localized to the plasma membrane in the wild type and μ2-adaptin mutant but not in α-adaptin mutants or α-μ2 adaptin double mutants .", "The oocyte cell surface is indicated by black arrows .", "The scale bar represents 20 μm .", "Bottom: Western blot for the expression level of σ2 adaptin-GFP in apa-2 ( ox422 ) and apm-2 ( e840 ) .", "The protein level is reduced more in apa-2 ( 91% ) than in apm-2 mutants ( 86% ) .", "Antibodies are mouse monoclonal anti-GFP and anti-tubulin .", "Endogenous σ2 adaptin is present in this experiment .", "Please refer to supplementary file 1 for detailed genotypes . DOI: http://dx . doi . org/10 . 7554/eLife . 00190 . 01510 . 7554/eLife . 00190 . 016Table 1 . GFP fluorescence of tagged AP2 subunits in the nerve ring ( quantification for Figure 6 ) .", "Average GFP intensity in the nerve ring ( percentage of the wild type ) DOI: http://dx . doi . org/10 . 7554/eLife . 00190 . 016wild typeapa-2apm-2σ2::GFP3543 ± 169 ( 100% ) 389 ± 28 ( 11% ) 1391 ± 51 ( 39% ) β1::GFP3881 ± 31 ( 100% ) 2329 ± 123 ( 60% ) 1360 ± 62 ( 35% ) μ2::GFP2783 ± 142 ( 100% ) 532 ± 73 ( 19% ) –α::GFP3230 ± 132 ( 100% ) –1296 ± 126 ( 40% ) The data are mean ± SEM of averaged fluorescence , n = 5 worms each .", "The p value for all pair-wise comparisons ( wild type vs mutants or apa-2 vs apm-2 ) is p<0 . 0001 .", "Student's t test .", "Note the beta subunit in C . elegans is shared by both the AP1 and AP2 complexes .", "Beta levels are reduced in apm-2 mutants compared to apa-2 mutants; however , beta is still present and stable in AP1 complexes in apm-2 mutants .", "In particular , beta is highly expressed in pharyngeal muscle , which is included in the region of interest .", "Conversely , α-adaptin and σ2-adaptin are localized in the absence of μ2-adaptin .", "In apm-2 mutants , tagged α-adaptin is still localized to the synapse ( Figure 6D ) and the plasma membrane of coelomocytes .", "α-adaptin levels are only reduced to 60% as assayed by western blot ( Gu et al . , 2008 ) or 40% as assayed by fluorescence .", "Tagged σ2-adaptin is still localized to the nerve ring ( Figure 6A ) and is reduced to 40% as assayed by fluorescence ( Figure 6 , Table 1 ) .", "On the other hand , the large β subunit , which is normally tightly bound to μ2-adaptin , is unstable in apm-2 mutants .", "The β subunit is shared by AP1 and AP2 in C . elegans , and tagged β subunit fluorescence is visible in cell bodies in apm-2 mutants .", "However , tagged β is no longer detectable in the synapse-rich region of the nerve ring in the absence of μ2-adaptin ( Figure 6B , Table 1 ) .", "Taken together , these data suggest that AP2 hemicomplexes are partially stable and can function in vivo in the absence of a complete AP2 complex .", "To study the function of AP2 components in neurons , we rescued the mutant defects in the epidermis .", "Providing AP2 function in the skin was necessary for two reasons: First , AP2 components are required in the epidermis to play non-autonomous roles in synaptic development ( Gu et al . , 2008; Pan et al . , 2008 ) .", "Second , due to the low viability of the double mutant , it is impossible to maintain as a homozygous strain .", "However , when α- and μ2-adaptins are simultaneously introduced back into the epidermis , 100% of the double mutants grow to adults .", "The skin-rescued worms have no detectable APA-2::GFP in the nervous system ( Figure 3—figure supplement 1 ) and the skin promoter Pdpy-7 is only expressed in larval stages during development ( Johnstone and Barry , 1996 ) .", "These rescued animals are still egg-laying defective and slow-growing , but they provide an opportunity to study synaptic vesicle endocytosis in AP2-deficient synapses .", "We assayed the synaptic localization of α-adaptin by expressing an apa-2::GFP fusion construct specifically in GABA neurons .", "α-adaptin colocalizes with a synaptic vesicle protein , synaptobrevin , in both the dorsal and ventral nerve cords ( Figure 7—figure supplement 1 ) .", "This result suggests that α-adaptin associates with synaptic varicosities , similar to μ2-adaptin ( Gu et al . , 2008 ) .", "We examined the requirement of AP2 for the recycling of several synaptic-vesicle proteins .", "In C . elegans mutants lacking particular adaptor proteins , the cognate cargo protein diffuses into axons .", "For example in AP180 mutants , synaptobrevin is no longer concentrated at synapses but is diffuse in axons ( Nonet et al . , 1999 ) .", "By contrast , in AP2 adaptin mutants , synaptic vesicle proteins are not grossly mislocalized .", "Synaptotagmin , the vesicular GABA transporter ( UNC-47 ) , and synaptogyrin are largely confined to synaptic varicosities in α-adaptin single mutants and α-μ2 adaptin double mutants , although the GFP signal is slightly diffuse in axons ( Figure 7A , B ) .", "These data are consistent with previous data demonstrating that the relevant adaptors for synaptotagmin and the GABA transporter are Stonin and BAD-LAMP/UNC-46 , respectively ( Schuske et al . , 2007; Maritzen et al . , 2010; Mullen et al . , 2012 ) .", "These results suggest that vesicle proteins are endocytosed properly in AP2 mutants , although it is possible that some proteins remain on the surface but are confined to the synapse . 10 . 7554/eLife . 00190 . 017Figure 7 . α-adaptin mutants exhibit weak defects in synaptic vesicle protein localization .", "( A ) All images are inverted to better visualize GFP fluorescence .", "Synaptic localization of synaptic vesicle proteins in apa-2 and apm-2 ( e840 ) apa-2 ( ox422 ) double mutants ( an escaper with no skin rescue ) .", "Synaptotagmin ( SNT-1::GFP ) is expressed in all neurons under its own promoter and imaged in ventral sublateral cords .", "VGAT ( UNC-47::GFP ) and synaptogyrin ( SNG-1::GFP ) are expressed in GABA neurons and imaged in the dorsal nerve cord .", "Presynaptic varicosities of neuromuscular junctions along the nerve cords of an adult hermaphrodite are visible as fluorescent puncta .", "The axon regions with increased fluorescence are indicated by black arrowheads .", "Images are confocal Z-stack projections through the worm nerve cord .", "The scale bar represents 10 μm .", "( B ) Quantification of the average fluorescence intensity ratio between axon region and synaptic region .", "Ratio of SNT-1::GFP mean ± SEM: wild type 0 . 041 ± 0 . 007 n = 10 , apa-2 ( ox422 ) 0 . 204 ± 0 . 029 n = 10 ( p<0 . 0001 ) , apa-2 ( b1044 ) 0 . 196 ± 0 . 017 n = 10 ( p<0 . 0001 ) , apa-2 ( ox422 ) apm-2 ( e840 ) 0 . 219 ± 0 . 032 n = 6 ( p<0 . 0001 ) .", "Ratio of UNC-47::GFP mean ± SEM: wild type 0 . 108 ± 0 . 004 n = 8 , apa-2 ( ox422 ) 0 . 239 ± 0 . 009 n = 8 ( p<0 . 0001 ) , apa-2 ( b1044 ) 0 . 220 ± 0 . 024 n = 7 ( p=0 . 0003 ) , apa-2 ( ox422 ) apm-2 ( e840 ) 0 . 247 ± 0 . 032 n = 6 ( p=0 . 0003 ) .", "Ratio of SNG-1::GFP mean ± SEM: wild type 0 . 077 ± 0 . 008 n = 8 , apa-2 ( ox422 ) 0 . 116 ± 0 . 019 n = 10 ( p=0 . 1026 ) , apa-2 ( b1044 ) 0 . 095 ± 0 . 011 n = 10 ( p=0 . 2252 ) , apa-2 ( ox422 ) apm-2 ( e840 ) 0 . 143 ± 0 . 032 n = 5 ( p=0 . 0308 ) .", "* p<0 . 05 , ** p<0 . 01 , *** p<0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 00190 . 01710 . 7554/eLife . 00190 . 018Figure 7—figure supplement 1 . α-adaptin colocalizes with synaptobrevin at synapses . Young adult hermaphrodites were used for imaging .", "Left: α-adaptin ( APA-2::GFP ) and synaptobrevin ( SNB-1::tagRFP ) colocalize at synapses in the dorsal nerve cord of GABA motor neurons .", "The fluorescent puncta correspond to synaptic varicosities along the dorsal muscles ( white arrow head ) .", "Right: α-adaptin and synaptobrevin localization in the ventral nerve cord of GABA motor neurons .", "A GABA neuron cell body is indicated by the white arrow .", "Images are confocal Z-stack projections through the worm nerve cord .", "The scale bar represents 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 00190 . 018 To determine if there is a defect in membrane endocytosis , we characterized the ultrastructure of neuromuscular junctions in α-adaptin mutants ( Figure 8A ) .", "In mutants lacking apa-2 in the nervous system , synaptic vesicle numbers are reduced to 71% in acetylcholine neurons and 59% in GABA neurons ( Figure 8B; Figure 8—figure supplement 1 ) .", "This moderate reduction in synaptic vesicle numbers is similar to the loss observed in μ2 mutants ( Gu et al . , 2008 ) .", "The synaptic vesicle defects observed in α-adaptin mutants can be fully rescued by expression of APA-2 in the nervous system .", "Defects in synaptic vesicle numbers are more severe in mutants lacking rescue in the skin ( 56% in acetylcholine neurons and 29% in GABA neurons compared to the wild type ) as was observed in μ2 mutants ( Gu et al . , 2008 ) .", "In summary , specific loss of just α-adaptin or just μ2-adaptin in neurons only leads to a moderate defect in synaptic vesicle number . 10 . 7554/eLife . 00190 . 019Figure 8 . Large vesicles accumulate at synapses in AP2 mutants .", "( A ) Representative images of acetylcholine neuromuscular junctions in the ventral nerve cord from the wild type , apa-2 ( ox422 ) , ubiquitously-rescued apa-2 ( ox422 ) , neuronally-rescued apa-2 ( ox422 ) , skin-rescued apa-2 ( ox422 ) , skin-rescued apa-2 ( ox422 ) apm-2 ( e840 ) in adult hermaphrodites .", "At apa-2 apm-2 synapses , at least one large vesicle was usually observed adjacent to the dense projection ( 13/21 synapses ) , and a large vacuole in the center of the varicosity ( 17/21 synapses ) .", "The scale bar represents 200 nm .", "Abbreviations: SV: synaptic vesicle; LV: large vesicle; dense proj: dense projection .", "( B ) Morphometry of acetylcholine neuromuscular junctions in adaptin mutants .", "The number of synaptic vesicles is reduced in neurons lacking α adaptin or both α and μ2-adaptins .", "Average number of synaptic vesicles per profile containing a dense projection ± SEM n = synapses: wild type 22 . 0 ± 1 . 4 n = 35 , apa-2 ( ox422 ) 12 . 3 ± 1 . 1 n = 66 ( p<0 . 0001 ) , ubiquitous rescued apa-2 ( ox422 ) 19 . 1 ± 1 . 1 n = 54 ( p=0 . 1052 ) , neuron-rescued apa-2 ( ox422 ) 25 . 1 ± 1 . 5 n = 49 ( p=0 . 1501 ) , skin-rescued apa-2 ( ox422 ) 15 . 6 ± 0 . 8 n = 97 ( p<0 . 0001 ) , skin-rescued apa-2 ( ox422 ) apm-2 ( e840 ) 6 . 2 ± 0 . 8 n = 47 ( p<0 . 0001; compared with skin rescued apa-2 ( ox422 ) p<0 . 0001 ) .", "( C ) Median size of synaptic vesicles per profile containing a dense projection n = synapses: wild type 28 . 4 nm n = 9 , apa-2 ( ox422 ) 29 . 9 nm n = 12 ( p=0 . 0007 ) , ubiquitous rescued apa-2 ( ox422 ) 27 . 4 nm n = 12 ( p=0 . 0001 ) , neuron- rescued apa-2 ( ox422 ) 27 . 9 nm n = 12 ( p=0 . 5538 ) , skin-rescued apa-2 ( ox422 ) 30 . 9 nm n = 23 ( p<0 . 0001 ) , skin-rescued apa-2 ( ox422 ) apm-2 ( e840 ) 31 . 9 nm n = 11 ( 0 . 0057 ) .", "Median is the middle line and box defines the 25th and 75th percentiles .", "The length of the whiskers indicates the span between the 10th and 90th percentiles .", "( D ) Average number of large vesicles ( clear core and the diameter > 35 nm ) per profile containing a dense projection ± SEM n = synapses: wild type 0 . 30 ± 0 . 11 n = 30 , apa-2 ( ox422 ) 3 . 1 ± 0 . 34 n = 72 ( p<0 . 0001 ) , ubiquitous rescued apa-2 ( ox422 ) 0 . 7 ± 0 . 13 n = 43 ( p=0 . 0302 ) , neuron-rescued apa-2 ( ox422 ) 0 . 6 ± 0 . 08 n = 62 ( p=0 . 0325 ) , skin-rescued apa-2 ( ox422 ) 1 . 8 ± 0 . 2 n = 97 ( p<0 . 0001 ) , skin-rescued apa-2 ( ox422 ) apm-2 ( e840 ) 2 . 2 ± 0 . 29 n = 53 ( p<0 . 0001 ) .", "( E ) Cumulative vesicle diameter in acetylcholine neurons .", "For all panels , the imaged synapses are from two young adult hermaphrodites for each genotype .", "Statistics are comparison with wild type , except where marked .", "* p<0 . 05 , ** p<0 . 01 , *** p<0 . 001 .", "( F ) 3D modeling of an acetylcholine synapse from a skin-rescued apa-2 ( ox422 ) apm-2 ( e840 ) animal .", "Structures were hand-traced from ten consecutive sections using an imageJ plugin , TrakEM2 ( Cardona et al . , 2012 ) .", "The transparent light-blue structures are synaptic vesicles , and the red structure is a dense projection .", "Large vesicles ( dark blue ) that accumulate in the terminal are typically severed from the surface . DOI: http://dx . doi . org/10 . 7554/eLife . 00190 . 01910 . 7554/eLife . 00190 . 020Figure 8—figure supplement 1 . Synaptic vesicles are reduced at GABA synapses in α-adaptin mutants and α-adaptin μ2-adaptin double mutants . The number of synaptic vesicles in GABA neurons n = synapses: wild type 36 . 9 ± 1 . 5 n = 36 , apa-2 ( ox422 ) 10 . 7 ± 0 . 7 n = 46 ( p<0 . 0001 ) , ubiquitously-rescued apa-2 ( ox422 ) 33 . 9 ± 1 . 4 n = 33 ( p=0 . 1504 ) , neuron-rescued apa-2 ( ox422 ) 32 . 5 ± 2 . 7 n = 40 ( p=0 . 1711 ) , skin-rescued apa-2 ( ox422 ) 21 . 7 ± 1 . 3 n = 45 ( p<0 . 0001 ) , skin-rescued apa-2 ( ox422 ) apm-2 ( e840 ) 11 . 5 ± 0 . 9 n = 52 ( p<0 . 0001; compared with skin-rescued apa-2 ( ox422 ) p<0 . 0001 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 00190 . 02010 . 7554/eLife . 00190 . 021Figure 8—figure supplement 2 . Synaptic vesicle diameters are larger in α-adaptin mutants . Median size of synaptic vesicles per GABA synapse profile containing a dense projection n = synapses: wild type 28 . 4 nm n = 8 , apa-2 ( ox422 ) 30 . 9 nm n = 9 ( p<0 . 0001 ) , ubiquitously-rescued apa-2 ( ox422 ) 27 . 4 nm n = 7 ( p=0 . 0140 ) , neuron-rescued apa-2 ( ox422 ) 28 . 4 nm n = 9 ( p=0 . 2359 ) , skin-rescued apa-2 ( ox422 ) 31 . 4 nm n = 11 ( p<0 . 0001 ) , skin-rescued apa-2 ( ox422 ) apm-2 ( e840 ) 31 . 9 nm n = 12 ( p<0 . 0001 ) .", "The center line indicates the median and the box defines the 25th and 75th percentiles .", "The upper and lower ends of the whiskers are the 90th and 10th percentiles respectively .", "Mann-Whitney U test was used for statistics .", "Statistical comparisons are to the wild type . DOI: http://dx . doi . org/10 . 7554/eLife . 00190 . 02110 . 7554/eLife . 00190 . 022Figure 8—figure supplement 3 . Large vesicles accumulate in α-adaptin mutants and α-adaptin μ2-adaptin double mutants . The number of large vesicles in GABA neurons n = synapses: Wild type GABA 0 . 9 ± 0 . 1 n = 41 , apa-2 ( ox422 ) GABA 4 . 9 ± 0 . 5 n = 43 ( p<0 . 0001 ) , ubiquitously-rescued apa-2 ( ox422 ) GABA 0 . 9 ± 0 . 2 n = 32 ( p=1 . 0000 ) , neuron-rescued apa-2 ( ox422 ) GABA 1 . 0 ± 0 . 2 n = 37 ( p=0 . 6464 ) , skin-rescued apa-2 ( ox422 ) GABA 3 . 6 ± 0 . 5 n = 45 ( p<0 . 0001 ) , skin-rescued apa-2 ( ox422 ) apm-2 ( e840 ) GABA 4 . 8 ± 0 . 4 n = 50 ( p<0 . 0001 ) .", "Statistics are in comparison with the wild type , except where indicated .", "*** p<0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 00190 . 02210 . 7554/eLife . 00190 . 023Figure 8—figure supplement 4 . In some cases , the large vacuole remains associated with the plasma membrane in α-adaptin μ2-adaptin double mutants .", "( A–C ) additional images of acetylcholine neuromuscular junctions in the ventral nerve cord from the skin-rescued apa-2 ( ox422 ) apm-2 ( e840 ) in adult hermaphrodites .", "( D–F )", "Zoomed-in images of large vacuoles indicated by black arrows in ( A–C ) .", "The scale bar represents 100 nm in ( A–C ) and 50 nm in ( D–F ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 00190 . 023 In contrast to the single mutants , complete loss of AP2 at synapses leads to a severe defect in synaptic vesicle number .", "In synapses of apa-2 ( ox422 ) apm-2 ( e840 ) double mutants ( but rescued in the epidermis ) the number of synaptic vesicles is reduced to 28% in acetylcholine neurons and 31% in GABA neurons ( Figure 8B; Figure 8—figure supplement 1 ) .", "It is likely that of loss of α and μ2 leads to a complete loss of AP2 since σ2 and β are lost at synapses in each of these mutants respectively ( Figure 6A , B ) .", "In summary , loss of μ2 alone leads to a 31% decrease in synaptic vesicles in acetylcholine neurons ( Gu et al . , 2008 ) , loss of α-adaptin alone leads to a 29% decrease , but a complete loss of AP2 leads to a 70% reduction in synaptic vesicle number .", "These data suggest that complete inactivation of AP2 requires removal of both the α and μ2 subunits .", "The diameter of the remaining synaptic vesicles is slightly increased in apa-2 and apm-2 apa-2 double mutants ( Figure 8C; Figure 8—figure supplement 2 ) .", "The median diameter of synaptic vesicles in the wild type is 28 . 4 nm , the diameter in apa-2 mutants ( skin rescued ) is 30 . 9 nm , and the diameter in apa-2 apm-2 double mutants ( skin rescued ) is 31 . 9 nm ( Figure 8C ) .", "These data suggest that the AP2 complex may play a role in regulating the size of synaptic vesicles .", "Alternatively , the effect on vesicle size may be indirect due to pleiotropic defects in endocytosis .", "Beyond the slight increase in diameter of synaptic vesicles , α-adaptin mutant synapses also exhibit an accumulation of large vesicles ( diameter > 40 nm , Figure 8A , D , E; Figure 8—figure supplement 3 ) .", "This phenotype is not apparent in μ2 mutants ( Gu et al . , 2008 ) , suggesting different roles for α and μ2 at synapses .", "In apa-2 apm-2 double mutants a large vesicle is often observed adjacent to the dense projection and very large vesicles occupy the center of the synaptic varicosity ( Figure 8A , E , F; Figure 8—figure supplement 4 ) .", "We speculate that these large vesicles could be endosomal intermediates generated by bulk endocytosis .", "Are these large vesicles bonafide synaptic vesicles ?", "Specifically , can they fuse and release neurotransmitter in an electrophysiological assay ?", "The increase in diameter of synaptic vesicles was accompanied by an increase in the amount of neurotransmitter released by a synaptic vesicle .", "Miniature postsynaptic currents ( ‘minis' ) were measured from motor neurons using voltage-clamp recordings from body muscles ( Figure 9A , B ) .", "In apa-2 ( ox422 ) mutants , the amplitude from miniature spontaneously released vesicles ( minis ) is increased by 40% ( Figure 9C ) .", "The mini amplitudes in the skin-rescued single and double mutants are also larger , although they do not reach statistical significance .", "The enhanced mini amplitude could have been caused by an increase in postsynaptic receptor density due to a defect in receptor endocytosis ( Kittler et al . , 2005; Kastning et al . , 2007; Vithlani and Moss , 2009 ) .", "However , the defect in mini amplitude was fully rescued by expressing apa-2 in neurons ( Figure 9C ) .", "Thus , the increase in mini current amplitude is consistent with the observed increase in the diameter of synaptic vesicles . 10 . 7554/eLife . 00190 . 024Figure 9 . Synaptic vesicle fusion is reduced in α adaptin mutants .", "( A ) Sample traces of miniature postsynaptic current ( minis ) recorded from the wild type , apa- 2 ( ox422 ) , apa-2 ( ox422 ) neuronal-rescued , apa-2 ( ox422 ) skin-rescued and apa-2 ( ox422 ) apm-2 ( e840 ) skin-rescued worms .", "( B ) Sample traces of evoked postsynaptic current ( electrically evoked ) recorded from same genotypes .", "( C ) Summary of mini amplitudes ( pA ± SEM n = animals ) : wild type 26 . 4 ± 2 . 5 n = 16 , apa-2 ( ox422 ) 36 . 9 ± 2 . 5 n = 19 ( p=0 . 0058 ) , apa-2 ( ox422 ) skin-res .", "35 . 2 ± 3 . 6 n = 9 ( p=0 . 0516 ) , apa-2 ( ox422 ) neur-res .", "28 . 1 ± 2 . 4 n = 21 ( p=0 . 6313 ) , apa-2 ( ox422 ) apm-2 ( e840 ) skin-res .", "33 . 3 ± 3 . 8 n = 9 ( p=0 . 1287 ) .", "( D ) Summary of mini frequency ( minis/sec ± SEM n = animals ) : wild type 43 . 9 ± 5 . 9 n = 16 , apa-2 ( ox422 ) 7 . 8 ± 1 . 5 n = 19 ( p<0 . 0001 ) , apa-2 ( ox422 ) skin-res .", "22 . 5 ± 4 . 5 n = 9 ( p=0 . 0206 ) , apa-2 ( ox422 ) neur-res .", "44 . 9 ± 4 . 8 n = 21 ( p=0 . 8951 ) , apa-2 ( ox422 ) apm-2 ( e840 ) skin-res .", "14 . 2 ± 4 . 0 n = 9 ( p=0 . 0019 ) .", "( E ) Summary of evoked amplitude ( pA ± SEM n = animals ) : wild type 2159 . 6 ± 131 . 1 n = 11 , apa-2 ( ox422 ) 1259 . 1 ± 274 . 9 n = 5 ( p=0 . 0044 ) , apa-2 ( ox422 ) skin-res .", "1627 . 3 ± 182 . 0 .", "n = 6 ( p=0 . 0303 ) , apa-2 ( ox422 ) neur-res .", "2090 . 7 ± 149 . 0 n = 6 ( p=0 . 7468 ) , apa-2 ( ox422 ) apm-2 ( e840 ) skin-res .", "1264 . 3 ± 323 . 7 n = 6 ( p=0 . 0082 ) .", "* p<0 . 05 , ** p<0 . 01 , *** p<0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 00190 . 024 The reduction in synaptic vesicle numbers was also paralleled by a reduction in the electrophysiological response of the neuromuscular junctions .", "There is a 25% reduction in the amplitude of evoked release in skin-rescued apa-2 mutants , and this defect can be rescued by expressing apa-2 in neurons .", "The double mutants exhibit a more severe , 42% reduction in the amplitude of the evoked responses ( Figure 9E ) .", "There is also a more severe reduction in the rates of tonic synaptic vesicle fusion .", "Skin-rescued apa-2 animals exhibit a 50% reduction in mini frequency , and the skin-rescued apa-2 apm-2 double mutants exhibit a 68% reduction in mini frequency ( Figure 9D ) .", "The reduction in vesicle fusions ( 68% reduction ) is proportional to the reduction in synaptic vesicle numbers at synapses ( 70% reduction ) , suggesting that the vesicles seen by electron microscopy are bonafide synaptic vesicles in the AP2 mutants .", "In summary , the loss of both α- and μ2-adaptin leads to a more severe synaptic defect than the single mutants , suggesting that these subunits can function independently in synaptic vesicle endocytosis ." ], [ "In this study , we genetically characterized AP2 function in C . elegans with a particular focus on the synapse .", "The results indicate that AP2 can function as two hemicomplexes comprised of either the α/σ subunits or μ/β subunits .", "The evidence for hemicomplexes is the following: First , in α-adaptin mutants , the μ2-β subunits are stable , but the small σ2 subunit is unstable .", "Second , in μ2-adaptin mutants , α-σ2 subunits are stable , but the β subunits are unstable .", "Third , specific cargoes require the cognate hemicomplex for endocytosis .", "Fourth , the subunits contribute genetically independent functions to viability , body morphology and synaptic vesicle biogenesis .", "Although our data suggest that AP2 hemicomplexes can function in C . elegans , it must be emphasized that they are not fully independent; each hemicomplex is less stable in the absence of the other .", "Nonetheless , a complete block of AP2 function requires the simultaneous removal of both α- and μ2-adaptins .", "Below , we discuss four aspects of these results: What is the structural basis for hemicomplex function ?", "What is the functional division of hemicomplexes ?", "Can hemicomplexes function in other organisms ?", "How can synapses function in the absence any AP2 function ?", "Recent structural studies support the possibility of stable hemicomplexes .", "Previous trypsin-sensitivity experiments suggested that AP2 undergoes a conformational change between the cytosolic and clathrin-bound states ( Matsui and Kirchhausen , 1990 ) .", "The crystal structures of both the closed and open conformations have been solved ( Collins et al . , 2002; Jackson et al . , 2010 ) .", "In the open state , μ2-adaptin is postulated to undergo a large-scale conformational change; this rearrangement brings the four PIP2 binding sites and two endocytic motif binding sites of AP2 into a single plane .", "In this open conformation , the interactions between the C-terminal μ2 domain and α and σ2 are lost , and the binding surface between the C-terminal domain of μ2 and β is doubled ( Jackson et al . , 2010 ) .", "This implies that upon cargo binding , the interaction between μ2 and β is strengthened while the contacts with the other half of the complex are weakened .", "It is possible that upon cargo binding the AP2 complex becomes two loosely connected hemi-complexes .", "What is the functional relationship between the hemicomplexes ?", "There are three possibilities: inseparable functions , separable functions , and redundant functions .", "First , some functions seem to require both hemi-complexes combined , and it is surprising that loss of one hemicomplex does not eliminate all AP2 function .", "For example , recruitment of AP2 to membranes in vitro requires both PIP2 binding sites on α and β subunits ( Jackson et al . , 2010 ) .", "On the other hand , PIP2 binding sites on each of the hemicomplexes may be sufficient for membrane association albeit with a lowered avidity .", "Second , other AP2 functions may be uniquely provided by each hemicomplex .", "Substrate binding in some cases is subunit-specific and loss of one hemicomplex preferentially affects a cargo protein , for example , MIG-14 is not recruited in a μ2 adaptin mutant .", "On the cytoplasmic side , the ear of the alpha subunit preferentially binds particular ancillary proteins like amphiphysin , synaptojanin , Numb , and stonin2 ( Owen et al . , 2000; Santolini et al . , 2000; Praefcke et al . , 2004; Jung et al . , 2007 ) , whereas clathrin heavy chain binds the appendage of β strongly and only binds the α appendage weakly ( Shih et al . , 1995; Owen et al . , 2000; Schmid et al . , 2006 ) .", "Thus , loss of a single hemicomplex will result in the loss of only a specific subset of AP2 functions .", "Third , some functions might be mediated by either subunit , and only a double mutant would lead to a severe phenotype .", "For example , the appendage domains of both large subunits bind some of the same proteins , for example AP180 , epsin , and eps15 ( Owen et al . , 2000; Mishra et al . , 2004 ) .", "Importantly , redundancy need not act only at the level of AP2 subunits but could be contained within the network of associated proteins .", "Although clathrin is largely recruited to AP2 by the β subunit , even in the absence of β , it could still be recruited indirectly to the complex via AP180—the web of interactions within the clathrin complex generates a redundant network ( Royle , 2006; Schmid et al . , 2006 ) .", "Thus , in contrast to un-networked hubs ( Jeong et al . , 2001 ) , the loss of the hub does not cause things to fall apart; the center can hold .", "Are functional hemicomplexes conserved ?", "Certainly the sequences of the AP2 subunits are strongly conserved .", "For example , the amino acid sequences of AP2 subunits in C . elegans and mouse are at least 64% identical ( α 65%; β2 64%; μ2 82%; σ2 95% ) .", "It is also possible that the ability of AP2 hemicomplexes to function is also conserved .", "A purified human α-σ2 hemicomplex can bind di-leucine motifs , suggesting that hemicomplexes can be stable and exhibit appropriate biochemical interactions ( Doray et al . , 2007 ) .", "Although double mutants have not been analyzed in other metazoans , a genome-wide genetic interaction analysis in S . pombe found that mutations in AP2 β2 and σ2 exhibited synthetic interactions in double mutants ( Frost et al . , 2012 ) , suggesting that functional hemicomplexes may be conserved in other organisms .", "On the other hand , knocking down the μ2 subunit in cultured hippocampal neurons caused a concomitant 96% reduction of α-adaptin suggesting that hemicomplexins are not stable in these cells ( Kim and Ryan 2009 ) .", "It is likely that the stability of hemicomplexes may vary in organisms depending on a variety of factors such as temperature , chaperones and degradation machinery .", "What is the molecular role of AP2 in synaptic vesicle biogenesis ?", "A reduction of synaptic vesicle numbers by 70% and the accumulation of large vesicles imply an important role of AP2 in endocytosis .", "These defects resemble those observed in synaptotagmin mutants in C . elegans or after acute disruption of synaptotagmin in Drosophila ( Jorgensen et al . , 1995; Poskanzer et al . , 2006 ) .", "Moreover , the synaptic phenotypes of mutants lacking stonin are similar ( Fergestad et al . , 1999; Mullen et al . , 2012 ) .", "One possibility is that AP2 nucleates synaptic vesicle endocytosis with stonin and synaptotagmin .", "The synaptotagmin C2B domain binds AP2 via the mu-homology domain of μ2-adaptin ( Zhang et al . , 1994; Haucke et al . , 2000 ) , and the C2A domain binds the mu-homology domain of stonin ( Jung et al . , 2007 ) .", "It is possible that these proteins work together in a single process .", "In fact analysis of double mutants suggest that stonin and AP2 act in a similar process ( Mullen et al . , 2012 ) .", "In the simplest model , synaptotagmin recruits stonin and AP2 to the plasma membrane to recover synaptic vesicle components .", "On the other hand , the AP2 double mutants lacking both α- and μ2-adaptins exhibit remarkably normal locomotion and evoked currents .", "One is forced to conclude that despite an important role in endocytosis , that synaptic vesicles are still being generated in the absence of AP2 .", "What process contributes to synaptic vesicle endocytosis when AP2 is missing ?", "One possibility is that AP1 or AP3 could compensate for the loss of AP2 .", "In the mouse , there is evidence that AP1could function at the synapse and substitute for AP2 in its absence ( Kim and Ryan , 2009; Glyvuk et al . , 2010 ) .", "Alternatively AP3 might be able to provide function in the absence of AP2 ( Blumstein et al . , 2001; Voglmaier et al . , 2006 ) .", "However , AP1 and AP3 are not likely to be recycling vesicles from the membrane at the C . elegans neuromuscular junctions .", "First , the presence of β-adaptin ( shared by AP1 ) in the nerve ring is completely dependent upon the presence of μ2; AP1 does not seem to be at the synapse ( Figure 6B ) .", "Second μ2-μ3 double mutants do not exhibit a synthetic phenotype , suggesting that AP3 does not substitute for AP2 at C . elegans synapses ( Gu et al . , 2008 ) .", "It is more likely that the adaptor protein associated with the AP2 complex , such as AP180 , mediates endocytosis in the absence of the AP2 complex .", "AP180 possesses functions remarkably similar to AP2 .", "AP180 can bind and stimulate clathrin assembly and bind PIP2 in the membrane ( Hao et al . , 1999; Ford et al . , 2001 ) .", "It acts as an adaptor for synaptic vesicle proteins since it can bind and recruit synaptobrevin to invaginating vesicles ( Nonet et al . , 1999; Miller et al . , 2011 ) .", "Finally AP180 mutants in C . elegans exhibit defects in synaptic vesicle endocytosis as analyzed by electron microscopy ( Nonet et al . , 1999 ) .", "It is possible that the remaining functional synaptic vesicles in the absence of AP2 are generated by AP180 .", "Where then does AP2 act ?", "Classic studies of synaptic ultrastructure of frog and fly synapses suggest that clathrin and the AP2 complex act at the plasma membrane ( Heuser and Reese , 1973; Gonzalez-Gaitan and Jackle , 1997 ) .", "The data presented here do not contradict those studies , but also suggest that synaptic vesicle proteins and membrane can be recovered from the membrane despite a loss of AP2 .", "The most prominent defect observed in AP2 mutants is the presence of large diameter vesicles and vacuoles in the synapses of the α-μ2 adaptin double mutants .", "In some cases these vacuoles appear to be attached to the plasma membrane at the adherens junctions ( Figure 8—figure supplement 4 ) , suggesting a defect in the formation and cleavage of vesicles from the plasma membrane .", "In other cases , reconstructions of these vacuoles from serial electron micrographs indicate that they are separated from the plasma membrane ( Figure 8F ) .", "These data suggest that AP2 has a late function; AP2 may be required to regenerate synaptic vesicles from endosomes .", "In summary , these data suggest two major conclusions: First , the AP2 complex can function as two semi-independent hemicomplexes , consistent with new structural data for the complex .", "Second , there are at least two mechanisms ( AP2-dependent and AP2-independent ) for endocytosis at synapses in C . elegans that regenerate synaptic vesicles and maintain synaptic function ." ], [ "The wild type is Bristol N2 .", "All other genotypes are described in supplementary file 1 .", "apa-2 ( b1044 ) was isolated by polymerase chain reaction and sibling selection from an ultraviolet- and trimethyl-psoralen-mutagenized ‘mutant library' generously provided by X Li , A Melendez and I Greenwald .", "Primers ATTTGTCGGTCGGTACTTGC and ATTCGCCTACGCCATTCTTC were used in the first round of amplification , whereas the nested primers ATCTGTCGTAATTGTCACGG and TTTGGATCCACGTCAGTCAG were used for the second round of amplification .", "The reference strain EG4739 for apa-2 ( b1044 ) X was outcrossed twice before phenotypic analysis .", "apa-2 ( ox422 ) was isolated from a non-complementation screen of b1044 from 4000 haploid genomes mutagenized by ENU .", "ox422 is an A to T transversion that creates a premature stop at lysine 215 .", "A second deletion allele ox421 was also isolated which removes the entire apa-2 ORF .", "ox421 is likely to be a deficiency since it removes at least 6 kb upstream and 3 kb downstream of the apa-2 ORF and thus deletes genes upstream and downstream of apa-2 .", "The reference strain EG6147 for apa-2 ( ox422 ) X was outcrossed seven times before phenotypic analysis .", "All oxSi single copy insertions were generated by MosSCI ( Frokjaer-Jensen et al . , 2008 ) .", "apa-2 translational reporter: 1 . 9 kb promoter and the coding sequence of apa-2 was cloned into pGEM-3Zf vector .", "This fragment was fused to GFP-unc-54 3′UTR at the XbaI and HindIII sites .", "Three-fragment Multisite Gateway vectors were used ( Invitrogen , Grand Island , NY; catalog no . 12537-023 ) for generating most other constructs .", "PENTRY4-1 was used as the slot 1 promoter entry vector .", "Promoters include Pdpy-30 , Pdpy-7 , Prab-3 and Punc-47; the promoter fragments do not include the initiating methionine codon ( ATG ) .", "PENTRY1-2 was used as the slot 2 ORF entry vector , ORFs include apa-2 ( cDNA ) , apm-2 ( cDNA ) , apb-1 ( cDNA ) , eGFP::CD4 ( di-leucine ) , and sng-1 ( cDNA ) , all of which have an ATG immediately following the att site at the beginning of the ORF; they do not have a stop codon at the 3′ end .", "PENTRY2-3 was used for the slot 3 C-terminal tag and 3′UTR entry vector , slot 3 clones include GFP-unc-54 3′UTR and mCherry-unc-54 3′UTR .", "The destination vectors are Gateway pDEST R4-R3 , pCFJ150 for MosSCI on chromosome II and pCFJ201 for MosSCI on chromosome IV ( Frokjaer-Jensen et al . , 2008 ) .", "The 1 . 2 kb aps-2 promoter and the 1 kb aps-2 genomic coding sequence were cloned by PCR from wild-type genomic DNA .", "GFP with the unc-54 3′UTR was fused to the C-terminus of aps-2 using a PstI site and the entire fusion fragment was dropped between the restriction sites BssHII and SpeI on pCFJ151 for MosSCI on chromosome II .", "The final DNA concentration of each injection mix was 100 ng/μl .", "This target concentration was obtained with the addition of Fermentas 1 kb DNA ladder ( #SM0311 ) .", "APA-2 translational GFP: pMG16 apa-2::GFP was injected into lin-15 ( n765ts ) X animals at 1 ng/μl .", "The coinjection marker lin-15 ( + ) was used at 50 ng/μl .", "APA-2 and synaptobrevin colocalization: pRH324 Punc-47::SNB-1::tagRFP was injected into the wild type ( N2 ) at 0 . 25 ng/μl .", "The co-injection marker Punc-122::GFP was used at 50 ng/μl .", "F1 transgenic worms were singled .", "One of the transgenic lines , oxEx1411 , was crossed into dkIs160[Punc-25::GFP::APA-2; unc-119 ( + ) ] .", "apa-2 apm-2 double mutant skin rescue: pMG50 Pdpy-7::APM-2::GFP and pMG40 Pdpy-7::APA-2::mCherry were coinjected into the adaptin double mutant-balanced strain EG6158 +/szT1[lon-2 ( e678 ) ] I; szT1/apm-2 ( e840 ) apa-2 ( ox422 ) X at 1 ng/μl each .", "The coinjection marker was Punc-122::GFP , at 50 ng/μl .", "In the next generation , rescued but egg-laying defective worms were singled .", "One of the lines , EG6151 , was used in the electron microscopy and electrophysiology assays .", "Worm samples were prepared by boiling 1 volume of worm pellet in 1 volume of 2× loading buffer for 5 min .", "Samples were run on a 10% SDS-PAGE gel and then transferred to PVDF transfer membrane ( Immobilon ) .", "The primary antibody for adaptin was a rabbit polyclonal anti-APA-2 ( Sato et al . , 2005 ) at a dilution of 1:500 .", "Primary antibody incubation was done in 5% BSA at 4°C overnight .", "The primary antibody for the anti-tubulin control was 12G10 mouse monoclonal anti-tubulin ( Developmental Studies Hybridoma Bank ) at a dilution of 1:10 , 000 .", "Primary antibody incubation was done in 5% BSA at room temperature for 1 hr .", "Secondary antibodies were anti-rabbit and mouse IgG fragments conjugated with HRP ( GE Healthcare , Pittsburgh , PA ) .", "Secondary incubations were done in 5% BSA at room temperature for 45 min .", "The detection reagent was SuperSignal West Dura ( Thermo Scientific , Waltham , MA ) .", "For anti-GFP western blot , the primary antibody for GFP was mouse monoclonal anti-GFP at a dilution of 1:5000 ( Clontech , Mountain View , CA; Cat . No . 632375 ) .", "Primary antibody incubation was done in 5% sea block blocking buffer ( Pierce , Rockford , IL; prod#37 , 527 ) at 4°C overnight .", "For each genotype , 10–12 L4 worms were singled to plates and were transferred to a fresh plate every 12 hr .", "The transfers stopped when the worm burst ( due to an egg-laying defect such as in AP2 mutants ) or the worm started laying unfertilized oocytes ( such as wild type ) .", "The fertilized embryos from each animal were counted to determine the brood size .", "If the worm was lost during the transfer , the data were discarded .", "apm-2 apa-2 double mutants were survivors from the balanced strain EG6158 +/szT1[lon-2 ( e678 ) ] I; szT1/apm-2 ( e840 ) apa-2 ( ox422 ) X .", "All embryos from the brood size quantification were scored for hatching .", "Hatching was checked after 12 hr .", "Unhatched embryos were marked and checked again after another 12 hr .", "The total dead embryos were divided by the brood size to determine the lethal fraction .", "L1 worms were picked to a plate and checked every 12 hr for the growth until they reached L4 stage .", "If the L4 stage was difficult to score due to the sickness of the worm , the scoring was confirmed 12 hr later to insure the animal had become an adult .", "Worms were immobilized using 2% phenoxypropanol and imaged on a Zeiss Pascal LSM5 confocal microscope using a plan-Neofluar 10× 0 . 3 NA , 20× 0 . 5 NA , 40× 1 . 3 NA oil or Zeiss plan-apochromat 63× 1 . 4NA oil objectives .", "Adult nematodes were prepared in parallel for transmission electron microscopy as previously described ( Hammarlund et al . , 2007 ) .", "To briefly summarize , 10 young adult hermaphrodites were placed into a freezing cup ( 100 µm well of type A specimen carrier ) containing space-filling bacteria , covered with a type B specimen carrier flat side down , and frozen instantaneously in the BAL-TEC HPM 010 ( BAL-TEC , Liechtenstein ) .", "The frozen animals were fixed in the Leica AFS device with 1% osmium tetroxide and 0 . 1% uranyl acetate in anhydrous acetone for 2 days at −90°C and for 38 . 9 more hr with a gradual increase in temperature ( 5 °C/hr to −20°C over 14 hr , constant temperature at −20°C for 16 hr , and 10 °C/hr to 20°C over 4 hr ) .", "The fixed animals were embedded in epon-araldite resin following the infiltration series ( 30% epon-araldite/acetone for 4 hr , 70% epon-araldite/acetone for 5 hr , 90% epon-araldite/acetone overnight , and pure epon-araldite for 8 hr ) .", "Mutant and control blocks were blinded .", "Ribbons of ultra-thin ( 33 nm ) serial sections were collected using an Ultracut six microtome at the level of the anterior reflex of the gonad .", "Images were obtained on a Hitachi H-7100 electron microscope using a Gatan digital camera .", "Two hundred and fifty ultra-thin , contiguous sections were cut , and the ventral nerve cord was reconstructed from two animals representing each genotype .", "Image analysis was performed using Image J software .", "The numbers of synaptic vesicles ( ∼30 nm ) , dense-core vesicles ( ∼40 nm ) and large vesicles ( >40 nm ) in each synapse were counted .", "Their distances from the presynaptic specialization and the plasma membrane , as well as their diameters , were measured in acetylcholine neurons VA and VB and the GABA neuron VD .", "A synapse ‘profile' is defined as a single section that passes through the dense projection at a neuromuscular junction .", "Profiles are used for quantifying synaptic vesicle numbers at synapses ( in fact , it uses a section that passes through the middle of the synapse as a representative section of the synapse ) .", "A ‘synapse' encompasses adjacent serial sections containing a dense projection ( usually four sections ) .", "Sections on either side of that density were also included if they contained synaptic vesicle numbers above the average number of synaptic vesicles per profile .", "‘Synapse' reconstructions are used for quantifying the presence of large vesicles or vacuoles associated with the dense projection; since there is usually only one such structure per synapse , partial reconstructions of the synapse are required to reliably identify these structures .", "Two-tailed Student's t-test was used for vesicle numbers and Mann-Whitney U test was used for vesicle diameters .", "For synaptic modeling , we aligned 10 consecutive sections of an acetylcholine neuron from apm-2 apa-2 double mutant using an imageJ plugin called TrakEM2 ( Cardona et al . , 2012 , http://www . ncbi . nlm . nih . gov/pubmed/22723842 ) .", "Plasma membranes , large vacuole membranes , and dense projections were traced using a paint brush tool .", "Synaptic vesicles and large vesicles are created using a ‘ball' tool .", "The size of each vesicle is set by its diameter .", "The reconstructed volume was displayed in the 3D viewer .", "The plasma membrane and vacuole membrane were smoothed multiple times .", "The transparency of synaptic vesicles was set to 20% .", "C . elegans were grown at room temperature ( 22–24 °C ) on agar plates with a layer of OP50 Escherichia coli .", "Adult hermaphrodite animals were used for electrophysiological analysis .", "Postsynaptic currents ( mPSCs and ePSCs ) at the NMJ were recorded as previously described ( Richmond et al . , 1999; Liu et al . , 2007 ) .", "To recapitulate , an animal was immobilized on a sylgard-coated glass coverslip by applying a cyanoacrylate adhesive along the dorsal side .", "A longitudinal incision was made in the dorsolateral region .", "After clearing the viscera , the cuticle flap was folded back and glued to the coverslip , exposing the ventral nerve cord and two adjacent muscle quadrants .", "A Zeiss Axioskop microscope equipped with a 40× water immersion lens and 15× eyepieces were used for viewing the preparation .", "Borosilicate glass pipettes with a tip resistance of 3–5 MΩ were used as electrodes for voltage clamping .", "The classical whole-cell configuration was obtained by rupturing the patch membrane of a gigaohm seal formed between the recording electrode and a body wall muscle cell .", "The cell was voltage-clamped at –60 mV to record mPSCs and ePSCs .", "ePSCs were evoked by applying a 0 . 5 ms square wave current pulse at a supramaximal voltage ( 25 V ) through a stimulation electrode placed in close apposition to the ventral nerve cord .", "Postsynaptic currents were amplified with a Heka EP10 amplifier ( InstruTECH ) and acquired with Patchmaster software ( HEKA ) .", "Data were sampled at a rate of 10 kHz after filtering at 2 kHz .", "The recording pipette solution contained the following ( in mM ) : 120 KCl , 20 KOH , 5 TES , 0 . 25 CaCl2 , 4 MgCl2 , 36 sucrose , 5 EGTA , and 4 Na2ATP .", "The pH was adjusted to 7 . 2 with KOH , and the osmolarity was 310–320 mOsm .", "The standard external solution included the following ( in mM ) : 150 NaCl , 5 KCl , 5 CaCl2 , 1 MgCl2 , 5 sucrose , 10 glucose and 15 HEPES , with the pH adjusted to 7 . 35 using NaOH and an osmolarity of 330–340 mOsm .", "The amplitude and frequency of mPSCs were analyzed using MiniAnalysis ( Synaptosoft , Decatur , GA ) .", "A detection threshold of 10 pA was used in initial automatic analysis , followed by visual inspections to include missed events ( ≥5 pA ) and to exclude false events resulting from baseline fluctuations .", "Amplitudes of ePSCs were measured with Fitmaster ( HEKA ) .", "The amplitude of the largest peak of ePSCs from each experiment was used for statistical analysis .", "Data were imported into Origin , version 7 . 5 ( OriginLab , Northampton , MA ) , for graphing and statistical analysis .", "An unpaired t test was used for statistical comparisons .", "A value of p<0 . 05 is considered statistically significant .", "All values are expressed as the mean ± the SEM n is the number of worms from which recordings were taken .", "A single worm was placed into a 50 μl drop of M9 solution .", "The worm was allowed to adapt to the liquid environment for 2 min .", "The number of body bends was counted for 60 s for each genotype ( n = 5 ) .", "A single body bend is considered a complete left to right and back to left bend .", "Two-tailed Student's t test was used for the statistics .", "Ten L4 stage worms were imaged for each genotype .", "The built-in measure function of LSM image browser ( Zeiss ) was used for the body-length quantification .", "Two-tailed Student's t test was used for the statistics .", "For fluorescence images , the figure panels were assembled as a single image then inverted and contrast adjusted evenly for better visualization .", "All individual images within the panel were treated identically .", "All nerve ring images were exported as 12-bit RGB files .", "ImageJ 1 . 43u was used for quantification .", "The region of interest of fixed size was placed over the center of the nerve ring and fluorescence quantified .", "A region outside of the worm was used to quantify background fluorescence and the value was subtracted from the fluorescence image .", "All worm nerve cord images were exported as 8-bit RGB files and ImageJ 1 . 43u was used for quantification .", "The region of interest was drawn by hand .", "The total pixel intensity and the total number of pixels were recorded to calculate the average fluorescence intensity at both synaptic regions and axonal regions .", "Each image gives a ratio of fluorescence intensity between synapses and axons .", "n refers to the number of images used for quantification ." ] ]

[ "The clathrin adaptor complex AP2 is thought to be an obligate heterotetramer .", "We identify null mutations in the α subunit of AP2 in the nematode Caenorhabditis elegans .", "α-adaptin mutants are viable and the remaining μ2/β hemicomplex retains some function .", "Conversely , in μ2 mutants , the alpha/sigma2 hemicomplex is localized and is partially functional .", "α-μ2 double mutants disrupt both halves of the complex and are lethal .", "The lethality can be rescued by expression of AP2 components in the skin , which allowed us to evaluate the requirement for AP2 subunits at synapses .", "Mutations in either α or μ2 subunits alone reduce the number of synaptic vesicles by about 30%; however , simultaneous loss of both α and μ2 subunits leads to a 70% reduction in synaptic vesicles and the presence of large vacuoles .", "These data suggest that AP2 may function as two partially independent hemicomplexes ." ]

[ "The cell membrane is a busy place , with cell-surface proteins continually added and removed according to the needs of the cell .", "Each protein extends a polypeptide tail into the cell cytoplasm .", "When a protein is to be removed from the cell surface , its tail recruits a protein complex known as the AP2 adaptor to the membrane .", "AP2 then recruits a coat protein called clathrin , which forms a spherical scaffold around the adaptor , the target protein and the surrounding membrane , enclosing them inside a vesicle that breaks off from the membrane and enters the cell .", "Endocytosis is particularly common in neurons , which use it as a means of recycling proteins at synapses—the contact points between nerve cells .", "However , it is unclear whether synaptic-vesicle recycling also involves clathrin and AP2 .", "To address this question , Gu et al . examined mutant nematode worms ( C . elegans ) in which the composition of AP2 had been altered .", "AP2 has four subunits , called α , β2 , μ2 and σ2 , and Gu et al . produced worms that lack either the α- or μ2-subunit , or both .", "Few worms that lacked both subunits survived .", "Surprisingly , however , worms that lacked just one subunit were viable , despite previous evidence that AP2 requires all four subunits to be functional .", "Nevertheless , these single mutants produced 30% fewer synaptic vesicles compared to wild-type worms .", "To examine the consequences of both subunits being absent , Gu et al . rescued the double mutants by selectively expressing AP2 in their skin .", "These animals—which still lack AP2 in their nervous systems—produced 70% fewer synaptic vesicles than their wild-type counterparts .", "The results show that AP2 does not need all four of its subunits and that it can exist as two semi-independent hemicomplexes .", "Moreover , Gu et al . show that C . elegans uses at least two endocytotic mechanisms ( AP2-dependent and independent ) to recycle vesicles and so maintain synaptic function ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "cell biology" ]

[ [ "During the development of the central nervous system , neuronal progenitor cells proliferate , migrate , and finally differentiate into functional units to form a multi-cellular neuronal network ( Ayala et al . , 2007 ) .", "In culture , differentiation of individual neurons occurs in a stereotypic pattern starting with the formation of an axon , followed by the creation of an elaborate dendritic tree and culminating with the initiation and maturation of trans-neuronal synaptic contacts ( Dotti et al . , 1988 ) .", "The formation of synaptic connections is often facilitated by dynamic exploratory filopodia that extend out of thicker dendritic branches to sample the environment and thereby increase the probability that selective pre-to-postsynaptic connections are established ( Ziv and Smith , 1996; Marrs et al . , 2001 ) .", "Exploratory filopodia are dynamic finger-like membrane structures containing actin cables formed out of actin patches along the dendritic shaft ( Lau et al . , 1999; Matus , 2000 ) .", "Extension of filopodia is driven by local activation of formins , Ena/VASP proteins , small GTPases , and likely other steps ( Krugmann et al . , 2001; Lebrand et al . , 2004 ) .", "Intriguingly , the frequency of filopodia formation dramatically drops once high synapse density is established ( Ziv and Smith , 1996 ) , suggesting that the initiation of these structures is controlled by opposing negative regulators .", "However , the identity of these inhibitors has remained elusive .", "Here , we provide evidence that ArhGAP44 limits the initiation of exploratory dendritic filopodia .", "Consistent with previous reports , we observe that formation of actin patches precedes filopodia extension .", "Notably , we find that within actin patches Myosin II-mediated pulling on plasma membrane ( PM ) -associated actin cables induces highly curved membrane sections that trigger ArhGAP44 recruitment .", "The resulting enrichment of ArhGAP44 then reduces local actin polymerization due to the Rac GAP activity of ArhGAP44 , preventing the formation of filopodia .", "ArhGAP44 expression increases as the neuronal network is established and the frequency of exploratory filopodia formation is diminished , suggesting that ArhGAP44 may facilitate the transition of neurons from a dynamic exploratory mode to a mature more static state , a hallmark of nervous system development ." ], [ "Formation of filopodia depends on proteins that regulate polymerization of actin filaments ( Krugmann et al . , 2001; Lebrand et al . , 2004 ) .", "To identify new regulators of exploratory dendritic filopodia formation , we performed a literature search and identified 286 genes that were previously associated either directly or indirectly with actin reorganization .", "We then clustered these genes according to expression pattern using published microarray data and found 89 of the 286 genes to be expressed predominantly in neuronal tissues ( Figure 1—figure supplement 1A and ‘Materials and methods’ and Supplementary file 1 ) .", "As we were interested in regulators of actin dynamics selective for the brain , we ranked these 89 genes for high expression in the brain compared to the spinal cord and tested the validity of the ranking using a set of control genes expressed only in one of the respective tissues ( Figure 1—figure supplement 1B and Table 1 and ‘Materials and methods’ ) .", "Among the five actin regulators with the highest brain vs spinal cord ratio , we found ArhGAP44 ( also known as Rich2 or Nadrin2 ) , a membrane-curvature-sensing GTPase Activating Protein ( GAP ) selective for the small Rho GTPases Rac1 and Cdc42 ( Richnau and Aspenstrom , 2001 ) .", "In previous studies , ArhGAP44 has been associated with the maintenance of apical microvilli in polarized epithelial cells as well as postsynaptic maturation and vesicle release ( Rollason et al . , 2009; Nahm et al . , 2010; Raynaud et al . , 2013 , 2014 ) .", "Considering the role of Rho GTPases during filopodia formation ( Krugmann et al . , 2001 ) , we decided to further investigate the role of ArhGAP44 in developing neurons . 10 . 7554/eLife . 03116 . 003Table 1 . Reference genes expressed predominantly in the adult brain or in the spinal cordDOI: http://dx . doi . org/10 . 7554/eLife . 03116 . 003GeneAdult brainSpinal cordRatioReferencePMP22*720 . 552516 . 350 . 286347 ( Snipes et al . , 1992 ) MPZ*5 . 5181 . 50 . 030303 ( Su et al . , 1993 ) BSN†56 . 73 . 4516 . 43478 ( tom Dieck et al . , 1998 ) GRIA2†355 . 111 . 5530 . 74459 ( Martin et al . , 1993 ) *enriched in the spinal cord .", "†enriched in the adult brain .", "To validate the microarray expression pattern of ArhGAP44 , we first isolated various organs and brain regions and probed protein levels with an antibody directed against ArhGAP44 .", "Consistent with previous work ( Richnau and Aspenstrom , 2001 ) , western blot analysis showed expression of ArhGAP44 in the brain while being below detection level in all other tested organs ( Figure 1A and Figure 1—figure supplement 2A ) .", "Within the brain , immunoblotting directed against ArhGAP44 showed increased protein levels in the frontal cortex and olfactory bulb ( Figure 1B ) .", "The same expression pattern was found in sagittal brain sections stained against ArhGAP44 ( Figure 1—figure supplement 2B ) . 10 . 7554/eLife . 03116 . 004Figure 1 . The brain-enriched ArhGAP44 regulates exploratory dendritic filopodia formation .", "( A ) Microarray data for ArhGAP44 across 74 tissue samples show predominant expression in neuronal tissues ( red box ) .", "Immunoblot to the right shows ArhGAP44 expressed in the brain while being below detection level in other tissues .", "Note that individual tissue samples are likely composed of a variety of different cell types .", "( B ) Total extracts of individual brain regions probed with an antibody directed against ArhGAP44 ( top ) and tubulin ( bottom ) .", "( C ) Expression of ArhGAP44 in cultured hippocampal neurons .", "Total extracts of individual neuronal samples were isolated at DIV3 , DIV10 , and DIV17 and probed with an antibody directed against ArhGAP44 ( top ) , the synaptic proteins PSD95 and Bassoon ( middle lanes ) , and tubulin ( bottom ) .", "( D ) Scanning electron micrographs of cultured neurons .", "Dendritic surface structures are classified based on morphology as convoluted nodes ( red ) , elongated protrusion without contact ( light gray ) , or stubby protrusions that contact adjacent neurites ( dark gray ) .", "Analysis is shown for DIV3 ( n = 10 neurons , 2 independent experiments ) , DIV10 ( n = 10 neurons , 2 independent experiments ) , and DIV17 ( n = 9 neurons , 2 independent experiments ) .", "( E ) Overexpression of ArhGAP44 decreases filopodia density .", "Representative examples of neurons ( green ) stained with anti-MAP2 antibody ( red ) are shown .", "Analysis of filopodia density upon overexpression of control ( black; n = 67 neurons , 3 independent experiments ) , ArhGAP44 ( wt ) ( dark blue; n = 73 neurons , 3 independent experiments ) , and mutant ArhGAP44 ( R291M ) ( light blue; n = 53 neurons , 3 independent experiments ) is shown below .", "( F ) Rac-GAP activity of individual ArhGAP44 mutants .", "Note that ArhGAP44 ( R291M ) shows higher GTP-Rac1 hydrolysis than control .", "( G ) Knockdown of ArhGAP44 increases filopodia density .", "Analysis of filopodia density upon expression of control ( black; n = 67 neurons , 3 independent experiments ) , control siRNA ( gray; n = 67 neurons , 3 independent experiments ) , and knockdown of ArhGAP44 ( siRNA #1 , light yellow; n = 83 neurons; siRNA #2 , dark yellow; n = 85 neurons; both 3 independent experiments ) is shown below .", "( H ) Control western blot analysis testing the effectiveness of individual siRNA pools .", "Scale bars ( D ) , 1 µm; ( E and F ) , 20 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 03116 . 00410 . 7554/eLife . 03116 . 005Figure 1—figure supplement 1 . Cluster analysis of putative actin-regulating genes .", "( A ) Identification of neuron-enriched putative regulators of actin dynamics .", "286 genes identified in a NCBI data-search ( see ‘Materials and methods’ ) were clustered according to expression pattern across 74 different tissue samples .", "A group of 89 genes enriched in neuronal tissue is highlighted in red .", "( B ) ArhGAP44 is enriched in the whole brain but not the spinal cord .", "The 89 genes identified to be enriched in neuronal tissues were sorted according to their relative whole brain vs . spinal cord expression ratio .", "Reference genes ( Table", "1 ) expressed selectively in Schwann cells ( green ) or the brain ( red ) is shown below the ranked list . DOI: http://dx . doi . org/10 . 7554/eLife . 03116 . 00510 . 7554/eLife . 03116 . 006Figure 1—figure supplement 2 . Ponceau loading control of various tissues and ArhGAP44 protein expression in the brain .", "( A ) Loading control of tissues used in Figure 1A .", "Same amounts of total protein were loaded into the different lanes .", "Note the non-uniform protein composition .", "( B ) ArhGAP44 is enriched in the cerebral cortex and olfactory bulb .", "Adult rat brain was sectioned and stained with DAPI ( blue ) and an antibody directed against ArhGAP44 ( yellow ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 03116 . 00610 . 7554/eLife . 03116 . 007Figure 1—figure supplement 3 . ArhGAP44 expression increases over time .", "( A ) ArhGAP44 is expressed in the adult but not the fetal human brain .", "The 89 genes previously identified to be enriched in neuronal tissues were sorted for strong expression in the adult brain vs . fetal brain .", "Reference genes ( Table", "2 ) expressed during neuronal migration ( green ) , trans-synaptic contact formation ( yellow ) , and synapse maturation ( red ) are shown along the x-axis .", "Note that ArhGAP44 ( red ) is expressed 20-fold higher in the adult compared to the fetal brain .", "( B ) Expression levels of ArhGAP44 in the prefrontal cortex increase with age .", "Individual samples from rat prefrontal cortex were fixed at the age of 6 , 18 , 24 , 34 , and 39 months and stained against ArhGAP44 .", "Scale bar ( B ) , 20 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 03116 . 00710 . 7554/eLife . 03116 . 008Figure 1—figure supplement 4 . Neuronal complexity increases over time . Scanning Electron Micrographs of neurons fixed at DIV3 ( n = 10 images , 2 independent experiments ) , DIV10 ( n = 10 images , 2 independent experiments ) , and DIV17 ( n = 9 images , 2 independent experiments ) as well as quantification of total dendrite length are shown .", "Scale bar , 20 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 03116 . 00810 . 7554/eLife . 03116 . 009Figure 1—figure supplement 5 . Electron micrographs of neurons .", "( A ) Dendritic surface analysis .", "Neurons were imaged at low resolution and individual neurons were identified .", "The proximal 100 µm of dendritic arbors were then imaged at high resolution ( boxes #1–#7 ) and surface structures in high resolution images were then grouped as nodes , elongated protrusions , and stubby protrusions .", "Note in image #6 ( red box ) protrusion emerging from the dendritic surface .", "( B ) Examples of nodes that form on the dendritic surface .", "Scanning electron micrographs of neurons that were fixed on DIV11 .", "Individual nodes are highlighted in yellow .", "Scale bar , 500 nm . DOI: http://dx . doi . org/10 . 7554/eLife . 03116 . 00910 . 7554/eLife . 03116 . 010Figure 1—figure supplement 6 . Overexpression phenotypes of ArhGAP44 in cultured neurons . Cells were transfected with control plasmid , ArhGAP44 ( wt ) and ArhGAP44 ( 291 ) , respectively , and cell morphology was assessed 12 hr ( n = 161 neurons , 3 independent experiments ) , 24 hr ( n = 112 neurons , 3 independent experiments ) , and 48 hr ( n = 160 neurons , 3 independent experiments ) after transfection .", "Neurons were classified based on cell morphology showing no effect ( white ) , reduced protrusion density ( red ) , and varicosity formation ( black ) .", "Scale bar , 10 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 03116 . 010 We then explored the expression of ArhGAP44 in the brain during development and found ArhGAP44 among the genes with the highest adult-to-fetal ratio ( Figure 1—figure supplement 3 , and Table 2 and ‘Materials and methods’ ) .", "To validate the observed increase in ArhGAP44 expression with age , we measured protein levels in rat primary hippocampal neurons by western blot , using samples isolated during neurite extension ( 3 days in vitro , i . e . , DIV3 ) , the peak of exploratory filopodia formation ( DIV10 ) , and after initial synaptic contacts were formed ( DIV17 ) .", "Consistent with the micro-array data , ArhGAP44 protein levels increased with time ( Figure 1C ) . 10 . 7554/eLife . 03116 . 011Table 2 . Reference genes expressed predominantly in the adult or in the fetal brainDOI: http://dx . doi . org/10 . 7554/eLife . 03116 . 011GeneAdult brainFetal brainRatioReferenceRELN*34 . 8593 . 350 . 373326 ( D'Arcangelo et al . , 1995 ) DCX*10 . 5525770 . 004094 ( des Portes et al . , 1998 ) NRXN1*72 . 1143 . 150 . 503667 ( Ushkaryov et al . , 1992 ) NLGN1*7 . 524 . 70 . 303644 ( Ichtchenko et al . , 1995 ) CAMK2B†818 . 15321 . 252 . 54677 ( Omkumar et al . , 1996 ) MUNC13†26 . 39 . 752 . 697436 ( Betz et al . , 1998 ) MECP2†818 . 15321 . 252 . 54677 ( Amir et al . , 1999 ) PSD95†362 . 112 . 728 . 51181 ( Kornau et al . , 1995 ) PACSIN1†90 . 820 . 14 . 517413 ( Qualmann et al . , 1999 ) *enriched in the fetal brain .", "†enriched in the adult brain .", "As N-BAR domains present in ArhGAP44 and other proteins can bind to inward-curved plasma membranes ( Galic et al . , 2012 ) , we used high-resolution Field Emission Scanning Electron Microscopy to examine potential changes to local neuronal membrane curvature during maturation .", "As expected , we observed an increase in overall neuronal complexity with time ( Figure 1—figure supplement 4 ) .", "High magnification micrographs further showed convoluted membrane sections ( i . e . , convoluted nodes ) along the dendrite at DIV10 that may reflect such local regions of high curvature ( Figure 1D and Figure 1—figure supplement 5 ) .", "To determine the function of ArhGAP44 in neurons , we transfected primary rat hippocampal neurons .", "Compared to control cells , overexpression of ArhGAP44 caused a significant reduction in the density of dendritic filopodia at DIV12 ( Figure 1E , dark blue ) .", "Prolonged expression increased the fraction of cells forming varicosities , likely due to increased RhoGAP activity associated with elevated ArhGAP44 levels ( Figure 1—figure supplement 6 ) .", "To decrease the GAP activity of ArhGAP44 , we substituted a conserved arginine in the catalytic cleft with a methionine , which was shown to reduce but not eliminate the enzymatic activity of GAP proteins ( Muller et al . , 1997; Graham et al . , 1999 ) .", "Consistent with partial activity , expression of the ArhGAP44 ( R291M ) mutant reduced but did not abolish GTP hydrolysis of the small GTPase Rac1 ( Figure 1F ) , showed a milder loss in filopodia density ( Figure 1E , light blue ) , and delayed the onset of varicosity formation ( Figure 1—figure supplement 6 ) .", "To further validate the observed effect on filopodia density , neurons were transfected with siRNA directed against ArhGAP44 .", "Markedly , knockdown of ArhGAP44 augmented the density of dendritic filopodia ( Figure 1G , yellow ) .", "Control experiments using a second siRNA pool directed against a different region of ArhGAP44 mRNA confirmed the phenotype , thus showing that siRNA knockdown and overexpression of ArhGAP44 have opposing effects .", "Both siRNAs were effective since protein levels of ArhGAP44 were significantly reduced in cells co-transfected with either one of the siRNA pools directed against ArhGAP44 ( Figure 1H ) .", "As dendritic filopodia frequently extend , reorient , and collapse ( Ziv and Smith , 1996 ) , filopodia density reflects the product of de novo formation frequency and stabilization rate .", "To determine which of these parameters are controlled by ArhGAP44 , we performed time-lapse imaging of dendritic filopodia dynamics ( Figure 2A and Figure 2—figure supplement 1 ) .", "Compared to control , neither knockdown of ArhGAP44 nor expression of ArhGAP44 ( R291M ) showed significant changes in the density of static protrusions that persisted longer than 10 min ( Figure 2B and Figure 2—figure supplement 2A and Video", "1 ) In contrast , expression of ArhGAP44 ( R291M ) reduced ( Figure 2B , blue and Video 2 ) , whereas knockdown of ArhGAP44 increased ( Figure 2B , yellow and Video 3 and Video 4 ) the formation of dynamic protrusions .", "Most of these newly formed protrusions were short-lived , while stabilization of extending protrusions or collapse of previously static protrusions was observed only infrequently ( Figure 2B and Figure 2—figure supplement 2B ) .", "Together , these results argue that ArhGAP44 primarily limits filopodia formation with little effect on the stabilization of existing filopodia . 10 . 7554/eLife . 03116 . 012Figure 2 . Knockdown of ArhGAP44 and overexpression of Rac both increase de novo filopodia formation .", "( A ) Color-coded overlay of image-series .", "Note increased protrusion dynamics upon knockdown of ArhGAP44 .", "( B and C )", "ArhGAP44 negatively regulates de novo protrusions .", "Analysis of protrusion dynamics in neurons transfected with control ( black; n = 85 protrusions , 10 neurons , 3 independent experiments ) , ArhGAP44 ( R291M ) ( blue; n = 106 protrusions , 11 neurons , 3 independent experiments ) , and upon knockdown of ArhGAP44 ( yellow; siRNA #1 = 131 protrusions , 12 neurons , 3 independent experiments; siRNA #2 = 215 protrusions , 15 neurons , 3 independent experiments ) .", "Compared to controls , overexpression of ArhGAP44 ( R291M ) reduces while knockdown of ArhGAP44 increases formation of transient protrusion ( B ) .", "Both increase node formation ( C ) .", "( D ) Color-coded overlay of image-series .", "Note increased protrusion dynamics upon overexpression of Rac1 .", "( E and F )", "Co-expression of Rac1 reverses ArhGAP44-dependent reduction in protrusion dynamics .", "Compared to control ( black; n = 85 protrusions , 10 neurons , 3 independent experiments ) , overexpression of Rac1 ( red , n = 123 protrusions , 9 neurons , 3 independent experiments ) increases the formation of transient protrusion ( E ) and nodes ( F ) .", "For both parameters , co-expression of Rac1 with ArhGAP44 ( R291M ) ( purple , n = 126 protrusions , 12 neurons , 3 independent experiments ) can compensate for the reduction observed for ArhGAP44 ( R291M ) alone ( blue , n = 106 protrusions , 11 neurons , 3 independent experiments ) .", "Scale bars ( A and D ) , 1 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 03116 . 01210 . 7554/eLife . 03116 . 013Figure 2—figure supplement 1 . Filopodia analysis tool and GTPase overexpression control . Analysis method used to measure filopodia kinetics .", "A set of images ( left column ) was binarized ( middle column ) and individual images were super-imposed to create a color-coded overlay of the image-series ( right image ) .", "Scale bar , 1 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 03116 . 01310 . 7554/eLife . 03116 . 014Figure 2—figure supplement 2 . Examples of prototypic dendritic protrusions .", "( A ) Protrusions that persist throughout the acquisition interval were considered static .", "( B ) Dynamic protrusions were divided into three groups: protrusions that extend and retract during acquisition ( top panel ) ; protrusions that extend and remain extended for at least 3 min ( middle panel ) ; and protrusions that persisted for at least 3 min before collapsing ( bottom panel ) .", "Scale bars ( A and B ) , 2 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 03116 . 01410 . 7554/eLife . 03116 . 015Figure 2—figure supplement 3 . Examples of dendritic nodes .", "( A ) Nodes are identified as thickened regions in the dendritic shaft .", "A section of the dendrite with a node but no filopodia ( red arrow ) and a node with a filopodium ( white arrow ) are shown enlarged .", "( B ) Dendritic nodes ( red arrow ) are often observed without a subsequent extension of a filopodia .", "Scale bars ( A ) , 20 µm; ( B ) , 2 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 03116 . 01510 . 7554/eLife . 03116 . 016Figure 2—figure supplement 4 . Acquisition interval of 60 s is sufficient to detect dynamic protrusions but not all nodes .", "( A ) Time lapse of a dendritic branch from which a protrusion ( red ) emerges and collapses .", "Note that not all nodes ( yellow arrows ) can be identified when using a 60 s interval ( e . g . , compare columns 2 and 4 ) .", "( B ) The majority of protrusions emerge from nodes .", "Quantification of percentage of filopodia that emerge from nodes using acquisition intervals of 60 s ( black; 45 nodes form 11 neurons , 3 independent experiments ) and 15 s ( red; 75 nodes form 12 neurons , 3 independent experiments ) , respectively .", "Note that the fraction of protrusions emerging from nodes increases with shorter intervals as nodes can be short-lived .", "Scale bar ( A ) , 2 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 03116 . 01610 . 7554/eLife . 03116 . 017Figure 2—figure supplement 5 . The ArhGAP44 knockdown phenotype is phenocopied by the small GTPase Rac1 but not Cdc42 . ( A ) ArhGAP44 is a RhoGAP with dual selectivity for Rac1 and Cdc42 .", "GTPase pull-down assay show hydrolysis of GTP-Cdc42 ( green ) and GTP-Rac1 ( red ) by ArhGAP44 .", "( B ) Overexpression of Rac1 but not of Cdc42 increases protrusion density .", "Neurons were transfected with the wild type form of human Rac1 and Cdc42 .", "Note that only Rac1 ( red ) increased protrusion density .", "( C ) Rac1 but not Cdc42 reverse ArhGAP44 ( R291M ) -dependent slow-down in protrusion dynamics .", "Neurons were transfected either with a control plasmid ( black , n = 18 neurons , 3 independent experiments ) , Cdc42 ( green , n = 15 neurons , 3 independent experiments ) , Rac1 ( red , n = 13 neurons , 3 independent experiments ) , ArhGAP44 ( R291M ) ( blue , n = 18 neurons , 3 independent experiments ) , or co-transfected with ArhGAP44 ( R291M ) together with Cdc42 ( green-blue , n = 11 neurons , 3 independent experiments ) or Rac1 ( violet , n = 13 neurons , 3 independent experiments ) .", "( D ) Overexpression of Rac1 but not of Cdc42 triggered node formation observed upon knockdown of ArhGAP44 .", "Time-lapse images of neurons co-transfected with a siRNA directed against ArhGAP44 together with a fluorescence marker ( top ) , expressing wild-type Rac1 ( second panel ) , expressing wild-type Cdc42 ( third panel ) , and expressing control plasmid ( bottom ) .", "Note that ectopic nodes are formed from which filopodia emerge upon knockdown of ArhGAP44 and overexpression of Rac1 but not after expression of Cdc42 .", "Scale bars ( D , left panels ) , 20 µm; ( D , right panels ) , 2 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 03116 . 01710 . 7554/eLife . 03116 . 018Video 1 . Example of a stable dendritic protrusion . Neuron was transfected with a fluorescence marker at DIV11 and imaged 24 hr later .", "Individual frames were taken every 60 s .", "Scale bar is 2 μm .", "Video is 720× real-time . DOI: http://dx . doi . org/10 . 7554/eLife . 03116 . 01810 . 7554/eLife . 03116 . 019Video 2 . ArhGAP44 ( R291M ) transfection causes increased dendritic node and reduced protrusion formation . Neuron was transfected with ArhGAP44 ( R291M ) at DIV11 and imaged 24 hr later .", "Individual frames were taken every 60 s .", "Scale bar is 2 μm .", "Video is 360× real-time . DOI: http://dx . doi . org/10 . 7554/eLife . 03116 . 01910 . 7554/eLife . 03116 . 020Video 3 . Knockdown of ArhGAP44 increases dendritic node and protrusion formation . Neuron was co-transfected with diced RNA directed against ArhGAP44 and a cytosolic reference at DIV7 and imaged at DIV12 .", "Individual frames were taken every 60 s .", "Scale bar is 2 μm .", "Video is 360× real-time . DOI: http://dx . doi . org/10 . 7554/eLife . 03116 . 02010 . 7554/eLife . 03116 . 021Video 4 . Knockdown of ArhGAP44 increases dendritic node formation . Neuron was co-transfected with diced RNA directed against ArhGAP44 and a cytosolic reference at DIV7 and imaged at DIV12 .", "Individual frames were taken every 60 s .", "Scale bar is 2 μm .", "Video is 360× real-time . DOI: http://dx . doi . org/10 . 7554/eLife . 03116 . 021 For both , overexpression and knockdown of ArhGAP44 , we further observed an increase in the number of transiently formed nodes along dendritic arbors ( Figure 2C and Figure 2—figure supplement 3 and Videos 2–5 ) .", "Intriguingly , we find that the majority ( 83% ± 7% ) of de novo protrusions extended from such node-like structures ( Figure 2—figure supplement 4 and Video 6 ) .", "We thus consider these nodal structures to represent nascent filopodia sites , where filopodia formation is either initiated or aborted .", "Given their spacing and their presence at the same time in culture , they likely correlate with the convoluted nodes along dendrites seen in electron microscopy studies ( Figure 1D ) . 10 . 7554/eLife . 03116 . 022Video 5 . Example of dendritic node formation . Neuron was transfected with a fluorescence marker at DIV11 and imaged 24hr later .", "Individual frames were taken every 60 s .", "Scale bar is 2 μm .", "Video is 360× real-time . DOI: http://dx . doi . org/10 . 7554/eLife . 03116 . 02210 . 7554/eLife . 03116 . 023Video 6 . Knockdown of ArhGAP44 triggers formation of protrusion from dendritic nodes . Neuron was co-transfected with RNA directed against ArhGAP44 and a cytosolic reference at DIV7 and imaged at DIV12 .", "Individual frames were taken every 60 s .", "Scale bar is 2 μm .", "Video is 360× real-time . DOI: http://dx . doi . org/10 . 7554/eLife . 03116 . 023 Consistent with previous reports ( Richnau and Aspenstrom , 2001 ) , we find that the GAP domain of ArhGAP44 inhibits GTPase activity of Rac and Cdc42 ( Figure 2—figure supplement 5A ) .", "We thus aimed to investigate to which extent the two possible targets of ArhGAP44 contribute to filopodia formation .", "We find that overexpression of wild-type Rac ( human Rac1 ) but not Cdc42 phenocopied protrusion density ( Figure 2—figure supplement 5B ) as well as protrusion kinetics ( Figure 2—figure supplement 5C and Videos 7–10 ) observed upon knockdown of ArhGAP44 .", "Similar to knockdown of ArhGAP44 , Rac1 overexpression increased protrusion dynamics and node formation ( Figure 2D–F and Figure 2—figure supplement 5D and Videos 7 , 8 ) .", "These observations are consistent with previous work showing Rac activity associated with increased actin patch formation and filopodia dynamics in axons ( Spillane et al . , 2012 ) and dendrites ( Korobova and Svitkina , 2010; Cheadle and Biederer , 2012 ) .", "We thus considered that ArhGAP44 might act by inhibiting Rac to limit initiation of exploratory filopodia formation . 10 . 7554/eLife . 03116 . 024Video 7 . Rac1 overexpression causes abnormal dendritic node and protrusion formation . Neuron was transfected with Rac1 ( wt ) at DIV11 and imaged 24 hr later .", "Individual frames were taken every 60 s .", "Scale bar is 2 μm .", "Video is 360× real-time . DOI: http://dx . doi . org/10 . 7554/eLife . 03116 . 02410 . 7554/eLife . 03116 . 025Video 8 . Rac1 overexpression causes abnormal dendritic node and protrusion formation . Neuron was transfected with Rac1 ( wt ) at DIV11 and imaged 24 hr later .", "Individual frames were taken every 60 s .", "Scale bar is 2 μm .", "Video is 360× real-time . DOI: http://dx . doi . org/10 . 7554/eLife . 03116 . 02510 . 7554/eLife . 03116 . 026Video 9 . Cdc42 overexpression causes abnormal dendritic protrusion formation . Neuron was transfected with Cdc42 ( wt ) at DIV11 and imaged 24 hr later .", "Note the elongation of a preexisting dendritic protrusion .", "Individual frames were taken every 60 s .", "Scale bar is 2 μm .", "Video is 360× real-time . DOI: http://dx . doi . org/10 . 7554/eLife . 03116 . 02610 . 7554/eLife . 03116 . 027Video 10 . Cdc42 overexpression causes abnormal dendritic protrusion formation . Neuron was transfected with Cdc42 ( wt ) at DIV11 and imaged 24 hr later .", "Note the elongated filopodia emerging from the dendrite .", "Individual frames were taken every 60 s .", "Scale bar is 2 μm .", "Video is 360× real-time . DOI: http://dx . doi . org/10 . 7554/eLife . 03116 . 027 To test this hypothesis , we performed a synthetic compensation experiment , co-expressing Rac1 with ArhGAP44 ( R291M ) .", "Although the resulting protrusions were shorter than control filopodia , co-expression of ArhGAP44 ( R291M ) together with Rac1 reversed the observed decrease in the frequency of protrusion formation and node formation caused by expression of ArhGAP44 ( R291M ) ( Figure 2E , F purple and Video 11 ) . 10 . 7554/eLife . 03116 . 028Video 11 . Rac1 synthetically rescues ArhGAP44 ( R291M ) -dependent reduction in protrusion formation . Neuron was co-transfected with Rac1 ( wt ) and ArhGAP44 ( R291M ) at DIV11 and imaged 24 hr later .", "Individual frames were taken every 60 s .", "Scale bar is 2 μm .", "Video is 360× real-time . DOI: http://dx . doi . org/10 . 7554/eLife . 03116 . 028 To analyze the subcellular protein localization , we cultured hippocampal neurons and stained against endogenous ArhGAP44 .", "We found ArhGAP44 to be absent from the nucleus and present in patches along dendrites ( Figure 3—figure supplements 1 and 2A ) .", "We then expressed fluorescently tagged ArhGAP44 in cultured neurons .", "Like the endogenous protein , fluorescently tagged ArhGAP44 was excluded from the nucleus , distributed uniformly through the cytosol , and formed distinct ArhGAP44 patches along dendrites ( Figure 3—figure supplement 2B , C ) , arguing that the fluorescent tag did not interfere with its localization .", "To investigate the molecular mechanism that caused ArhGAP44 patch formation , we compared the subcellular localization of various deletion mutants of ArhGAP44 ( Figure 3A ) .", "ArhGAP44 contains an N-BAR domain that has been reported to bind to positively ( i . e . , inward ) curved lipid membranes ( Peter et al . , 2004 ) .", "Full-length protein and the isolated N-BAR domain of ArhGAP44 both enriched in patches along dendritic arbors , while deletion of the amino-terminal amphipathic helix ( ΔN-BAR ) , a critical sequence motif for binding and stabilization of curved membranes ( Peter et al . , 2004 ) , showed no enrichment over a cytosolic reference ( Figure 3B ) .", "Intriguingly , none of the ArhGAP44 constructs enriched in extended filopodia ( Figure 3C ) .", "Time-lapse imaging showed that filopodia often emerged from ArhGAP44 patches ( Figure 3D ) .", "We considered that ArhGAP44 patches might reflect the convoluted , node-like dendritic membrane sections we previously observed in electron micrographs ( Figure 1D ) and fluorescence images ( Figure 2—figure supplement 3A ) .", "Consistently , measurement of individual membrane folds within nodes ( Figure 3—figure supplement 2D ) showed sufficient deformation to trigger curvature-dependent protein recruitment to the plasma membrane ( Bhatia et al . , 2009 ) .", "Together with the previous overexpression and knockdown experiments ( Figure 2A , B ) , the transient localization of ArhGAP44 to patches but not to extended filopodia argues that ArhGAP44 limits initiation rather than elongation of newly formed filopodia . 10 . 7554/eLife . 03116 . 029Figure 3 . ArhGAP44 recruitment to dendritic nodes precedes filopodia extension .", "( A ) ArhGAP44 protein structure and deletion mutants .", "ArhGAP44 is composed of an amino-terminal curvature-sensing N-BAR domain ( AA 1–254 ) , followed by the RhoGAP domain ( AA 255–445 ) and a stretch of ∼350 amino acids ( AA 445–818 ) with no annotated domain structure .", "Full length ( left , dark red ) , the isolated N-BAR domain ( middle , red ) , and the N-BAR domain lacking the initial 18 amino acids ( right , white ) were tested .", "( B ) Enrichment of full length ( dark red; 25 patches , 12 neurons , 3 independent experiments ) and the isolated N-BAR domain of ArhGAP44 ( red , n = 34 patches , 11 neurons , 3 independent experiments ) in dendritic patches .", "No enrichment is observed for the N-BAR domain lacking the first 18AA that encode an amphipathic helix critical for curvature sensing ( white; 20 patches , 11 neurons , 3 independent experiments ) .", "Note , ArhGAP44 ( R291M ) was used to reduce the compromised cell health caused by overexpression of equal levels of active wild-type protein .", "( C ) No enrichment of full length ( dark red; 26 filopodia , 14 neurons , 3 independent experiments ) , the isolated N-BAR domain of ArhGAP44 ( red , n = 40 filopodia , 13 neurons , 3 independent experiments ) , or the N-BAR domain lacking the first 18AA ( white; 22 patches , 10 neurons , 3 independent experiments ) in dendritic filopodia .", "( D ) Filopodia emerge from ArhGAP44-rich patches .", "Scale bars ( B , C , D ) , 1 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 03116 . 02910 . 7554/eLife . 03116 . 030Figure 3—figure supplement 1 . ArhGAP44 antibody controls .", "( A ) Western blot of whole rat brain with antibody directed against ArhGAP44 ( 818AA ) ( unspecific bands are visible ) .", "( B ) Increase immunostaining directed against ArhGAP44 after overexpression of ArhGAP44 .", "Primary hippocampal neurons were transfected with a fluorescently tagged ArhGAP44 ( R291M ) and fixed 24 hr later .", "Quantification of fluorescence intensity of non-transfected neurons ( black , n = 20 cells , 2 independent experiments ) and neurons transfected with ArhGAP44 ( red , n = 12 cells , 2 independent experiments ) after staining with an antibody directed against ArhGAP44 is shown below .", "( C ) Control experiment showing reduced immunostaining of ArhGAP44 after knockdown of ArhGAP44 .", "Primary hippocampal neurons were transfected at DIV10 with diced RNA directed against ArhGAP44 and a fluorescence reference ( empty pEYFP plasmid ) and fixed 48 hr later .", "Relative fluorescence intensity was measuerd in dendritic stretches and normalized to the adjacent background ( arrows ) .", "Quantification of fluorescence intensity after staining with an antibody directed against ArhGAP44 of cells transfected with control siRNA ( black , n = 16 cells , 2 independent experiments ) and siRNA directed against ArhGAP44 ( red , n = 18 cells , 2 independent experiments ) is shown below .", "Scale bars ( B and C ) , 20 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 03116 . 03010 . 7554/eLife . 03116 . 031Figure 3—figure supplement 2 . ArhGAP44 localization in neurons .", "( A ) Immunostaining directed against endogenous ArhGAP44 in cultured neurons ( top ) .", "Primary hippocampal neurons were cultured for 11 days and stained with antibodies directed against ArhGAP44 .", "ArhGAP44 is absent from the nucleus and enriched in distinct patches along the dendritic shaft ( red arrows ) .", "( B ) Overexpressed ArhGAP44 is absent from the nucleus and enriched at distinct patches along the dendritic arbors ( bottom ) .", "Primary hippocampal neurons were transfected with ArhGAP44 ( R291M ) ( red ) and a cytosolic reference ( green ) at DIV10 , and imaged 24 hr after transfection .", "ArhGAP44 ( R291M ) is absent from the nucleus ( bottom panels ) , but present at distinct patches along the dendrite ( top panels , red arrow ) .", "( C ) Overexpression of the isolated N-BAR domain of ArhGAP44 for 48 hr causes bright protein aggregates that are distinct from ArhGAP44 patches .", "Colocalization analysis ( right panels ) indicates that expression of ArhGAP44 for extended time causes formation of bright protein aggregates ( yellow , n = 24 aggregates , 9 neurons , 2 independent experiments ) that do not co-localize with actin patches ( white , 34 actin patches form 25 neurons; 3 independent experiments ) .", "( D ) Nodes that form along the dendrite surface display highly curved membrane sections .", "Average diameter of individual membrane ruffles in nodes is shown next to it .", "Scale bars ( A and B ) , 20 µm; ( C ) , 2 µm; ( D ) , 500 nm . DOI: http://dx . doi . org/10 . 7554/eLife . 03116 . 031 Next , we aimed to investigate what caused the convoluted membrane surface at dendritic nodes .", "Consistent with previous reports ( Lau et al . , 1999; Spillane et al . , 2012 ) , ratio-imaging of the filamentous actin marker f-tractin ( Johnson and Schell , 2009 ) to a cytosolic reference showed formation of actin patches that preceded extension of exploratory filopodia ( Figure 4—figure supplement 1A and Video 12 ) .", "This was the case in 89% ± 6% of all filopodia ( Figure 4—figure supplement 1B ) .", "To directly test whether actin patches cause convoluted node-like PM subsections ( Figure 3—figure supplement 2D ) , we performed correlative light and electron microscopy ( Figure 4—figure supplement 2 ) .", "Consistent with a role of actin in node-formation , we find that actin intensity in nodes was significantly higher compared to adjacent dendritic sections ( Figure 4A ) .", "We then expressed f-tractin together with the isolated N-BAR domain of ArhGAP44 and found significant enrichment of f-tractin in ArhGAP44 patches ( Figure 4B and Figure 4—figure supplement 3 ) , arguing that ArhGAP44 and actin localize to the same structure .", "Consistently , the N-BAR domain of ArhGAP44 enriched in dendritic actin-patches by 80% ± 8% compared to adjacent dendritic regions ( Figure 4—figure supplement 3B ) .", "To test for Myosin II-dependent contractile forces in ArhGAP44 patches , we co-expressed the isolated N-BAR domain of ArhGAP44 and non-muscle Myosin Heavy Chain IIB ( NMHC-2B ) and found a significant enrichment of NMHC-2B in ArhGAP44 patches ( Figure 4C ) .", "Together , this suggests that myosin-dependent contraction of actin patches triggers inward membrane deformations within nodes to which ArhGAP44 is enriched .", "Consistently , we find increased staining for the phosphorylated form of myosin light chain ( pMLC ) , a marker for active Myosin II ( Tan et al . , 1992 ) , in dendritic actin patches ( Figure 4D ) . 10 . 7554/eLife . 03116 . 032Video 12 . Actin enriches at patches that precede exploratory filopodia initiation . Neurons were transfected with a marker for filamentous actin ( f-tractin , red ) and a cytosolic reference ( green ) .", "Note the formation of actin patches prior to filopodia formation ( white boxes ) .", "Individual frames were taken every 5 s .", "Video is 50× real-time . DOI: http://dx . doi . org/10 . 7554/eLife . 03116 . 03210 . 7554/eLife . 03116 . 033Figure 4 . ArhGAP44 is recruited to convoluted dendritic PM sections enriched in polymerized actin and myosin .", "( A ) Correlative fluorescence and scanning electron microscopy shows actin enrichment in convoluted nodes that form along dendritic arbors .", "Quantification of the relative fluorescent intensity of phalloidin in individual nodes compared to adjacent dendritic sections is shown to the right .", "( B ) F-tractin and ArhGAP44 co-localize .", "Neuron was transfected with the N-BAR domain of ArhGAP44 ( red ) , f-tractin ( blue ) , and a cytosolic reference ( green ) .", "A magnified section ( white box ) and quantification of relative f-tractin intensity in ArhGAP44 patches are shown next to it ( 34 patches form 25 neurons; 3 independent experiments ) .", "( C ) NMHC-2B and ArhGAP44 co-localize .", "Neurons were transfected with the N-BAR domain of ArhGAP44 ( red ) , NMHC-2B ( blue ) , and a cytosolic reference ( green ) .", "Quantification of relative NMHC-2B intensity in ArhGAP44 patches is shown next to it ( n = 20 patches form 13 neurons; 3 independent experiments ) .", "( D ) Phosphorylated regulatory myosin light chain ( green ) is enriched in dendritic actin patches ( red ) .", "Scale bars ( A , C , D ) , 5 µm; ( B ) , 10 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 03116 . 03310 . 7554/eLife . 03116 . 034Figure 4—figure supplement 1 . Time-lapse of exploratory dendritic filopodia emerging from actin patches .", "( A ) Neurons were transfected at DIV10 with the filamentous actin marker f-tractin ( red ) together with a cytosolic reference ( green ) and imaged 24 hr later .", "Red arrows indicate relative accumulation of f-tractin compared to a cytosolic reference at nodes that form before filopodia initiation and persist after filopodia collapse .", "( B ) Filopodia emerge from pre-existing actin patches .", "Quantification using an acquisition interval of 15 s show that 89% ± 6% of filopodia emerge from actin patches ( 24 patches form 10 neurons; 3 independent experiments ) .", "Scale bar ( A ) , 1 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 03116 . 03410 . 7554/eLife . 03116 . 035Figure 4—figure supplement 2 . Workflow to identify individual neurons for correlative SEM/IF microscopy . Example of pattern used for navigation on the glass slide is shown at the top .", "Gold deposit can be detected on the SEM using the back-scatter or inlens detector as well as in immunofluorescence .", "Scale bar , 10 mm . DOI: http://dx . doi . org/10 . 7554/eLife . 03116 . 03510 . 7554/eLife . 03116 . 036Figure 4—figure supplement 3 . ArhGAP44 is enriched at actin patches .", "( A ) Neurons were fixed and stained with an antibody directed against ArhGAP44 ( red ) and phalloidin ( green ) .", "Note that the antibody directed against ArhGAP44 is enriched in phalloidin-rich patches .", "( B ) The isolated N-BAR domain of ArhGAP44 is enriched in dendritic actin-patches .", "Neurons were transfected with the isolated N-BAR domain of ArhGAP44 , a marker for filamentous actin ( f-tractin ) and a cytosolic reference .", "The average enrichment of the isolated N-BAR domain of ArhGAP44 over a cytosolic reference in dendritic actin patches is 80% ± 8% ( 34 patches form 25 neurons; 3 independent experiments ) .", "Scale bar , 2 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 03116 . 036 To test the hypothesis that ArhGAP44 enrichment relies on acto-myosin-dependent pulling forces to the PM , we altered either actin integrity or myosin-dependent actin contraction .", "Notably , both treatments led to a significant reduction of ArhGAP44 concentration in actin patches ( Figure 5A , B and Figure 5—figure supplement 1 ) , suggesting that Myosin II-dependent forces within dendritic actin patches trigger local inward deformation of the PM , mediating a recruitment of curvature-sensing protein ArhGAP44 .", "Notably , we find ArhGAP44 to be also enriched at inward plasma membrane deformation created by retracting lamellipodia ( Figure 5—figure supplement 2 and Video 13 ) , as well as in response to membrane bending by artificial nanocone structures ( Figure 5—figure supplements 3 , 4; and Videos 14 , 15 ) , arguing that inward membrane deformation is sufficient for binding of ArhGAP44 to the plasma membrane . 10 . 7554/eLife . 03116 . 037Figure 5 . Myosin dependent recruitment of the N-BAR domain of ArhGAP44 to actin patches .", "( A ) Inhibition of acto-myosin dependent forces decreases relative concentration of the N-BAR domain of ArhGAP44 in actin patches .", "Neurons were transfected with the N-BAR domain of ArhGAP44 ( red ) , f-tractin ( blue ) , and a cytosolic reference ( green ) .", "Neuron is shown before ( left panels ) and after addition of the MLCK inhibitor ML-7 ( right panels ) .", "( B ) Cumulative distribution and average values of the relative intensity of the N-BAR domain of ArhGAP44 to a cytosolic reference in actin patches are shown before ( black ) and after ( red ) addition of CytoD ( n = 49 patches; 12 cells , 3 independent experiments ) , LatA , ( n = 27 patches; 10 cells , 3 independent experiments ) , ML-7 ( n = 118 patches; 15 cells , 3 independent experiments ) , or the vehicle DMSO ( n = 62 patches; 12 cells , 3 independent experiments ) .", "Scale bar , 5 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 03116 . 03710 . 7554/eLife . 03116 . 038Figure 5—figure supplement 1 . Inhibition of actin polymerization decreases relative concentration of the N-BAR domain of ArhGAP44 but does not completely dissolve actin patches . Neurons were transfected with the N-BAR domain of ArhGAP44 ( red ) , f-tractin ( blue ) , and a cytosolic reference ( green ) , and imaged 24 hr later .", "Note that actin patches persist treatment with CytoD ( red arrow ) .", "Scale bar , 5 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 03116 . 03810 . 7554/eLife . 03116 . 039Figure 5—figure supplement 2 . ArhGAP44 is recruited to collapsing artificial lamellipodia in neurons .", "( A ) Rapamycin-induced dimerization assay .", "Neurons were quadruple-transfected with a membrane-anchored FRB , a fluorescently labeled FKBP that was associated with the Rac-GEF TIAM , a cytosolic reference , and the fluorescently labelled N-BAR domain of ArhGAP44 .", "Addition of rapamycin triggered dimerization of FRB and FKBP , which led to a rapid translocation of TIAM to the plasma membrane .", "Enrichment of TIAM at the plasma membrane augmented local Rac activity and actin dynamics .", "( B ) Neurons quadruple-transfected with the constructs before and after addition of rapamycin .", "Note the formation of ectopic actin-rich structures .", "( C ) Time lapse images show enrichment of the N-BAR domain of ArhGAP44 at retracting actin-rich structures .", "Cells were quadruple-transfected with Lyn-FRB , CFP-FKBP-TIAM , YFP-ArhGAP44 ( N-BAR ) , and the empty mCherry plasmid as a cytosolic reference .", "Note the enrichment of the N-BAR domain of ArhGAP44 at the collapsing lamellipodia .", "( D ) Kymograph of ArhGAP44 ( N-BAR ) ( top panel ) and the cytosolic reference mCherry ( middle panel ) show relative enrichment of the N-BAR domain of ArhGAP44 at a retracting actin-rich structures ( bottom panel ) .", "( B ) , 10 µm; ( C ) , 2 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 03116 . 03910 . 7554/eLife . 03116 . 040Figure 5—figure supplement 3 . Artificial membrane deformations recruit ArhGAP44 to the plasma membrane .", "( A ) Inward membrane deformation by acto-myosin-dependent contraction of membrane-associated actin cables ( top panel ) is mimicked by artificially applied external forces via cone-shaped nanostructures ( bottom panel ) .", "( B ) Brightfield image of glass slide where cone-shaped nanostructures ( i . e . , nanocones ) were deposited in 3-µm wide stripes .", "Nanocones are depicted as small triangular structures at the bottom of the image .", "( C ) Atomic force microscope images of the surface of such cone-shapes nanostructures .", "( D and E )", "Control experiment showing cells cultured on stripes of nanocones transfected with a cytosolic marker ( D ) and with a membrane marker ( E ) .", "( F ) Control experiments testing for correlation between ArhGAP44 ( R291M ) and membrane ( CFP-CAAX ) puncta over individual nanocones .", "Since positions and amplitudes of the respective puncta show low correlation , an increase in total membrane cannot account for the formation of YFP-ArhGAP44 ( R291M ) puncta ( G ) Control experiments testing for a possible local actin polymerization induced by nanocones .", "Cells expressing the actin marker Ruby-LifeAct ( left ) together with CFP-ArhGAP44 ( R291M ) ( right ) over nanocones .", "Individual LifeAct and ArhGAP44 ( R291M ) puncta show no significant correlation .", "This is consistent with the hypothesis that nanocone-induced membrane deformation and not binding to local actin structures is responsible for the observed N-BAR translocation .", "( H ) Control experiment testing for co-localization of ArhGAP44 ( R291M ) with itself .", "Scale bars ( B , D , E ) , 3 µm; ( C ) , 5 µm; ( F–H ) , 1 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 03116 . 04010 . 7554/eLife . 03116 . 041Figure 5—figure supplement 4 . N-BAR domain of ArhGAP44 enriched at nanocone-induced dendritic plasma membrane deformations in neurons .", "( A and B )", "The curvature-sensitive N-BAR domain of ArhGAP44 is enriched to nanocone-induced membrane deformation at the basal membrane .", "Neurons were cultured on a glass-slide patterned with nanocones and transfected with the isolated N-BAR domain of ArhGAP44 .", "Note in ( B ) that puncta are present only on the dendritic stretch to the left that is in contact with nanocones but not in the section of the dendrite to the right that is not touching the nanomaterial .", "( C ) Neurons cultured on a glass-slide patterned with stripes of nanocones and transfected with the isolated N-BAR domain of ArhGAP44 .", "Quantification of N-BAR puncta on glass ( black , n = 17 neurons , 2 independent experiments ) and on nanocones ( red , n = neurons , 2 independent experiments ) is shown below .", "Scale bars ( A and B ) , 10 µm; ( C ) , 50 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 03116 . 04110 . 7554/eLife . 03116 . 042Figure 5—figure supplement 5 . ArhGAP44 is enriched at dendritic tips . Primary hippocampal neurons were transfected with a fluorescently tagged N-BAR domain of ArhGAP44 ( red ) , a marker for filamentous actin ( blue ) , and a cytosolic reference ( green ) at DIV6 and imaged 24 hr later .", "Red arrows depict dendritic tips .", "Quantification of fluorescence intensity show a relative enrichment of the isolated N-BAR domain over a cytosolic reference at actin-rich structures at the tip of dendrites ( n = 14 dendritic tips from 10 neurons; 2 independent experiments ) .", "Scale bar , 5 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 03116 . 04210 . 7554/eLife . 03116 . 043Figure 5—figure supplement 6 . ArhGAP44 is enriched at dendritic spines . Primary hippocampal neurons were transfected with fluorescently tagged ArhGAP44 ( red ) , a marker for filamentous actin ( blue ) , and a cytosolic reference ( green ) at DIV16 and imaged 24 hr later .", "Quantification of fluorescence intensity show a relative enrichment of the full-length ArhGAP44 ( R291M ) ( dark red ) and for the isolated N-BAR domain of ArhGAP44 ( red ) over a cytosolic reference at spine-shaped , actin-rich dendritic structures ( n = 21 structures form 13 neurons for ArhGAP44 ( R291 ) and 24 structures from 12 neurons for the N-BAR domain of ArhGAP44; both from 2 independent experiments ) .", "Scale bar , 1 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 03116 . 04310 . 7554/eLife . 03116 . 044Figure 5—figure supplement 7 . Knockdown of ArhGAP44 alters protrusion morphology and dynamics in aged neurons .", "( A ) Increased filopodia dynamics after knockdown of ArhGAP44 .", "Primary hippocampal neurons were transfected at DIV14 with diced RNA directed against ArhGAP44 ( or a control ) and a fluorescence marker and imaged 72 hr later .", "Note the increase in node and filopodia formation ( red arrows ) upon knockdown of ArhGAP44 .", "( B ) Reduction of spine-shaped protrusions upon knockdown of ArhGAP44 ( n = control , 18 neurons; siRNA ArhGAP44 ( diced pool #1 ) , 20 neurons; siRNA ArhGAP44 ( diced pool #2 ) , 23 neurons; both from 2 independent experiments ) .", "Scale bar , 2 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 03116 . 04410 . 7554/eLife . 03116 . 045Video 13 . Enrichment of the N-BAR domain of ArhGAP44 at retracting actin-rich structures in neurons . Neuron was transfected with Lyn-FRB , CFP-FKBP-Tiam1 ( blue ) , the N-BAR domain of ArhGAP44 ( red ) , and a cytosolic reference ( green ) at DIV11 .", "Images show formation and retraction of artificial lamelipodia-like structures that formed along the dendritic shaft upon rapamycin-triggered recruitment of the Rac GEF Tiam1 to the plasma membrane .", "Individual frames were taken every 15 s .", "Scale bar is 2 μm .", "Video is 120× real-time . DOI: http://dx . doi . org/10 . 7554/eLife . 03116 . 04510 . 7554/eLife . 03116 . 046Video 14 . Enrichment of the N-BAR domain of ArhGAP44 at basal membrane sections indented by nanocones . 3D rotation of neuron plated on nanocone-coated glass slide and transfected with the N-BAR domain of ArhGAP44 .", "Note the punctate enrichment at the basal membrane below the soma .", "Scale bar is 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 03116 . 04610 . 7554/eLife . 03116 . 047Video 15 . Enrichment of the N-BAR domain of ArhGAP44 at the dendritic section that is in contact with nanocones . 3D rotation of neuron plated on nanocone-coated glass slide and transfected with the N-BAR domain of ArhGAP44 .", "Note the punctate enrichment at the top where the dendrite is touching the nanocone surface .", "Scale bar is 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 03116 . 047 Finally , we investigated whether ArhGAP44 is also enriched to other actin-rich structures in neurons .", "We find the N-BAR domain enriched at the end of dendrites ( dendritic tips; Figure 5—figure supplement 5 ) , as well as in spine-shaped dendritic protrusions ( Figure 5—figure supplement 6 ) .", "Knockdown of ArhGAP44 in aged neurons facilitates re-emergence of exploratory dendritic protrusions ( Figure 5—figure supplement 7 ) .", "Thus , considering that ArhGAP44 expression increases with time , this suggests that ArhGAP44 may facilitate the transition of neurons from a dynamic exploratory mode to a mature more static state ." ], [ "Our study shows that recruitment of ArhGAP44 to actin patches , which seed exploratory filopodia along dendritic branches , is mediated by Myosin II-dependent contraction of membrane-associated actin cables .", "These actin patches have been shown to serve as precursors for the formation of filopodia in axons ( Lau et al . , 1999; Spillane et al . , 2012 ) and dendrites ( Korobova and Svitkina , 2010 ) .", "Studies in non-neuronal cells showed that individual actin filaments within the actin cortex form bundles prior to filopodia elongation ( Svitkina et al . , 2003 ) .", "Consistently , electron micrographs of neurons showed that these filopodial actin bundles are embedded in the underlying dendritic actin meshwork ( Korobova and Svitkina , 2010 ) .", "It has been proposed that once enough actin filaments are bundled to generate the force required to protrude the PM , the resulting outward membrane deformation triggers recruitment of actin bundling/Cdc42 activating proteins , which then further increase the polymerization rate within the extending filopodia ( Krugmann et al . , 2001; Disanza et al . , 2006 ) .", "Given the localization of Myosin II in actin patches ( Figure 4D ) , it is reasonable to conjecture that Myosin II-dependent contraction of individual actin filaments within actin patches provides structural integrity to counter the force generated by the extending filopodia .", "However , considering that individual actin filaments within a bundle are oriented with the barbed end to the PM , Myosin II-dependent contraction also exerts an inward directed pull forces that curve the PM inward which triggers increased recruitment of ArhGAP44 .", "We propose that the highly convoluted membrane topography associated with actin patches ( Figure 4A ) reflect such Myosin II-dependent contraction of membrane-associated actin cables , at which ArhGAP44 becomes enriched in a curvature-dependent manner .", "In support of this hypothesis , we find not only that inward membrane deformation is sufficient for ArhGAP44 and N-BAR domain recruitment ( Figure 5—figure supplements 3 and 4 ) , but that deletion of ArhGAP44 curvature-sensitivity ( Figure 3A , B ) or reducing action-myosin-dependent contractile forces ( Figure 5A ) both prevented ArhGAP44 enrichment .", "A second major finding of our study is that recruitment of ArhGAP44 to plasma membrane deformations in nodes ( i . e . , acto-myosin patches ) limits initiation of exploratory filopodia .", "We show that ArhGAP44 can hydrolize the small GTPase Rac and Cdc42 ( Figure 2—figure supplement 5 ) .", "Thus , a likely function of local ArhGAP44 at actin patches is to suppress GTPase-mediated local actin polymerization .", "We propose that ArhGAP44 is limiting Rac-dependent formation of actin patches , which provide the structural integrity required for filopodia to protrude outwards ( Figure 6 ) .", "This is consistent with previous reports in neurons , showing that dynamic rearrangements within actin patches relies on Rac activity ( Andersen et al . , 2005; Spillane et al . , 2012 ) , and that elevated Rac levels increase filopodia dynamics ( Luo et al . , 1996; Nakayama et al . , 2000; Zhang and Macara , 2006; Cheadle and Biederer , 2012 ) .", "In support of this notion , we find Rac1 localized at actin patches ( Figure 6—figure supplement 1A ) and show that artificial decrease of ArhGAP44 concentration at actin patches either by reducing overall ArhGAP44 levels by knockdown ( Figure 1F ) or by preventing ArhGAP44-recruitment to actin patches via inhibition of acto-myosin contraction ( Figure 6—figure supplement 1B , see also [Ryu et al . , 2006; Hodges et al . , 2011] ) both increased the number of exploratory filopodia . 10 . 7554/eLife . 03116 . 048Figure 6 . Proposed model of ArhGAP44-dependent regulation of exploratory dendritic filopodia initiation . Scanning electron micrographs of dendritic protrusions with and without filopodial protrusions aligned in a hypothetical time-line .", "Proposed model that myosin-dependent contractions in actin patches triggers local plasma membrane indentations ( i . e . , node formation ) with curved membranes to which ArhGAP44 is recruited ( frame 4–6 , red ) .", "Increased local ArhGAP44 concentration limits Rac1-dependent actin polymerization and weakens or dissociates the node .", "Low ArhGAP44 concentration at nodes allows filopodia initiation that relies on Cdc42 and other factors ( frame 7–11 , gray ) .", "Scale bar , 500 nm . DOI: http://dx . doi . org/10 . 7554/eLife . 03116 . 04810 . 7554/eLife . 03116 . 049Figure 6—figure supplement 1 . Control experiments supporting the proposed model .", "( A ) Rac1 is enriched in dendritic actin patches .", "Neurons were fixed at DIV12 and stained with an antibody directed against Rac1 ( green ) and phalloidin ( red ) .", "( B ) Inhibition of myosin light chain kinase increases filopodia density .", "Filopodia density on neurons was measured before ( black ) and 120 min after ( red ) the addition of the MLCK inhibitor ML-7 ( n = 21 neurons , 3 independent experiments ) .", "Scale bars ( A and B ) , 10 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 03116 . 049 Cdc42 acts as an activator of Irsp53 ( Kast et al . , 2014 ) , promoting IRSp53-dependent enrichment and clustering of VASP and other factors to drive actin assembly in elongating filopodia ( Disanza et al . , 2013 ) .", "Consistently , knockdown of Cdc42 substantially reduces filopodia formation in neurons ( Garvalov et al . , 2007 ) .", "Intriguingly , overexpression of Cdc42 is not sufficient to initiate filopodia formation in neurons ( Figure 2—figure supplement 5 , see also [Hotulainen et al . , 2009] ) or in other cell lines ( Kast et al . , 2014 ) .", "This has led to the hypothesis that elongation of filopodia is a combinatorial process requiring multiple factors ( Kast et al . , 2014 ) .", "We propose that signal integration at actin patches controls this decision of filopodia elongation .", "Considering that actin-patch formation occurs before filopodia elongation , this argues for a 2-step process where Rac1-induced patch formation ( and ArhGAP44-dependent regulation thereof ) precedes Cdc42-induced filopodia elongation ( Figure 6 ) .", "However , since ArhGAP44 shows dual specificity for Rac1 and Cdc42 , both steps will be limited by recruitment by ArhGAP44 to actin patches .", "Taken together , we propose that ArhGAP44 mediates a localized negative feedback that becomes upregulated as neurons mature to reduce the frequency with which neurons initiate new exploratory filopodia .", "Considering that acto-myosin initiated PM deformation is a ubiquitous process and that a high number of curvature-sensing proteins are known to modify actin dynamics , this suggests that the local feedback mechanism for ArhGAP44 described in our study likely exemplifies a more general principle for receptor-independent signaling whereby signal transduction is initiated by the transient recruitment of regulatory proteins to actin- and force-dependent nanoscale PM indentations ." ], [ "A set of 286 genes relating to the actin cytoskeleton and GTPase signaling was identified with a search on the NCBI Gene database with the query ‘actin AND GTPase AND human[orgn]’ .", "The Human U133A/GNF1H Gene Atlas data set ( gnf1h-gcrma unaveraged ) was downloaded from the biogps . org website ( Su et al . , 2004 ) ( only the U133A data were analyzed ) .", "The data were renormalized using the median intensity on each array .", "To generate a focused expression data set for actin- and GTPase-related genes , we extracted the data from all probe sets corresponding to the 286 genes described above .", "The data were then log-transformed , and the mean log-expression for each probe across all tissue types was subtracted to yield relative expression values .", "The values were then hierarchically clustered using the Cluster 3 . 0 software , with the Pearson correlation distance , and average linkage .", "To test the validity of the brain vs spinal cord ranking in Figure 1—figure supplement 1B , we characterized genes known to be expressed exclusively in the brain or in the spinal cord ( Table 1 and [Snipes et al . , 1992; Martin et al . , 1993; Su et al . , 1993; tom Dieck et al . , 1998] ) .", "We found for the Schwann cell-specific genes MPZ and PMP22 an adult brain/spinal cord ratio smaller than 0 . 3 while the brain enriched presynaptic vesicle fusion protein Bassoon ( BSN ) , and the postsynaptic AMPA receptor 2 ( GRIA2 ) both showed an adult brain/spinal cord ratio greater than 15 .", "Of the 89 neuron-enriched genes identified in Figure 1—figure supplement 1B the top five hits were: the RhoGEF Kalirin that was shown to contribute to EphB receptor-dependent spine maturation ( Penzes et al . , 2003 ) , the synaptic vesicle-associated protein Amphiphysin ( David et al . , 1996 ) , the small GTPase K-Ras that translocates from the PM to the Golgi complex , and early/recycling endosomes in response to neuronal activity ( Fivaz and Meyer , 2005 ) , the microtubule tip-tracking protein EB3 that is a modulator of spine morphology ( Jaworski et al . , 2009 ) , and ArhGAP44 .", "To test the validity of the ranking for genes critical for specific steps during neuronal developmental in Figure 1—figure supplement 3A , we characterized where genes known to be involved in various aspects of neuronal maturation ( Table 2 and [Ushkaryov et al . , 1992; D'Arcangelo et al . , 1995; Ichtchenko et al . , 1995; Kornau et al . , 1995; Omkumar et al . , 1996; Betz et al . , 1998; des Portes et al . , 1998; Amir et al . , 1999; Qualmann et al . , 1999] ) .", "We observed a clear separation of DCX and RELN , which are involved in neuronal migration , and NLGN1 and NRXN1 , which initiate trans-synaptic contact , from MUNC13 , MECP2 , PSD95 , and PACSIN1which all contribute to synapse function .", "Of the 89 neuron-enriched genes identified in Figure 1—figure supplement 3A , genes with the highest adult-to-fetal ratio included the microtubule tip-tracking protein EB3 ( Jaworski et al . , 2009 ) , the MAP kinase ERK1 as well as the MAP kinase kinase MEK1 which both control dendrite development ( Crino et al . , 1998 ) and synaptic plasticity ( Dash et al . , 1990 ) , the Armadillo-like protein PKP4 ( Wolf et al . , 2006 ) , the Lowe syndrome protein OCRL ( Attree et al . , 1992 ) , and ArhGAP44 .", "Tissue samples were isolated from female Wistar rat and suspended in ice-cold lysis buffer containing 1% Tween and protease inhibitors ( Roche [Indianapolis , IN] , 11873580001 ) .", "Each sample was homogenized and absolute protein concentration was measured , using the BCA Protein Assay Kit ( Thermo Scientific [Rockford , IL] , 23225 ) , and adjusted to equal levels for each sample .", "Next , 6× SDS was added , and the samples were heated to 90°C for 5 min .", "Finally , the samples were vortexed and loaded on a gel .", "20 µg total protein was loaded for each sample in Figure 1A and probed with an antibody directed against ArhGAP44 ( Sigma-Aldrich [St . Louis , MO] , HPA038814 ) .", "No single protein was used as reference for comparison of ArhGAP44 expression level across organs , as the expression of conventional housekeeping proteins ( e . g . , tubulin or GAPDH ) can vary between tissues by up to an order of magnitude ( Figure 1—figure supplement 2A and http://biogps . org/#goto=genereport&id=37238 ) .", "In Figure 1B 20 µg total protein was loaded and probed with an antibody directed against ArhGAP44 as well as beta-tubulin ( Sigma , T8578 ) as a reference .", "For detection , secondary antibodies from Invitrogen and SuperSignal West Femto Maximum Sensitivity Substrate ( Pierce [Thermo Scientific , Rockford , IL] , 34095 ) were used .", "Neurons were harvested at DIV3 , DIV10 , and DIV17 in ice-cold lysis buffer containing 1% Tween and protease inhibitors ( Roche , 11873580001 ) .", "Absolute protein concentration was immediately measured using the BCA Protein Assay kit ( Thermo 23225 ) and adjusted to equal levels for each time point .", "Relative protein levels were probed using specific antibodies directed against ArhGAP44 ( Abcam [Cambridge , MA] , ab93627 ) , the postsynaptic marker PSD95 ( EMD Millipore [Billerica , MA] , MAB1596 ) , the presynaptic protein Bassoon ( Abcam , 76065 ) , and the loading control beta-tubulin ( Sigma , T8578 ) .", "For detection , we used secondary antibodies from Invitrogen and SuperSignal West Femto Maximum Sensitivity Substrate ( Pierce , 34095 ) .", "Neurons were cultured on Poly-L-Lysine-coated glass coverslips and fixed using 2% Glutaraldehyde ( 8% stock-EM grade ) and 4% p-Formaldehyde in NaCacodylate buffer pH 7 . 4 for 10 min .", "Neurons were rinsed with 0 . 1 M NaCacodylate buffer ( pH 7 . 4 ) after primary fixation and post-fixed for 1 hr with aqueous 1% OsO4 , washed briefly with water and dehydrated in an ascending ethanol series ( 50 , 70 , 90 , and 100% [twice] for 20 min each ) before critical point drying with liquid CO2 in a Tousimis 815B ( Tousimis , Rockville , MD , USA ) .", "Samples were mounted on colloidal Graphite on 15-mm aluminum stubs ( Ted Pella , Redding , CA , USA ) and sputter-coated with 70A of Au/Pd using a Denton Desk 11 Sputter Coater .", "Visualization of samples was performed with a Zeiss Sigma FESEM ( Zeiss Microscopy LLC , Thornwood , NY ) operated at 2–3 kV , working distance 4–6 mm and an in-lens SE detector under high vacuum conditions .", "Images were captured in TIFF format .", "Neurons were cultured on glass slides for various periods of time ( 3 , 10 , and 17 days ) , fixed and prepared for SEM as described above .", "Using low resolution ( 1000× magnification ) , individual neurons were identified ( Figure 1—figure supplement 5A , left panel ) .", "Starting from the soma , initial segments of the dendritic arbors were imaged at high resolution ( 10 , 000× ) , and individual protrusions were classified based on morphology ( Figure 1—figure supplement 5A , right panel ) .", "Only the proximal 50–60 μm of the dendritic arbors that can clearly be associated to a particular neuron were analyzed .", "Examples of dendritic nodes are shown in Figure 1—figure supplement 5B .", "Rat hippocampal neurons were prepared as previously described ( Fink et al . , 2003 ) .", "Neurons were transfected using Lipofectamine 2000 ( Invitrogen , Carlsbad , CA ) according to the manufacturer's protocol .", "For live imaging , neurons were plated in chambers ( Lab-Tek 155383; Thermo , Rockford , IL ) using NBM ( Neurobasal Medium , Gibco [Life Technologies , Carlsbad , CA] , 21103-049 ) , supplemented with SM1 ( StemCell Technologies [Vancouver , Canada] , 05711 ) , Pen/Strep ( Gibco , 15070-063 ) and 20 mM HEPES ( Gibco , 15630 ) .", "For immunostaining , cells were fixed in PBS ( Gibco , 10010-023 ) containing 4% Formaldehyde ( Ted Pella , 18505 ) and 120 mM sucrose , stained , and imaged .", "ArhGAP44 antibody was from Sigma ( 1:150 , HPA038814 ) , MAP2 antibody was from Chemicon ( 1:1000 , AB 5622; Chemicon , EMD Millipore , Billerica , MA ) , pMLC antibody was from Cell Signaling ( 1:200 , 3671S; Cell Signalling Technology , Danvers , MA ) , and Rac1 antibody was from Cytoskeleton ( 1:200 , ARC03; Cytoskeleton , Denver , CO ) .", "All experiments were performed on a spinning disc confocal microscope .", "CFP , YFP , and mCherry excitations were obtained by a 442-nm helium cadmium laser ( 100 mM; Kimmon Electrics , Centennial , CO ) , a 514-nm argon laser ( 300 mW; Melles Griot , Carlsbad , CA ) , and a 594-nm solid-state laser ( 80 mW; CNI Laser , Changchun , China ) , respectively .", "Images were captured using an EMCCD camera ( QuantEM 512SC [Photometrics , Tucson , AZ] ) , driven by Micromanager mounted on the side port of an inverted microscope ( model IX-71; Olympus , Center Valley , PA ) .", "Primary cultured rat hippocampal neurons were transfected 7 days after plating with siRNA directed against ArhGAP44 together with a fluorescent marker and fixed at DIV12 .", "Fluorescently tagged ArhGAP44 ( R291M ) was transfected at DIV11 and fixed at DIV12 .", "For each condition , the sample was fixed and individual neurons were imaged .", "Filopodia density was measured manually , analyzing only the proximal 100 µm of each dendrite .", "For each condition tested , >20 cells were used .", "HeLa cells were grown in DMEM ( high glucose ) supplemented with 10% FCS and Pen/Strep until they reached 80% confluency and then transfected either with CFP-ArhGAP44 ( wt ) , CFP-ArhGAP44 ( R291M ) , or empty CFP plasmid ( control ) using LF2000 according to the manufacturer's protocol for 4 hr in DMEM in the absence of FCS and Pen/Strep .", "Cells were then serum starved for 18 hr .", "For all conditions , live cell fluorescence 18 hr post transfection showed transfection efficiency of >80% .", "Cells were stimulated with 50 ng/ml EGF for 5 min .", "Next , cells were scratched and protein levels were measured and adjusted for all samples to equal levels .", "Of each sample 900 µl were used for pulldown and 100 µl for loading control .", "GTP levels were probed using Rac1 Activation Assay Kit ( Cell Biolabs [San Diego , CA] , STA-404 ) according to the manufacturer's protocol .", "In brief , GTP-bound Rac was eluted from cell lysates using PAK PBD agarose beads and detected by western blot using α-Rac1 ( Cell Biolabs , 240106 ) antibody .", "Relative intensities were compared to loading controls .", "The protocol used to synthesize siRNA has been previously reported ( Liou et al . , 2005 ) .", "Specific primers for ArhGAP44 were automatically designed and used to amplify from a cDNA library an approximate 600-bp PCR fragment of the 3′ region of the coding sequence .", "A second amplification was performed with a set of nested primers bearing a T7 promoter sequence on their 5′ extension .", "Nested PCR products were transcribed in vitro ( T7 MEGA script kit; Ambion , Austin , TX ) and the resulting double-stranded RNAs were annealed and processed with 30 units per reaction of human recombinant Dicer ( Invitrogen ) for 15 hr at 37°C .", "The 21mer siRNAs were separated from incompletely digested fragments using a succession of isopropanol precipitations and filtration on glass fiber plates ( Nunc , Rochester , NY ) .", "Neurons were imaged for 10 min every 60 s using a 63× objective .", "Changes in filopodia dynamics were assessed manually using ImageJ .", "In detail , dynamic protrusions ( i . e . , nodes and dynamic filopodia ) were counted and normalized to filopodia that remained over the course of the acquisition ( i . e . , static filopodia ) .", "Dynamics was visualized using the Temporal Color-Code designed by Kota Miura ( http://fiji . sc/wiki/index . php/Temporal-Color_Code ) .", "The software used for ratio-metric imaging has been previously described ( Tsai and Meyer , 2012 ) .", "In brief , a low-pass Gaussian filter was first applied to all images to suppress the noise while retaining the details of the fluorescent signals .", "Background subtraction was subsequently performed by ( the value of each pixel ) — ( the mean value of the background within 40 μm of that pixel ) .", "To determine relative intracellular ArhGAP44 levels , ratio images were created dividing ArhGAP44 fluorescence over the cytosolic fluorescence .", "To measure the relative protein concentration at patches , average fluorescent intensities of the protein of interest ( POI ) and the cytosolic reference were measured in the patch and in the adjacent dendritic stretch .", "The background ( BG ) was determined separately for both channels using the average of four sectors outside the cell adjacent to the region of interest .", "The relative intensity of POI’s was determined as [ ( POIPatch − POIBG ) / ( CytosolPatch − CytosolBG ) ]/[ ( POIDendrite − POIBG ) / ( CytosolDendrite − CytosolBG ) ] .", "A micro-pattern was generated on glass slides and coated over night with PLL ( 0 . 1 mg/ml ) .", "Neurons were plated for 11 days and then fixed as described above for the FESEM analysis .", "Using the micro-pattern as reference points , individual neurons were then labeled with fluorescently tagged phalloidin and imaged on a 63× objective with a 1 . 5 Optovar .", "Cells were then sputter-coated with 70A of Au/Pd using a Denton Desk 11 Sputter Coater .", "Using the backscatter detector , individual micro-patterns were identified and used to navigate and identify individual previously imaged neurons as shown in Figure 4—figure supplement 2 .", "SEM Images were then taken at 10 , 000× magnification and aligned with the immunofluorescent images .", "Finally , individual nodes were identified using the SEM images , and the average fluorescent intensity of phalloidin was measured in nodes and the adjacent dendritic stretches .", "Full-length and the N-BAR domain constructs of ArhGAP44 , F-tractin , NMHC-2B ( Addgene Plasmid No . 11348 ) , Rac1 and Cdc42 were previously described ( Wei and Adelstein , 2000; Heo and Meyer , 2003; Johnson and Schell , 2009; Galic et al . , 2012 ) .", "The point mutation in ArhGAP44 ( R291M ) was introduced using the site-specific mutagenesis kit ( 200518; Stratagene , Cedar Creek , TX ) .", "All constructs were sequenced prior to use .", "ML-7 ( sc-200557; Santa Cruz Biotechnology ) was used at 10 µM ( Figure 5A ) and 50 µM ( Figure 6—figure supplement 1B ) .", "Latrunculin A ( 428026; Cal Biochem , EMD Millipore , Billerica , MA ) was used at 4 µM .", "Cytochalasin D ( PHZ1063; Invitrogen ) was used at 5 µM .", "For individual neurons , z-stacks were acquired before and 20–30 min after addition of drugs .", "For the analysis , a maximal projection was made and individual actin patches ( = sites along the dendrite where the actin/cytosol ratio was >200% above the average ratio ) were identified .", "Using a mask , the ArhGAP44/cytosol intensity within the actin patches was determined and normalized to ArhGAP44/cytosol ratio in the dendritic shaft .", "Dynamic translocation of Tiam1 with the FRB-FKB system in non-neuronal cells has been previously described ( Inoue et al . , 2005 ) .", "Here , we cultured hippocampal neurons were quadruple-transfected with Lyn-FRB , CFP-FKBP-Tiam1 the YFP-tagged N-BAR domain of ArhGAP44 and the cytosolic reference mCherry using Lipofectamine 2000 ( according to manufacturer's protocol ) .", "24 hr later , individual cells were imaged before and after addition of 100 nM rapamycin ( B0560; Sigma–Aldrich ) with a 63× objective and a 1 . 5× Optovar module .", "Nanocone production has been previously described ( Jeong et al . , 2011; Galic et al . , 2012 ) .", "In brief , a 35–50 nm thin film of tin was deposited by heat evaporation on a glass coverslip at room temperature .", "The glass with the deposited tin was then exposed to a nitrogen gas environment with a low concentration of oxygen ( about 1 part per million ) at 350°C for 90 min .", "The annealing to the glass and the formation of the replicate nanocone shapes occurred during this heating step .", "In order to make the nanocone structures transparent , the glass coverslip with nanocones was further heated to a temperature 400°C for 3 hr in air .", "Labtek chambers were then mounted with nanocoated glass slides as previously described ( Jeong and Galic , 2014 ) , and neurons were subsequently cultured , transfected on DIV10 , and imaged alive 24 hr later .", "3D rotation ( Videos 14 , 15 ) of confocal stacks was done in ImageJ .", "AFM images of nanocones were done in tapping mode using commercial cantilevers on a JPK Nanowizard II instrument .", "Height analysis was performed using JPK image processing software .", "p-values in all figures depict pair-wise comparisons and were evaluated using the Student's t test , with two tails and two-sample unequal variance .", "Error bars in all images represent SEM of the mean value .", "**p < 0 . 01 ." ] ]

[ "In the vertebrate central nervous system , exploratory filopodia transiently form on dendritic branches to sample the neuronal environment and initiate new trans-neuronal contacts .", "While much is known about the molecules that control filopodia extension and subsequent maturation into functional synapses , the mechanisms that regulate initiation of these dynamic , actin-rich structures have remained elusive .", "Here , we find that filopodia initiation is suppressed by recruitment of ArhGAP44 to actin-patches that seed filopodia .", "Recruitment is mediated by binding of a membrane curvature-sensing ArhGAP44 N-BAR domain to plasma membrane sections that were deformed inward by acto-myosin mediated contractile forces .", "A GAP domain in ArhGAP44 triggers local Rac-GTP hydrolysis , thus reducing actin polymerization required for filopodia formation .", "Additionally , ArhGAP44 expression increases during neuronal development , concurrent with a decrease in the rate of filopodia formation .", "Together , our data reveals a local auto-regulatory mechanism that limits initiation of filopodia via protein recruitment to nanoscale membrane deformations ." ]

[ "Our brains contain a vast network of many billions of cells that communicate with , and are connected to , each other .", "Each brain cell , or neuron , can form connections with as many as 10 , 000 other neurons—and signals pass from one neuron to the next at sites known as synapses .", "A neuron's surface has numerous finger-like protrusions known as filopodia that are important for sensing the environment around the cells .", "Filopodia are highly changeable and constantly extend and retract as the filaments that support them—which are made up of a protein called actin—grow and shrink back .", "Neurons use their filopodia to explore and seek out other neurons in the brain , and when they make contact with the right neuron , it leads to the formation of a synapse .", "However , how filopodial extensions begin to grow—and what stops a neuron from forming too many filopodia—is not fully understood .", "Galic et al . now show that a protein called ArhGAP44 limits the formation of new filopodia in neurons .", "The ArhGAP44 protein is recruited to patches of the surface membrane that have a lot of actin and that curve inwards .", "ArhGAP44 then locally inhibits other proteins that are normally required to extend the actin filaments and drive the growth of filopodia out from the surface of the cell .", "Galic et al . also show that more ArhGAP44 is produced with age—levels are low in embryos and high in adults—and this increase in the amount of protein correlates with a decrease in the number of filopodia formed .", "When Galic et al . engineered rat neurons to produce more of the ArhGAP44 protein , fewer filopodia formed on the surface of the neurons .", "Decreasing the amount of this protein had the opposite effect .", "Moreover , ArhGAP44 was shown to mainly stop new filopodia from forming and had little effect on existing filopodia .", "Together , these findings suggest that ArhGAP44 may help neurons transition from a dynamic exploratory mode to a mature , more static , state; this is a characteristic of the development of the nervous system ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "neuroscience" ]

[ [ "Neuronal circuits originating in the hypothalamus direct behavioral responses to match an organism's perception of scarce or excess energy environments ( Morton et al . , 2006 ) .", "For example , scarcity challenges organisms to modulate energy homeostasis , conserving the resources they have and seeking out additional food sources around them .", "A maladaptive relationship between such energy cues and appropriate behavioral responses is a key feature of many eating disorders ( Becker et al . , 1999 ) .", "Homeostatic food intake is also impacted by time of day , with laboratory housed mice normally eating most of their food during their nighttime active phase ( Challet , 2019 ) .", "Conversely , scarcity due to a limited window of food availability ( e . g . a prey species emerges to forage for only a few hours ) is capable of inducing adaptation of this ostensibly circadian feeding circuit .", "The context of this timing information is so significant that regularly recurring cycles of food availability can lead organisms to modify their behavior and physiology , changing their locomotor activity , glucocorticoid levels , and body temperature to better match the predicted time of food availability ( Patton and Mistlberger , 2013 ) .", "A growing body of evidence suggests that desynchronization of feeding relative to the normal circadian time of eating adversely impacts metabolic health ( Challet , 2019; Hatori et al . , 2012; Pan et al . , 2011; Sutton et al . , 2018 ) .", "While many of the peripheral responses induced by caloric scarcity are known ( e . g . elevated glycogenolysis , increased ketone body production ) , there is a significant gap in our understanding of the neural and molecular mechanisms leading to scarcity-associated behaviors .", "In response to time restricted feeding ( TRF ) , mice increase their activity in the time window preceding feeding , a phenomenon known as food anticipatory activity ( FAA ) ( Richter , 1922 ) .", "This is the hypothesized output of a putative food entrainable oscillator ( FEO ) , which functions in a comparable manner for entrainment to food as the suprachiasmatic nucleus ( SCN ) does for entrainment to light ( Stephan , 2002 ) .", "Despite the recognition of FAA , the identification and characterization of the anatomic and molecular correlates of the FEO have remained elusive ( Pendergast and Yamazaki , 2018 ) .", "Recently , it has been hypothesized that the FEO may be anatomically dispersed , with at least one component embedded within hypothalamic circuits to alter feeding behavior in response to peripheral energy status ( Pendergast and Yamazaki , 2018 ) .", "One of the hypothalamic drivers of feeding that has been implicated in FAA are AgRP neurons of the arcuate hypothalamus .", "These cells respond to hunger and satiety factors released from peripheral organs and neighboring neurons to drive feeding and associated behaviors ( Aponte et al . , 2011; Dietrich et al . , 2015; Krashes et al . , 2011 ) .", "Strikingly , neonatal ablation of AgRP neurons leads to diminished FAA , and more prominently so during the daytime ( Tan et al . , 2014 ) .", "The basis for how AgRP neurons alter FAA , and indeed how the FEO can be impacted by time of day , as observed in the AgRP neuron ablated animals ( Tan et al . , 2014 ) , remains unknown .", "Herein , we examined the role of the p75 neurotrophin receptor ( p75NTR , Ngfr ) in modulating behavioral response to homeostatic challenge .", "p75NTR is one of the two receptors for brain derived neurotrophic factor , BDNF , which is known to promote satiety and prevent weight gain through its actions in the hypothalamus ( An et al . , 2015; Liao et al . , 2015; Xu et al . , 2003 ) .", "While genetic deletion of p75NTR does not alter locomotor activity or metabolism under normal conditions ( normal chow , ad lib ) , its expression cycles in a circadian manner in the SCN , with an increase during the rest phase , and it regulates the oscillation of glucose and lipid homeostasis genes in the liver ( Baeza-Raja et al . , 2013 ) .", "Interestingly , metabolically relevant functions of p75NTR have been unmasked when animals are homeostatically challenged .", "Loss of p75NTR improves glucose and insulin regulation during glucose or insulin tolerance tests ( Baeza-Raja et al . , 2012 ) .", "In response to high fat diet this unmasking effect is even more evident , with resistance to diet-induced obesity observed when p75NTR is ablated in adipocytes ( Baeza-Raja et al . , 2016 ) .", "The involvement of neurotrophic factors in hypothalamic feeding circuits , and the identified roles of p75NTR in circadian and metabolic regulation , together suggest that p75NTR may be a candidate to influence food entrainable behaviors such as FAA .", "To better understand the role of p75NTR in feeding behavior we studied the response of Ngfr-KO mice to energy deficiency .", "We found that p75NTR is required to consume normal post-fasting levels of food .", "Unexpectedly , we also find that p75NTR is necessary for circadian expression of daytime , but not nighttime , FAA .", "Furthermore , we show that these effects do not require any developmental role of p75NTR , instead requiring it’s function in AgRP neurons .", "This work documents a novel role of p75NTR in the CNS circuitry controlling feeding behavior , and posits it as a sought after molecular player to elucidate circadian regulation of feeding behavior ." ], [ "To establish whether p75NTR is involved in any gross processes of energy homeostasis , we first examined body weight , food intake , and locomotor activity in ad libitum fed mice harboring germline knockout alleles of p75NTR ( Ngfr-KO ) ( Lee et al . , 1992 ) .", "Ngfr-KO mice have similar body weight to control animals ( Ngfr-WT ) into adulthood , with increased variability as wildtype mice gain weight with age ( Figure 1—figure supplement 1A; Tables 1 and 2 ) , and with no differences in total daily activity ( Figure 1—figure supplement 1B ) .", "Additionally , Ngfr-KO mice increase nighttime food intake by 4% but total daily ad libitum food intake is not significantly different compared to littermate controls ( Figure 1—figure supplement 1C ) .", "In line with previous reports , ad libitum fed Ngfr-KO mice also have similar serum chemistry markers compared to controls , including insulin , leptin and corticosterone , during either the day or night ( Tables 1 and 2; Baeza-Raja et al . , 2012 ) .", "These data suggest that p75NTR does not influence energy homeostasis under ad libitum conditions .", "Previously reported metabolic phenotypes of Ngfr-KO mice have been identified during homeostatic challenges , such as consumption of high fat foods that normally result in diet-induced obesity .", "For example , loss of p75NTR from white adipose depots results in excess lipolysis and resistance to weight gain on an energy dense diet ( Baeza-Raja et al . , 2016 ) .", "We thus sought to examine the role of p75NTR under homeostatic challenge , but with energy deficiency rather than energy excess .", "Weight-matched Ngfr-KO mice and wildtype littermate controls were fasted overnight ( 16 hr ) and then refed for 3 hr during the day ( Figure 1A ) .", "During daytime refeeding , we found that Ngfr-KO mice consumed ~21% less food than wild-type littermate controls ( Figure 1A ) .", "Since mice eat ~85% of their food during the night ( Figure 1—figure supplement 1C ) and p75NTR expression is under circadian control , we repeated the experiments with daytime food deprivation and nighttime refeeding ( Figure 1B ) .", "Unlike daytime refeeding , Ngfr-KO mice consumed similar amounts of food with nighttime refeeding as controls ( Figure 1B ) .", "As mice normally increase their activity during fasting , purportedly as an effort to forage for food , we additionally monitored locomotor activity during the initial 12 hr of food deprivation to assess the activity response of Ngfr-KO mice ( Gelegen et al . , 2006; Jensen et al . , 2013 ) .", "In agreement with previous literature we found that wildtype mice trended towards increased nighttime locomotor activity when they were fasting as compared to their activity during ad libitum feeding ( Figure 1C ) .", "However , we observed that Ngfr-KO mice decreased their fasted nighttime activity relative to controls ( Figure 1C ) .", "In contrast , both wildtype and Ngfr-KO mice decreased their fasted daytime activity ( Figure 1D ) .", "These results suggest that Ngfr-KO mice may have defective responses to hunger that extend beyond feeding behavior .", "We next sought to determine whether the perturbations observed in feeding and locomotor activity reflect changes of peripheral hunger signals that would lead to alterations of central hunger responses .", "To this end , we assessed serum hormone and nutrients in both the daytime and nighttime fasted state .", "We found comparable levels of most of the measured peripheral metabolites between wildtype and Ngfr-KO mice ( Tables 1 and 2 ) , with similar regulation between fed and fasted states .", "Of note , there is a lack of a drop in leptin levels in overnight fasted Ngfr-KO mice , which may contribute towards the observed feeding defect .", "Together with the observed behavioral alterations , these data suggest that p75NTR is necessary for appropriate responses to changes in energy balance , and suggest the feeding and activity responses may be functioning in a time-of-day dependent capacity .", "The aforementioned phenotypes of decreased nighttime activity during fasting and daytime refeeding prompted the question: Is p75NTR required for food anticipatory activity ( FAA ) ?", "FAA is measured as an increase in locomotor activity in the timeframe preceding a scheduled meal , indicating a preparedness to consume food quickly in times of caloric scarcity ( Gallardo et al . , 2014; Pendergast and Yamazaki , 2018 ) .", "To investigate FAA behavior of mice we subjected Ngfr-KO mice and littermate controls to the paradigm illustrated in Figure 1A for 5 days .", "Body weight , blood glucose , and ketones were measured at ZT0 ( Figure 2A ) .", "In agreement with our finding that Ngfr-KO mice have diminished refeeding following an overnight fast ( Figure 1A ) , on each of the five days of daytime TRF , Ngfr-KO mice consumed significantly less food and lost more body weight than littermate controls ( Figure 2B ) , with no detectable differences in blood glucose or ketone levels ( Figure 2—figure supplement 1 ) .", "When we assessed activity , littermate controls showed a robust increase in the proportion of their daytime activity that occurred before feeding ( FAA ) , while Ngfr-KO mice showed no such increase in their FAA after five days ( Figure 2C ) .", "Furthermore , this lack of activity is specific to the FAA time period , as we do not observe any significant changes in dark period activity after 5 days of TRF ( Figure 2—figure supplement 2A ) .", "As the strength of FAA differs in response to feeding during the day versus the night ( Davidson et al . , 2003; Tan et al . , 2014 ) , and since p75NTR has been implicated as a clock gene in the control of circadian and metabolic transcript levels ( Baeza-Raja et al . , 2013 ) , we next modified our 5 day TRF paradigm to give access to food from ZT15-18 , during the night ( Figure 2D ) .", "In contrast to daytime TRF , we found that Ngfr-KO mice exhibited unaltered food intake and body weight on nighttime TRF ( Figure 2E ) with intact FAA ( Figure 2F ) , albeit with a reduction of total activity ( Figure 2—figure supplement 2B ) .", "These data indicate p75NTR plays a role in the expression of FAA in a circadian phase dependent manner .", "p75NTR has been implicated in nervous system development , acting as either a death signal or a survival cue depending on the cell type ( Bamji et al . , 1998; Cheng et al . , 2018 ) .", "As such , the behavioral/feeding phenotypes observed could be due to developmental miswiring of central circuits necessary for energy detection and appropriate responses .", "To address this possibility , we generated adult specific p75NTR knockout mice by treating inducible Ndor1Tg ( UBC-Cre/ERT2 ) ::Ngfr-floxed mice with tamoxifen in young adulthood ( Adult-Ngfr-KO ) ( Ruzankina et al . , 2007 ) .", "During ad libitum feeding we observed no significant difference in body weight or food intake between Adult-Ngfr-KO and wildtype controls ( Figure 3A ) .", "However , Adult-Ngfr-KO mice exhibited a similar defect in daytime refeeding ( Figure 3B ) and FAA during daytime TRF with a significant reduction in food intake ( Figure 3C , D ) .", "Furthermore , a third of the Adult-Ngfr-KO mice had to be removed early due to weight loss in excess of 30% of baseline weight , suggesting that the phenotype of Adult-Ngfr-KO mice is more severe than that of the germline knockout ( Figure 3C , arrows ) .", "These results together demonstrate that the Ngfr-KO phenotype can be ascribed to an adult function of p75NTR , rather than a developmental one .", "In addition , this implies that there may be a degree of developmental compensation in germline Ngfr-KO mice that rescues homeostatic feeding and food anticipatory behavior .", "Previously identified roles of p75NTR in modulating whole body metabolism have been attributed to its function in white adipocytes and other peripheral tissues ( Baeza-Raja et al . , 2016; Baeza-Raja et al . , 2012 ) .", "Since feeding behavior is tightly controlled by central feeding circuits , we next investigated whether p75NTR may also be necessary in neurons of the hypothalamus .", "Indeed , we detected p75NTR immunofluorescence through an antibody against p75NTR within the arcuate nucleus of the hypothalamus , and quantified co-expression with NPY/AgRP neurons ( Figure 4A , 69 . 97 ± 0 . 022% of NPY+ neurons co-express p75NTR , n = 3 ) .", "To address whether p75NTR has a functional role in activating these arcuate neurons in response to fasting , we measured expression of the immediate early gene c-Fos in a region of interest masked on Npy-GFP expressing neurons of the arcuate of fasted Ngfr-KO mice and littermate controls .", "We found a 33% reduction of Npy-GFP c-Fos activation of fasted Ngfr-KO mice , suggesting that p75NTR may be necessary for central detection of energy status ( Figure 4B ) .", "We observed a similar phenotype in arcuate c-Fos induction after fasting in Adult-Ngfr-KO mice ( Figure 4—figure supplement 1B ) , suggesting a similar defect in these two models .", "This reduction in c-Fos activation suggests that p75NTR in arcuate neurons themselves may be required for robust neuronal activation during fasting .", "As a major driver of homeostatic feeding , NPY/AgRP neurons integrate peripheral and central metabolic information related to energy needs and availability of food ( Betley et al . , 2013; Su et al . , 2017 ) .", "In particular , mice with neonatal ablation of AgRP neurons are phenotypically normal during ad libitum feeding , but are unable to increase food intake and express FAA during daytime , but not nighttime , TRF ( Luquet et al . , 2005; Tan et al . , 2014 ) .", "This is a remarkably similar phenotype to what we have observed in our Ngfr-KO mice on TRF , and led us to suspect that these two mouse models may have defects in the same pathway .", "Given defective feeding behavior ( Figures 1A and 2B ) , expression in NPY/AgRP neurons ( Figure 4A ) , and decreased fasting activation of arcuate neurons ( Figure 4B ) , we hypothesized that p75NTR might be necessary for robust function of AgRP neurons .", "To address this hypothesis , we generated mice with an Agrp-specific knockout of p75NTR ( AgRP-Ngfr-KO ) .", "During ad libitum feeding these mice similarly show no defect in food intake or body weight ( Figure 5A ) , however exhibit a similar reduction in refeeding food intake following an overnight fast ( Figure 5B ) .", "Furthermore , during daytime TRF , AgRP-Ngfr-KO mice behaved similarly to germline Ngfr-KO and Adult-Ngfr-KO mice , exhibiting no FAA and having reduced food intake ( Figure 5C , D ) .", "However , we do not observe a statistically significant decrease in body weight .", "Lastly , as AgRP neurons have been shown to be necessary to promote feeding in response to ghrelin , we tested whether p75NTR in AgRP neurons is necessary for ghrelin responsiveness .", "We found that intracerebroventricular infusion of 1 ug ghrelin at ZT4 in ad libitum fed mice significantly increased food intake and c-Fos expression in both AgRP-Ngfr-KO and control mice , suggesting that p75NTR is not required for ghrelin-mediated AgRP neuron activity and feeding behavior ( Figure 5—figure supplement 2 ) .", "Taken together , these data suggest that p75NTR is required postnatally and in AgRP neurons for robust behavioral responses to food deficit , while p75NTR in other cell populations may be required for weight loss .", "Roles for p75NTR in the modulation of numerous intracellular signaling pathways have been well documented ( Kraemer et al . , 2014; Reichardt , 2006; Vilar et al . , 2009 ) .", "One of the primary pathways through which p75NTR has been shown to function is the c-Jun N-terminal kinase ( JNK ) signaling cascade ( Harrington et al . , 2002; Yoon et al . , 1998 ) .", "We first sought to assess whether JNK signaling is perturbed in the arcuate of AgRP-Ngfr-KO mice by measuring levels of a JNK signaling target c-Jun through immunofluorescence .", "We find in the fed state that there is no significant difference in phospho-c-Jun levels between genotypes ( Figure 6A , B ) , and that both wildtype and AgRP-Ngfr-KO mice show a similar reduction in phospho-c-Jun in response to fasting ( Figure 6A , B ) .", "This suggests that p75NTR is not necessary for JNK activation in AgRP neurons and is in agreement with previous findings that phospho-c-Jun is decreased in AgRP neurons following a fast ( Unger et al . , 2010 ) .", "We next turned our attention to other signaling pathways that could be altered in p75NTR-deficient AgRP neurons .", "Phosphorylation of cAMP response element binding protein ( CREB ) in AgRP neurons is required for adaptive feeding behavior in response to fasting ( Morikawa et al . , 2004; Yang and McKnight , 2015 ) .", "While levels of phospho-CREB are comparable between fed wildtype and AgRP-Ngfr-KO mice , we find a significant blunting of fasting-induced phospho-CREB in the arcuate hypothalamus of AgRP-Ngfr-KO mice ( Figure 6C , D ) , and show this as a specific defect within AgRP neurons ( Figure 6—figure supplement 1 ) .", "These data agree with our previous finding showing blunted c-Fos induction in Ngfr-KO mice ( Figure 4B ) , and suggest that p75NTR is necessary for activation of AgRP neuron CREB signaling in response to fasting ." ], [ "While hunger has long been recognized as a strong motivator of feeding , it has become clear that circadian inputs may also contribute significantly to driving feeding behavior .", "In Ngfr-KO mice , we show a striking phenotypic difference in the ability to consume equivalent post-fasting amounts of food depending on time of day , with a 21% deficit during the normal rest phase ( Figure 1 ) .", "Similarly , the distinction between intact nighttime FAA and lost daytime FAA ( Figure 2 ) is intriguing , and suggests that food anticipation may depend on separate mechanisms depending on the phase of TRF .", "These data suggest that p75NTR may be engaged by hunger cues to override inhibitory signals of feeding that are normally present during the daytime , but which may be absent at night .", "However , it is unknown how the presence of light or the time-of-day may influence a food entrained clock .", "These signals could ultimately be derived from the central pacemaker in the SCN , which is known to be more active during the normal rest phase ( Inouye and Kawamura , 1979 ) , to connect to feeding control centers in the hypothalamus , including the arcuate ( Guzmán-Ruiz et al . , 2014 ) , and function as the master circadian clock in response to changes in light ( Güler et al . , 2008; Mistlberger , 2011 ) .", "However , it is unresolved how the SCN may interact with a food entrained clock ( Storch and Weitz , 2009 ) .", "AgRP neurons have a diurnal firing pattern ( higher during the evening and lower during the morning ) which accompanies differential transcriptional profiles in response to refeeding during the day versus at night ( Cedernaes et al . , 2019 ) .", "Interestingly , among these changes were a significant enrichment in neurotrophin signaling pathway components ( e . g . BDNF , Rac1 , Ripk2 , Frs2 , Rap1 etc . ) as determined by the KEGG database .", "p75NTR has been shown to be important in controlling the expression of core clock genes in the SCN and liver ( Baeza-Raja et al . , 2013 ) , and may have a similar role in AgRP neurons .", "It is possible that some of these transcriptional changes may also be mediated by the modulation of CREB signaling that we observed in AgRP neurons .", "p75NTR has been suggested to interact with PKA to alter CREB activity , and , interestingly , CREB has been shown to have roles in both the core circadian clock machinery in the SCN , and in the peripheral metabolic alterations associated with time restricted feeding ( Asher and Schibler , 2011; Baeza-Raja et al . , 2016; Ginty et al . , 1993; Hatori et al . , 2012; O'Neill et al . , 2008 ) .", "However , our understanding is limited on how changes in clock genes in metabolically sensitive neurons , such as AgRP neurons , could alter behavioral responses .", "Neurotrophins function broadly in the development and maintenance of nervous system wiring .", "It could be considered that p75NTR , canonically involved in synaptic plasticity , may influence feeding by altering some broad measure of AgRP neuron remodeling in response to fasting .", "Indeed , fasting induced activation of AgRP neurons has been shown to require NMDA receptors and spinogenesis ( Liu et al . , 2012 ) .", "Additionally , CREB signaling , which we show is altered in fasted AgRP-Ngfr-KO mice ( Figure 6 ) , has been shown to be important for long-term changes in neuronal plasticity ( Sakamoto et al . , 2011 ) .", "Meanwhile , p75NTR can localize to dendritic spines , and loss of p75NTR has been shown to impair NMDA-dependent LTD in the hippocampus ( Woo et al . , 2005 ) .", "It is intriguing to speculate that the requirement of p75NTR for proper activation of arcuate neurons in response to fasting ( Figures 4 and 6 ) is due to a role in dendritic remodeling of AgRP neurons , which will be explored in the future .", "It is also possible that neurotrophin family members like p75NTR play non-canonical roles as essential detectors of energy state and in turn regulate feeding behavior .", "p75NTR , along with TrkB , is one of the two receptors for the neurotrophic factor BDNF , which has been previously implicated in hypothalamic circuits to suppress feeding ( Ozek et al . , 2015 ) .", "Here we report that p75NTR acts oppositely to BDNF-TrkB signaling to promote feeding .", "Additionally , mice lacking the ability to form mature BDNF have intact FAA ( Krizo et al . , 2018 ) , suggesting that BDNF signaling , either through TrkB or p75NTR , may not be required for FAA .", "Interestingly , previous work in the peripheral nervous system has delineated several dichotomous functions for p75NTR and TrkB ( Lu et al . , 2005 ) .", "The results presented here further support this notion by demonstrating that p75NTR acts in support of feeding , as opposed to TrkB’s previously documented suppression of feeding ( Ozek et al . , 2015 ) .", "It is plausible that p75NTR and TrkB play opposing roles in the coordination of hypothalamic mediated energy coordination .", "This distinction is made even more notable by recent work demonstrating a time-of-day dependent action of hypothalamic TrkB neurons ( Liao et al . , 2019 ) .", "Silencing TrkB neurons in the dorsomedial hypothalamus ( DMH ) during the day , for example , significantly increases food intake , whereas their silencing at night has no effect ( Liao et al . , 2019 ) .", "While it remains unknown whether p75NTR and TrkB might interact at either a cellular or circuit level in the hypothalamus , we can conclude that neurotrophin receptors have critical functions in time-of-day dependent feeding behaviors .", "Understanding the connections between circadian rhythms and metabolism could lead to delineation of how food entrained clocks impact metabolic health .", "First , deciphering the neuronal regions that mediate diurnal control of feeding and food anticipatory behavior would greatly improve our ability to study this phenomenon .", "Interactions between the SCN , DMH , and arcuate hypothalamus have long been hypothesized as mediators of circadian control of feeding .", "Extending our understanding of how neuronal activity in these regions changes normally across the 24 hr light/dark cycle , and how this is altered by changes in feeding patterns , would lend great insight into their circadian regulation .", "Second , studying the role of important molecular players in circadian regulation of feeding , such as p75NTR , holds promise to find viable targets for modulating these behaviors .", "Exploring the role of p75NTR in other hypothalamic neuronal populations will allow further elucidation of the mechanisms of FAA , thereby allowing us to define the neural correlates of how caloric scarcity and time of day work in concert to influence feeding and related behaviors ." ], [ "All experiments were carried out in compliance with the Association for Assessment of Laboratory Animal Care policies and approved by the University of Virginia Animal Care and Use Committee .", "Animals were housed on a 12 hr light/dark cycle with food ( Teklad Diet 8664 ) and water ad libitum unless otherwise indicated .", "Ngfr-KO mice were purchased from Jackson Labs ( Bar Harbor , Maine ) ( #002213 ) ( Lee et al . , 1992 ) , and were maintained on a B6;129 s mixed background and genotyped with primers against intron II in Ngfr - Intron II ( Ngfr -IntII , 5′-CGA TGC TCC TAT GGC TAC TA ) , Intron III ( Ngfr -IntIII , 5′-CCT CGC ATT CGG CGT CAG CC ) , and the pGK-Neo cassette ( pGK , 5′-GGG AAC TTC CTG ACT AGG GG ) .", "Ngfr fl/fl mice were acquired as a generous gift from Brian Pierchala ( University of Michigan ) ( Bogenmann et al . , 2011 ) and were maintained on a 129/S2/SvPas; C57Bl/6J mixed background and genotyped with a three primer system to detect the wildtype , floxed , and delta alleles ( which are generated from unintended germline excision of the loxP sites ) using two forward primers ( 5′-TGC AGA AAT CAT CGA CCC TTC CC ) , ( 5′-CCT CCG CCA GCT GTC TGC TTC CT ) and a reverse primer ( 5′-TCC TCA CCC CGT TCT TTC CCC ) .", "Ndor1Tg ( UBC-Cre/ERT2 ) mice ( expressing Cre recombinase fused to ERT2 from the ubiquitin C promoter ) were purchased from Jackson labs ( #008085 ) ( Ruzankina et al . , 2007 ) .", "Nuclear translocation of the Cre fusion protein was induced by tamoxifen injections once daily for 5 days in both Adult-Ngfr-WT and Adult-Ngfr-KO mice ( 75 mg tamoxifen/kg body weight , Sigma ) when mice were 8–9 weeks of age , followed by a 2 week waiting period to ensure excision of floxed alleles ( Heffner , 2011 ) .", "Agrp-IRES-Cre mice ( expressing Cre recombinase from AgRP neurons ) were purchased from Jackson labs ( #012899 ) ( Tong et al . , 2008 ) .", "All Cre recombinase expressing lines were genotyped with primers against the Cre allele ( 5′-GCA TTA CCG GTC GAA CGA GTG ATG AG and 5′-GAG TGA ACG AAC CCG AAA TCA GTG CG ) and an internal control sequence ( 5′-TGG GCT GGG TGT TAG CCT TA and 5′-TTA CGT CCA TCG TGG ACA GC ) .", "Npy-GFP mice were purchased from Jackson labs ( #006417 ) ( van den Pol et al . , 2009 ) .", "Ai9 tdTomato mice were purchased from Jackson labs ( #007909 ) ( Madisen et al . , 2010 ) .", "All experiments were performed on male mice 12–16 weeks old unless otherwise indicated .", "Food intake was performed on individually housed male mice that were acclimated for 7 days , followed by food and body weight measurements weekly ( for development curves ) , twice daily for day/night food intake measures , or daily during restricted feeding experiments .", "Total and ambulatory activity levels were measured using IR beam interruption ( Columbus Instruments ) .", "For scheduled feeding , mice were first acclimated to single housing for 7 days , followed by acclimation to IR beam interruption chambers ( Columbus Instruments ) for 72 hr .", "For daytime scheduled feeding , mice were fasted at lights off ( ZT12 ) on day 0 .", "Mice were weighed , and glucose ( one touch ultra 2 , Bayer , Leverkusen , Germany ) and β-ketone ( Precision xtra , Abbott , Chicago , Illinois ) measures were taken 12 hr later at lights on ( ZT0 ) .", "Mice were then refed 4 hr later at ZT4 , with food removed 3 hr later at ZT7 .", "Mice were fed between ZT4-7 on each of the next four days .", "For nighttime scheduled feeding , mice were fasted at lights on ( ZT0 ) on day 0 .", "Mice were weighed 12 hr later prior to lights off ( ZT11-12 ) .", "Mice were then refed 4 hr later at ZT15 , with food removed 3 hr later at ZT18 .", "Mice were fed between ZT15-18 on each of the next four days .", "All groups were age and weight-matched .", "In accordance with University of Virginia Animal Care and Use Committee guidelines , any mouse that lost 30% or more of their body weight was removed from the experiment .", "This is indicated with an arrow in the figure .", "Glucose levels were measured using the one touch ultra two glucometer ( Bayer ) .", "B-ketone levels were measured using the precision xtra meter ( Abbott ) .", "Insulin was measured using the ultra sensitive mouse insulin ELISA kit according to manufacturer's instructions ( Crystal Chem , Elk Grove Village , Illinois ) .", "Leptin was measured using the mouse/rat leptin EIA kit according to manufacturer's instructions ( Cayman Chemical , Ann Arbor , Michigan ) .", "Corticosterone was measured using the corticosterone ELISA kit according to manufacturer's instructions ( Cayman Chemical ) .", "Mice were transcardially perfused by first anesthetizing with ketamine/xylazine , then perfusing with ice cold 1x PBS , followed by ice cold 4% paraformaldehyde ( PFA ) .", "Brains were removed and placed into 4% PFA overnight , before transitioning to 30% sucrose to dehydrate the brains for 48–72 hr .", "Brains were then frozen and sliced into 30 micron free-floating sections on a cryostat into 1x PBS with 0 . 002% sodium azide .", "Antibody staining was performed as follows: sections were washed in 1x PBS with 0 . 5% Triton , blocked in 1x PBS with 0 . 5% Triton and 5% donkey serum , incubated in block with primary antibody overnight at 4C ( p75NTR , goat , 1:5000 , Neuromics cat# GT15057 , Edina , Minnesota; c-Fos , 1:1000 , rabbit , Synaptic Systems cat# 226 003 , Goettingen , Germany [Grippo et al . , 2017]; phospho-c-Jun , 1:800 , rabbit , Cell Signaling Technologies cat# 3270; phospho-CREB , 1:800 , rabbit , Cell Signaling Technologies cat# 9198 ) , again washed , incubated in block with secondary antibody for 2 hr at room temperature ( AF 568 donkey anti-goat , 1:500 ( for p75NTR ) ; AF 488 or 568 donkey anti-rabbit , 1:500 ( for c-Fos ) , AF 488 donkey anti-rabbit , 1:500 ( for phospho-c-Jun and phospho-CREB ) ) , again washed , and mounted with DAPI-fluoromount G ( Southern Biotech ) .", "Sections were imaged and quantified by a researcher blinded to the genotype and treatment , and unblinded once all analysis was completed .", "For sections with Npy-GFP or Agrp-tdTomato reporter co-expression , FIJI software was used to first set a threshold that accounted for reporter expression , and then create a mask of the thresholded region that was overlaid onto c-Fos expression .", "Surface area of c-Fos expression within the designated region above a threshold determined by a blinded observer was then measured , and normalized to the masked area of the reporter expressing region of interest ( Rieux et al . , 2002 ) .", "Briefly , animals were anesthetized with isoflurane ( induction 5% , maintenance 2–2 . 5%; Isothesia ) and placed in a stereotaxic apparatus ( Kopf ) .", "A heating pad was used for the duration of the surgery to maintain body temperature and ocular lubricant was applied to the eyes to prevent desiccation .", "AgRP-Ngfr-KO and littermate control mice were stereotactically implanted with a 6 mm cannula targeted to the mediobasal hypothalamus ( Plastics one , Roanoke , VA ) .", "Stereotaxic coordinates relative to Bregma: 0 mm lateral , 1 . 46 mm posterior and −5 . 8 mm below the surface of the skull .", "Cannulas were maintained in place by dental cement anchored to one stainless steel screw fixed to the skull .", "A dummy cannula was inserted to prevent clogging of the cannula .", "After the surgery , the animals were housed individually for 1 week .", "All surgical procedures were performed in sterile conditions and in accordance with University of Virginia IACUC guidelines .", "Ad libitum fed , cannulated , AgRP-Ngfr-KO and littermate control mice were centrally infused with vehicle or ghrelin ( 0 . 5 ug/ul ) ( Phoenix Pharmaceuticals , cat# 031–31 ) at a rate of 1 µl/min for a final volume of 2 µl .", "Infusions occurred from ZT3-5 , and food intake was measured hourly for three hours .", "At least 1 week after initial infusions of vehicle and ghrelin , mice were randomly selected to recieve either vehicle or ghrelin at the same concentration and volume , and were intracardially perfused 90 min after infusion for verification of implant target site and immunofluorescence , as described above .", "Data are presented as mean ± SEM .", "Statistical analysis was carried out using Graphpad Prism version 8 . 0 .", "Outliers were detected using the ROUT method , Q = 1% .", "Student’s t-test was used to compare single means between genotypes , and 2-way Anova was used to compare genotype by time interactions .", "Differences were considered significant if p<0 . 05 ." ] ]

[ "Networks of neurons control feeding and activity patterns by integrating internal metabolic signals of energy balance with external environmental cues such as time-of-day .", "Proper circadian alignment of feeding behavior is necessary to prevent metabolic disease , and thus it is imperative that molecular players that maintain neuronal coordination of energy homeostasis are identified .", "Here , we demonstrate that mice lacking the p75 neurotrophin receptor , p75NTR , decrease their feeding and food anticipatory behavior ( FAA ) in response to daytime , but not nighttime , restricted feeding .", "These effects lead to increased weight loss , but do not require p75NTR during development .", "Instead , p75NTR is required for fasting-induced activation of neurons within the arcuate hypothalamus .", "Indeed , p75NTR specifically in AgRP neurons is required for FAA in response to daytime restricted feeding .", "These findings establish p75NTR as a novel regulator gating behavioral response to food scarcity and time-of-day dependence of circadian food anticipation ." ]

[ "In many animals , specific types of neurons , such as the hypothalamic hunger neurons , are tasked with regulating food consumption , integrating internal signals of hunger .", "In general , individuals eat if food becomes available when they are hungry; if food is absent , they will start moving to find new resources .", "Finally , if food always comes at the same time , animals will increase their activity just before it is delivered .", "Neurotrophins are a family of proteins that have many essential roles in the brain .", "In recent years , they have been shown to interact with the circadian clock , the built-in mechanism that helps animals stay synchronized with the cycle of day and night .", "A protein known as p75NTR is present in nerve cells , including hypothalamic hunger neurons: there , it helps to relay messages from a neurotrophin which , amongst other roles , controls food intake .", "However , it was unclear whether p75NTR played a role in regulating feeding behaviors , especially in a circadian manner .", "To investigate this question , Podyma et al . genetically engineered a group of mice lacking p75NTR , and a group missing the protein only in their hypothalamic hunger neurons .", "Both types of mutants had abnormal control of their feeding behavior: compared to normal mice , they fed less ( and lost more weight ) after they had been deprived of food overnight , or when they faced food shortage over multiple days .", "In addition , the mutants failed to move more before being fed .", "However , these feeding patterns were only affected during daytime , while they were preserved at night .", "These results reveal a new role for p75NTR in hypothalamic hunger neurons .", "Dissecting the biological processes that control food intake is key since obesity levels are increasing around the world .", "In particular , the relationship between food intake and the circadian clock is an important avenue of research as time-restricted diets ( where food intake is only allowed during specific periods of the day ) are growing in popularity ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "medicine", "cell biology" ]

[ [ "Low back pain is a global health problem that is experienced by ~80% of individuals at some point in their lifetime ( Vos et al . , 2012 ) .", "This problem is the number one single cause of years lived with disability with enormous personal and health system related costs ( Institute of Medicine ( US ) Committee on Advancing Pain Research , Care , and Education , 2016; Hartvigsen et al . , 2018 ) .", "Intervertebral disc ( IVD ) degeneration is a major factor contributing to low back pain ( Vos et al . , 2012; Adams and Hutton , 1983; Vergroesen et al . , 2015 ) .", "The cellular pathogenesis of IVD degeneration and the mechanisms leading to pain are not fully understood .", "One novel approach to treat painful degeneration is to target cellular senescence , a state of irreversible growth arrest occurring in response to cellular stress ( Tchkonia et al . , 2013 ) .", "Stress-induced premature senescence is caused by factors such as oxidative and genotoxic stresses ( Toussaint et al . , 2000; Campisi and d'Adda di Fagagna , 2007 ) .", "Increasing evidence suggests that accumulation of senescent cells during tissue degeneration contributes directly to initiation and development of musculoskeletal degenerative diseases like osteoarthritis ( Jeon et al . , 2017 ) and IVD degeneration ( Le Maitre et al . , 2007; Feng et al . , 2016; Wang et al . , 2016; Patil et al . , 2018; Cherif et al . , 2019 ) .", "Senescent cells secrete a range of cytokines , chemokines , growth factors , and proteases termed as the senescence-associated secretory phenotype ( SASP ) ( Herbig et al . , 2006; Kuilman et al . , 2008; Coppé et al . , 2008; Xu et al . , 2015 ) .", "These SASP factors are suggested to further induce senescence in a paracrine manner ( Acosta et al . , 2013 ) , to promote matrix catabolism and sterile inflammation in IVDs , thereby accelerating the degenerative process ( Parrinello et al . , 2005; Tominaga , 2015 ) .", "Elimination of senescent cells enhances disc tissue homeostasis in genetically modified progeroid Ercc1−/Δ22 and p16‐3MR ( Patil et al . , 2019 ) mice suggesting that senotherapeutic drugs have great potential to treat low back pain resulting from IVD degeneration .", "The effect could potentially be mediated by apoptotic ( senoptosis ) or nonapoptotic ( senolysis ) mechanisms ( Schmitt , 2017; Soto-Gamez and Demaria , 2017; Kirkland et al . , 2017; Niedernhofer and Robbins , 2018 ) or by modulating the SASP , indirectly suppressing senescence ( senomorphics ) ( Zhu et al . , 2015; Soto-Gamez and Demaria , 2017; Kirkland et al . , 2017; Childs et al . , 2017 ) .", "Further , interest is growing toward the use of natural senotherapeutic compounds such as quercetin , fisetin and piperlongumine , curcumin and o-Vanillin ( Cherif et al . , 2019; Li et al . , 2019 ) ; their key advantages being low toxicity and great potential to be translated into clinical applications .", "Cell-cycle arrest of senescent disc cells is mainly mediated by the two pathways: p53-p21-Rb and p16-Rb .", "During disc degeneration , both pathways are activated simultaneously to induce senescence ( Feng et al . , 2016 ) .", "The FDA-approved drug RG-7112 ( RO5045337 ) ( Weber , 2010; Laberge et al . , 2018 ) is a highly potent and selective MDM2 antagonist ( Weber , 2010; Tovar et al . , 2013 ) that restores the physiological activity of p53 .", "RG-7112 is the first nutlin family member to be assessed clinically ( Weber , 2010; Laberge et al . , 2018 ) showing evidence of acceptable safety ( Ray-Coquard et al . , 2012; Constantinidou et al . , 2012 ) .", "RG-7112 was reported to selectively kill senescent lung fibroblasts ( IMR90 ) where senescence was induced by ionizing radiation ( IR ) ( Weber , 2010; Laberge et al . , 2018 ) .", "The natural compound o-Vanillin , known for its antioxidant and anti-inflammatory effects ( Santosh Kumar et al . , 2002; Oliveira et al . , 2014; Shah et al . , 2019; Marton et al . , 2016 ) , has recently been described for its senotherapeutic activity in human IVD cells ( Cherif et al . , 2019 ) .", "One caveat in the field is that there are currently no large bipedal animal models that mimic naturally occurring IVD degeneration and human genetic background ( Alini et al . , 2008; Jin et al . , 2018 ) .", "One solution to this caveat is the use of an intact disc culture model ( Gawri et al . , 2011; Krock et al . , 2014; Gantenbein et al . , 2015; Rosenzweig et al . , 2016 ) of human IVDs to test and develop new senotherapeutic drugs suitable for human disc degeneration .", "Here , we utilized in vitro and ex vivo models , to assess the senotherapeutic effects of RG-7112 and o-Vanillin on naturally occurring senescent cells in degenerating human IVDs .", "We have previously shown that o-Vanillin has senolytic effects on senescent human IVD cells .", "o-Vanillin is a natural compound that in addition to its senolytic effect has antioxidant and anti-inflammatory properties .", "We compared the effect of o-Vanillin to RG-7112 , a pure senolytic drug , that we found to efficiently kill senescent annulus fibrosus ( AF ) and nucleus pulposus ( NP ) cells .", "The objective was to evaluate if o-Vanillin with its dual function could further reduces inflammatory factors released by non-senescent cells thus enhancing the therapeutic effect .", "We also aimed to establish if one or both drugs could reach and kill senescent IVD cells in their native environment of intact human disc with naturally occurring degeneration ." ], [ "We aimed to compare the senotherapeutic activity of the natural senolytic o-Vanillin a natural senolytics with antioxidant and antiinflammatory properties with a pure synthetic senolytic drug .", "We chose the commercially available FDA approved drug RG-7112 as a candidate .", "A concentration of 5 μM RG-7112 , shown as a safe and effective dose for human IMR90 lung fibroblasts , was used to evaluate its effect on senescent and non-senescent human IVD cells ( Weber , 2010; Laberge et al . , 2018 ) .", "Pellet cultures of IVD cells from degenerate NP and AF regions were exposed to RG-7112 for 4 days , the drug was removed , and the treated pellets were maintained for 21 days in standard media .", "Then , evaluated for cytotoxicity , senolytic and therapeutic activity .", "No cytotoxicity was observed following treatment .", "In contrast , both NP ( 9 . 87%; p<0 . 05 ) and AF ( 11 . 87%; p<0 . 05 ) cells showed a significant increase in metabolic activity ( Figure 1—figure supplement 1A ) .", "Pellet cultures were treated as before and the presence of p16Ink4a , Ki-67 and caspase-3 were analyzed after 21 days ( Figure 1A ) .", "RG-7112 significantly decreased the percentage of p16Ink4a positive NP ( 11 . 48%; p<0 . 001 ) and AF ( 20 . 03%; p<0 . 0001 ) cells compared to untreated control cultures ( Figure 1B ) .", "Similarly , RG-7112 significantly increased the percentage of Ki-67-positive NP ( 23 . 07%; p<0 . 05 ) and AF ( 34 . 92%; p<0 . 0001 ) cells ( Figure 1C ) .", "Caspase-3-positive AF cells increased significantly ( 17 . 88%; p<0 . 01 ) while a non-significant increase ( 2 . 94%; p=0 . 51 ) was observed in NP cells following treatment ( Figure 1D ) .", "Caspase 3 and caspase 3/7 activity was confirmed by fluorescence microscopy and activity assays , respectively ( Figure 1 —figure supplement 1B-C and d-f ) .", "Confocal immunofluorescence confirmed co-localization of p16Ink4a and caspase-3 , whereas proliferating ( Ki-67-positive ) cells did not co-localize with p16Ink4a ( Figure 1 —figure supplement 1E ( a–c ) .", "RG-7112 selectively increased apoptosis ( Figure 1 —figure supplement 1E ) while it maintained comparable metabolic activity ( Figure 1 —figure supplement 1F ) in NP cells from both degenerate and non-degenerate IVDs .", "Finally , we investigated proteoglycan synthesis using the DMMB assay following treatment .", "A significant increase in proteoglycan release in conditioned media was observed after 14 days of treatment ( Figure 1 —figure supplement 1G ) .", "To identify molecular pathways affected by RG-7112 and o-Vanillin in human disc cells , gene expression analysis of 96 selected cellular senescence genes was performed and 91 of the 96 genes were expressed at a detectable level .", "Pellet cultures were treated with RG-7112 ( 5 μM ) or o-Vanillin ( 100 μM ) for 4 days .", "The relationship between the differentially up- and downregulated genes are depicted in Figure 2A .", "Compared with the control group , the number of differentially expressed genes for o-Vanillin were 40 ( 30 upregulated and 10 downregulated ) and eight for RG-7112 ( 6 upregulated and two downregulated ) .", "In upregulated DEGs , three are common to both drugs , mitogen-activated protein kinase 14 ( MAPK14 ) , cell division cycle 25 c ( CDC25c ) and cyclin dependant kinase 2D ( CDKN2D or p19ARF ) .", "No down-regulated DEGs were common to the drugs .", "Although cyclin B1 ( CCNB1 ) is significantly expressed following the treatment with the two compounds , it is upregulated in RG-7112 and downregulated in o-Vanillin ( Figure 2A ) .", "Of the 91 genes identified , 44 were differentially expressed with a p<0 . 05 in one or both treatments ( Figure 2B ) .", "The 47 genes that did not meet the significance criteria of p<0 . 05 are shown in ( Figure 2—figure supplement 1A ) .", "Next , we compared gene expression profiles of the o-Vanillin group with the RG-7112 group .", "Of the 91 evaluated , only eight genes were significantly affected by RG-7112 treatment , four were common to o-Vanillin ( MAPK14 , CCNB1 , CDC25c and CDKN2D ( p19ARF ) ) , and four were exclusive to RG-7112 ( MDM2 , CDKN1A ( p21ARF ) , E2F1 and RBL1 ) .", "In contrast , 40 genes were significantly affected by o-Vanillin treatment .", "Cell cycle and senescence genes were significantly downregulated including cyclin dependent kinase 2A ( CDK2A or p16Ink4a ) , cyclin dependent kinase 2C ( p18ARF ) , Cyclin A2 , CCNB1 , CDC25c , Vimentin , Mitogen-activated protein kinase 6 ( MAPK6 ) and Checkpoint kinase 1 ( CHEK1 ) ( Figure 2C ) .", "Examples of apoptotic and proliferative genes significantly upregulated by o-Vanillin include B-cell lymphoma 2 ( Bcl-2 ) , Bcl-2-like 1 , Bcl-2-like 2 , interferon regulatory factor 5 ( IRF5 ) , IRF7 and receptor tyrosine-protein kinase ERBB2 .", "Interestingly , MAPK14 , CDC25c and CDKN2D ( p19ARF ) were the only genes significantly upregulated after the treatment with both compounds , while CCNB1 showed opposite regulation patterns .", "In conjunction , apoptotic pathways are significantly upregulated to kill senescent cells while proliferation related pathways are activated in non-senescent cells .", "Detailed fold change difference of differentially expressed genes and respective p value are included in Supplementary file 3 ( a-d ) .", "To gain a further insight into a potential mechanism of action , the differentially expressed genes were mapped to networks in the Ingenuity Pathways Analysis ( IPA ) database .", "The scores take into account the number of focus genes and the size of the network to approximate the relevance of the network to the original list of focus genes .", "The IPA core analysis features allowed identification and determination of one network connecting the cell cycle , cell death and survival , connective tissue development and function pathways ( RG-7112 , network 1 ) of NP cells treated with RG-7112 ( Figure 2D ) .", "In the o-Vanillin-treated pellets , the highest-scoring network revealed a significant link with cell death and survival , neurological disease and organismal injury and abnormalities ( o-Vanillin , network 1 ) ( Figure 2E ) .", "Furthermore , connective tissue development and function , cell cycle ( o-Vanillin , network 2 ) , cancer , cellular movement ( o-Vanillin , network 3 ) , were shown to be influenced in the other two networks ( Figure 2F–G ) .", "All networks were identified and ranked by the score of the calculated p-value of the IPA assay within the selected set of 91 genes , the scores and molecules used to order these networks are shown in Supplementary file 4 ( a-b ) .", "These results confirmed the expected mode of action for RG-7112 and provide new insights to the predicted pathways involved in senescence and the responses to treatment with o-Vanillin .", "Transcriptomic results following the treatment with the two senolytics predicted the activation of apoptotic ( in senescent cells ) and proliferative ( in non-senescent cells ) pathways which suggest a reduction in the senescence burden likeley affecting SASP factor release .", "To verify an effect on SASP , media of untreated NP cell pellets , RG-7112 and o-Vanillin-treated pellets were used .", "The media collected were from the same donors used for gene expression analysis in Figure 2 .", "Samples were analyzed using an antibody array that simultaneously screen 80 factors .", "Out of the 80 factors present , 50 were detected in at least four donors and were included in further analysis ( Figure 3 —figure supplement 1A ) .", "The 25 most affected SASP factors are divided into four classes: Cytokines ( Figure 3A–a ) , chemokines CC ( Figure 3A–b ) , chemokines CXC ( Figure 3A–c ) , growth and neurotrophic factors ( Figure 3A–d ) .", "The most reduced factors following RG-7112 treatment compared with control were for cytokines ( TNF-α; −78 . 05 ± 4 . 69% ) ( p=0 . 71 ) , CC-chemokines ( CCL26; −66 . 69 ± 7 . 09% ) ( p=0 . 93 ) , CXC-chemokines ( CXCL13; −29 . 02 ± 17 . 26% ) ( p=0 . 68 ) , growth and neurotrophic factors ( HGF; −65 . 24 ± 5 . 9% ) ( p=0 . 87 ) .", "The top four factors decreased by o-Vanillin treatment were: IL-7 ( −60 , 37 ± 11 . 46% ) ( p=0 . 5 ) , CCL26 ( −40 , 14 ± 3 . 41% ) ( p=0 . 99 ) , CXCL10 ( −18 , 11 ± 6 , 15% ) ( p=0 . 62 ) and HGF ( −52 , 42 ± 5 , 45% ) ( p=0 . 96 ) ( Figure 3A ( a-d ) ) .", "To better visualize the overall effect of the two drugs on SASPs release , a scatter plot of average changes was generated demonstrating a similar overall effect of the two senolytics ( Figure 3A–e ) .", "The trends for all 80 factors are visualized in ( Figure 3—figure supplement 1B-C ) .", "The semi-quantitative cytokine array results were used to select 19 factor that were quantified with the Luminex assay .", "The 19 factors were selected based on the following criteria: only factors detected in all conditions and in at least four donors were chosen .", "These factors were selected to represent all the SASP classes and to include the most variably expressed cytokines , chemokines and growth factors .", "The 19 factors selected for further investigation were ( INF-γ , TNF-α , IL1-α , IL1-b , IL-6 , IL-8 , CCL5 , CCL7 , CCL11 , CCL24 , CCL26 , CXCL1 , CXCL5 , CXCL9 , CXCL10 , CXCL11 , CX3CL1 , Angiogenin and VEGF-A ) .", "The concentrations were measured and expression heatmap showed an overall decrease in the level of proinflammatory factors from both RG-7112 and o-Vanillin when compared to non-treated cultures .", "( Figure 3B ) .", "The levels of INF-γ , IL-6 , IL-8 and CCL24 were significantly decreased following both treatments whereas the decrease in CCL7 , CXCL1 , CXCL5 , CXCL9 , CXCL10 and VEGF significance was reached only following o-Vanillin treatment ( Figure 3C ) .", "However , significance was not reached for TNF-α , IL1-α , IL1-β , CCL5 , CCL11 , CCL26 , CXCL11 , CX3CL1 and Angiogenin in treated cultures compared to untreated cultures ( Figure 3—figure supplement 1C ) .", "Although , o-Vanillin reduced more SASP factors than RG-7112 at a significant level when compared to untreated cultures , there was no significant difference in concentrations between o-Vanillin and RG-7112-treated cultures , validating the similar overall effect the two drugs have on SASP factor release observed by cytokine array in Figure 3A–f .", "These results demonstrate that both RG-7112 and o-Vanillin decreased the overall inflammatory environment in pellet cultures of degenerating tissue and suggest a broader anti-inflammatory effect of o-Vanillin treatment .", "RG-7112 or o-Vanillin was injected into the central region of intact human IVDs to verify that the drugs can reach and kill the target cells in native tissue .", "Discs were pre-cultured for 4–6 days .", "The IVDs were treated with a single injection of vehicle , o-Vanillin , or RG-7112 , delivered to the centre of the NP and were cultured for another 28 days as outlined in Figure 4A .", "An adapted MRI protocol ( Rosenzweig et al . , 2018 ) was used to assess T1ρ-weighted MRI signal that directly correlates with proteoglycan content ( Rosenzweig et al . , 2018; Mulligan , 2015 ) .", "The method used to assess uniform NP regions of interest is shown in ( Figure 2—figure supplement 1B-D ) .", "The same NP regions of interest were scanned in the same orientation pre- and post-treatment .", "Increased T1ρ values were found in RG-7112 and o-Vanillin-treated discs while vehicle-treated discs displayed decreased T1ρ values .", "( Figure 4B ( a-d ) ) Quantification of pre- versus post- treatment intensity values showed a non-significant decrease of 13 . 2 ± 4 . 7% ( p=0 . 058 ) in vehicle-treated control IVDs .", "In RG-7112 treated discs , there was a significant 6 . 8 ± 1 . 5% ( p=0 . 024 ) increase in the T1ρ value post-treatment .", "Discs that were treated with o-Vanillin , displayed a significant increase of 11 . 1 ± 1 . 2% ( p=0 . 001 ) in T1ρ values following treatment ( Figure 4C ) .", "The red dye safranin-O , binds negatively charged molecules , which are primarily represented by proteoglycan in the NP .", "Histological evaluation post-treatment using Safranin-O/fast green staining showed strong ( intense red ) staining in treated discs ( Figure 4D ) .", "Finally , p16Ink4a and Ki-67 immunohistochemistry were performed to verify senolytic activity of the two compounds ( Figure 4E ) .", "Indeed , immunohistochemical assessment of p16Ink4a showed a significant decrease in the number of senescent cells of 17 . 38 ± 1 . 6% ( p=0 . 0017 ) in RG-7112 and 22 . 65 ± 3 . 6% ( p=0 . 008 ) in o-Vanillin-treated IVDs compared with vehicle-treated control discs ( Figure 4F ) .", "Quantification of Ki-67 staining showed a non-significant increase in both RG-7112 and o-Vanillin 3 . 05 ± 3 . 4% ( p=0 . 4 ) treated IVDs ( Figure 4G ) .", "The data suggest that both senolytics can reach and kill naturally occurring senescent human IVDs cells situated in their native environment and at the same time promote tissue repair and regeneration .", "Culture media from treated and untreated discs ( in Figure 4 ) was analyzed using cytokine arrays and were compared to their respective pre-treatment media .", "Interestingly , a single injection with RG-7112 strongly decreased secretion of several proinflammatory cytokines and chemokines including IL-7 ( −60 . 63% ( p=0 . 01 ) ) , IL-6 ( −59 . 39% ( p=0 . 14 ) ) , CXCL1 ( −36 . 72% ( p=0 . 26 ) ) , GRO-abg ( −32 . 84% ( p=0 . 21 ) ) and CCL24 ( −30 . 57% ( p=0 . 15 ) ) .", "o-Vanillin also strongly decreased IL-7 ( −83 . 37% ( p=0 . 09 ) ) , CXCL1 ( −65 . 45% ( p=0 . 12 ) ) , IL-6 ( −58 . 68% ( p=0 . 10 ) ) , CXCL6 ( −52 . 47% ( p=0 . 009 ) ) , IGFBP-2 ( −44 . 51% ( p=0 . 15 ) ) , GRO-abg ( −41 . 83% ( p=0 . 03 ) ) and CCL2 ( −39 . 91% ( p=0 . 05 ) ) .", "Both compounds significantly decreased IL-8 by 18 . 72% ( p=0 . 004 ) for RG-7112 and by 11 . 75% ( p=0 . 04 ) for o-Vanillin .", "We also detected a moderate increase of CXCL9 , CCL22 , NT3 and EGF for o-Vanillin and only in CCL2 and OPN for RG-7112-treated media ( Figure 5A ) .", "The level of the SASP factors released from vehicle-treated discs showed an increase in IL-6 ( 530 . 85% ( p=0 . 15 ) ) , CXCL6 ( 196 . 89% ( p=0 . 07 ) ) , CXCL1 ( 99 . 83% ( p=0 . 18 ) ) , OPN ( 92 . 36% ( p=0 . 01 ) ) , CCL2 ( 53 . 37% ( p=0 . 18 ) ) , IGFBP-2 ( 39 . 26% ( p=0 . 13 ) ) , IL-7 ( 23 . 52% ( p=0 . 53 ) , GRO-abg , ( 14 . 25% ( p=0 . 91 ) ) , CCL24 ( 9 . 64% ( p=0 . 97 ) ) , EGF ( 3 . 18% ( p=0 . 85 ) ) , TNSF-14 ( 2 . 09% ( p=0 . 66 ) ) , IL-8 ( 1 . 61% ( p=0 . 82 ) ) and a decrease in CXCL9 ( −35 . 23% ( p=0 . 26 ) ) , CCL22 ( −11 . 95% ( p=0 . 56 ) ) and NT3 ( −13 . 46% ( p=0 . 25 ) ) ( Figure 5B ) .", "The complete set of factors from all groups are presented in Figure 5—figure supplement 1 ( A-C ) .", "Luminex quantification of the same 19 selected SASP factors that were analyzed in pellet cultures showed an overall decrease in inflammatory mediators in culture media from both RG-7112 or o-Vanillin-treated discs compared with that of vehicle ( Figure 5C ) .", "All factors decreased in RG-7112 and o-Vanillin-treated discs ( Figure 5D and Figure 5—figure supplement 1D ) .", "Six factors in RG-7112 and five in o-Vanillin-treated discs were significantly decreased in the post-treatment media compared with their respective pre-treatment media .", "For RG-7112 , these proteins were TNF-α ( mean 12 . 4 pg/ml in pre , 6 . 8 pg/ml in post , p=0 . 03 ) , CCL11 ( mean 13 . 1 pg/ml in pre , 7 . 8 pg/ml in post , p=0 . 009 ) , CCL24 ( mean 201 . 7 pg/ml in pre , 101 . 1 pg/ml in post , p=0 . 04 ) , CXCL1 ( mean 261 . 8 pg/ml in pre , 148 . 2 pg/ml in post , p=0 . 001 ) , CXCL10 ( mean 197 . 1 pg/ml in pre , 158 . 7 pg/ml in post , p=0 . 04 ) and Angiogenin ( mean 51 . 7 pg/ml in pre , 37 . 6 pg/ml in post , p=0 . 02 ) .", "o-Vanillin significantly reduced the levels of INF-γ ( mean 1 . 6 pg/ml in pre , 1 . 1 pg/ml in post , p=0 . 04 ) , CCL24 ( mean 232 . 7 pg/ml in pre , 73 . 7 pg/ml in post , p=0 . 02 ) , CXCL1 ( mean 313 . 9 pg/ml in pre , 130 . 2 pg/ml in post , p=0 . 01 ) , Angiogenin ( mean 47 . 4 pg/ml in pre , 35 . 5 pg/ml in post , p=0 . 02 ) and VEGF-A ( mean 24802 . 1 pg/ml in pre , 20905 . 5 pg/ml in post , p=0 . 04 ) ( Figure 5D ) .", "Finally , we found a significant increase in the release of four factors in vehicle treated discs: INF-γ ( mean 1 pg/ml in pre , 1 . 45 pg/ml in post , p=0 . 02 ) , CCL11 ( mean 7 pg/ml in pre , 19 . 2 pg/ml in post , p=0 . 04 ) , CXCL1 ( mean 104 . 3 pg/ml in pre , 476 . 9 pg/ml in post , p=0 . 03 ) and CXCL9 ( mean 60 . 4 pg/ml in pre , 162 . 4 pg/ml in post , p=0 . 01 ) ." ], [ "We have previously demonstrated that curcumin and its metabolite o-Vanillin have senolytic activity toward senescent human IVD cells ( Cherif et al . , 2019 ) .", "Treatment with curcumin and o-Vanillin reduced SASP factors released and enhanced matrix synthesis in a pellet culture model ( Cherif et al . , 2019 ) .", "In this study , we aimed to compare the effects of a synthetic pure senolytic compound with a natural anti-inflammatory and senolytic compound , to determine a potential enhanced therapeutic effect with the latter .", "We used o-Vanillin instead of curcumin , as o-Vanillin has higher specificity and better bioavailability .", "For a synthetic and pure senolytic we chose RG-7112 , a drug with documented senotherapeutic effects in fibroblasts ( Weber , 2010; Laberge et al . , 2018 ) .", "Cells from degenerate discs or intact degenerate discs that correspond with the tissue targeted for treatment were used .", "Cells from degenrating tissue was used as it would be difficult to evaluate an effect of removing senescent cells in non-degenerate discs since they have have very few senescent cells .", "Here , we demonstrated that RG-7112 has a potent senotherapeutic and a strong proliferative effect on human IVD cells in vitro .", "Metabolic activity and extracellular matrix production were also increased in treated cultures .", "We verified that treatment specifically targets senescent cells by activating the caspase-3 apoptotic pathway .", "This is similar to the effect of UBX0101 , an analogue of RG-7112 that triggers apoptosis of senescent chondrocytes in a murine osteoarthritis model ( Jeon et al . , 2017 ) .", "Currently , phase I and II clinical trials are in progress to assess safety , tolerability and clinical effects of single dose ( NCT03513016 and NCT04129944 ) and both single and repeated doses ( NCT04229225 ) of intra-articular administration of UBX0101 in patients with moderate to severe painful knee osteoarthritis ( van Deursen , 2014; Vassilev et al . , 2004; Vu et al . , 2013 ) .", "Cellular senescence can be induced by replicative senescence or stress-induced premature senescence ( Wang et al . , 2016 ) .", "The pathway and resulting changes in the microenvironment surrounding the cells depends on inducing factor ( Frolov and Dyson , 2004 ) .", "In this study , we found gene expression modulation for a number of inflammatory and cell cycle genes following treatment with the two senolytics , o-Vanillin and RG-7112 .", "We evaluated differential gene expression in 96 pre-selected genes to determine the mechanisms by which the compounds mediate their effects .", "Our data demonstrate that 91 of the genes were expressed at a detectable level and a significant effect was found on 50% of the expressed genes , supporting the senolytic activity of the two compounds in vitro .", "RG-7112 decreased gene expression of CDK1A and MDM2 and increased expression of the E2F1 , RB factors , MAPK-14 , CDK2D , CCNB1 and CDC25c .", "o-Vanillin modulated gene expression of 40 genes including upregulation of cell cycle genes such as CDK6 , CDK2C , CDK2D , and CDC25c while expression levels of CDK2A , Cyclin A2 , Cyclin D1 and CCNB1 were decreased .", "Collectively , the data support the mechanistic action of RG-7112 to stabilize p53 and p21 by attenuating the MDM2-p53 interaction ( Weber , 2010; Tovar et al . , 2013; Henley and Dick , 2012; Che et al . , 2020 ) .", "Also , it confirms regulation of the RB–E2F1 pathway that releases E2F1 and activates genes involved in cell cycle regulation , DNA synthesis , and cell proliferation ( Andreeff et al . , 2016; Xu et al . , 2018 ) .", "Another example of a senolytic drug that interferes with the E3 ubiquitin ligase-MDM2-p53 mechanism is UBX0101 , which triggers apoptosis of senescent cells in articular cartilage and synovium in a murine osteoarthritis model ( Jeon et al . , 2017 ) .", "o-Vanillin treatment affected multiple pathways suggesting that o-Vanillin eliminates senescent cells both by apoptosis and non-apoptotic means .", "Recently , p16 deletion in NP cells from mouse discs with induced degeneration was shown to also upregulate the expression of cyclin-dependent kinases 4/6 , phosphorylated retinoblastoma protein , and transcription factor E2F1/2 ( Che et al . , 2020 ) .", "Clinical trials with RG-7112 for cancer treatment were limited by the high incidence of hematological toxicities ( Ray-Coquard et al . , 2012; Jung et al . , 2008 ) .", "Here , we used a lower concentration ( 5 μM ) and short exposure time of RG-7112 , in contrast to the high and toxic doses used in cancer therapy ( 20–1400 mg/m2 administrated daily for 10 days ) .", "This may prevent these side effects in patients treated for IVD degeneration .", "This hypothesis was verified by the clinical study of the RG-7112 analogue UBX0101 in patients diagnosed with painful osteoarthritis of the knee .", "An administered dose of 4 mg was safe and well-tolerated , and it improved pain scores , reduced SASP factors and disease-related biomarkers after a single dose ( Weber , 2010; Laberge et al . , 2018 ) .", "The activation of multiple pathways following o-Vanillin treatment and within the selected set of 91 genes implies that diverse biological processes are affected , which was also reported for other natural senolytics like Quercetin , Fisetin , and Dasatinib ( Pezet and McMahon , 2006; Wang et al . , 2008 ) .", "Although it is difficult to determine how these natural senolytics eliminate senescent cells and drive their therapeutic effects , the activation of several mechanisms increases their capacity to target the heterogeneous cellular states acquired by senescent cells after the initial growth arrest ( Frolov and Dyson , 2004 ) .", "We further sought to determine if the two senolytics had apparent effects on the SASP secretome .", "Conditioned media of pellet cultures was analyzed by cytokine array following treatment .", "Although donor variation was observed , notable changes between treatment groups were found indicating an overall decrease in the majority of the SASP factors analyzed .", "Similar decrease in the SASP factors was observed following p16 deletion in NP cells in mice with induced disc degeneration ( Yousefzadeh et al . , 2018 ) .", "An antiinflammatory effect was expected for o-Vanillin from previous studies ( Cherif et al . , 2019; Santosh Kumar et al . , 2002; Oliveira et al . , 2014; Shah et al . , 2019; Marton et al . , 2016 ) .", "However , our study documents , for the first time , antiinflammatory properties of RG-7112 in human IVD cells .", "We selected 19 factors from the initial screening associated with SASP and measured their concentrations using a Luminex immunoassay .", "In general , concentrations were decreased in response to treatment .", "RG-7112 significantly reduced the concentrations of IFN-γ , IL-6 and CCL24 while o-Vanillin in addition reduced factors IL-8 , CCL7 , CXCL1 , CXCL5 , CXCL9 , CXCL10 and VEGF-A .", "These factors are known for their implication in painful IVD degeneration ( Acosta and Gil , 2009; Acosta et al . , 2008; Phillips et al . , 2013 ) and for promoting senescence of surrounding cells ( Coppé et al . , 2010; Adams et al . , 2015 ) .", "For example , IL-8 and chemokines binding to the C-X-C motif chemokine receptors is needed for the establishment and maintenance of senescence .", "CCL7 expression has been reported to be concordant with IVD degeneration ( Binch et al . , 2014 ) and it has previously been described as a SASP factor involved in IVD cell senescence ( Burke et al . , 2002 ) .", "Finally , the decrease of VEGF-A indicates a possible role of o-Vanillin to block the mechanism of neovascularization .", "VEGF and its receptors have been proposed to be closely correlated with inflammation , chronic back pain and accelerated IVD degeneration ( Freemont et al . , 1997; Lu et al . , 2013; Peng et al . , 2006; Daly et al . , 2016; Gruber et al . , 2009; Thompson et al . , 1991 ) .", "These results consolidate the senolytic effect of the two compounds and suggest a potential role in reducing degenerative factors in human discs with o-Vanillin having a possible stronger and broader antiinflammatory effect .", "Animal models that replicate human disc pathology are limited due to the differences in anatomy , disc size , cell type , and loading ( Jin et al . , 2018 ) .", "Moreover , notochord cells are retained longer in the majority of animal species ( Grezella et al . , 2018 ) , further increasing the difference between humans and animals .", "Thus , we previously developed and validated an ex vivo intact human disc culture system to study the potential for biologic repair and regeneration ( Gawri et al . , 2011 ) .", "Matrix differences were quantified pre- and post-treatment by T1ρ weighted MRI that directly correlates with proteoglycan content in the IVDs ( Rosenzweig et al . , 2016; Mulligan , 2015 ) .", "When comparing the intensity in RG-7112 and o-Vanillin-treated IVDs , we observed an increased intensity post-treatment , while a decrease was observed in vehicle-treated control .", "This suggests that treatment with senolytics could increase proteoglycan content in human patients .", "It also validates our results from the pellet cultures where the proteoglycan content also increased significantly in the treated cultures .", "The improvement of T1ρ MRI values was validated by the consistent high levels of proteoglycan content in the treated IVDs .", "We further verified that the improved matrix was linked to removal of senescent cells .", "Indeed , both compounds significantly reduced the number of p16Ink4a-positive cells in treated discs .", "Moreover , we observed a slight but non-significant increase in proliferating cells .", "These findings correlate with the negative impact previously reported between the number of senescent cells and cell proliferation during IVD degeneration ( Hwang et al . , 2018 ) .", "Furthermore , culture media from control discs , showed no change in cytokine release whereas both compounds decreased levels of IL-6 , IL-7 , IL-8 , CXCL1 , CXCL6 , CCL2 , CCL22 , GROa/b/g , IGFBP-2 , TNSF-14 and OPN .", "Moreover , we observed a significant increase of EGF in the media from o-Vanillin treated discs .", "The positive effects of EGF on proteoglycan synthesis were first reported by Thompson et al . ; these effects were more pronounced in the NP ( Zhu et al . , 2017 ) .", "Together the results demonstrate an overall decrease in SASP factors following treatment .", "The difference in SASP compounds affected by the two drugs could be explained by the difference in selectivity and the specificity of the drugs ( Zhu et al . , 2017; Zhu et al . , 2016; Wilke et al . , 2006; Thompson et al . , 1990; Gawri et al . , 2014 ) .", "Finally , we compared the SASP factors release in pellet and disc culture media .", "The common SASP factors that were downregulated include INF-γ , IL-6 , CCL24 , CXCL1 , CXCL10 and Angiogenin .", "Interestingly , o-Vanillin also significantly reduced the levels of IL8 , CCL7 , CXCL5 , CXCL9 and VEGF-A .", "This could be due to the effect on the neighboring non-senescent cells .", "Consistent with prior literature , this decrease highlights the antioxidant and antiinflammatory properties of o-Vanillin ( Cherif et al . , 2019; Santosh Kumar et al . , 2002; Oliveira et al . , 2014; Shah et al . , 2019; Marton et al . , 2016 ) .", "Also , these decreases have been reported in several other natural compounds such as quercetin , fisetin , and piperlongumine ( Wang et al . , 2008; Thompson et al . , 1990 ) .", "Our findings suggest that both o-Vanillin and RG-7112 have the potential to be translated to treatment of painful IVD degeneration .", "As well , this study can be used as a base for the development of additional senolytic agents for lower back pain ." ], [ "Human lumbar IVDs were harvested from organ donors through a collaboration with Transplant Quebec .", "All procedures are approved by and performed in accordance with the ethical review board at McGill University ( IRB#s A04-M53-08B ) .", "Familial consent was obtained for each subject .", "Table 1 provides an overview of donor demographics and Table 2 provides detailed description of the used discs .", "Lumbar spinal columns were removed from organ donors , they were imaged radiographically and visually , and signs of degeneration were noted ( Page et al . , 1993; Mort and Roughley , 2007 ) .", "Discs were then dissected from the spinal column and used for cell and organ cultures .", "Nucleus pulposus ( NP ) and annulus fibrosis ( AF ) cells were isolated separately as described previously ( Cherif et al . , 2019; Kirkland and Tchkonia , 2017 ) .", "All cultures were mycoplasma free as verified by Mycoplasma PCR Detection Kit ( ZmTech Scientific ) .", "10 , 000 cells were seeded per well in 96-well tissue culture plates for 12 hr .", "Then , cells were incubated in the presence or absence of RG-7112 ( 5 μM ) for 6 h .", "Metabolic activity was evaluated by the Alamar blue assay as previously described ( Livak and Schmittgen , 2001 ) .", "We measured the metabolic activity of treated and untreated NP cells cultured in monolayer or in pellet from degenerate and non-degenerate discs to verify a non cytotoxic window of RG-7112 .", "Results are presented as a percentage of metabolic activity compared to their control .", "Experiments were performed ( 3–6 ) times in triplicate wells for each compound and concentration .", "300 , 000 NP cells/tube were collected by centrifugation at 500 × g for 5 min .", "Pellets were incubated in 1 mL DMEM ( 2 . 25 g/L glucose , 5% FBS , ascorbic acid ( 5 μM ) ( Sigma-Aldrich , Oakville , ON , Canada ) ) at 37°C and 5% CO2 .", "Culture media was changed every 3 days and collected for analysis .", "After 4 days , the pellet culture is stabilized and a single dose of the senolytics or vehicle was added to the culture media .", "Metabolic activity measures on pellet culture media from degenerate NP and AF cells , treated or not with RG-7112 , was performed to evaluate a potential cytotoxic effect of RG-7112 by comparing , in the same pellet culture , the effect of the treatment ( at day 21 ) to the control before treatment ( at day 4 ) .", "RNA was collected in TRIzol ( Thermo Fisher Scientific ) for gene expression experiments ( as described in Krock et al . , 2014 ) .", "For immunohistochemistry , pellets were fixed in 4% paraformaldehyde and cryopreserved , 5 μm sections were prepared and stained overnight at 4°C for p16Ink4a and 1 hr at room temperature for Ki-67 and caspase-3 primary antibodies as described ( Cherif et al . , 2019 ) .", "Coverslips were mounted using Aqua Polymount , and bright-field images were visualized .", "Ten fields , randomly distributed across the well , were analyzed , and the number of positive ( brown stained ) and total cells were counted to calculate the percentage of senescent , proliferative and apoptotic cells .", "Senescence , Apoptosis and proliferation assessments were performed on cells pellet cultured for 21 days by comparing , in the same subject , the senolytics-treated pellet to their respective controls .", "Isolated cells were expanded to Passage 1 ( P1 ) in monolayer cultures .", "P1 cells were then seeded at 20 , 000 or 10 , 000 cells per well in eight-well chamber slides ( Nunc Lab-Tek II Chamber Slide System ) and 96- well flat clear bottom black microplates ( Corning , NY ) respectively .", "Cells were serum-starved in DMEM with ITS ( 1X ) ( Thermo Fisher , Waltham , MA ) for 2 hr prior to treatment with 5 μM RG-7112 ( Selleck Chemicals , TX ) , 100 μM o-Vanillin ( Sigma-Aldrich , Oakville , ON , Canada ) or vehicle ( DMSO ( 0 . 01% , ( Sigma-Aldrich , Oakville , ON , Canada ) for 6 hr .", "Immunocytochemistry was performed as previously described ( Cherif et al . , 2019 ) .", "Apoptosis was detected using a commercial kit ( ab176749 , Abcam , Cambridge , MA ) according to the manufacturer’s instructions .", "Photomicrographs were acquired with a fluorescent Olympus BX51 microscope equipped with an Olympus DP71 digital camera ( Olympus , Tokyo , Japan ) .", "Caspase 3/7 activity of treated and untreated NP cells from degenerate and non-degenerate IVDs was measured using the Amplite Fluorimetric Caspase 3/7 Assay Kit ( AAT Bioquest , Sunnyvale , CA ) according to the manufacturer’s protocol .", "Cells were incubated with the caspase 3/7 assay solution , which contained caspase substrate ( Z-DEVD-R110 ) , at room temperature for 1 hr in the dark .", "Fluorescence intensity was then measured at 490 nm excitation and 525 nm emission .", "The results are expressed as a percentage of the mean of the control group ( set at 100% ) .", "Each experiment was performed in triplicate and carried out three times from each round of cell isolation .", "Sulphated glycosaminoglycans ( GAGs ) were quantified using the DMMB assay in the media of degenerate NP and AF pellets with or without RG-7112 treatment , performed as previously described ( Wickham , 2016 ) .", "Chondroitin sulfate was used to generate the standard curve .", "Conditioned media samples from days 7 , 10 , 14 and 21 were evaluated separately in triplicate into clear 96-well plates ( Costar , Corning , NY ) .", "sGAG release in media was normalized to the sGAG concentration in media at day 0 and then normalized to the untreated group .", "Following treatment , RNA was extracted using the TRIzol chloroform extraction method , as previously described ( Krock et al . , 2014 ) .", "Briefly , 500 ng of RNA was reverse transcribed using a qScript cDNA Synthesis Kit ( Quanta Biosciences , Beverly , MA ) with an Applied Biosystems Verti Thermocycler ( Thermo Fisher , Waltham , MA ) .", "RT-qPCR was performed using an Applied Biosystems StepOnePlus machine with TaqMan Fast Universal PCR Master Mix ( 2× ) and Custom TaqMan Array 96-Well Fast Plates ( Thermo Fisher , Waltham , MA ) .", "The evaluated genes are senescent and apoptotic genes included in the Human TaqMan Array , for Human Cellular Senescence ( Thermofisher scientific , Array ID: RPU62T7 , Catalog number: 4413255 ) .", "The Array Plate was customized to include 12 additional recently identified senescence and anti-apoptotic pathway genes84 .", "The 96 included genes are described in Supplementary file 1 .", "Each well of the TaqMan Array Plate was reconstituted using a mix of Fast Master Mix and a cDNA sample ( 20 ng ) to a set final volume ( 10 μl ) .", "Five plates were used for each group ( CTRL , RG-7112 and o-Vanillin ) , and fold-change in gene expression was calculated using the 2−ΔΔCt method 85 after normalizing to the housekeeping gene and vehicle-treated cells .", "We conducted a gene expression study of a pre-specified set of apoptotic and senescence-genes of interest ( Custom Taqman 96-Well Fast Plates , Thermofisher scientific ) in nucleus pulposus cells .", "This single-gene approach offers the advantage that highly relevant genes can be identified and tested first .", "The candidate genes examined with this approach allowed us also to identify the genes within the selected group that together carry out the drug response in disc cells .", "In order to mine the feature genes from different datasets , a Venn diagram analysis was conducted using Venny version 2 . 1 . 0 software ( Oliveros , 2007 ) .", "Differences in the expression levels of DEGs for treated and untreated groups were obtained , and the number of DEGs upregulated and downregulated were calculated .", "The odds ratio ( OR ) was calculated according to OR = ( DEGs_o-Vanillin * DEGs_RG-7112/DEGs_o-Vanillin * NonDEGs_RG-7112 ) / ( NonDEGs_o-Vanillin * DEGs_RG-7112/nonDEGs_o-Vanillin * NonDEGs_RG-7112 ) .", "Heatmaps of gene expression pattern were constructed using unsupervised hierarchical clustering using Euclidean distance metric and complete linkage clustering method of the 43 differentially expressed genes in response to either RG-7112 or o-Vanillin ( p<0 . 05 ) treatment of degenerate NP cells pellet .", "Differences in gene expression between o-Vanillin , RG-7112 and control groups were respectively compared via unpaired t-tests using the R package .", "Genes for which met the p<0 . 05 cut-off point were selected as DEGs , following which gene expression profiles of DEGs were visualized ( heatmaps ) via the ‘ggplots’ in R package 87 and were represented in the heatmaps as Z-scores , which is: ( expression value - mean expression value across samples ) /divided by the standard deviation .", "Colors , ranging from blue to grey then red , for each treatment represents the average fold change of each subject in that group .", "Differentially expressed genes were subjected to Ingenuity Pathways Analysis ( IPA ) ( Ingenuity Systems , Redwood City , CA ) and used as a starting point for building biological networks .", "This analysis uses computational algorithms to identify networks consisting of focus genes ( genes that were present in our list of 91 genes ) and their interactions with other genes ( ‘non-focused’ ) in the knowledge base .", "Scores were calculated for each network according to the fit of the network to the set of focus genes and used to rank networks on the Ingenuity analysis .", "IPA uses the genes from the highest-scoring network to extract a connectivity pathway that relates candidate genes to each other based on their interactions .", "The involved function and disease significantly associated with these candidates’ genes were shown .", "To generate the networks , significant pathways were filtered by p-value ( α ) <0 . 05 and activation Z-score > −2 or >2 , set as the cut-off values and representing a significant deactivation or activation , respectively .", "Colors are based on log2 fold changes on these genes .", "To rank networks of the IPA , p-scores were calculated from p-values .", "For example , for n genes in the network and f of them are Focus Genes .", "The p-value is the probability of finding f or more Focus Genes in a set of n genes randomly selected from the Global Molecular Network calculated using Fisher’s exact test .", "Since interesting p-values are typically quite low , it is visually easier to concentrate on the exponent and the p-score is defined as p-score = -log10 ( p-value ) .", "Networks with a score ≥2 have at least 99% confidence that indicate a 1/100 chance that the focus genes are in a network because of random chance .", "Intact lumbar spines were x-rayed , and discs were selected for the study based on the grading system described by Wilke et al . ( Page et al . , 1993 ) .", "Discs with a Wilke grade of 2 were included for this study .", "Three IVDs from the same spine ( n = 4 spines , 12 discs ) were isolated and cultured as previously described ( Gawri et al . , 2011; Krock et al . , 2014; Rosenzweig et al . , 2016 ) .", "Disc characteristics are described in Table 3 .", "Isolated discs were scanned by MRI before and after treatment at day 28 as described by Rosenzweig et al . , 2018 .", "Images were obtained on a 7T Bruker BioSpec 70/30 USR ( Bruker Biospin , Milton , ON , Canada ) with the high-performance mini-imaging kit gradient upgrade AVIII electronics ( Bruker ) and a Bruker-issued T1ρ-RARE pulse sequence , as previously established ( Rosenzweig et al . , 2018; Mulligan , 2015 ) .", "Briefly , 3D images were acquired and T1ρ values were quantified using the MIPAV software ( NIH Center for Information Technology , Bethesda , MD , USA ) .", "T1ρ values of the same region of interest ( ROI ) of ʹbeforeʹ and ʹafterʹ treatment scans of each disc were normalized to the surrounding culture medium using editing features in MIPAV software .", "After 4 days of culture and one media change , single injection of the discs with vehicle , RG-7112 ( 5 μM/g disc ) or 100 μM o-Vanillin ( 100 μM/g disc ) in a total volume of 200 μl PBS was performed as previously described ( Krock et al . , 2014; Rosenzweig et al . , 2016 ) .", "Discs were then cultured in DMEM supplemented with 1x Glutamax , 50 μg/ml gentamicin and 1% FBS for 28 days .", "Media was changed and collected every 4 days .", "On day 28 and after MRI imaging , discs were prepared for immunohistochemistry as described below ( See Intact IVD Tissue Immunohistochemistry ) .", "Conditioned media was collected at each change and frozen as individual samples at −80°C for protein analysis .", "Preliminary measures to delimitate the NP region of interest ( ROI ) were calculated using 11-disc images from random organ donors .", "A contour was drawn around the disc using a Wacom Intuos Pro tablet and stylus ( Wacom , Japan ) .", "This polygon was used to measure total disc voxel intensity .", "The NP area was created by reshaping the original disc contour to 30% of its frontal size and 40% of its sagittal size centered around the middle of NP area , which was calculated to be 10% shift below the center of the disc ( Figure 2—figure supplement 1B-D ) .", "The T1ρ images were manually cropped around the perimeter of the IVDs .", "The average of the T1ρ values was calculated within the ROIs for the vehicle and the injected discs slices per image .", "Heat maps representing signal intensity were created using the MIPAV software .", "Age of an individual donor is specified in years .", "The level of the disc injected with either DMSO , O-Vanillin , and RG-7112 from the lumbar region is indicated .", "Discs were graded based on Wilke et al . , 2006 ( Page et al . , 1993 ) .", "Disc height was determined by averaging the dorsal , ventral and midsection disc height .", "No differences in grade or height were found between groups prior to treatment .", "Media from the Degenerate NP Pellet used in gene expression analysis was collected from day 4 to day 21 and pooled separately for each group ( vehicle , o-Vanillin and RG-7112 ) and subject .", "Then , they were analyzed and compared to evaluate the effect of the two senolytics on SASP release .", "Disc media collected from day 4 ( pre-treatment ) and day 28 ( post-treatment ) were analyzed separately for each disc and each factor .", "The Human Cytokine Antibody Array C5 ( RayBiotech , Inc ) was used for semi quantitative detection of 80 proteins according to manufacturer’s instructions and as previously described ( Krock et al . , 2014 ) .", "Intensity units were detected by the chemiluminescence using an ImageQuant LAS4000 Image Analyzer ( GE Healthcare , Baie d'Urfe , QC , Canada ) and analyzed with ImageQuant TL array analysis software ( GE Healthcare ) .", "The relative quantity of each factor present in each media sample was normalized to the positive and negative controls included on the array .", "Mean relative concentration of each factor of treated and control groups were then calculated .", "Data was normalized to secretion of vehicle injected discs from the same donor spine .", "A list of included cytokines is provided in Supplementary file 2 .", "Nineteen proteins were selected for analysis by Luminex multiplex assay according to manufacturer's instructions .", "A limitation in the number of factors we could measure was the incompatibility of some factors to be measured simultaneously as indicated by the supplier .", "Concentrations ( pg/mL ) ( INF-γ , TNF-α , IL-1α , IL-1β , IL6 , IL8 , CCL5 , CCL7 , CCL11 , CCL24 , CCL26 , CXCL1 , CXCL5 , CXCL9 , CXCL10 , CXCL11 , CX3CL1 , VEGF-A and Angiogenin ) were measured in 40 μl media .", "Median fluorescence intensity ( MFI ) from microspheres was acquired with a BD FACSCanto II and analyzed in FlowCytomix Pro2 . 2 . 1 software ( eBioscience ) .", "Concentration of each analyte was obtained by interpolating fluorescence intensity to a seven-point dilution standard curve supplied by the manufacturer .", "For intact IVDs , post-MRI analysis , a 2 mm wide sagittal tissue segment from the center of the IVD was fixed in periodate lysine paraformaldehyde ( PLP ) fixative overnight at 4°C .", "Samples were then washed in PBS and decalcified using Shandon TBD1 Decalcifier solution ( ThermoFischer Scientific ) over 72 hr at 4°C , changing solution each day .", "Tissue segments were washed in PBS and placed in 70% ethanol prior to paraffin embedding .", "Sections of 5 µm were cut and mounted on glass slides .", "All sections were heated on a hot plate at 55°C for 45 min and deparaffinized and rehydrated .", "Next , sections were stained with safranin-O/fast green ( Sigma-Aldrich , Oakville , ON , Canada ) and with antibodies against p16Ink4a and Ki-67 and counter stained using the DAB detection IHC Kit ( ab64264 , Abcam , Cambridge , MA ) following the manufacturer’s instructions .", "All images were acquired using a Zeiss Axioskop 40 and an AxioCam MR ( Zeiss ) and processed using AxioVision LE64 software ( Zeiss ) .", "The data was analyzed using Graph Prism 8 ( Graph Pad , La Jolla , CA ) .", "Analysis was performed by two-tailed Student's t test for comparison between two groups and a multiple pairwise comparison ( Analysis of Variance ( ANOVA ) was used to evaluate the variance between multiple groups with Turkey’s post hoc test .", "A p value < 0 . 05 was considered statistically significant ." ] ]

[ "Cellular senescence is a contributor to intervertebral disc ( IVD ) degeneration and low back pain .", "Here , we found that RG-7112 , a potent mouse double-minute two protein inhibitor , selectively kills senescent IVD cells through apoptosis .", "Gene expression pathway analysis was used to compare the functional networks of genes affected by RG-7112 , a pure synthetic senolytic with o-Vanillin a natural and anti-inflammatory senolytic .", "Both affected a functional gene network related to cell death and survival .", "O-Vanillin also affected networks related to cell cycle progression as well as connective tissue development and function .", "Both senolytics effectively decreased the senescence-associated secretory phenotype ( SASP ) of IVD cells .", "Furthermore , bioavailability and efficacy were verified ex vivo in the physiological environment of degenerating intact human discs where a single dose improved disc matrix homeostasis .", "Matrix improvement correlated with a reduction in senescent cells and SASP , supporting a translational potential of targeting senescent cells as a therapeutic intervention ." ]

[ "Pain in the lower back affects about four in five people during their lifetime .", "Over time , the discs that provide cushioning between the vertebrae of the spine can degenerate , which can be one of the major causes of lower back pain .", "It has been shown that when the cells of these discs are exposed to different stress factors , they stop growing and become irreversibly dormant .", "Such ‘senescent’ cells release a range of proteins and small molecules that lead to painful inflammation and further degeneration of the discs .", "Moreover , it is thought that a high number of senescent cells may be linked to other degenerative diseases such as arthritis .", "Current treatments can only reduce the severity of the symptoms , but they cannot prevent the degeneration from progressing .", "Now , Cherif et al . set out to test the effects of two different compounds on human disc cells grown in the laboratory .", "One of the molecules studied , RG-7112 , is a synthetic drug that has been approved for safety by the US Food and Drug Administration and has been shown to remove senescent cells .", "The other , o-Vanillin , is a natural compound that has anti-inflammatory and anti-senescence properties .", "The results showed that both compounds were able to trigger changes to that helped new , healthy cells to grow and at the same time kill senescent cells .", "They also reduced the production of molecules linked to inflammation and pain .", "Further analyses revealed that the compounds were able to strengthen the fibrous matrix that surrounds and supports the discs .", "Cherif et al . hope that this could form the basis for a new family of drugs for back pain to slow the degeneration of the discs and reduce pain .", "This may also have benefits for other similar degenerative diseases caused by cell senescence , such as arthritis ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "ecology", "plant biology" ]

[ [ "Many flowering plants require pollen transport by animals to ensure reproductive success ( Ollerton et al . , 2011 ) .", "These pollinating animals are often insects ( Kevan and Baker , 1983 ) , such as bees .", "To encourage pollinator visits flowering plants create floral displays ( Raguso , 2004; Leonard et al . , 2012 ) which produce diverse floral cues in different sensory modalities ( Kevan and Lane , 1985; Bhagavan and Smith , 1997; Whitney et al . , 2009; Hempel de Ibarra and Vorobyev , 2009; von Arx et al . , 2012; Lawson et al . , 2017b ) .", "These signals allow pollinators to find and locate flowers ( Spaethe et al . , 2001; Chittka and Spaethe , 2007 ) , and also allow pollinators to learn and recognise them ( Heinrich , 1979; Raine and Chittka , 2008 ) .", "Bees and other pollinators adjust their foraging behaviour to favour visits to more rewarding species found in their environment ( Heinrich , 1979 ) , avoiding ‘mistake visits’ to less rewarding flowers in order to enhance their foraging success ( Raine and Chittka , 2008 ) .", "Similarly , a floral display that is easily learnt and distinguished from others in its environment ensures greater visitation to the flower ( Galen and Newport , 1988; Lynn et al . , 2005 ) and thus greater reproductive success ( Ashman et al . , 2004; Bell et al . , 2005; Schiestl and Johnson , 2013 ) .", "Identifiable floral cues are therefore critical to both plant and pollinator .", "One flower cue bees can use to recognise flowers is floral temperature ( Whitney et al . , 2008; Hammer et al . , 2009; Norgate et al . , 2010 ) .", "Warming of flowers can occur due to floral thermogenesis ( Seymour and Schultze-Motel , 1997; Seymour and Matthews , 2006; Seymour et al . , 2009 ) , but is more frequently the result of captured solar radiation ( Totland , 1996; Sapir et al . , 2006; Rejšková et al . , 2010; Zhang et al . , 2010; Atamian et al . , 2016 ) .", "The absorption of sunlight and heat loss is influenced by pigmentation ( Kay et al . , 1981; Sapir et al . , 2006 ) , structure ( Rejšková et al . , 2010; Whitney et al . , 2011 ) and heliotropism ( Totland , 1996; Zhang et al . , 2010; Atamian et al . , 2016 ) , all of which will contribute to how much a certain flower will heat up in given conditions .", "This can create differences in temperature between different flower species ( Rejšková et al . , 2010; Kovac and Stabentheiner , 2011 ) .", "Using thermal detectors in their antennae and tarsi ( Heran , 1952 ) , bumblebees ( Dyer et al . , 2006; Whitney et al . , 2008 ) , honeybees ( Hammer et al . , 2009; Kovac and Stabentheiner , 2011 ) and stingless bees ( Norgate et al . , 2010 ) can distinguish flowers based on differences in overall temperature .", "Greater differences in temperature between flowers appear to be easier for bees to detect ( Hammer et al . , 2009 ) , although bees have been shown to be able to detect differences in temperature as little as 2°C ( Heran , 1952 ) .", "Floral temperature can also function as a floral reward by keeping pollinators warm while they feed ( Rands and Whitney , 2008; Herrera , 1995 ) .", "Warmer flowers help insect visitors maintain a body temperature above their minimum threshold for flight ( Heinrich , 1979a; Heinrich , 1979c ) .", "This allows pollinators to forage and collect nectar in colder conditions ( Herrera , 1995 ) , and avoid the metabolic costs they might incur if they have to warm themselves for flight ( Rands and Whitney , 2008 ) .", "Therefore , floral temperature cues are likely to be salient to insect pollinators .", "As well as being sensitive to differences between the flower and its environment ( Whitney et al . , 2008; Hammer et al . , 2009 ) , insects should also be sensitive to differences within a floral display .", "When flowers are observed using infrared thermography ( thermal imaging ) , it is apparent that floral temperature is not necessarily distributed uniformly across the flower surface ( Rejšková et al . , 2010; Dietrich and Körner , 2014; Ladinig et al . , 2015; Atamian et al . , 2016 ) .", "It has not been investigated whether any pollinators can learn to recognise flowers based on which parts of the flower are hotter or colder , which will determine the flower’s temperature pattern .", "Understanding whether pollinators can detect temperature patterns within the flower will improve our understanding of how pollinators interact with flowers , and how floral displays have evolved .", "In this study , we investigate the capacity of these floral temperature patterns to function as a floral cue .", "We demonstrate floral temperature patterns are common by taking thermographs of the displays of 118 plant species , that are visited by a range of pollinator groups and show a variety of flower forms , under good weather conditions .", "We further ask whether bumblebees , frequently a generalist pollinator group ( Heinrich , 1976; Williams , 1989; Goulson et al . , 2005 ) , can learn to distinguish rewarding from non-rewarding artificial flowers , based solely on temperature pattern differences comparable to those observed in real flowers ." ], [ "The thermographs of flowers of 118 species in different taxa reveal the variety of temperature patterns of different shapes , sizes and locations that pollinators may encounter ( Figure 1 and Supplementary file 1 ) .", "Some species had little to no detectable temperature differences across their surface , for example Dahlia coccinea and Pelargonium echinatum ( Figure 1 ) .", "However , most species observed showed some part of the flower that differed in temperature from the rest of the flower , thus displaying a temperature pattern ( Figure 1 and Supplementary file 1 ) .", "Most often there was a temperature contrast between the flower centre and its periphery , although the extent and shape of contrasting regions varied greatly .", "In such cases the centre of the flower was often hotter , as in Bellis perennis and Geranium psilostemon , ( but not always , as with Papaver rohoeas or Hydrangea macrophylla ) ( Figure 1 ) .", "Warming or cooling of a protruding section of the flower , such as ‘landing pad’ petals of zygomorphic flowers such as Crinum , or the reproductive structures of Papaver ( Figure 1 ) , also frequently created contrasting regions of temperature .", "Flowers of all sizes showed temperature patterns , such as the large Hermerocallis ‘autumn red’ and small Bellis perennis flowers ( Figure 1 ) .", "Of the 118 species thermographed , 65 species ( 55% ) showed within-flower temperature differences of at least 2°C ( Supplementary file 1 ) .", "So more than half the species observed show temperature contrasts which at least bees would be able to detect ( Heran , 1952 ) .", "Within these 65 species the average temperature difference was 4 . 89°C ± 2 . 28 ( mean ± SD ) .", "While the temperature patterns that can vary greatly between species , we must determine whether pollinators can use such differences in temperature patterns to inform foraging in order to show these differences can be used as floral cues .", "We carried out two conditioning experiments investigating the ability of bumblebees to detect temperature patterns .", "Bumblebees are an appropriate choice of pollinator for investigating whether any pollinators can respond to the observed diversity of temperature patterns .", "Many bees are generalist pollinators ( Waser et al . , 1996; Fenster et al . , 2004 ) , and it is known that generalist bees will visit many flower forms and families ( Heinrich , 1976; Heinrich , 1979a; Williams , 1989; Fenster et al . , 2004; Fontaine et al . , 2008; de Vere et al . , 2017 ) .", "Bees also visit flowers which they may not pollinate to carry out larceny ( Inouye , 1980; Manning et al . , 2002; Castellanos et al . , 2004; Fenster et al . , 2004 ) .", "There is great variation in size and tongue length both within and between bumblebee species , with long tongued species tending to be specialist , and shorter tongued bumblebees ( such as Bombus terrestris ) tending to be generalist ( Heinrich , 1976; Heinrich , 1979a; Williams , 1989; Goulson et al . , 2005 ) .", "Bumblebees also occur all over the globe ( Heinrich , 1979a ) .", "Thus , bumblebees , both as individual species and as a large functional group , will experience a large portion of the diversity of floral temperature patterns observed in our survey .", "This includes some of the species with the most contrasting temperature patterns , and flowers showing near to no temperature pattern at all ( Williams , 1989; Goulson et al . , 2005; Larsson , 2005; Fontaine et al . , 2008; Smith , 2010 ) .", "Additionally , the temperature sensitivities of bumblebees are understood better than many other pollinators ( Heinrich , 1979a; Dyer et al . , 2006; Rands and Whitney , 2008; Whitney et al . , 2008 ) and techniques for bumblebee conditioning experiments used here are well established ( Dyer and Chittka , 2004; Raine and Chittka , 2008 ) , making them ideal for investigating pollinator responses to floral displays .", "In each of the two experiments bumblebees B . terrestris were presented with artificial flowers , either small ( 40 mm in diameter ) or large size ( 85 mm in diameter ) depending on experiment ( Figure 2a and b ) .", "The two experiments with different sized flowers allow us to determine the impact of the size of temperature patterns on the identification of rewarding flowers .", "By using electrical heating elements , we were able to present differing temperature patterns on both sets of artificial flowers .", "On each flower size , these temperature patterns had two variants in layout and shape , but did not differ in either overall flower temperature , within-flower temperature contrast , or total area heated , to exclude other means by which bees could recognise variants .", "Small artificial flowers produced temperature patterns where either the edges of the flower’s lid were hotter than the rest of the flower ( the ‘circle pattern’ ) , or a rectangular section across the middle of the centre of the flower’s lid was hotter than the rest of the flower , ( the ‘bar pattern’ ) ( Figure 2c ) .", "These circle- and bar-shaped temperature patterns were comparable to those displayed by real flowers: flowers with colder centres and hotter peripheries , such as Papaver rhoeas and Hydrangea macrophylla ( Figure 1 ) , relating to the circle pattern; flowers with hot centre and colder periphery , such as Bellis perennis , Geranium psilostemon or Eschscholzia californica ( Figure 1 ) relating to the bar pattern .", "Consequently , the differences in temperature patterns between small artificial flowers reflected a large aspect of temperature pattern diversity .", "Both of the large artificial flower variants used had hotter flower centres: one where the heated parts radiated out from the centre in a ‘cross pattern’ , and one where heated parts spanned across the flower centre in another ‘bar pattern’ ( Figure 2d ) .", "These larger temperature patterns are similar to those of flowers with hotter centres but differ in the size and shape of the hotter regions of which there are several ( compare varieties of Cistus and Geranium , Figure 1 and supplementary materials ) .", "Flower naïve bumblebees , Bombus terrestris audax , were allowed to visit artificial flowers which provided a drop of sucrose solution ( rewarding flowers ) , or water ( nonrewarding flowers ) , in the centre of the flower hidden in a small well ( Figure 2e and f ) .", "There were three test groups:", "( i ) ‘Bar rewards’ group where the bar temperature pattern was rewarding , and the distractor pattern nonrewarding ( cross in large or circle in small flowers ) ;", "( ii ) ‘Circle/cross rewards’ group where reciprocally the circle or cross temperature pattern was rewarding , and the distractor nonrewarding ( bar pattern ) ;", "( iii ) ‘Control’ group where heating elements in the flowers were disconnected , and thus neither rewarding or nonrewarding flowers showed temperature patterns .", "The relationship between foraging success ( probing or feeding from flowers rewarding with sucrose solution , as well as not probing when visiting on nonrewarding flowers offering water ) and the experience bees had of the flowers ( number of flower visits the bees made ) was compared between the three test groups .", "When foraging on small artificial flowers , bumblebees learnt to identify rewarding flowers when they differed in temperature patterns ( Figure 3 ) , but did not learn in the control group .", "When models of bumblebee foraging success in the learning phase were compared , the relationship between success and experience varied between test groups ( Figure 3a ) , with models that allowed test group to have an interacting effect with experience producing a lower AIC ( −226 . 3 vs . −216 . 9 , ΔAIC = 9 . 4 ) and a better fit ( Δdeviance = 13 . 4 , df = 2 , p < 0 . 01 ) than models that did not .", "Bees from the control group foraged randomly throughout the experiment maintaining a 50% success rate , experience having no effect on success ( AIC −83 . 3 vs . −83 . 0 ΔAIC = 0 . 3; Δdeviance = 1 . 6 , df = 1 , p = 0 . 201 ) .", "When flowers differed in temperature pattern , bees began with a success rate comparable to the control group but improved with experience; this occurred regardless of which temperature pattern corresponded with rewards ( Circle rewards: AIC −92 . 1 vs . −68 . 5 , ΔAIC = 23 . 6; Δdeviance = 25 . 6 , df = 1 , p < 0 . 001 . Bar rewards: AIC −50 . 5 vs . −28 . 0 ΔAIC = 22 . 5; Δdeviance = 24 . 5 , df = 1 , p < 0 . 001 ) .", "When the conditioned preference was tested in nonrewarding tests , bees in the bar and circle reward groups made more correct visits than the control group ( F2 , 33 = 23 . 8 , p < 0 . 001 , Figure 3b ) .", "These results demonstrate that bumblebees can learn and alter foraging decisions based on differences in temperature patterns .", "Bumblebees also showed the ability to perceive temperature patterns in large-sized flowers ( Figure 4 ) , although test groups showed similar shaped relationships between success and experience .", "Models including an interaction between test groups and those that did not , were comparable in terms of AIC ( Richards , 2008 ) ( AIC −290 . 88 vs . −287 . 72 ΔAIC = 3 . 16 ) , but were a better fit ( Δdeviance = 7 . 16 , df = 2 , p = 0 . 03 ) .", "Nevertheless , which test group bees were in still had a significant effect on the level of success achieved ( AIC −287 . 72 vs . −266 . 71 ΔAIC = 21 . 01 , Δdeviance = 25 . 01 , df = 2 , p < 0 . 001 ) , with Bar and Cross reward groups achieving a greater level of success than the control .", "Thus , the presence of temperature patterns improved bumblebee foraging success , indicating their ability to use these larger patterns to distinguish flowers .", "The increase in success rate in the control group in this experiment ( unlike in the previous small flower experiment ) can be explained by the experimental set-up leading to spatial preferences within the arena that developed during training .", "Three rewarding and three nonrewarding flowers were present in the arena during the large flower experiment due to space constraints , and there was a reduced ability for random re-arrangement of each flower due to wiring constraints .", "Bees have a great capacity for spatial learning ( Burns and Thomson , 2006; Robert et al . , 2017 ) , and therefore the control group may have learnt to identify within each foraging bout which regions of the arena contained more rewarding flowers .", "However , even with this spatial learning , the presence of temperature patterns improved bumblebee foraging success on the large artificial flowers ." ], [ "The results of both of the conditioning experiments showed that temperature pattern differences improved the ability of bumblebees to distinguish between rewarding and nonrewarding artificial flowers ( Figures 3 and 4 ) .", "This suggests that floral temperature patterns can function as a floral cue .", "The main variation observed in floral temperature patterns were between flowers with hot centres and cold edges and vice versa ( see Figure 1 and Supplementary file 1 ) , and bees foraging on the small artificial flowers were observed to be able to distinguish similar differences ( Figure 3 ) .", "Furthermore , bees foraging on large artificial flowers could distinguish between two differently-shaped patterns where the centre of the flower was hotter ( Figure 4 ) , demonstrating that bumblebees can detect more detailed aspects of temperature signals .", "Artificial flowers showed within-flower temperature differences similar to that of real flowers ( Figure 2 and Supplementary file 1 ) .", "Real flowers can show a greater degree of variety in the temperature differences than those used in our experiments , which represented flowers showing the clearest temperature patterns ( Supplementary file 1 ) .", "However , bees have been shown to have a high sensitivity to differences in temperature ( Heran , 1952; Dyer et al . , 2006 ) and are therefore likely to detect the lower temperature differences as well as the higher .", "The use of floral temperature cues might not be limited to bumblebees , since other pollinating insects have been observed to detect and respond temperature differences between different flowers ( Sapir et al . , 2006; Kleineidam et al . , 2007; Hammer et al . , 2009; Kovac and Stabentheiner , 2011 ) , and therefore may also be able to use temperature patterns as cues .", "Furthermore , it did not appear that temperature patterns were limited to flowers associated with bumblebees .", "Temperature patterns appear to be a floral phenomenon , rather than a ‘bee flower’ phenomenon .", "Several of the Asteraceae ( which are known to be visited by a variety of insects including bumblebees , Goulson et al . , 2005 ) , as well as primarily bee pollinated flowers such as Eschscholzia californica ( Smith , 2010 ) , were among those that produced the most contrasting temperature patterns ( Supplementary file 1 ) .", "However , other plants attracting similar pollinators were also observed to produce little temperature contrast across their surface .", "Additionally , some plants associated with moths and hummingbirds , such as Crinium and Crocosmia ( Manning et al . , 2002; Goldblatt and Manning , 2006 ) , were also observed to produce contrasting temperature patterns ( Supplementary file 1 ) .", "Demonstrating that floral temperature patterns could present a floral cue raises the question as to how they might be generated , and there are several potential mechanisms .", "Different flower species differed in which structures generated temperatures patterns ( Figure 1 and Supplementary file 1 ) .", "Some patterns are created by hotter or colder parts of the petals , and others by hotter or colder reproductive structures .", "The variation in shape and contrast of temperature patterns between different plants derived from the same species ( i . e . cultivars , subspecies ) suggest that small changes in floral characters can influence temperature patterns .", "This is perhaps most evident in the various Cistus , Gazania and Knautia flowers thermographed ( Supplementary file 1 ) .", "Floral morphology appears to influence temperature pattern generation , as structures in a position more likely to capture light tended to be warmer ( e . g . the exposed petals in the landing pad of Crinum ) .", "Structures that were more densely packed , and might retain heat better were often warmer ( such as the florets of composite inflorescences ) .", "Likewise , colour differences in the visible spectrum often appeared to occur alongside temperature differences ( Figure 1 and Figure 1—figure supplement 1 ) .", "Such observations are in agreement with our understanding of the influence of solar radiation ( Totland , 1996; Sapir et al . , 2006; Rejšková et al . , 2010; Kovac and Stabentheiner , 2011 ) and floral structure ( Miller , 1986 ) on floral temperature .", "Additional potential influences on temperature include floral metabolism ( Seymour and Schultze-Motel , 1997; Seymour and Matthews , 2006 ) , active heat loss by transpiration ( Gates , 1968; Tsukaguchi et al . , 2003 ) and petal epidermal cell shape effects ( Whitney et al . , 2011 ) .", "Further study of how these influences differ across the floral surface will help us gain a greater understanding of floral temperature pattern generation .", "Ecological factors might influence a flower’s capability to generate temperature patterns pollinators can detect .", "The amount of sunlight captured limits floral warming in non-thermogenic plants ( Totland , 1996; Rejšková et al . , 2010; Zhang et al . , 2010 ) .", "While Rejšková et al . , 2010found that artificially shaded Bellis perennis flowers maintained temperature patterns , overall temperature of the flower and temperature contrast between regions decreased , and shaded Anemone nemorosa cooled to even temperature across the flower .", "Pollinators may only be able to use temperature pattern cues during sunny weather and when flowers grow in open non-shady environments .", "Understanding how floral temperature patterns change with environmental conditions , and the sensitivity of pollinators to changing temperature patterns ( including how small a contrast in temperature that pollinators are able to detect ) , will reveal the level of influence that environmental factors have on temperature patterns .", "It may be that flowers that grow in less sunny climates and in shadier habitats may not be under strong selection to produce complex thermal cues such as temperature patterns .", "Plants in these conditions may seldomly generate temperature patterns ( Rejšková et al . , 2010 ) , and pollinators may not be able to detect or respond to these patterns .", "Several of the flower species that produced the greatest contrasts in temperature within the flower are associated with hot and dry climates ( e . g . Osteospermum and Dimorphotheca species ) or with more open environments ( e . g . Geranium psilostemon and Eschscholzia californica , Supplementary file 1 ) , even though all samplings took place in similar conditions .", "This may reflect such plants experiencing greater selection to produce thermal cues .", "Flowers are multimodal displays - they produce many different kinds of cues simultaneously ( Raguso , 2004; Leonard et al . , 2012 ) , despite pollinators often being able to distinguish flowers based on a single cue ( Bhagavan and Smith , 1997; Dyer and Chittka , 2004; Clarke et al . , 2013 ) .", "The benefits of this multimodality are only just starting to be understood ( Leonard et al . , 2012; Kaczorowski et al . , 2012; Leonard and Masek , 2014; Lawson et al . , 2017b; Lawson et al . , 2017a ) .", "‘Novel’ sensory cues , such as floral electrostatic fields , have been found to be equally beneficial in foraging maintaining accuracy ( Clarke et al . , 2013 ) .", "The discovery of another floral cue that bumblebees can use to recognise flowers , temperature patterns , encourages further investigation into this apparent redundancy in floral signalling and the potential benefits multimodal signalling confers .", "The frequent overlap of temperature patterns with structural and visual elements of the floral display perhaps makes them ideal for investigation of how floral signals interact within multimodal displays .", "Thermal imaging of floral temperature reveals that flowers show a diversity of temperature patterns .", "It is known that bees can distinguish differences in temperature between flowers ( Whitney et al . , 2008 ) and using temperature as a reward ( Rands and Whitney , 2008 ) , and we have shown here that bumblebees can use these floral temperature patterns as a cue to recognise flowers and make informed foraging choices based upon them .", "This ability does not seem to be influenced by the size of the flower and its floral temperature pattern .", "Thus , floral temperature patterns may be added to the growing number of floral cues ( Raguso , 2004; Leonard et al . , 2012 ) that pollinators , at least bumblebees , may be able to utilise to identify more rewarding flowers in their environment ." ], [ "Thermographs of floral blooms ( flowers or flowering heads ) were taken in Royal Fort Gardens and the University Botanic Garden , Bristol and in the National Botanic Garden of Wales , Carmarthen .", "Species were selected with the aim of sampling flowers visited by a wide range of floral visitor groups and as broad range of floral shapes , colours and phylogeny as possible .", "Due to thermal camera limitations in minimum area of measurement ( I . T . C , 2008; Usamentiaga et al . , 2014 ) very small flowers , when not part of a compound inflorescence , could not be sampled .", "Cultivars and subspecies were also thermographed .", "Any additional cultivars and subspecies were counted as the same species as the one they were derived from when calculating temperature pattern occurrence or average within flower temperature difference .", "In such cases whichever variant showed the lowest temperature difference was used , providing more conservative estimates .", "Thermographs were taken on clear and sunny days , or inside a controlled glasshouse with near-UV permeable windows , while in sunlight .", "Mean ambient temperature for during sampling was 14 . 3°C ( SD 4 . 7 ) .", "More details on the weather conditions are available in Supplementary files 2 and 3 .", "All thermographs were taken with a FLIR E60bx thermal camera ( FLIR systems , Inc . , Wilsonville , USA ) , to a standard acceptable for I . T . C . guidelines ( I . T . C , 2008; Usamentiaga et al . , 2014 ) .", "The thermal infrared emissivity was set at 0 . 98 .", "This value is the estimate for vegetation ( Rubio , 1997; López et al . , 2012 ) and has been used for floral tissue ( Rejšková et al . , 2010; Dietrich and Körner , 2014 ) .", "For the sake of efficiency , reflected temperature was kept at 23°C for all thermographs , due to the high emissivity of floral tissue this would have a minimal effect on temperature readings .", "All thermographs were viewed and measurements taken using in FLIR tools software ( Flir Systems INC , 2015 ) .", "Using the point measurement functions , the temperature differences between the hottest and coldest points on the flower were measured and used to calculate the temperature range across each flower .", "Established bumblebee differential conditioning techniques ( Dyer and Chittka , 2004; Raine and Chittka , 2008; Whitney et al . , 2008; Whitney et al . , 2009; Clarke et al . , 2013 ) were used to investigate whether bumblebees could learn to tell apart flowers based on differences in temperature patterns .", "All experiments were carried out in lab conditions , using flight arenas as described in Clarke et al . ( 2013 ) .", "Ambient temperature was maintained at 21°C and flight arenas were ventilated regularly when access hatches were opened to insert artificial flowers .", "Flower naïve bumblebees , Bombus terrestris audax , were supplied by Biobest ( Westerlo , Belgium ) via Agralan ( Swindon , UK ) or Syngenta-Bioline ( Clacton-on-Sea , UK ) .", "Before bees began foraging they were assigned to one of three test groups described above ( Circle/Cross rewards , Bar rewards , Control ) .", "This was done with the goal of balancing occurrence of bees from the same nest across test groups , although this was subject to bee activity .", "An individual bee only foraged in one test group and were not used in both experiments .", "Both conditioning experiments began with a learning phase , where bees were presented with a choice of flowers placed randomly about the flight arena floor .", "Bees were allowed to freely forage on the artificial flowers , and return to their nest .", "This time between a bee departing the nest to forage and returning was classified as a single foraging bout .", "During the learning phase feeding wells of the rewarding artificial flowers ( as determined by the bee’s test group ) were filled with 25 μl of 30% sucrose solution and the feeding well of nonrewarding artificial flowers with 25 μl of water .", "In small flower tests , sixteen flowers ( eight of each temperature pattern ) were presented to the bee .", "In large flower tests , six flowers ( three of each temperature pattern ) were presented .", "Typically , bees made contact with the flower top while hovering above it before quitting flight and landing .", "If a bee landed on the flower it would normally approach the feeding well and extend its proboscis and attempt to feed from the sucrose solution presented in rewarding flowers ( Figure 2e and f ) .", "It could also decide to depart without attempting to feed .", "As bees detect temperature via touch ( Heran , 1952 ) , physical contact with the top of the flower was considered a landing , even if the bee did not quit flying .", "Bees were each observed for 60 flower landings .", "Bees completed the learning phase in 5 . 69 ± 1 . 79 and 8 . 60 ± 2 . 63 foraging bouts ( mean ± SD ) for the small and large flower experiments , respectively , making 10 . 53 ± 6 . 58 and 6 . 97 ± 3 . 96 landings per bout .", "At each landing , we monitored whether the bee fed from the feeder or left without feeding .", "For small flower experiments the learning phase was followed by a test phase .", "In the test phase , bees were allowed to forage freely as discussed above .", "Here bees were presented with a fresh set of sixteen small temperature pattern flowers with 25 μl of water in feeding wells but presenting the same temperature patterns , or lack of patterns in control group , the bee had experienced in the training phase .", "Bees were observed for twenty flower landings in this test phase .", "A test phase was not carried out in the large flower experiment as the large flowers limited the number that could be sensibly placed within the arena .", "In small temperature pattern experiments , flowers were not interfered with by the experimenters while the bee was in the flight arena foraging .", "This was to minimise disturbance of the foraging bees .", "Once a bee had emptied the feeder of a flower any subsequent returns to that flower during the same bout were not counted .", "This was done so that a bee’s foraging success was not influenced by encounters with empty feeding wells .", "It is not possible to distinguish whether a bee withholds its probing response because it is correctly responding to a nonrewarding flower ( or incorrectly responding to a rewarding flower ) or because the feeding well is empty .", "In large temperature pattern experiments , flowers were topped up after the bee departed and moved to a different point in the arena , as the small number of flowers meant bees often had to visit flowers more than once in a bout .", "Return visits were not counted unless the flower had been moved to a different location and refilled whilst the bee was flying elsewhere in the arena .", "In both experiments after a bee returned to the nest , the end of a foraging bout , all the artificial flowers were taken out of the arena .", "Flower feeders were emptied and refilled to prevent differences in reward temperature developing .", "The flower tops were then wiped down with ethanol removing any scent marks the bees could have left .", "Thus , flowers were cleaned regularly preventing the bee from using these to recognise rewarding flowers .", "Temperature patterns were then checked with the thermal camera before placing flower feeders back in the arena , replacing any flower that ceased to present the temperature pattern due to a fault .", "Each flower landing was classed as correct or incorrect , as described in previous bee conditioning studies ( Whitney et al . , 2008; Whitney et al . , 2009; Clarke et al . , 2013 ) .", "In the learning phase experiments extending their proboscis into the feeding well ( probing and/or feeding ) on a rewarding flower , or not doing so when landing on a nonrewarding flower , was deemed a correct action .", "Doing otherwise was deemed incorrect .", "In the test phase all flowers were non-rewarding , therefore scoring flowers as ‘rewarding’ and ‘nonrewarding’ was determined by the reward scheme in the preceding learning phase .", "So , probing the feeding well of flowers with the temperature pattern that had been rewarding in that bee’s test phase , or not probing after landing on a flower showing the temperature pattern that had been non-rewarding were correct actions in the test phase .", "Success over the previous 10 visits ( starting at visit 10 , then 20 , 30 , etc . ) in the learning phase and overall success rate in the test phase were calculated for each bee .", "Comparing foraging success between the control bees and bees that had foraged on flowers with temperature patterns differences allows us to evaluate if temperature patterns aided bumblebee learning .", "Data were analysed using R 3 . 1 . 1 ( R Core Team , 2008 ) .", "The success rate data from the learning and test phase underwent an arcsine transformation in order to account for it being bound between 0 and 1 .", "The arcsine of success probability across the whole test phase was compared between the three test groups using analysis of variance .", "Bee identity was included as a random factor .", "Generalised linear model techniques and AIC model simplification were used in our analysis of bumblebee foraging success during the learning phase of our experiments .", "While differential conditioning data are often analysed by t-tests on the first and last 10 visits bees make during learning ( Clarke et al . , 2013 ) , the model simplification technique used here has the advantage of including all visits made throughout the learning phase in comparisons and allows more specific comparisons of the influences on learning between each test group .", "For this reason , we feel the following model simplification technique is more appropriate and informative for the learning data collected in this study .", "Not counting revisits to emptied flowers while scoring foraging success does mean the balance between rewarding and non-rewarding flowers could change as flowers are emptied , especially during the learning phase , as bees are more likely to empty the wells of rewarding flowers .", "This effect was minimal in the large flower experiments , as flowers were refilled shortly after bees departed from them .", "In the small flower experiments , there was a much larger number of flowers in the flight arena and bees seldom visited all of them in a bout .", "Small flowers were refilled at the end of each bout , on average every 10 . 53 visits .", "So , any changes in the balance of rewarding and unrewarding flowers did not persist for long .", "Furthermore , bees can carry out correct foraging actions on rewarding and unrewarding flowers showing probing , or not , as described above .", "Thus , the capacity of bees to forage correctly does not change as flowers are emptied , as long as some flowers still have sucrose or water in their feeding wells .", "Consequently , the impact of a changing balance of rewarding and non-rewarding flowers on scoring of pollinator foraging success is likely small and short-lived , thus was not included within our analysis .", "The following represents our full model before any simplification was applied:ynx=i+lnx*l+Tst+lnx*ct+Csc+lnx*cc+bn+lnx*rn Where ynx is the arcsine success rate of bee n over the previous 10 visits to the artificial flowers , at x flower visits .", "x is the number of the visits the bee has made to the artificial flowers , the data for y is calculated in blocks of 10 ( 10 , 20 , 30 , 40 , 50 , 60 ) .", "i is the initial arcsine success rate , the intercept , for bees in the bar rewards test group when x=0 .", "l dictates the change in arcsine success rate with increased x in the bar test group , thus l is effectively the learning speed parameter and allows bee’s experience to affect success rate .", "T and C are Boolean parameters which allow the model to alter y depending on which test group the bee is in .", "C indicates whether the bee is in the control group , where: ( 2 ) C={0 , bee is not in the control group;1 , bee is in the control group; .", "T indicates whether the bee is in the circle rewards or cross rewards test group , depending on the experiment ( see above and main text ) , where: ( 3 ) T={0 , bee is not in the cross or circle test group;1 , bee is in the cross or circle test group; .", "sc and st are the change in initial arcsine success rate , relative to i , for bees in the control and circle or cross test groups respectively .", "cc and ct are the change in learning speed , relative to l , for bees in the control and circle or cross test groups respectively .", "Variation between individual bees was included in our model as a random factor .", "bn and rn represent the change in initial arcsine success rate and learning speed , for bee number n .", "In the model described in Equation ( 1 ) parameters i , l , sc , st , cc , ct , bn and rn are parameters to be estimated .", "Model simplification procedure involved paired comparisons between the standing ‘best model’ , beginning with the full model described in Equation ( 1 ) , with a simpler model .", "Simpler models were constructed from the standing best model but with further parameters removed ( effectively forcing the relevant parameters to equal 0 ) , in the order described below .", "Should the removal of the parameter has no significant effect on AIC , as laid out by Richards , 2008 , this simpler model would become the best model for the next comparison .", "If removal of a parameter led to a significant increase in AIC the standing best ( more complex ) model would remain the best for the next comparison .", "Initially , the effects of random factors were compared , a model without rn was compared to the complete model .", "This allowed testing of whether individual bees differed only in intercepts or intercepts and learning speed ( as in the full model ) .", "In both experiments rn had no significant effect on the model , and is thus not included in subsequent models below .", "Secondly interaction effects were investigated by removing cc and ct .", "This created a model where the shape of the relationship between x and y in all test groups was dictated only by l .", "Should the best model according to AIC , find no significant interaction the effects of the test groups would be investigated by removing T and C creating a model where all test groups both showed the same intercepts and learning .", "Finally , the impact of experience on success was compared by removing the learning parameter l .", "Should the best fitting interaction model include interaction effects individual models for each test group would be fitted as follows: ( 4 ) ynx=i+ ( lnx∗l ) +bn .", "For each test group , using the model described in Equation ( 4 ) , we tested whether bee foraging success changed with the number of visits the bee has made by removing l ." ] ]

[ "Pollinating insects utilise various sensory cues to identify and learn rewarding flower species .", "One such cue is floral temperature , created by captured sunlight or plant thermogenesis .", "Bumblebees , honeybees and stingless bees can distinguish flowers based on differences in overall temperature between flowers .", "We report here that floral temperature often differs between different parts of the flower creating a temperature structure or pattern .", "Temperature patterns are common , with 55% of 118 plant species thermographed , showing within-flower temperature differences greater than the 2°C difference that bees are known to be able to detect .", "Using differential conditioning techniques , we show that bumblebees can distinguish artificial flowers differing in temperature patterns comparable to those seen in real flowers .", "Thus , bumblebees are able to perceive the shape of these within-flower temperature patterns .", "Floral temperature patterns may therefore represent a new floral cue that could assist pollinators in the recognition and learning of rewarding flowers ." ]

[ "Bees experience the world in a different way to humans .", "The plants that they visit exploit the bee’s senses to make sure that a searching bee can easily find , handle and pollinate flowers .", "For example , bumblebees can learn to choose between flowers that are different temperatures , using heat as a way of identifying the best flowers .", "Some wild flowers are warmer than others when they grow in their natural environment .", "Recent advances in technology mean that scientists are now able to take a more detailed look at flower temperature than ever before .", "Harrap et al . used this technology to look at 118 species of plant , including daisies , rockroses and poppies .", "Over half of the plants examined had flowers with complex patterns of heat across their petals , echoing the colourful patterns that we see with our own eyes .", "On average , some parts of the petals were 4–5°C warmer than the rest .", "In further experiments , artificial flowers that replicated these patterns showed that bumblebees are able to tell apart flowers with different temperature patterns across their petals .", "These newly discovered floral heat patterns appear widespread in nature .", "It is likely that these patterns are a hidden signal to pollinators that , together with other cues like colour and scent , attracts them to the flowers and helps them locate any reward , like nectar .", "As well as opening up a new field of research in understanding the interactions between plants and their pollinators , these findings are potentially important given current concerns about climate change .", "If pollinators are partly reliant on subtle differences in temperature across the surface of a petal , then even small changes in the temperature of the environment could have a large and unanticipated influence on how efficient bees and other pollinators are when they are visiting flowers with hidden heat patterns ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "cell biology", "neuroscience" ]

[ [ "Parkinson's disease ( PD ) is the second most common neurodegenerative disorder and there is no cure for this progressive illness ( Guo , 2012 ) .", "Mutations in PINK1 , a mitochondria-localized serine–threonine kinase , and Parkin , an E3 ubiquitin ligase , lead to autosomal recessive forms of the disease ( Kitada et al . , 1998; Valente et al . , 2004 ) .", "Genetic studies in Drosophila first demonstrated that PINK1 and parkin act in the same genetic pathway , with PINK1 positively regulating parkin , to regulate mitochondrial integrity and function ( Clark et al . , 2006; Park et al . , 2006; Yang et al . , 2006 ) .", "Mitochondrial morphology is maintained by a balance between two opposing actions , mitochondrial fusion that is promoted by mitofusin ( mfn ) and mitochondrial fission that is controlled by Dynamin-related protein 1 ( Drp1 ) ( Chan , 2012; Nunnari and Suomalainen , 2012 ) .", "Genetic studies in Drosophila have shown that downregulation of mfn or overexpression of drp1 suppresses multiple phenotypes associated with lack of PINK1 or parkin , including defects in mitochondrial integrity , cell death , tissue health , and flight ability ( Deng et al . , 2008; Poole et al . , 2008; Yang et al . , 2008 ) .", "Parkin ubiquitinates Mfn and promotes Mfn degradation ( Poole et al . , 2010; Ziviani et al . , 2010 ) .", "However , it is not clear if increased mfn or decreased drp1 levels are sufficient to cause the phenotypes observed in PINK1 or parkin mutants .", "In addition to mitochondrial dynamics , the PINK1/Parkin pathway promotes mitophagy , selective autophagic degradation of defective mitochondria in mammalian cells .", "Accumulation of mitochondrial damage can result in loss of mitochondrial membrane potential .", "This leads to recruitment of Parkin to the depolarized mitochondria , ultimately resulting in autophagic degradation of these mitochondria ( Narendra et al . , 2008; Ding et al . , 2010; Gegg et al . , 2010; Geisler et al . , 2010; Matsuda et al . , 2010; Narendra et al . , 2010; Okatsu et al . , 2010; Tanaka et al . , 2010; Vives-Bauza et al . , 2010; Chan et al . , 2011 ) .", "Parkin-mediated mitophagy also occurs in mouse cortical neurons and heart muscle ( Cai et al . , 2012; Chen and Dorn , 2013 ) .", "An important step during this process is Parkin-dependent ubiquitination of Mfn and other substrates , followed by their proteasome-dependent degradation ( Tanaka et al . , 2010; Chan et al . , 2011 ) .", "Relevant to PD , PINK1 and parkin mutant fibroblasts from PD patients also show deregulation of mitochondrial dynamics and modest defects in the clearance of mitochondria ( Rakovic et al . , 2011 , 2013 ) .", "An important puzzle in the field of PD research is why mice lacking PINK1 or parkin bear only subtle phenotypes related to dopaminergic neuronal degeneration or mitochondrial morphology change ( Palacino et al . , 2004; Perez and Palmiter , 2005; Perez et al . , 2005; Kitada et al . , 2007; Frank-Cannon et al . , 2008; Gautier et al . , 2008; Gispert et al . , 2009; Kitada et al . , 2009; Akundi et al . , 2011 ) .", "This raises the possibility that other mechanisms may compensate for loss of PINK1 or parkin .", "Indeed , when parkin is knocked down in adult dopaminergic neurons rather than during development , more striking neuronal degeneration is observed ( Dawson et al . , 2010; Shin et al . , 2011; Lee et al . , 2012 ) .", "However , the molecular mechanisms by which loss of PINK1/parkin function can be compensated are not known .", "Mitochondrial ubiquitin ligase 1 ( MUL1 ) , also known as mitochondrial-anchored protein ligase ( MAPL ) ( Neuspiel , 2008 ) , mitochondrial ubiquitin ligase activator of NF-kB ( MULAN ) ( Li et al . , 2008 ) , or growth inhibition and death E3 ligase ( GIDE ) ( Zhang et al . , 2008 ) , was identified as an E3 protein ligase by three independent groups .", "Work in mammalian systems shows that MUL1 has small ubiquitin-like modifier ( SUMO ) ligase activity , stabilizing Drp1 ( Harder et al . , 2004; Braschi et al . , 2009 ) , or ubiquitin ligase activity , degrading Mfn ( Lokireddy et al . , 2012 ) .", "As expected from a protein with these proposed biochemical activities , MUL1 expression in mammalian cells results in smaller and more fragmented mitochondria ( Li et al . , 2008; Neuspiel , 2008 ) .", "However , the consequences of loss of MUL1 in vivo have not been reported in any organism .", "In this study , we show that overexpression of mfn is sufficient to recapitulate many PINK1/parkin mutant phenotypes , underlining the central importance deregulation of this protein has for PD pathogenesis .", "Expression of wild-type MUL1 , but not a ligase-dead version , suppresses PINK1 or parkin mutant phenotypes , and those due to mfn overexpression in Drosophila .", "Conversely , removing MUL1 in PINK1 or parkin null mutants results in enhanced phenotypes as compared with the single mutants , suggesting that MUL1 acts in parallel to the PINK1/parkin pathway .", "MUL1 physically binds to Mfn and promotes its ubiquitin-dependent degradation .", "MUL1 , but not a ligase-dead version , also regulates Mfn levels and mitochondrial morphology in human cells .", "Experiments in Drosophila and mammalian systems suggest that MUL1 regulates mfn through a pathway parallel to that of PINK1/parkin pathway .", "Finally , knockdown of MUL1 from parkin knockout mouse cortical neurons augments mitochondrial damage and induces neurodegeneration-like phenotypes than does removing either gene alone .", "Together , these results suggest that MUL1 plays an important compensatory function in organisms or cells lacking PINK1/parkin ." ], [ "We identified MUL1 as a novel suppressor of PINK1/parkin mutant phenotypes .", "Human MUL1 contains two transmembrane ( TM ) domains and a highly conserved C-terminal ring finger ( RNF ) domain .", "Topological studies suggest that the two TM domains anchor the protein to the mitochondrial outer membrane , with the RNF domain facing the cytosol ( Li et al . , 2008 ) .", "Drosophila MUL1 ( CG1134 ) encodes a protein with a similar domain structure , and 52% amino acid similarity to human MUL1 ( Figure 1A , B ) . 10 . 7554/eLife . 01958 . 003Figure 1 . Overexpression of MUL1 , but not MUL1 LD , suppresses PINK1/parkin mutant phenotypes .", "( A ) Protein domain organization of Drosophila MUL1 .", "TM1 , TM2 , and RNF represent transmembrane domains 1 and 2 , and the RING Finger domain , respectively .", "The position of the mutation in the ligase dead ( LD ) version of MUL1 is marked with a red asterisk .", "( B ) Sequence alignment of MUL1 in various species in the highly conserved RNF domain .", "A highly conserved histidine residue ( marked as red ) was mutated to alanine in MUL1 LD , ablating ligase activity .", "( C–C″ )", "Dopaminergic neurons stained with an anti-TH antibody in red and mitochondria labeled with mitoGFP in green .", "Neurons in the PPL1 cluster are shown .", "While mitochondria in wild-type dopaminergic neurons are dispersed ( C ) , mitochondria in PINK1 mutant dopaminergic neurons are clumped ( C′ , white arrow heads ) .", "This phenotype is suppressed by MUL1 overexpression driven by TH-Gal4 ( C″ ) .", "Scale bars: 10 µm .", "( D–E″’’ and J–M″’’ )", "Confocal images of the IFM from thoraces double labeled with mitoGFP and phalloidin ( red ) ( D–D″ , J–J″ , L–L″ ) , or double labeled with mitoGFP and TUNEL ( red ) with lower magnification ( E–E″ , K–K″ , M–M″ ) .", "Scale bars: 5 µm .", "MUL1 overexpression is driven by Mef2-Gal4 .", "In wild-type ( D ) , mitochondria have a regular size and shape , and are localized in between myofibrils .", "In PINK1 mutants ( D′ ) , mitochondrial size becomes irregular , and the GFP signal is reduced .", "Large mitochondrial clumps also appear .", "PINK1 mutant muscle is TUNEL-positive ( E′ ) .", "( F–F″ )", "Touidine blue staining of muscle .", "Compared with the wild-type ( F ) , PINK1 mutant muscle shows vacuolation indicating muscle degeneration ( F′ ) .", "These PINK1 mutant phenotypes ( D′ , E′ , F′ ) are almost completely suppressed by MUL1 overexpression ( D″ , E″ , F″ ) .", "( Ga–Ga″ , Gb–Gb″ )", "EM images of mitochondria in muscle .", "( Gb–Gb″ )", "Single mitochondrion ( outlined with dashed lines ) from white boxes in Ga–Ga″ .", "Scale bars: 1 µm ( Ga–G″a ) 0 . 5 µm ( Gb–G″b ) .", "In wild-type ( Ga and Gb ) , mitochondria have compact and organized cristae whereas mitochondria from PINK1 mutants ( Ga′ , Gb′ ) are swollen with fragmented cristae , and this is rescued by MUL1 overexpression ( Ga″ , Gb″ ) .", "( H ) Images of thoraces .", "Arrows point to thoracic indentations due to muscle degeneration .", "Compared with WT , PINK1 mutants have thoracic indentation due to muscle degeneration .", "MUL1 overexpression , but not MUL1 LD overexpression , suppresses PINK1 mutant thoracic indentation .", "( I ) qPCR analysis shows that MUL1 and MUL1 LD mRNA are expressed at similar levels in muscles .", "The data are shown as the mean ± SEM from three experiments ( RNA from ten 5-day-old fly thoraces for each genotype ) .", "The statistical analysis was done using One-way ANOVA with Tukey' multiple comparisons test .", "ns: not statistically significant .", "MUL1 LD overexpression in the PINK1 mutant background does not suppress the formation of mitochondrial clumps ( J″ ) or TUNEL-positivity ( K″ ) .", "( L–M″ )", "Overexpression of MUL1 , but not MUL1 LD , suppresses parkin mutant phenotypes . DOI: http://dx . doi . org/10 . 7554/eLife . 01958 . 00310 . 7554/eLife . 01958 . 004Figure 1—figure supplement 1 . MUL1 , but not its ligase-dead version ( MUL1 LD ) , is able to self-ubiquitinate in vitro . In vitro ubiquitination assay using purified Drosophila MUL1 and MUL1 LD .", "Western blot probed with anti-GST antibody detects unmodified forms of both MUL1 and MUL1 LD ( lower panel , arrowhead ) .", "In addition , MUL1 has high molecular weight bands suggesting self-ubiquitination of MUL1 .", "When the blot was probed with antibodies against poly-ubiquitinylated protein , only MUL1 show a smear of high-molecular-weight ubiquitinated bands , but not MUL1 LD ( upper panel ) .", "The result confirms that mutation in MUL1 LD completely abolishes its ligase activity . DOI: http://dx . doi . org/10 . 7554/eLife . 01958 . 004 We overexpressed MUL1 in various tissues using the UAS/GAL4 system ( Brand and Perrimon , 1993 ) .", "Drosophila contains clusters of dopaminergic ( DA ) neurons in the adult brain .", "In wild-type DA neurons , mitochondria are dispersed in the cytosol ( Figure 1C ) .", "In contrast , PINK1 mutant DA neurons show abnormally clumped mitochondria ( Figure 1C′ , arrowheads ) ( Park et al . , 2006 ) , which can be suppressed by overexpression of MUL1 ( Figure 1C″ ) .", "We further characterized MUL1's effects on PINK1/parkin mutants in thoracic indirect flight muscle ( IFM ) , which consists of well-organized muscle fibers , in which mitochondria fill spaces between myofibrils .", "PINK1 null mutant flies have severe defects in mitochondrial morphology , including an overall reduction in mitochondria-targeted GFP ( mitoGFP ) signal and the presence of large mitoGFP clumps ( Figure 1D–D′ , E–E′ ) .", "PINK1 mutant muscle also shows extensive TUNEL-positive cell death ( Figure1E–E′ ) , muscle vacuolation , and degeneration ( Figure 1F–F′ ) .", "In addition , when examined under the electron microscopy ( EM ) level , many mitochondria are swollen with broken cristae ( Figure 1Ga–Ga′ , Gb–Gb′ ) .", "At the level of the whole organism , PINK1 mutants show a thoracic indentation due to IFM degeneration ( Figure 1H ) .", "Strikingly , MUL1 overexpression almost completely rescues all of the above PINK1 mutant phenotypes ( Figure 1D″–F″ , Ga″–Gb″ , H ) .", "To determine if the E3 ligase activity of MUL1 is required for suppression of PINK1 mutant phenotypes , we generated a ligase-dead form of Drosophila MUL1 ( MUL1 LD ) in which histidine 307 , a highly conserved residue within the RNF domain , was mutated to alanine ( Figure 1A , B ) .", "This mutation has been shown to abolish ligase activity of mammalian MUL1 ( Zhang et al . , 2008 ) ; in vitro ubiquitination assays confirm that Drosophila MUL1 LD lacks ligase activity ( Figure 1—figure supplement 1 ) .", "The expression levels of MUL1 and MUL1 LD in muscles are comparable ( Figure 1I ) , and no mitochondrial clumps or muscle cell death are observed when MUL1 or MUL1 LD is overexpressed in wild-type animals ( Figure 1J , J′ , K , K′ ) .", "Expression of MUL1 LD does not suppress PINK1 mutant phenotypes ( Figure 1H , J″ , K″ ) .", "Overexpression of MUL1 ( Figure 1L′ , M′ ) , but not MUL1 LD ( Figure 1L″ , M″ ) , also suppressed parkin null mutant phenotypes .", "Thus , MUL1 is a robust suppressor of PINK1/parkin mutants and this requires MUL1's ligase activity .", "As an E3 ligase anchored onto the mitochondrial outer membrane , MUL1 has been shown to have multiple substrates including Drp1 and Mfn ( Braschi et al . , 2009; Lokireddy et al . , 2012 ) .", "However , the consequences of loss of MUL1 have not been reported in any organism .", "The P element , MUL1EY12156 ( MUL1EY ) , inserted at 20 bp upstream of the MUL1 start codon ( Figure 2A ) , is a partial loss-of-function allele with reduced mRNA expression ( Figure 2B–C ) .", "We performed imprecise excision of this P element and generated a large deletion allele , MUL1A6 .", "MUL1A6 , hereafter called the MUL1 mutant , produces no detectable transcript ( Figure 2B–C ) , and therefore is a null allele .", "Flies homozygous for MUL1A6 are viable .", "We also generated two independent RNAi constructs that target two different locations in the MUL1 coding region .", "Flies expressing these constructs ( MUL1 RNAi lines ) show the same phenotypes ( see below ) and reverse the suppression of PINK1 mutant phenotypes observed upon MUL1 overexpression ( Figure 2D ) . 10 . 7554/eLife . 01958 . 005Figure 2 . MUL1 regulates mitochondrial morphology .", "( A ) A schematic depicting the Drosophila MUL1 genomic region ( cytological location 64A4 ) .", "MUL1 coding and untranslated regions ( dark and open rectangles , respectively ) are depicted .", "The P element , MUL1EY , inserted in the 5′ UTR , is shown as an inverted triangle .", "The deleted region in the MUL1A6 allele is indicated by parentheses .", "( B ) RT PCR shows that flies carrying the MUL1EY allele have detectable but reduced levels of MUL1 transcripts .", "However , no MUL1 transcript is detected in flies homozygous for the MUL1 deletion , MUL1A6 .", "( C ) qPCR shows that MUL1EY allele has approximately a 60% reduction of MUL1 transcript compared to the wild-type ( WT ) .", "No MUL1 transcript is detected in flies homozygous for MUL1A6 .", "( D ) MUL1 RNAi line reverses the suppression of PINK1 mutant mitochondrial phenotypes due to MUL1 overexpression .", "( E ) Muscle fibers stained with mitoGFP in green and actin in red .", "Compared with the WT , flies homozygous for the MUL1 deletion or expressing MUL1 RNAi show slightly elongated mitochondria .", "In contrast , when MUL1 is overexpressed using the Mef2-Gal4 driver , mitochondria are significantly smaller .", "( F ) Salivary glands , with cell boundaries labeled with rhodamine phalloidin in red , and mitoGFP in green .", "In WT , mitochondria are tubular and evenly distributed .", "In contrast , in cells expressing MUL1 RNAi ( driven by OK6-Gal4 ) mitochondria are fewer in number and found in clumps .", "In contrast , MUL1 overexpression ( also driven by OK6-Gal4 ) results in fragmented mitochondria and irregular cell boundaries .", "( G ) Quantification of mitochondrial number and size in salivary glands ( mean ± SEM , n > 6 larvae for each genotype ) .", "* Significantly different from wild-type , p<0 . 05 ( One-way ANOVA with Tukey's multiple comparisons test ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 01958 . 005 We examined phenotypes due to loss-of-function and overexpression of MUL1 in the IFM , which is a cellular syncytium , and in salivary glands , in which a number of individual cells contain an extensive tubular mitochondrial reticulum .", "Cells from MUL1 null mutant flies , or flies in which MUL1 RNAi is expressed , have mildly elongated mitochondria , while those from flies overexpressing MUL1 have small and fragmented mitochondria ( Figure 2E–G ) .", "Thus , Drosophila MUL1 has a mild pro-fission function , as with mammalian MUL1 ( Braschi et al . , 2009 ) .", "Next , we asked whether Drp1 , Mfn or both serve as MUL1 targets .", "Previous work suggested that MUL1 positively regulates Drp1's pro-fission activity through sumoylation-dependent protein stabilization ( Harder et al . , 2004; Braschi et al . , 2009 ) .", "Surprisingly , overexpression of MUL1 did not change Drp1 levels ( Figure 3A ) .", "In contrast , overexpression of MUL1 led to a reduction in Mfn levels ( Figure 3B ) .", "Analysis of larval lysates from mutants showed that loss of MUL1 results in an increase in Mfn levels , as does loss of PINK1 or parkin , which serve as positive controls ( Figure 5—figure supplement 1 ) .", "Knockdown of MUL1 in Drosophila S2 cells also resulted in an increase in Mfn levels ( Figure 3C ) . 10 . 7554/eLife . 01958 . 006Figure 3 . MUL1 physically binds to Mfn , and promotes ubiquitination-mediated Mfn degradation .", "( A and B )", "Western blots and quantifications of Drp1 and Mfn levels in vivo .", "Analysis of lysates from thoraces show that MUL1 overexpression reduces Mfn levels ( B ) but not Drp1 levels ( A ) .", "The data are shown as the mean ± SEM from three experiments ( each experiment was done with lysate from 8 thoraces for each genotype ) .", "The statistical analysis was done using One-way ANOVA with Tukey's multiple comparisons test .", "ns: not statistically significant .", "** Significantly different , p<0 . 01 .", "( C ) Western blots of Mfn levels in S2 cells either not treated or treated with control , PINK1 , parkin or MUL1 RNAi .", "Quantification of relative Mfn levels shows that there is an increase in Mfn levels in cells treated with RNAi to PINK1 , parkin , or MUL1 ( mean ± SEM , ** Significantly different from cells not treated with RNAi , p<0 . 01 , One-way ANOVA with Tukey's multiple comparisons test ) .", "( D ) Co-immunoprecipitation using lysates from S2 cells transfected with the indicated constructs .", "The INPUT represents 2% of total lysate to monitor protein expression ( top panel ) .", "MUL1-GFP is co-immunoprecipitated with Mfn-myc using both anti-GFP and anti-Myc antibodies .", "Mfn-myc also co-immunoprecipitates with HA-Parkin , which serves as a positive control .", "The interaction between Mfn-Myc and MUL1-GFP was specific , as confirmed by separate immunoprecipitation control experiments ( Figure 3—figure supplement 1 ) .", "( E ) Mfn ubiquitination levels in S2 cells .", "S2 cells are treated with dsRNA designed to silence various genes and transfected with Mfn-Flag .", "Immunoprecipitation was performed with anti-Flag antibody , and Western blots were probed with anti-Ubiquitin antibody and an anti-Flag antibody .", "Relative ubiquitination levels compared to control are shown below ( mean ± SEM ) .", "** Significantly different from control , p<0 . 01 ( One-way ANOVA with Tukey's multiple comparisons test ) .", "In S2 cells , Mfn is highly ubiquitinated .", "RNAi of MUL1 or parkin results in reduced levels of ubiquitnated Mfn .", "Two independent MUL1 RNAs are utilized to knockdown MUL1 , which yield the same results .", "( F ) In PINK1 mutant thoraces , where Mfn levels are increased , MUL1 overexpression ( driven by Mef2-Gal4 ) reduces the increased Mfn levels .", "Relative Mfn levels compared to control are shown below ( mean ± SEM ) .", "** Significantly different , p<0 . 01 ( One-way ANOVA with Tukey's multiple comparisons test ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 01958 . 00610 . 7554/eLife . 01958 . 007Figure 3—figure supplement 1 . MUL1 co-immunoprecipitates with Mfn in S2 cells .", "( A ) Western blot analysis of co-immunoprecipitation .", "S2 cells were transfected with empty vector , Mfn-myc , GFP , or MUL1-GFP as indicated .", "Cells were harvested and lysed , and 2% of lysates were subjected to Western blot to monitor protein expression , shown as INPUT .", "For the rest of lysates , immunoprecipitations were performed with antibodies to Myc or GFP , and Western blots were probed with antibodies to GFP or Myc .", "In lysates from S2 cells transfected with both MUL1-GFP and Mfn-myc , MUL1-GFP is co-immunoprecipitated with Mfn-myc using both anti-GFP and anti-Myc antibodies .", "However , GFP is not co-immunoprecipitated with Mfn-myc . DOI: http://dx . doi . org/10 . 7554/eLife . 01958 . 007 Next , we determined if MUL1 and Mfn interact physically , and if MUL1 regulates Mfn levels through ubiquitination .", "Overexpressed MUL1 co-immunoprecipitated with overexpressed Mfn from Drosophila S2 cell lysates .", "Parkin was used as a positive control and co-immunoprecipitated with Mfn as previously reported ( Poole et al . , 2010; Ziviani et al . , 2010 ) .", "These physical interactions are specific to MUL1 and Mfn rather than the tags utilized ( Figure 3—figure supplement 1 ) .", "To assay ubiquitination , Flag-tagged Mfn was expressed in S2 cells exposed to dsRNA targeting MUL1 or parkin in the presence of the proteasome inhibitor MG132 ( Figure 3E ) .", "In control cells , highly ubiquitinated Mfn was observed .", "When cells were treated with two different dsRNAs against MUL1 , ubiquitinated Mfn levels were dramatically reduced , similar to those observed in parkin RNAi-treated cells , which serve as a positive control .", "Finally , we observed that the increased Mfn levels seen in the PINK1 mutant flies were reduced when MUL1 was overexpressed ( Figure 3F ) , strengthening our argument that MUL1 suppresses PINK1 mutant phenotypes through reduction of Mfn levels .", "Together , these results suggest that MUL1 suppresses PINK1/parkin phenotypes by reducing Mfn levels through its ubiquitination-dependent degradation .", "Previous studies showed that downregulation of mfn or overexpression of drp1 could suppress PINK1 and parkin mutant phenotypes in Drosophila ( Deng et al . , 2008; Poole et al . , 2008; Yang et al . , 2008 ) .", "Parkin has also been shown to bind and ubiquitinate Mfn , promoting Mfn degradation ( Gegg et al . , 2010; Poole et al . , 2010; Tanaka et al . , 2010; Ziviani et al . , 2010; Chan et al . , 2011; Glauser et al . , 2011 ) .", "While increased Mfn levels are observed in PINK1 or parkin mutants ( Poole et al . , 2010; Ziviani et al . , 2010 ) , it is unclear if these increased mfn levels are sufficient to cause the phenotypes observed in PINK1 or parkin mutants .", "It is also unclear if a decrease in the levels of drp1 , which can result in increased mitochondrial size through loss of fission , results in a phenotypically equivalent effect .", "To address these questions , we generated transgenic flies carrying UAS-mfn ( also called Marf , CG3869 ) and obtained two drp1 null alleles , drp11 and drp12 ( Verstreken et al . , 2005 ) .", "Overexpression of mfn under the control of the muscle-specific ( mef2 ) GAL4 driver resulted in organismal lethality .", "To circumvent this lethality , we also generated a new Gal4 driver , IFM-GAL4 , in which GAL4 expression is driven specifically in the IFM ( Figure 4F–J for IFM-GAL1 , vs Figure 4A–E for mef2-GAL4 ) , using regulatory sequences from the flightin gene .", "Since the IFMs are not required for viability , knockdown of essential genes using IFM-GAL4 does not cause lethality in flies ( data not shown ) . 10 . 7554/eLife . 01958 . 008Figure 4 . Generation and expression of the IFM-GAL driver; mfn overexpression , but not loss of drp1 , induces PINK1/parkin-mutant like pathology .", "( A–J )", "Different developmental stages of flies expressing GFP under Mef2-Gal4 ( A–E ) or IFM-Gal4 ( F–J ) .", "( A ) Third instar larvae show GFP expression in whole body muscles .", "( B ) At the early pupal stage , GFP is expressed in a similar pattern as in larvae .", "However , the GFP expression pattern become more specific at the late pupal stage ( C ) , in which the strongest GFP signal is seen in the thorax , and a weaker signal is observed in the head and abdomen ( arrows ) .", "( D ) In an adult fly , dorsal view shows GFP signal in the thorax , upper abdomen and legs .", "( E ) GFP is also expressed in adult head and legs , marked with arrows .", "( F ) Flies expressing GFP under IFM-Gal4 show no GFP expression in third instar larvae , or in early pupae ( G ) .", "( H ) GFP is strongly expressed only in the thorax at the late pupal stage , but not in other areas ( arrows ) .", "( I ) In the adult fly , GFP signal is highly concentrated in the thorax .", "No GFP expression in abdomen and legs is observed , arrows .", "( J ) In contrast to GFP expression under Mef2-Gal4 , IFM-Gal4 does not express in adult head or legs , as indicated with arrows .", "( K–P , T–Y )", "Confocal images of muscle double labeled with mitoGFP ( green ) and phalloidine ( red ) ( K–M , T–V ) , or those labeled with mitoGFP and TUNEL ( red ) at lower magnification ( N–P , W–Y ) , respectively .", "( Qa–Sb )", "EM images of mitochondria in muscle .", "Single mitochondrion from the black-boxed area in Qa , Ra , Sa is shown in Qb , Rb , Sb .", "Scale bars: 1 µm ( Qa , Ra , Sa ) and 0 . 5 µm ( Qb , Rb , Sb ) .", "Compared with wild-type ( K and N ) , parkin null mutant ( L and O ) shows overall reduced levels of mitoGFP signal , large mitochondrial clumps , and muscle cell death .", "Similar phenotypes are observed with mfn overexpression ( M and P ) , and these phenotypes are suppressed by MUL1 overexpression ( T and W ) .", "As a control , parkin overexpression also suppresses phenotypes due to mfn overexpression ( U and X ) .", "Importantly , drp1 null ( drp11/drp12 ) mutant muscle does not have any mitochondrial clumping or TUNEL-positivity seen in loss of parkin function or mfn overexpression ( V and Y ) .", "mfn overexpression is driven by IFM-Gal4 .", "Scale bars: 5 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 01958 . 008 Interestingly , overexpression of mfn in the IFM results in phenotypes ( Figure 4M , P , Sa , Sb ) similar to those of PINK1 or parkin mutants; mitoGFP clumps , TUNEL-positive muscle cell death , and broken mitochondrial cristae when examined at the EM level ( Figure 1D′ , E′ , G′a , G′b ) .", "In contrast , while loss of drp1 results in an increase in mitochondrial size , no muscle cell death or degeneration is observed ( Figure 4V , Y ) .", "Importantly , MUL1 overexpression ( Figure 4T , W ) , as with parkin overexpression ( Figure 4U , X ) , suppressed the phenotypes associated with mfn overexpression .", "Together , these results show that overexpression of mfn , but not loss of drp1 , leads to phenotypes similar to those due to lack of PINK1 or parkin , suggesting a direct link between increased Mfn levels and pathology .", "Our observations that MUL1 overexpression suppresses PINK1 or parkin mutant phenotypes , and that both Parkin and MUL1 promote Mfn degradation , suggest two possible scenarios of how MUL1 and PINK1/parkin interact .", "MUL1 may be a downstream target of the PINK1/parkin pathway and upstream of mfn .", "Alternatively , MUL1 could function in a parallel pathway to PINK1/Parkin , but with action on a common target such as Mfn .", "Characterization of double null mutants provides an effective way of distinguishing these possibilities .", "If MUL1 functions in the same pathway as PINK1 , double null mutants of PINK1 and MUL1 would be expected to show the same phenotype as the single mutant alone , as is observed in the case of PINK1 parkin double mutants ( Clark et al . , 2006; Park et al . , 2006 ) .", "Conversely , if MUL1 and PINK1/parkin act in parallel pathways , the phenotypes of double null mutants may be stronger than those of single mutants .", "We generated PINK1 MUL1 and parkin MUL1 double mutants .", "Several lines of evidence show that double mutants have significantly enhanced phenotypes as compared to those of single mutants alone .", "First , PINK1 MUL1 and parkin MUL1 double null mutants show a high frequency of pupal lethality as compared with single mutants ( data not shown ) , while double null mutants of PINK1 parkin have the same level of viability as single mutants ( Clark et al . , 2006; Park et al . , 2006 ) .", "Second , a thoracic indentation observed in PINK1 or parkin null mutants is much more severe in PINK1 MUL1 and parkin MUL1 double null mutants .", "In contrast , PINK1 parkin double null mutants show the same degree of thoracic indentation as PINK1 or parkin single mutants alone ( Figure 5A–G ) .", "Third , at the cellular level , PINK1 MUL1 and parkin MUL1 double null mutants have highly elongated and interconnected mitochondria , as determined using anti-mitochondrial ATPase antibodies .", "These mitochondrial phenotypes are very different from those of PINK1 , parkin , or MUL1 mutants ( Figure 5I–O ) .", "PINK1 parkin double null mutants show similar mitochondrial morphology phenotypes as PINK1 or parkin single mutants alone ( Figure 5O vs Figure 5J , M ) .", "Fourth , ATP levels in parkin MUL1 double null mutants were further reduced compared to those of parkin or MUL1 single null mutants ( Figure 5Q ) .", "Fifth , the ability of parkin overexpression to rescue PINK1 mutants is not dependent on MUL1 , and MUL1 overexpression can still suppress PINK1 mutants in the absence of parkin ( Figure 5—figure supplement 1 ) .", "Sixth , knockdown of both MUL1 and parkin in S2 cells further reduces Mfn ubiquitination below levels seen with knockdown of MUL1 or parkin alone ( Figure 5R ) .", "Seventh , PINK1 MUL1 and parkin MUL1 double null mutants have higher Mfn levels as compared to single null mutants of MUL1 , PINK1 , or parkin ( Figure 5—figure supplement 1 ) .", "Finally , knockdown of mfn in the background of parkin MUL1 double mutants almost completely rescues the thoracic indentation and mitochondrial phenotypes of parkin MUL1 double mutants ( Figure 5H , P ) .", "These genetic observations , in combination with biochemical findings that MUL1 physically interacts with Mfn , and that loss of MUL1 results in decreased ubiquitination of endogenous Mfn and increased Mfn levels , indicate that MUL1 acts in parallel to the PINK1/parkin pathway to regulate a common target Mfn . 10 . 7554/eLife . 01958 . 009Figure 5 . MUL1 acts in parallel to the PINK1/parkin pathway .", "( A–H )", "Images of thoraces of various mutants .", "Arrows point to thoracic indentations due to muscle degeneration .", "PINK1 MUL1 and parkin MUL1 double mutants have more severe thoracic indentation compared to either mutant alone .", "Remarkably , the severe thoracic indentation phenotype in parkin MUL1 double mutants is almost completely suppressed when mfn is also knocked down .", "( I–P )", "Mitochondria are labeled using an anti-ATP synthase antibody in the IFM .", "While PINK1 , parkin , and MUL1 mutant show slightly elongated mitochondrial morphology , PINK1 MUL1 and parkin MUL1 double mutants exhibit highly elongated and interconnected mitochondria .", "These phenotypes can be suppressed by mfn knockdown .", "Instead of using mitoGFP , we utilized anti-ATPase antibodies that allow better visualization of the enhancement phenotypes seen with double mutants .", "( Q ) Relative ATP levels in whole flies of various mutants ( mean ±SEM from three experiments , five 5-day-old flies for each genotype ) .", "** and *** significantly different from wild-type , p<0 . 01 and p<0 . 001 , respectably ( One-way ANOVA with Tukey's multiple comparisons test ) .", "# Significantly different from parkin mutant and MUL1 mutant , both p<0 . 01 ( Two-way ANOVA with Tukey's multiple comparisons test ) .", "( R ) In vivo ubiquitination assay of Mfn .", "S2 cells were treated with the indicated RNAi , transfected with Flag-Mfn , and treated with proteasome inhibitor MG132 .", "Immunoprecipitations were performed using anti-Flag antibody , and western blots were probed with antibodies against anti-Ubiquitin antibody ( P4D1 ) or anti-Flag antibody .", "Relative ubiquitination levels compared to control are shown in the lower panel ( mean ± SEM ) .", "** and *** Significantly different from control , p<0 . 01 and p<0 . 001 , respectably ( One-way ANOVA with Tukey's multiple comparisons test ) .", "# Significantly different from MUL1 RNAi #1 and parkin RNAi , both p<0 . 01 .", "& Significantly different from MUL1 RNAi #2 and parkin RNAi , p<0 . 001 and p<0 . 01 , respectably ( Two-way ANOVA with Tukey's multiple comparisons test ) .", "( S ) Western blot analysis of Mfn levels in vivo and quantification ( mean ± SEM from three experiments , eight third instar larvae for each genotype ) .", "* and ** significantly different from wild-type , p<0 . 05 and p<0 . 01 , respectably ( One-way ANOVA with Tukey's multiple comparisons test ) .", "# Significantly different from parkin mutant and MUL1 mutant , both p<0 . 01 .", "& Significantly different from PINK1 mutant and MUL1 mutant , both p<0 . 01 ( Two-way ANOVA with Tukey's multiple comparisons test ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 01958 . 00910 . 7554/eLife . 01958 . 010Figure 5—figure supplement 1 . MUL1 acts in a parallel pathway to the PINK1/parkin pathway .", "( A–H )", "Confocal images of muscle fibers labeled with mitoGFP ( green ) and rhodamine phalloidin ( red ) .", "Compared with wild-type ( A ) , PINK1 null mutant ( B ) or parkin RNAi ( C ) flies show large mitoGFP clumps .", "The abnormal mitochondrial phenotype in parkin RNAi flies is suppressed by MUL1 overexpression ( D ) .", "In PINK1 null mutant muscles , overexpression of MUL1 suppresses mitochondrial phenotypes ( E ) , and parkin knockdown does not affect the suppression of PINK1 mutant mitochondrial phenotype by MUL1 overexpression ( F ) .", "Similarly , parkin overexpression in PINK1 mutant muscles suppresses mitochondrial phenotypes ( G ) , and MUL1 knockdown fails to reverse the suppression of PINK1 mutant phenotype by parkin overexpression ( H ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 01958 . 010 Next , we asked if MUL1-mediated mitochondrial morphology and Mfn regulation is conserved in human cells .", "We expressed human MUL1 and MUL1 LD in HeLa cells ( Figure 6F ) .", "Cells expressing MUL1 or MUL1 LD are GFP-positive and marked with asterisks ( Figure 6A–D″ ) .", "Cells expressing GFP-MUL1 showed peri-nuclear mitochondrial clustering ( Figure 6A–B , asterisks ) , and mitochondria appeared small and globular in shape as compared to those in untransfected , GFP-negative cells ( Figure 6B–B″ ) .", "MUL1 LD neither causes mitochondrial clustering nor alters mitochondrial morphology ( Figure 6C–D″ , asterisks ) . 10 . 7554/eLife . 01958 . 011Figure 6 . MUL1’s function in mitochondrial morphology and Mfn levels is conserved in human cells .", "( A–D‴ )", "HeLa cells transfected with GFP-MUL1 ( A–B″ ) or GFP-MUL1 LD ( C–D″ ) are marked with asterisks , while cells not transfected serve as internal controls .", "Mitochondria are labeled with mitotracker in red ( B and D ) .", "( B′ and B″ , D′ and D″ )", "Higher magnification images of mitochondria within white boxes in B and D . Cells expressing GFP-MUL1 have clustered mitochondria in the perinuclear region ( B ) .", "Mitochondria are also small and fragmented ( B″ ) , as compared to cells not expressing GFP-MUL1 ( B′ ) .", "Importantly , GFP-MUL1 LD does not result in localization of mitochondria to the perinuclear region ( D ) or in mitochondrial fragmentation ( D′ ) .", "( E ) Western blot analysis of Mfn1 and Mfn2 levels after CHX treatment .", "HeLa cells expressing scrambled shRNA or MUL1 shMUL1 are treated with CHX .", "Mfn1 and 2 levels at each time point are normalized with Actin .", "The relative portion of remaining Mfn1 and 2 as compared to time point 0 was calculated and plotted ( E ) .", "In cells expressing MUL1 shRNA , Mfn1 and 2 levels after CHX treatment are more stable than those in cells expressing scrambled shRNA .", "( F ) Expression of transfected GFP-MUL1 and GFP-MUL1 LD in HeLa cells , as detected using anti-GFP antibody .", "( G ) Western blot analysis of endogenous MUL1 levels in HeLa cells stably expressing scrambled shRNA and MUL1 shRNA .", "MUL1 shRNA expressing cells have reduced levels of endogenous MUL1 .", "( H ) Human MUL1 sequence and deletion in MUL1 knockout ( MUL1−/− ) HeLa cells , generated using the CRISPR/Cas 9 system .", "Sequences targeting MUL1 are highlighted in blue .", "Red letters indicate start codon .", "Red dashes represent deleted bases .", "Deleted eight base pairs include the start codon of MUL1 .", "( I ) Western blot analysis of Mfn1 and Mfn2 levels in wild-type and MUL1−/− HeLa cells treated with CHX for the indicated time .", "Remaining Mfn1 and Mfn2 levels at each time point were plotted below .", "( J ) Western blot showing no MUL1 expression in MUL1−/− HeLa .", "Arrowhead points to MUL1 protein .", "Asterisk indicates a non-specific band . DOI: http://dx . doi . org/10 . 7554/eLife . 01958 . 011 Mammals have two Mfn proteins , Mfn1 and Mfn2 , both able to promote mitochondrial fusion ( Chen et al . , 2003; Eura et al . , 2003 ) .", "We monitored the fate of Mfn1 and Mfn2 in control HeLa cells and HeLa cells stably expressing small hairpin RNA against MUL1 ( MUL1 shRNA ) ( Figure 6E , G ) .", "When cells were exposed to the protein synthesis inhibitor cycloheximide ( CHX ) , Mfn1 and Mfn2 levels gradually decreased in control cells , but were dramatically stabilized in cells with decreased levels of MUL1 ( Figure 6E ) .", "To confirm the above result , we generated MUL1 knockout HeLa cells ( Figure 6H ) , which contain a deletion including the start codon in the MUL1 genomic region , using the CRISPR/Cas 9 system ( Cong et al . , 2013; Jinek et al . , 2013; Mali et al . , 2013 ) .", "Two independent anti-MUL1 antibodies confirmed no MUL1 expression in MUL1 knockout cells ( Figure 6J and data not shown ) .", "Similar to what was observed for the MUL1 shRNA , Mfn1 and Mfn2 levels were also dramatically stabilized in MUL1 knockout cells ( Figure 6I ) .", "Together , these results suggest that the role of MUL1 in regulating Mfn stability and mitochondrial morphology is conserved in human cells .", "The PINK1/Parkin pathway mediates mitophagy in HeLa cells ( Narendra et al . , 2008 , 2010 ) , mouse cortical neurons , and heart muscle ( Cai et al . , 2012; Chen and Dorn , 2013 ) .", "When cells are treated with a mitochondrial uncoupler , mitochondria lose their membrane potential .", "This leads to recruitment of Parkin to the depolarized mitochondria , ultimately resulting in autophagic degradation of these mitochondria .", "Because of the genetic interactions observed between MUL1 and PINK1/parkin in Drosophila , we asked if MUL1 was able to modulate Parkin-mediated mitophagy .", "We induced mitophagy by exposing HeLa cells to antimycin A , which inhibits electron transport and depolarizes the mitochondrial membrane .", "Wild-type , MUL1 knockout , and PINK1 knockout ( as a control ) HeLa cells were transfected with YFP-Parkin and treated with DMSO or antimycin A for indicated time .", "In wild-type , without antimycin A treatment , Parkin mainly localizes in the cytosol ( Figure 7A ) .", "However , following antimycin A treatment for 3 hrs , most Parkin was translocated to the mitochondria ( Figure 7B , D ) .", "After 24 hrs of antimycin A treatment , Parkin was found dispersed in the cytosol and mitochondria were no longer observed , indicating that mitophagy had occurred ( Figure 7C , E ) .", "To ensure there was no delay in mitophagy , we also assayed cells that were treated with antimycin A for 6 , 12 , 16 and 20 hrs ( Figure 7F , data not shown ) .", "In all cells , mitophagy was observed at least 16 hrs after antimycin A treatment .", "No significant differences were observed in the fraction of Parkin recruited to mitochondria , or the fraction of mitochondria that underwent mitophagy , at any of these time points , for wild-type and MUL1 knockout cells ( Figure 7B–F ) .", "Knockdown of MUL1 using shRNA also had no effect on Parkin translocation or mitophagy ( Figure 7—figure supplement 1 ) .", "As a control , PINK1 knockout HeLa cells showed almost no Parkin localization to mitochondria and lack of mitophagy ( Figure 7B–F ) .", "Similar results were obtained when cells were treated with carbonyl cyanide m-chlorophenylhydrazone ( CCCP ) , which uncouples mitochondrial membrane potential ( data not shown ) .", "Finally , MUL1 overexpression also had no effect on Parkin translocation ( Figure 7G–H ) and did not block mitophagy ( data not shown ) .", "Thus , neither loss of MUL1 nor its overexpression altered Parkin translocation or mitophagy .", "These results are consistent with MUL1 acting in parallel to PINK1/parkin in Drosophila , and suggest that MUL1 regulates mitochondrial health through a distinct pathway . 10 . 7554/eLife . 01958 . 012Figure 7 . Neither MUL1 knockout nor overexpression affects Parkin-mediated mitophagy .", "( A–C )", "HeLa cells ( control , MUL1 knockout or PINK1 knockout ) were transfected with YFP-Parkin , treated with either DMSO or antimycin A , and immunostained with an anti-Tom20 antibody which labels mitochondria .", "( A ) HeLa cells treated with DMSO as a control .", "( B ) Following treatment with antimycin A for 3 hrs , Parkin is recruited to mitochondria , as shown by co-localization of Parkin and the mitochondrial marker .", "In MUL1 null cells , Parkin recruitment to mitochondria is not affected , whereas in PINK1 null cells ( positive control ) , Parkin recruitment to mitochondria is abolished .", "( C ) After 24 hrs of antimycin A treatment , Parkin returns to the cytosol and the mitochondrial signal disappears .", "In MUL1 null cells , mitochondrial disappearance occurs similarly as with WT , whereas in PINK1 null cells ( positive control ) , mitochondria are not eliminated .", "( D–E )", "Quantification of cells with Parkin recruited to mitochondria after 3 hrs of antimycin A treatment ( D ) and with few or no mitochondria after 24 hr of antimycin A treatment ( E ) and after 16 and 20 hrs of antimycin A treatment ( F ) .", "The data are shown as the mean ± SEM from three experiments ( n ≥ 100 for each genotype ) .", "*** Significantly different from wild-type , p<0 . 001 .", "ns: not statistically significant ( One-way ANOVA with Tukey's multiple comparisons test ) .", "While Parkin translocation and mitochondrial disappearance are significantly blocked in PINK1 knockout cells , there is no significant difference between HeLa cells and MUL1 knockout cells in these processes .", "( G ) HeLa cells stably expressing YFP-Parkin and mito-RFP are transfected with Flag-MUL1 , treated with DMSO or antimycin A , and immunostained with anti-Flag antibody .", "3-hour antimycin A treatment causes Parkin localization to mitochondria in cells with or without MUL1 expression .", "( H ) Quantification of cells with Parkin recruited to mitochondria after 1 . 5 or 3 hrs Antimycin A treatment .", "Both 1 . 5 and 3 hrs of antimycin A treatments results in similar levels of Parkin recruitment to mitochondria .", "The data are shown as the mean ± SEM from three experiments ( n ≥ 100 for each genotype ) .", "ns: not statistically significant ( One-way ANOVA with Tukey's multiple comparisons test ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 01958 . 01210 . 7554/eLife . 01958 . 013Figure 7—figure supplement 1 . MUL1 knockdown does not affect Parkin-mediated mitophagy .", "( A ) In contrast to what is observed with antimycin A , DMSO treatment does not result in a change in Parkin localization or mitochondrial morphology in HeLa cells expressing scrambled shRNA or MUL1 shRNA .", "Parkin mainly localizes to the cytosol , shown in green , and does not co-localize with mitochondria , labeled in red .", "( B ) HeLa cells that stably express scrambled shRNA or MUL1 shRNA are transfected with YFP-Parkin , treated with antimycin A , and immunostained with anti-Tom20 antibodies , which label mitochondria .", "Following treatment with antimycin A for 3 hr , Parkin is recruited to mitochondria , as shown by co-localization of Parkin and the mitochondrial marker .", "After 24 hr of antimycin A treatment , Parkin returns to the cytosol and the mitochondrial signal disappears .", "( C ) Quantification of cells with Parkin recruited to mitochondria after 1 . 5 or 3 hr of antimycin A treatment shows that there is no significant difference between HeLa cells that stably express scrambled shRNA and MUL1 shRNA .", "( D ) Quantification of the number of cells with few or no mitochondria after 24 or 48 hr of antimycin A treatment shows that there is no significant difference between the cell lines .", "The data are shown as the mean ± SEM from three experiments ( n ≥ 100 for each genotype , One-way ANOVA with Tukey's multiple comparisons test ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 01958 . 013 To further test the hypothesis that MUL1 functions in parallel to the PINK1/parkin pathway in mammalian cells , we investigated the effects of depleting both MUL1 and parkin .", "As HeLa cells do not express Parkin , we turned to cultured mature cortical neurons .", "GFP-MUL1 localizes to the mitochondria in cell bodies and axons of the primary cortical neurons ( Figure 8A , Figure 8—figure supplement 1 ) . 10 . 7554/eLife . 01958 . 014Figure 8 . Loss of both MUL1 and parkin aggravates mitochondrial damage and induces degeneration-like phenotypes in mouse cortical neurons .", "( A ) MUL1 targets mitochondria in the cell bodies and axons of mouse primary cortical neurons .", "Neuronal mitochondria were labeled by DsRed-Mito or stained with an antibody against mitochondrial marker , TOM20 or Cytochrome C ( Figure 8—figure supplement 1 ) .", "( B ) Levels of endogenous MUL1 in neurons transfected with scrambled or MUL1 shRNA .", "Note that partial suppression of endogenous MUL1 may reflect relative low transfection rate ( 20% ) in the neuronal culture .", "( C–F′ )", "Mitochondria in live cortical neurons were co-labeled by expressing CFP-mito , which targets all mitochondria , and by loading fluorescent dye TMRE , which stains healthy mitochondria dependent upon membrane potential ( Δψm ) .", "Loading TMRE also labels mitochondria in glia in the culture .", "The edges of neuron cell bodies are marked with white solid lines , and the nuclei are outlined with white dashed lines .", "In contrast to other neurons , parkin knockout neurons with MUL1 knockdown show reduced TMRE intensity ( F and F′ ) , indicating decreased Δψm .", "Scale bars: 10 µm .", "( G ) Quantification of relative TMRE intensity .", "TMRE intensity measured from each group of neurons was normalized to WT neurons transfected with scrambled shRNA .", "The data are shown as the means ± SEM from three experiments .", "( n ≥ 12 for each group ) .", "*** Significantly different from wild-type neurons transfected with scrambled shRNA , p<0 . 001 .", "ns: not statistically significant ( One-way ANOVA with Tukey's multiple comparisons test ) .", "# Significantly different from wild-type neurons transfected with MUL1 shRNA and parkin KO neurons transfected with scrambled shRNA , p<0 . 001 and p<0 . 01 , respectively ( Two-way ANOVA with Tukey's multiple comparisons test ) .", "( H–M )", "MUL1 knockdown in parkin KO neurons results in enhanced fragmentation of neurites .", "Representative wild-type ( H and I ) and parkin KO ( J–K″ ) cortical neurons transfected with scrambled or MUL1 shRNA and labeled with GFP ( confirming transfection of shRNA and labeling axons and dendrites ) .", "( K′–K″ )", "Higher magnification of a white box in K showing the soma and proximal dendrites labeled with an anti-MAP2 antibody ( red ) .", "Arrows point to the GFP- and MAP2-labeled dendrites , and arrowheads indicate GFP-labeled but MAP2-negative fragmented axons .", "Scale bars: 20 µm .", "( L and M )", "Quantitative analysis showing enhanced process fragmentation ( L ) and dendritic retraction ( M ) .", "The data are shown as the means ± SEM from three experiments ( process fragmentation phenotype: n ≥ 115 for each genotype , dendritic retraction: n ≥ 127 for each phenotype ) .", "* , ** , and *** Significantly different from wild-type neurons transfected with scrambled shRNA , p<0 . 05 , p<0 . 01 , and p<0 . 001 , respectively .", "ns: not statistically significant ( One-way ANOVA with Tukey's multiple comparisons test ) .", "# Significantly different from wild-type neurons transfected with MUL1 shRNA and parkin KO neurons transfected with scrambled shRNA , both p<0 . 001 .", "& Significantly different from wild-type neurons transfected with MUL1 shRNA and parkin KO neurons transfected with scrambled shRNA , both p<0 . 001 ( Two-way ANOVA with Tukey's multiple comparisons test ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 01958 . 01410 . 7554/eLife . 01958 . 015Figure 8—figure supplement 1 . MUL1 localizes to mitochondria in mouse cortical neurons .", "( A ) Representative images showing co-localization of MUL1 with mitochondrial markers , TOM20 ( upper panels ) and Cytochrome C ( lower panels ) , in the cell bodies and proximal dendrites of mouse cortical neurons . DOI: http://dx . doi . org/10 . 7554/eLife . 01958 . 01510 . 7554/eLife . 01958 . 016Figure 8—figure supplement 2 . MUL1 knockdown increases Mfn2 levels in mouse cortical neurons .", "( A–D′ )", "Representative images of mouse cortical neurons ( the cell bodies and proximal dendrites ) labeled with anti-Cytochrome C ( A–D ) and Mfn2 ( A′–D′ ) antibodies .", "( E ) Quantitative analysis of relative Mfn2 levels normalized by Cytochrome C . The data are shown as the means ± SEM , n > 20 for each genotype .", "* , ** , and *** Significantly different from wild-type neurons transfected with scrambled shRNA , p<0 . 05 , p<0 . 01 , and p<0 . 001 , respectively ( One-way ANOVA with Tukey's multiple comparisons test ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 01958 . 016 The proper maintenance of the mitochondrial inner membrane potential ( Δψm ) depends on the physiological function of the mitochondrial respiratory chain , and is crucial for generating ATP .", "Dissipation of the membrane potential is a strong indication of unhealthy mitochondria , which can lead to severe mitochondrial dysfunction and subsequent cell death .", "The Δψm can be measured using a fluorescent dye tetramethyl rhodamine ethyl ester ( TMRE ) .", "We used two independent MUL1 shRNAs to suppress endogenous MUL1 expression in cortical neurons ( Figure 8B ) .", "Cortical neurons expressing CFP-mito , from either wild-type mice co-expressing two independent MUL1 shRNA ( Figure 8D–D′ ) , or parkin gene KO mice co-expressing a scrambled shRNA ( Figure 8E–E′ ) , showed no significant decrease in the intensity of TMRE fluorescence ( Figure 8C , C′ , G ) .", "However , parkin KO neurons co-expressing MUL1 shRNA showed a significant reduction of Δψm ( Figure 8F–F′ , G ) , suggesting that proper MUL1 expression in primary cortical neurons is required to compensate for loss of parkin in maintaining mitochondrial integrity .", "Next , we asked if MUL1 knockdown alters Mfn2 levels in mouse cortical neurons .", "Neurons from wild-type or parkin KO mice were transfected with either scrambled shRNA or MUL1 shRNA , followed by co-staining with anti-Cytochrome C and anti-Mfn2 antibodies .", "Relative Mfn2 intensity in individual neurons was analyzed by calculating the ratio of Mfn2 to Cytochrome C ( Figure 8—figure supplement 2 ) .", "Compared to wild-type neurons transfected with scrambled shRNA , wild-type neurons with MUL1 shRNA , or parkin KO neurons with either scrambled shRNA or MUL1 shRNA had an increased intensity ratio of Mfn2 to Cytochrome C . This suggests that MUL1's role in regulating Mfn2 levels is also conserved in neurons .", "Finally , we investigated if loss of either MUL1 or parkin , or loss of both , has any impact on cultured primary cortical neuron morphology .", "MUL1 knockdown in cells from wild-type mice resulted in a minor increase in dendritic retraction but no significant process fragmentation as compared with wild-type cells ( Figure 8H , L , M ) .", "Cortical neurons from parkin KO mice showed slightly increased process fragmentation but no dendritic retraction ( Figure 8J , L , M ) , as compared with wild-type cells .", "In contrast , MUL1 knockdown in parkin KO neurons resulted in a dramatic increase in the number of neurons with dendritic and axonal fragmentation and retraction ( Figure 8K–K″ , L–M; process fragmentation: total number of neurons examined: n > 115 and n > 127 each genotype for fragmentation and dendritic retraction analysis , respectively ) , indicative of early neurodegeneration .", "The observed phenotypes were confirmed using a second MUL1 shRNA in parkin KO neurons ( data not shown ) .", "These observations suggest that MUL1 acts in parallel to the PINK1/parkin pathway to ensure mitochondrial integrity and function , thus maintaining neuronal health in primary cortical neurons ." ], [ "In summary , we identified MUL1 as a robust suppressor of PINK1/parkin mutant phenotypes in Drosophila .", "MUL1 overexpression , but not expression of a ligase-dead version , strongly suppresses PINK1 and parkin mutant phenotypes .", "The mechanism of this suppression is unique in that MUL1 does not act on PINK1 or parkin , nor does it function as a downstream target .", "Rather , MUL1 acts by suppressing mfn in parallel to the PINK1/parkin pathway ( Figure 9B ) .", "mfn is crucial for actions downstream of PINK1/parkin to maintain mitochondrial function and tissue health ( Figure 9B ) , as overexpression of mfn leads to pathology similar to lack of PINK1/parkin function .", "We hypothesize that the increase in the Mfn level needs to reach a threshold , such as that observed in the PINK1/parkin mutant backgrounds , but not in the MUL1 null background , in order for overt muscle cell degeneration to occur ( Figure 9B ) .", "Biochemically , MUL1 binds to Mfn , and loss of MUL1 results in decreased ubiquitination of Mfn and increased Mfn levels .", "These observations suggest that MUL1 may directly ubiquitinate Mfn , leading to its degradation ( Figure 9A ) .", "Alternatively , MUL1 may act via an intermediary that promotes Mfn ubiquitination and degradation .", "In Drosophila , overexpression of MUL1 almost completely suppresses all aspects of the PINK1/parkin null phenotypes .", "Thus , treatments that manipulate MUL1 expression or activity may have potential as therapeutics strategies . 10 . 7554/eLife . 01958 . 017Figure 9 . Models for how MUL1 interacts with PINK1/parkin .", "( A ) Schematic depictions of how MUL1 , PINK1 , Parkin , and Mfn interact in the mitochondria .", "In mammalian cells , upon mitochondrial damage ( CCCP or antimycin A treatment ) , PINK1 is stabilized onto the mitochondrial OM of damaged mitochondria , with its kinase domain facing the cytosol ( Zhou et al . , 2008 ) .", "PINK1 recruits Parkin onto the OM , either through direct phosphorylation or indirect interaction with other proteins ( not depicted here ) ( Jin and Youle , 2012 ) .", "Parkin then ubiquitinates multiple substrates on the OM , including Mfn .", "MUL1 , a mitochondrial OM-anchored ligase with its RNF domain facing the cytosol , also mediates ubiquitination of Mitofusin .", "( B ) The PINK1/parkin pathway and MUL1 act in parallel to regulate mfn , and maintain mitochondrial function and tissue health .", "Reducing either PINK1/parkin or MUL1 leads to increased levels of Mfn .", "Significant elevation of Mfn leads to mitochondrial dysfunction and tissue damage , similar to what is observed in PINK1/parkin mutants .", "Loss of both PINK1/parkin and MUL1 leads to significantly higher Mfn levels , associated with severe mitochondrial dysfunction and tissue damage .", "OM: mitochondrial outer membrane; IM: mitochondrial inner membrane . DOI: http://dx . doi . org/10 . 7554/eLife . 01958 . 017 In addition to showing that overexpression of MUL1 compensates for lack of PINK1/parkin by downregulating Mfn levels , we have identified an evolutionarily conserved pathway and provide compelling evidence showing that endogenous levels of MUL1 normally compensates for lack of PINK1 or parkin in Drosophila and in mammals .", "Removal of MUL1 in the PINK1 or parkin null background significantly aggravates the phenotypes due to lack of PINK1 or parkin alone .", "Flies lacking MUL1 , PINK1 or parkin are viable , but PINK1/MUL1 or parkin/MUL1 double mutants manifest increased lethality with much more severe muscle degeneration , reduced ATP levels , defective mitochondrial morphology and increased Mfn levels .", "In addition , while parkin KO mature mouse cortical neurons or MUL1 knockdown neurons show only mild neuronal phenotypes , neurons with parkin KO and MUL1 KD show significantly diminished mitochondrial membrane potential , indicating mitochondrial dysfunction .", "They also show neurodegeneration-like phenotypes including axonal and dendritic fragmentation , and reduced mitochondrial distribution along processes .", "Finally , human HeLa cells , which have little or no endogenous Parkin , show a dramatic stabilization of Mfn when MUL1 is eliminated .", "Our findings may help to address an important puzzle in the field of PD research: why do PINK1 or parkin knockout mice , or even parkin/DJ-1/PINK1 triple knockout mice , bear only subtle phenotypes related to dopaminergic neuronal degeneration or mitochondrial morphology changes ( Palacino et al . , 2004; Perez and Palmiter , 2005; Perez et al . , 2005; Kitada et al . , 2007; Frank-Cannon et al . , 2008; Gautier et al . , 2008; Gispert et al . , 2009; Kitada et al . , 2009; Akundi et al . , 2011 ) .", "Our studies provide genetic and cellular clues that suggest compensation by MUL1 may contribute to the subtle phenotypes in PINK1 or parkin mutant mice .", "It will be interesting to determine whether PINK1/MUL1 or parkin/MUL1 double knockout mice show more severe PD-related pathology .", "Regarding PD therapies , optimizing the function of MUL1 is likely to be beneficial for PINK1/PARKIN patients; upregulating MUL1 may rescue the pathology due to lack of PINK1 or PARKIN .", "In contrast , downregulating MUL1 and/or mutations in MUL1 may lead to disruption of this compensatory pathway in maintaining mitochondrial integrity and function , and result in accelerated disease progression .", "Why do cells have multiple E3 ubiquitin ligases acting on a common target ?", "Mfn is localized to the mitochondrial outer membrane ( OM ) ( Figure 9A ) and is a key molecule that regulates mitochondrial fusion in response to various cellular processes .", "Due to its importance , the level of Mfn is expected to be tightly regulated , and this may require several E3 ubiquitin ligases and deubiquitinases that respond to different stimuli ( Gegg et al . , 2010; Park et al . , 2010; Leboucher et al . , 2012; Lokireddy et al . , 2012; Anton et al . , 2013; Fu et al . , 2013 ) .", "In the case of mitochondrial damage , Parkin translocates to depolarized mitochondria before it degrades Mfn ( Figure 9A ) , thus preventing damaged mitochondria from fusing with healthy ones .", "As an E3 ligase anchored on the OM ( Li et al . , 2008; Figure 9A ) , MUL1 is constantly present in the vicinity of Mfn , thus mediating Mfn clearance either constitutively or in a regulated manner in response to different stress signals .", "It is also possible that multiple E3 ligases work in a concerted way to ensure constant Mfn levels .", "In our study , CHX treatment leads to the stabilization of Mfn levels in HeLa cells lack of MUL1 .", "However , steady-state Mfn levels in these cells are not strongly affected .", "This may result from the existence of other pathways for Mfn regulation , such as direct transcriptional feedback regulation on Mfn expression , activities of deubquitinases , and additional E3 ligases .", "Similar considerations may explain the viability and apparently mild phenotypes of MUL1 mutant flies .", "More severe phenotypes may be uncovered in flies lacking MUL1 in response to specific stresses that cannot be buffered by other components .", "A recent study reports that MUL1 promotes mitophagy , when muscle wasting is stimulated in mice ( Lokireddy et al . , 2012 ) .", "To monitor mitophagy , this study measured mitochondrial DNA content and emission of a mitochondrial fluorescent protein that changes color in an acidic environment such as the lysosome ( Lokireddy et al . , 2012 ) .", "However , since these methods do not directly visualize mitochondrial fate , it is possible that the observations may reflect early signs of mitochondrial dysfunction or turnover of the indicator protein , rather than clearance of the mitochondria .", "Also , it is unknown whether MUL1 interacts with the PINK1/parkin pathway to regulate the mitochondrial clearance .", "Our results show that overexpression or lack of MUL1 does not affect Parkin-mediated mitophagy induced by mitochondrial damage in HeLa cells .", "This further strengthens our hypothesis that MUL1 acts in PINK1/parkin-independent pathway for regulating mitochondrial quality control .", "Given our observations , it will be interesting to ask if human mutations in mfn1 or mfn2 that decrease their abilities to be targeted for ubiquitin-dependent degradation , or mutations in MUL1 , result in susceptibility for PD .", "It will also be interesting to see if polymorphisms in MUL1 that affect MUL1 expression levels or activity occur in PD patients .", "In this regard , it is worth noting that MUL1 forms a complex with VPS35 and VPS26 ( Braschi et al . , 2010 ) .", "Since mutations in VPS35 have been identified in multiple PD families ( Vilarino-Guell et al . , 2011; Zimprich et al . , 2011; Kumar et al . , 2012; Lesage et al . , 2012 ) , it will be particularly interesting to determine if PD-associated mutations in VPS35 have effects on MUL1-dependent degradation of Mfn .", "Finally , our observation that overexpression of mfn alone is sufficient to recapitulate key phenotypes associated with loss of PINK1 or parkin suggests that inhibition of mfn may have important therapeutic potential for PD ." ], [ "To generate UAS-MUL1 , an EST clone from the Drosophila Genome Research Center ( DGRC ) , AT15655 , was subcloned into the UASt vector using EcoR1 and Xho1 .", "The Drosophila MUL1 ligase-dead mutant ( MUL1 LD ) was generated by mutating H307 to A via site-specific mutagenesis ( Stratagene QuikChange II XL Kit; Stratagene , La Jolla , CA ) .", "To generate UAS-mfn , the EST clone from DGRC , RE04414 , was subcloned into the UASt vector .", "For UAS-MUL1-GFP , UAS-mfn-myc , and UAS-HA-parkin , each gene's coding region was fused to a different tag using the gateway cloning system ( Invitrogen , Carlsbad , CA ) .", "To silence MUL1 and drp1 , the coding regions of MUL1 and drp1 transcripts were targeted using a synthetic microRNA-based technology ( Chen et al . , 2006; Ganguly et al . , 2008 ) .", "PCR products of these miRNA precursors were cloned into pUASt .", "To generate IFM-GAL4 , the regulatory region of the flightin gene was used .", "All constructs made were confirmed by DNA sequencing .", "To map MUL1 imprecise excision lines , breakpoints were determined by genomic PCR followed by DNA sequencing .", "pAC-mfn-Flag was a gift from Dr Alexander J Whitworth ( Ziviani et al . , 2010 ) .", "Human MUL1 cDNA ( BC010101 ) was purchased from ATCC and cloned into a pEGFP vector ( Clontech , San Jose , CA ) to generate GFP-MUL1 .", "Flag-MUL1 was generated by replacing the GFP tag with a Flag tag .", "Human MUL1 LD was generated by mutating H319 to A , which corresponds to Drosophila MUL1 H307A .", "Human MUL1 shRNA constructs were purchased from OriGene .", "The MUL1 shRNA sequences are 5′-CTTCAAGTCCTGCGTCTTTCTGGAGTGTG-3′ and 5′-GAAGGAGCTGTGCGGTCTGTTAAAG AAAC-3′ .", "CaSpeR-HA-drp1 flies were a gift from Dr Hugo J Bellen ( Verstreken et al . , 2005 ) .", "MUL1EY12156 , TRiP parkin RNAi , UAS-mitoGFP , Mef2-GAL4 , OK6-GAL4 and TH-GAL4 flies were obtained from the Bloomington Drosophila Stock Center .", "PINK15 , parkin25 , dpk21 , UAS-drp1 and UAS-mfn RNAi flies have been previously described ( Clark et al . , 2006; Deng et al . , 2008 ) .", "For experiments involving transgenic flies , constructs were injected into w1118 and multiple independent fly lines were generated and analyzed ( Rainbow Transgenic Flies , Inc . ) .", "The deletion mutant MUL1A6 was generated by imprecise excision of MUL1EY12156 using previously described methods ( Gross et al . , 2013 ) .", "Drosophila strains were largely maintained in a 25°C humidified incubator .", "RNA was isolated from whole flies using the Macherry-Nagel Nucleospin RNA II kit .", "cDNA synthesis was performed using the Clontech RNA to cDNA EcoDry Premix Kit , using a combination of Oligo-dT and random hexamer priming .", "Quantitative PCR was performed using the BioRadiTaq Fast Sybr Green enzyme mix , 10 µl reactions in triplicate , on a Roche Light Cycler 480 .", "Standard curves were generated for MUL1 and two control genes , rpl32 and eIF1α .", "Table 1 . 10 . 7554/eLife . 01958 . 018Table 1 . Primer sequences for qPCRDOI: http://dx . doi . org/10 . 7554/eLife . 01958 . 018PrimersSequenceMUL1-FGCTATTGGTGAACTGGAGTTGGAMUL1-RAGCTTGAGTATCGTCGTTGTCTTrpl32-FTATGCTAAGCTGTCGCACAAATGrpl32-RGAACTTCTTGAATCCGGTGGGCeIF1α-FACTTCGCAAGAAGGTGTGGATTAeIF1α-RGTACGTCTTCAGGTTCCTGGC Total RNA was prepared as described above .", "RT-PCR was performed using Titanium One-Step RT-PCR Kit according to the manufacturer's instructions ( Promega , Madison , WI ) .", "Primers used for RT-PCR are as follows in Table 2 . 10 . 7554/eLife . 01958 . 019Table 2 . Primer sequences for RT-PCRDOI: http://dx . doi . org/10 . 7554/eLife . 01958 . 019PrimersSequenceMUL1 RT-FACACGAATCCGT TGCACTGMUL1 RT-RGCTCGTAGTTGTCGTAGACC For analysis of muscle , thoraces of 1- to 2-day-old-adult flies were dissected and fixed in 4% paraformaldehyde in phosphate buffered saline ( PBS ) .", "After thoraces were washed three times in PBS , muscle fibers were isolated and stained with rhodamine phalloidin ( Invitrogen , 1:1000 ) in PBS+1% Triton X-100 .", "For antibody staining , muscle fibers were permeabilized in PBS+0 . 1% Triton X-100 , blocked in 5% normal goat serum in PBS , and incubated in primary and secondary antibodies diluted in 5% normal goat serum in PBS .", "For analysis of dopaminergic neurons , brains of 3-day-old male flies were dissected and fixed in 4% paraformaldehyde in PBS .", "Blocking , primary and secondary antibody staining were performed as described previously ( Yun et al . , 2008 ) .", "To analyze mitochondria in salivary glands , salivary glands of third instar larvae were dissected , fixed in 4% paraformaldehyde in PBS , and stained with rhodamine phalloidin .", "The following primary antibodies were used: mouse anti-ATP Synthase ( Mitosciences , Eugene , OR ) , chicken anti-HA ( Millipore , Billerica , CA ) , mouse anti-Tyrosine Hydroxylase ( Immunostar Hudson , WI ) .", "All images were taken on a Zeiss LSM5 confocal microscope .", "Adult male flies were aged for 5 days at 25°C .", "Thoraces of the flies were dissected and fixed in 4% paraformaldehyde in PBS .", "Muscle fibers were dissected and subsequently permeabilized and blocked in T-TBS-3% BSA ( T-TBS: 0 . 1% Triton X-100 , 50 mM Tris-Cl [pH 7 . 4] , 188 mM NaCl ) .", "After blocking , TUNEL staining was carried out using an In Situ Cell Death Detection Kit according to the manufacturer's instructions ( Roche , Switzerland ) .", "Thoraces from 3-day-old male flies were dissected , fixed in paraformaldehyde/glutaraldehyde , postfixed in osmium tetraoxide , dehydrated in ethanol , and embedded in Epon .", "After polymerization of Epon , blocks were cut to generate 1 . 5-µmthick sections using a glass knife , or 80-nm thick sections using a diamond knife on a microtome ( Leica , Germany ) .", "Toluidine blue was used to stain 1 . 5-µm -thick tissue sections .", "Thin sections ( 80-nm thick ) were stained with uranyl acetate and lead citrate , and examined using a JEOL 100C transmission electron microscope ( UCLA Brain Research Institute Electron Microscopy Facility ) .", "At least six thoraces were examined in each sample .", "Images were taken on a Zeiss LSM5 confocal microscope .", "Each cell in the image was outlined , and the outlined area was analyzed for mitochondrial number , average size and total area using the Analyze Particles function in ImageJ software ( NIH ) .", "N = 8 Thoraces from adult flies or whole animals were homogenized in RIPA buffer containing protease inhibitors ( Roche ) .", "Total protein concentration was measured using a Bradford assay kit ( Bio-Rad , Hercules , CA ) , and the same amount of protein was loaded onto SDS-polyacryamide gels .", "The following primary antibodies were used for Western blots: mouse anti-myc ( Millipore ) , mouse anti-HA ( Millipore ) , mouse anti-Tubulin ( Sigma , St . Louis , MO ) , rabbit anti-Actin ( Sigma ) , mouse anti-Porin ( mitosciences ) , and rabbit anti-Mfn ( a generous gift from Dr Alexander J Whitworth ) .", "S2 cells were cultured in Schneider's Drosophila Medium ( Gibco , Grand Island , NY ) with 10% fetal bovine serum ( Invitrogen ) and 1% penicillin/streptomycin ( Invitrogen ) .", "Cells were seeded a day before transfection , and transfections were performed using the Effectene kit according to the manufacturer's recommendations ( Qiagen , Valencia , CA ) .", "pAC-GAL4 was transfected along with UAS-Mfn-myc , UAS-HA-parkin , and UAS-MUL1-GFP for protein expression .", "UAS vector was used as empty vector .", "Cells were harvested 2 days after transfection .", "Double-stranded RNA ( dsRNA ) against coding regions of GFP , PINK1 , parkin , MUL1 , and mfn were generated using the T7 RiboMax express RNAi system ( Promega ) .", "Primers that were used to generate dsRNAs are described below .", "S2 cells were seeded and treated with dsRNAs in serum-free medium for 40 min .", "After dsRNA treatment , complete medium was added to the culture , and the culture was incubated for 2–3 days .", "Table 3 . 10 . 7554/eLife . 01958 . 020Table 3 . Primer sequences for the generation of dsRNA templatesDOI: http://dx . doi . org/10 . 7554/eLife . 01958 . 020PrimerSequenceGFP-F ( control ) TAATACGACTCACTATAGGGTGAACCGCATCGAGCTGAAGFP-R ( control ) TAATACGACTCACTATAGGGACTTGTACAGCTCGTCCATGPINK1-FTAATACGACTCACTATAGGGAATGTGACTTCTCCAGCGAPINK1-RTAATACGACTCACTATAGGGTCGTAGCGTTTCATCAGCAGparkin-FTAATACGACTCACTATAGGGGTACGCAAAATGCTGGAGCTparkin-RTAATACGACTCACTATAGGGTAGAGGCTTGGAGGCTTCATMUL1 #1-FTAATACGACTCACTATAGGGCCACCAAGTCCACGCTTATTMUL1 #1-RTAATACGACTCACTATAGGGTGATCCTGGGACAGAGTGTGMUL1 #2-FTAATACGACTCACTATAGGGGATTGTGAAGCTGCATGAGCMUL1 #2-RTAATACGACTCACTATAGGGAACACATGGTCGAAGAGGGA S2 cells were lysed in RIPA buffer containing protease inhibitors ( Roche ) , and Western blots were performed with 2% of lysates to check protein expression .", "Immunoprecipitations were performed with the rest of lysate using Dynabeads ( Invitrogen ) according to the manufacturer's instructions .", "Proteins bound to beads were eluted in SDS sample buffer , and Western blots were performed .", "Primary antibodies used include mouse anti-Myc ( Millipore ) , rabbit anti-GFP ( Invitrogen ) , rabbit anti-HA ( Sigma ) , and rabbit anti-Actin ( Sigma ) .", "After treatment with dsRNA for 2 days , S2 cells were transfected with Mfn-Flag and incubated for 24 hr .", "Before harvest , cells were treated with the proteasome inhibitor MG132 ( Millipore ) for 4 hr .", "Cells were lysed and boiled in SDS lysis buffer ( 1% SDS , 150 mM NaCl , 10 mM Tris–HCl , pH 8 . 0 ) with protease inhibitors ( Roche ) for 10 min .", "Dilution buffer ( 10 mM Tris–HCl , pH 8 . 0 , 150 mM NaCl , 2 mM EDTA , 1% Triton ) was added , and immunoprecipitations were performed using mouse anti-Flag antibody ( Sigma ) .", "After immunoprecipitations , Western blots were probed with mouse anti-ubiquitin ( Covance ) .", "Mouse anti-FK1 ( Enzo Life Sciences , Farmingdale , NY ) and anti-FLAG ( Sigma ) antibodies were used .", "For in vitro ubiquitination assay , the glutathione S-transferase ( GST ) -tagged expression vectors pGex-MUL1 and pGEX-MUL1 LD were generated .", "GST fusion proteins ( GST-MUL1 and GST-MUL1 LD ) were expressed in E . coli and purified from inclusion body .", "The in vitro ubiquitination assay was performed using the following buffer: 25 mM Tris ( pH 7 . 5 ) , 5 mM MgCl2 , 100 mM NaCl , 1 mM DTT , 0 . 05 mM MG132 , 2 mM ATP , and 0 . 125 µg/µl Ubiquitin , with E1 ( Rabbit , 0 . 5 µg/ml ) , E2 ( extract from E . coli expressing UbcH5C ) , and presence or absence of GST-MUL1 , or GST-MUL1 LD ( as indicated ) .", "Reaction mixtures were incubated at 30°C for 2 hr , and reactions were terminated by boiling in SDS loading buffer .", "HeLa cells that did or did not overexpress parkin were generous gifts from Dr David C Chan ( Chan et al . , 2011 ) .", "Cells were cultured in Dulbeco's modified Eagle's medium ( DMEM , Gibco ) containing 10% fetal bovine serum ( Invitrogen ) and 1% penicillin/streptomycin ( Invitrogen ) .", "Cells were plated a day before transfections , and transfections were performed using the Effectene kit ( Qiagen ) or X-tremeGENE 9 DNA Transfection Reagent ( Roche ) according to the manufacturer's recommendations .", "After transfections , Z-VAD-FMK ( Santa Cruz Biotechnology , Santa Cruz , CA ) was added to cultures every 24 hr to inhibit apoptosis .", "Cells were harvested 48 hr later and lysed in RIPA buffer containing protease inhibitors ( Roche ) .", "Western blots were performed with the following primary antibodies: rabbit anti-human MUL1 ( Sigma ) , mouse anti-Mfn1 ( Abcam ) , mouse anti-Mfn2 ( Abcam ) , rabbit anti-Actin ( Sigma ) , and mouse anti-Porin ( Mitosciences ) .", "HeLa cells that did or did not stably express MUL1 shRNA were seeded in chamber slides , respectively , and transfected with YFP-Parkin one day later .", "24 hrs after transfection , cells were treated with DMSO or 40 µg/ml Antimycin A ( Sigma ) for 1 . 5 , 3 , 24 , or 48 hrs as indicated to dissipate mitochondrial membrane potential .", "For MUL1 overexpression , HeLa cells stably expressing YFP-Parkin and mitoRFP ( a kind gift from Dr . Mark R Cookson ) were seeded and transfected with Myc-MUL1 1 day later .", "Cells were treated with DMSO or 80 µg/ml Antimycin A for 1 . 5 or 3 hrs .", "After treatment of Antimycin A , cells were fixed in 10% Formalin solution ( Sigma ) , permeabilized with 0 . 1% Triton X-100 in PBS , and blocked in PBS containing 5% fetal bovine serum .", "Primary and secondary antibody staining were performed in 5% fetal bovine serum + PBS .", "The following primary antibodies were used: mouse anti-Tom20 ( BD ) , mouse anti-Flag ( Sigma ) , rabbit anti-GFP ( Invitrogen ) , and rabbit anti-Parkin ( Abcam , Cambridge , MA ) .", "More than 100 cells for each experiment were counted for quantification , and the experiments were repeated twice .", "PINK1 knockout cells were a generous gift from Dr . Richard Youle .", "Western blot analysis confirmed that there is no PINK1 expression in PINK1 knockout cells ( Narendra et al . , 2013; personal communication ) .", "MUL1 knockout HeLa cells were generated using the CRISPR/Cas system as previously described ( Cong et al . , 2013 ) .", "Briefly , MUL1 targetting sequence 5′-GCCGCCGTCA TGGAGAGCGG-3′ was inserted into pX330-U6-Chimeric_BB-CBh-hSpCas9 ( Addgene ) .", "HeLa cells were seeded a day before transfection .", "Cells were transfected with the construct using X-tremeGENE 9 DNA transfection reagent ( Roche ) following manufacturer's instructions .", "2 days after the transfection , cells were diluted and split into 48 well plates .", "Each colony was screened for deletions in MUL1 by PCR and sequencing using a set of primers 5′-CGCCTCGAACCTGACACATAATAGG-3′ and 5′-GTCTGTAAAGCAAGGAGTG GAGTGG-3′ .", "Two MUL1 knockout cells were isolated .", "Both MUL1 knockout cells have deletions including the start codon of MUL1 , one with 228 base pair deletion and another with 8 base pair deletion .", "Both deletions result in frame shift and early termination of protein translation .", "Further western blot analysis using two different anti-MUL1 antibodies ( Sigma ) confirmed that there is no MUL1 expression in MUL1−/− cells .", "HeLa cells that express scrambled shRNA or MUL1 shRNA were treated with cycloheximide ( Sigma ) for 0 , 2 , 4 , 6 hrs .", "After cycloheximide treatment , cells were harvested and lysed .", "Protein concentration of each lysate was determined by Bradford assay ( Bio-Rad ) , and an equal amount of total protein was subjected to Western blot .", "Blots were probed with anti-Mfn1 ( Abcam ) , anti-Mfn2 ( Abcam ) and Actin ( Sigma ) antibodies .", "Levels of Mfn2 and Actin were quantified using ImageJ .", "Animal care and use were carried out in accordance with NIH guidelines , NIH Manual 3040-2 , Guide for the Care and Use of Laboratory Animals ( National Research Council ) , Institutional Animal Care and Use Committee Guidebook ( ARENA and OLAW ) and approved by the NIH , NINDS/NIDCD Animal Care and Use Committee on 3/5/2012 ( ASP# 1303-9 ) .", "The work and submission for publication was approved by the Intramural Program of NINDS , NIH .", "Dissection of embryonic mouse brains and isolation of cortical neurons and plating were designed to be very quick with minimal enzymatic , mechanical , chemical , and oxidative damage , as described by Cai et al . ( 2012 ) .", "Cortices were dissected from E18-19 mouse embryos .", "Cortical neurons were dissociated by papain ( Worthington ) and plated on glial beds at a density of 50 , 000 cells per cm2 on polyornithine ( Sigma ) and Matrigel ( BD Biosciences ) -coated coverslips .", "Neurons were grown overnight in plating medium ( 5% FBS , insulin , glutamate , G5 , 1 x B27 and beta-mercaptoethanol ) supplemented with 100 x L-glutamine in Neurobasal ( Invitrogen ) .", "Starting at DIV2 , cultures were maintained in conditioned medium with half-feed changes of neuronal feed ( 1 × B27 , 100 × L-glutamine and beta-mercaptoethanol in Neurobasal ) every 3 days .", "Neurons were transfected with various constructs at DIV7-8 using calcium phosphate and processed for immunocytochemistry 72 hr ( DIV10-11 ) post transfection .", "For immunostaining , cultured cells were fixed with 4% formaldehyde ( Electron Microscopy Sciences ) and 4% sucrose ( Sigma ) in 1X phosphate-buffered saline ( PBS ) at 4°C for 30 min , washed three times with PBS for 5 min each , and then incubated in 0 . 2% saponin , 5% normal goat serum ( NGS ) , and 2% bovine serum albumin ( BSA ) in PBS for 1 hr .", "Fixed cultures were incubated with primary antibodies in PBS with 1% BSA and 0 . 05% saponin at 4°C overnight .", "Cells were washed four times with PBS at RT for 5 min each , incubated with secondary fluorescent antibodies at 1:400 dilution in PBS with 1% BSA and 0 . 05% saponin for 60 min , re-washed with PBS , and then mounted with Fluoro-Gel anti-fade mounting medium ( EMS ) for imaging .", "Sources of antibodies are as follow: polyclonal antibodies against TOM20 ( Santa Cruz ) , MUL1 ( Sigma ) , Mfn2 ( Cell Signaling , Danvers , MA ) ; monoclonal antibodies against MAP2 ( Millipore ) , GAPDH ( Research Diagnostic , Hackensack , NJ ) , CytC ( BD Biosciences , San Jose , CA ) ; Alexafluor 546 and 633-conjugated secondary antibodies ( Invitrogen ) .", "Confocal images were obtained using an Olympus Fluoview FV1000 microscope , oil immersion 63X objective ( NA-1 . 45 ) with sequential-acquisition settings .", "Images were acquired using the same settings below saturation at a resolution of 1024X1024 pixels ( 12 bit ) .", "Z stacks were acquired using a step size of 0 . 37 µm from top to bottom , and brightest point projections were made .", "For quantification , data were obtained from at least three independent experiments and the number of cells used for quantification is indicated in the figures .", "All statistical analyses were performed using One-way or Two-way ANOVA with Tukey's multiple comparison test and are presented as mean ± SEM .", "To access mitochondrial potential on a single cell basis , mature cortical neurons DIV ( 10–11 ) , both from wild-type ( WT ) and parkin knockout ( KO ) , were incubated with the cationic lipophilic compound TMRE ( 50 nM ) for 20 min in a 37°C CO2 incubator .", "Post treatment , cells were washed three times with imaging media and mounted for imaging .", "Confocal images were obtained using an Olympus confocal oil immersion 63x objective with the sequential-acquisition setting .", "The images were acquired within 30 min .", "For fluorescent quantification , image acquisition settings were below saturation at a resolution of 1024 × 1024 pixels ( 12 bit ) .", "Five to six sections were taken from the top-to-bottom of the specimen and brightest point projections were made .", "Morphometric measurements were performed using NIH ImageJ .", "Measured data were imported into Excel software for analysis .", "The thresholds in all images were set to similar levels .", "Fluorescence intensity of TMRE was expressed in corrected total cell fluorescence ( CTCF ) values .", "The mean intensity of TMRE in the soma of each group was normalized as a percentile ratio relative to that in WT cells expressing scrambled shRNA .", "Data were obtained from at least three independent experiments and the number of cells used for quantification is indicated in the figures .", "All statistical analyses were performed using one-way or two-way ANOVA with Tukey's multiple comparisons test and are presented as mean ± SEM ." ] ]

[ "Parkinson's disease ( PD ) genes PINK1 and parkin act in a common pathway that regulates mitochondrial integrity and quality .", "Identifying new suppressors of the pathway is important for finding new therapeutic strategies .", "In this study , we show that MUL1 suppresses PINK1 or parkin mutant phenotypes in Drosophila .", "The suppression is achieved through the ubiquitin-dependent degradation of Mitofusin , which itself causes PINK1/parkin mutant-like toxicity when overexpressed .", "We further show that removing MUL1 in PINK1 or parkin loss-of-function mutant aggravates phenotypes caused by loss of either gene alone , leading to lethality in flies and degeneration in mouse cortical neurons .", "Together , these observations show that MUL1 acts in parallel to the PINK1/parkin pathway on a shared target mitofusin to maintain mitochondrial integrity .", "The MUL1 pathway compensates for loss of PINK1/parkin in both Drosophila and mammals and is a promising therapeutic target for PD ." ]

[ "Parkinson's disease is the second most common neurodegenerative disorder .", "Symptoms include tremors , rigidity , and slowness , as well as dementia and depression .", "While most cases of Parkinson's disease have no known genetic cause , mutations in either of two genes—PINK1 or parkin—are known to lead to the disease .", "PINK1 and parkin belong to a single pathway that regulates the structure and function of mitochondria , the organelles that generate energy inside cells .", "Identifying inhibitors of this pathway is critically important for development of future therapies .", "In addition , previous studies showed that mice with mutations in PINK1 or parkin , as opposed to those in humans and flies , display subtle signs of Parkinson's disease: the fact that these are weak suggests that other unknown proteins or cellular pathways might compensate for loss of the genes .", "Yun et al . have now identified one such protein by showing that an increase in the level of a Protein called MUL1 counteracts the deleterious effects due to the loss of PINK1 or parkin in fruit flies .", "MUL1 is a mitochondrial protein that regulates another protein called mitofusin; the role of mitofusin is to promote the fusion of mitochondria .", "Conversely , removing MUL1 from PINK1 or parkin mutant worsens the symptoms because MUL1 is no longer present to compensate for the defects .", "Yun et al . also show that MUL1 operates through a pathway that is independent of PINK1/parkin .", "Moreover , this pathway is found in both flies and mouse neurons , which suggest that it has been conserved during evolution .", "The work of Yun et al . also suggests that MUL1 as a potential therapeutic target for Parkinson's disease ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "biochemistry and chemical biology", "cell biology" ]

[ [ "Nearly 1/3 of the human proteome is targeted to the endoplasmic reticulum ( ER ) for folding and trafficking to downstream secretory pathway environments , including the extracellular space .", "These secreted proteins engage ER-localized protein homeostasis ( or proteostasis ) factors such as chaperones and folding enzymes to facilitate their folding ( Brodsky and Skach , 2011; Powers et al . , 2009 ) .", "Proteins unable to fold in the ER are identified by ER proteostasis factors that direct them towards the proteasome ( via ER-associated degradation ) or lysosome for degradation ( Brodsky and Skach , 2011; Smith et al . , 2011 ) .", "This partitioning toward degradation pathways prevents the folding and trafficking of destabilized , misfolding-prone proteins from the ER , which could aggregate into proteotoxic conformations in downstream secretory environments ( Brodsky and Skach , 2011; Chen et al . , 2011; Powers et al . , 2009 ) .", "Many degenerative diseases result from imbalances in ER proteostasis ( Hipp et al . , 2014; Luheshi et al . , 2008 ) .", "The aberrant secretion of properly folded , but destabilized aggregation-prone proteins facilitates extracellular proteotoxic aggregation associated with many amyloid diseases ( Tipping et al . , 2015 ) .", "Examples include the transthyretin ( TTR ) or light chain ( AL ) amyloidoses ( Blancas-Mejia and Ramirez-Alvarado , 2013 ) .", "Similarly , the inability to efficiently degrade destabilized , aggregation-prone variants of secreted proteins such as rhodopsin and α-1-antitrypsin ( A1AT ) facilitates their proteotoxic intracellular aggregation linked to retinitis pigmentosa and liver disease , respectively ( Gooptu et al . , 2014; Lin and Lavail , 2010 ) .", "One strategy to ameliorate the above-mentioned and related diseases is to transcriptionally reprogram the ER proteostasis network ( ER PN ) , which has the potential to increase the ER’s capacity to identify destabilized , misfolding-prone proteins and prevent their secretion to downstream secretory environments ( Calamini and Morimoto , 2012; Chen et al . , 2015; Gestwicki and Garza , 2012 ) .", "ER PN remodeling can be achieved by activation of the unfolded protein response ( UPR ) , comprising three signaling pathways activated downstream of the ER stress sensing proteins IRE1 , ATF6 , and PERK ( Walter and Ron , 2011 ) .", "In response to the accumulation of misfolded proteins within the ER ( i . e . , ER stress ) , these sensors are activated , resulting in ER PN reprogramming predominantly mediated by the UPR-associated transcription factors XBP1s and ATF6 .", "These transcription factors induce distinct , but overlapping , transcriptional programs that alter the composition of ER PN pathways ( Adachi et al . , 2008; Shoulders et al . , 2013; Yamamoto et al . , 2004 ) .", "Intriguingly , preferential ATF6-mediated reprogramming of the ER PN offers a unique opportunity to alter the fate of disease-associated proteins .", "Genetic activation of the ATF6 transcriptional program attenuates intracellular accumulation of mutant rhodopsin and the Z-variant of A1AT ( Chiang et al . , 2012; Smith et al . , 2011 ) .", "Furthermore , stress-independent chemical genetic activation of a ligand-regulated ATF6 attenuates the secretion and extracellular aggregation of destabilized , amyloidogenic variants of TTR or immunoglobulin light chains ( LCs ) , without significantly impacting secretion of stable , non-amyloidogenic variants of these proteins or the global endogenous secreted proteome ( Chen et al . , 2014; Cooley et al . , 2014; Shoulders et al . , 2013 ) .", "These results suggest that small molecules that similarly induce preferential ER PN remodeling through pharmacologic activation of the ATF6 transcriptional program should attenuate the aberrant secretion and aggregation of these proteins linked to degenerative diseases ( Chen et al . , 2015 ) .", "Traditionally , UPR signaling is activated using small molecule ER stressors such as the SERCA inhibitor thapsigargin ( Tg ) or tunicamycin ( Tm ) , an inhibitor of N-linked glycosylation ( Chen et al . , 2015 ) .", "However , chronic addition of these ER stressors induces apoptosis , predominantly through activation of the PERK arm of the UPR ( Hetz , 2012; Tabas and Ron , 2011 ) .", "Small molecule kinase inhibitors and ATP mimetics have been shown to activate the IRE1/XBP1s arm of the UPR ( Mendez et al . , 2015; Papa et al . , 2003; Wang et al . , 2012 ) .", "The small molecule BIX induces expression of the ATF6-target gene BiP through an ATF6-dependent mechanism , but does not significantly induce expression of other ATF6 target genes such as GRP94 , p58IPK and PDIA4 , likely limiting its utility for ER PN reprogramming in the above-mentioned diseases ( Kudo et al . , 2008 ) .", "These results highlight the need for further development of molecules that preferentially activate UPR-associated transcriptional programs , such as that regulated by ATF6 , to define the benefit of ER PN reprogramming to ameliorate protein aggregation diseases and protein misfolding diseases .", "A challenge in identifying such compounds is the difficulty associated with efficiently assessing the preferential activation of a sub-transcriptional pathway that is part of a highly integrated signaling network ( e . g . , the ATF6 arm of the UPR ) .", "An increasing proportion of failed clinical trials have highlighted the importance of the early elimination of compounds functioning through off-target effects , which can be achieved using multidimensional assays such as gene expression profiling ( Feng et al . , 2009; Verbist et al . , 2015 ) .", "The use of a transcriptional profiling approach early in the drug discovery process and before functional assessment allows for the identification of promising small molecules that preferentially activate a target transcriptional program , instead of global network activation .", "However this strategy has only been employed in a small number of drug development programs and has not been used to identify small molecule activators of a sub-UPR transcriptional program , such as that regulated by ATF6 ( Antipova et al . , 2008; Stegmaier et al . , 2004; Stegmaier et al . , 2007 ) .", "Here , we employ a cell-based high-throughput screen ( HTS ) in combination with transcriptional profiling to identify non-toxic small molecules that preferentially activate the ATF6 transcriptional program , independent of global UPR activation .", "Small molecule ER proteostasis regulators identified by our screening strategy mimic the ER PN reprogramming afforded by chemical genetic ATF6 activation , through a process dependent on endogenous ATF6 .", "Furthermore , our small molecule ER proteostasis regulators phenocopy the ability of chemical genetic ATF6 activation to preferentially decrease the secretion and proteotoxic aggregation of destabilized , amyloidogenic proteins without affecting the secretion of stable , non-amyloidogenic proteins or the global endogenous secreted proteome .", "These results highlight the utility of transcriptional profiling upstream of functional studies as part of a multi-tiered screening strategy to identify small molecule ER proteostasis regulators that can be employed to ameliorate imbalances in ER function involved in aggregation-associated degenerative diseases and possibly loss-of-function protein misfolding diseases ." ], [ "We employed a three-tiered screening strategy to identify small molecules that preferentially activate the ATF6 UPR transcriptional program for ER PN reprogramming ( Figure 1A ) .", "First , a cell-based HTS was performed using a transcriptional reporter that includes a fragment of the UPR-inducible BiP promoter driving expression of firefly luciferase ( ERSE-FLuc; Figure 1B ) ( Yoshida et al . , 1998 ) .", "BiP is preferentially induced by ATF6 ( Shoulders et al . , 2013 ) , indicating that the ERSE-FLuc reporter should preferentially report on activation of the ATF6 transcriptional program .", "We tested the dependence of ERSE-FLuc activation on XBP1s and ATF6 in HEK293DAX cells that stably express tet-inducible XBP1s and a trimethoprim ( TMP ) -regulated dihydrofolate reductase ( DHFR ) -ATF6 fusion , hereafter referred to as chemical genetic ATF6 activation ( Shoulders et al . , 2013 ) .", "As predicted , the ERSE-FLuc reporter was preferentially activated by ATF6 , relative to XBP1s ( Figure 1—figure supplement 1A ) in HEK293DAX cells .", "We then stably transfected the ERSE-FLuc reporter into HEK293T-Rex cells and selected a single clone exhibiting dose-dependent reporter activation upon treatment with the ER stressors Tg or Tm ( Figure 1C , D ) .", "This assay was further miniaturized for 1536-well high-throughput screening at the Scripps Research Institute Molecule Screening Center ( SRIMSC ) ( Supplementary file 1 ) . 10 . 7554/eLife . 15550 . 003Figure 1 . High-throughput screen to identify small molecule ER proteostasis regulators .", "( A ) Illustration showing the three-tiered screening strategy implemented to identify small molecules that preferentially activate the ATF6 transcriptional program .", "( B ) Schematic of the ERSE-firefly luciferase ( FLuc ) reporter used in our HTS approach .", "( C ) Activation of FLuc luminescence in HEK293T-Rex cells stably expressing ERSE-FLuc treated with the indicated concentrations of thapsigargin ( Tg ) for 18 hr .", "Error bars represent standard deviation for n = 3 replicates .", "( D ) Activation of FLuc luminescence in HEK293T-Rex cells stably expressing ERSE-FLuc treated with the indicated concentrations of tunicamycin ( Tm ) for 18 hr .", "Error bars represent standard deviation for n = 3 replicates .", "( E ) Plot showing ERSE-FLuc activation in HEK293T-Rex cells stably expressing ERSE-FLuc treated with the 13 , 748 small molecule ER proteostasis activators identified in the primary screen ( 6 . 8 µM; 18 hr ) .", "Luminescence is shown as % signal relative to Tg treatment ( 500 nM; 18 hr ) .", "Error bars show standard deviation for n = 3 replicates .", "The dashed red line shows 25 . 1% Tg activity . DOI: http://dx . doi . org/10 . 7554/eLife . 15550 . 00310 . 7554/eLife . 15550 . 004Figure 1—figure supplement 1 . Selectivity of the ERSE-FLuc reporter for the ATF6 UPR arm and highly represented chemical substructures in the top 281 ER proteostasis regulators .", "( A ) Activation of ERSE-FLuc in HEK293DAX cells stably expressing trimethoprim ( TMP ) -regulated DHFR-ATF6 and doxycycline ( dox ) inducible XBP1s .", "Dox ( 1 µM; 12 h ) was added to selectively activate XBP1s ( red ) and TMP ( 10 µM; 12 h ) was added to activate DHFR-ATF6 ( blue ) , or both were added to activate both XBP1s and ATF6 ( green ) .", "Error bars show standard error for n = 3 .", "***p<0 . 001 .", "( B ) Network plot depicting the 12 overrepresented structural moieties ( A-K ) identified by performing a hierarchical maximum common substructure search of the top 281 small molecule ER proteostasis regulators .", "The 8 prioritized small molecule ER proteostasis regulators are shown in red .", "( C ) Bar graph showing the prevalence of the 12 most represented chemical moieties identified in the top 281 ER proteostasis regulators .", "JKlustor was used for clustering and diversity analysis of these molecules , JChem 6 . 2 . 2 , 2014 , ChemAxon ( http://www . chemaxon . com ) .", "A hierarchical maximum common substructure search of the top 281 small molecule ER proteostasis regulators was carried out using LibraryMCS within JKlustor .", "A substructure search of the 12 most represented moieties ( including derivatives indicated by small letters “a” and “b” was then carried out in the 281 compound set to account for the presence of multiple moieties within the same compound .", "See the network plot in B for visualization of compounds containing multiple highly represented moieties . DOI: http://dx . doi . org/10 . 7554/eLife . 15550 . 004 We screened the 644 , 951-small molecule Scripps Drug Discovery Library ( SDDL ) at SRIMSC to identify molecules that activate the ERSE-FLuc reporter .", "The performance of this assay was consistent across all experimental plates ( Z’ = 0 . 58 ± 0 . 05 ) and exhibited a robust signal to noise ratio ( signal/background = 6 . 21 ± 0 . 73 ) ( Supplementary file 1 ) .", "Small molecule activation of ERSE-FLuc was normalized to Tg ( assigned to be 100% activation ) , allowing comparisons between screening plates .", "This screen identified 13 , 799 molecules that activated the ERSE-FLuc reporter >25 . 1% .", "These hits were then filtered against results from a previous screen of the SDDL to remove 49 small molecules that activate the cytosolic heat shock response ( Calamini et al . , 2012 ) .", "Confirmation screening of the remaining 13 , 750 compounds identified 12 , 376 molecules that activated the ERSE-FLuc reporter 3 standard deviations above the DMSO control ( hit cutoff 5 . 7% activation ) –a 90% hit confirmation ( Figure 1E ) .", "To decrease the number of compounds for follow-up , we increased the cutoff stringency to that used in the primary screen ( 25 . 1% activation ) , which narrowed the list of ERSE-FLuc activators to 281 compounds ( Figure 1E , Figure 2—source data 1 ) .", "These include the ER stressors Tg and Tm , which were both present in the SDDL .", "All 281 confirmed hits were subjected to quality control at SRIMSC to confirm identity and purity using liquid chromatography/mass spectrometry .", "A maximum common substructure search identified 12 chemical substructures that were highly represented in these 281 ERSE-FLuc activators ( Figure 1—figure supplement 1B , C ) .", "These include catechols ( 64/281 ) , anilides ( 61/281 ) and benzylidene hydrazines ( 33/281 ) .", "We next sought to identify small molecule ER proteostasis regulators that preferentially activate the ATF6 transcriptional program independent of global ER stress .", "To remove molecules that induce ER stress and global UPR activation , the top 281 compounds were counterscreened using an alternative luciferase reporter , signifying activation of the IRE1/XBP1s arm of the UPR ( Figure 1A ) .", "This reporter contains Renilla luciferase ( RLuc ) expressed out of frame downstream of the XBP1 splice site , preventing RLuc translation in the absence of ER stress ( Figure 2A ) ( Back et al . , 2006; Iwawaki et al . , 2004 ) .", "In response to ER stress-dependent IRE1 activation , the 26-nt XBP1 intron is removed , producing a frame shift that allows for RLuc translation and luminescence .", "Robust dose-dependent activation of the XBP1-RLuc reporter upon addition of the ER stressors Tg and Tm was confirmed in HEK293T-Rex cells stably expressing the XBP1-RLuc reporter , producing a Z’ score of 0 . 75 for Tg ( Figure 2—figure supplement 1A , B ) . 10 . 7554/eLife . 15550 . 005Figure 2 . Counterscreening and transcriptional profiling identifies preferential activators of the ATF6 transcriptional program .", "( A ) Schematic of the XBP1-RLuc splicing reporter used to monitor activation of the IRE1/XBP1s arm of the UPR in HEK293T-Rex cells treated with small molecule ER proteostasis regulators .", "( B ) Plot of ERSE-FLuc activation and XBP1-RLuc activation for the top 281 small molecule ER proteostasis regulators ( 6 . 8 µM; 18 hr ) in HEK293T-Rex cells stably expressing ERSE-FLuc or XBP1-RLuc .", "Activation for each axis is shown as the percent signal relative to Tg treatment ( 500 nM; 18 hr ) .", "Prioritized ER proteostasis regulators described in Table 1 are shown in red .", "Error bars show standard deviation for n = 3 .", "( C ) Heat map of multiplex gene expression ( MGE ) profiling for the top 281 ER proteostasis regulator compounds .", "HEK293T-Rex cells were incubated with 10 μM of each proteostasis regulator for 6 hr , then cells were lysed and MGE profiling was performed .", "Genes probed constitute transcriptional targets preferentially induced by the ATF6 , XBP1s or PERK arms of the UPR or other stress-responsive signaling pathways , as indicated .", "Heat map is shown with compounds clustered according to their activity .", "The green section represents global ER stressors while the pink sections represent molecules that preferentially activate the ATF6 transcriptional program .", "Prioritized ER proteostasis regulators identified in Table 1 are highlighted . DOI: http://dx . doi . org/10 . 7554/eLife . 15550 . 00510 . 7554/eLife . 15550 . 006Figure 2—source data 1 . Excel spreadsheet showing the structure , % activation ERSE-FLuc , and % activation XBP1-RLuc for the top 281 compounds identified through primary HTS screening and confirmation screening . These data were used to prepare Figure 2B . DOI: http://dx . doi . org/10 . 7554/eLife . 15550 . 00610 . 7554/eLife . 15550 . 007Figure 2—source data 2 . Excel spreadsheet showing the Multiplex Gene Expression ( MGE ) expression data used to prepare Figure 2C and Figure 2—figure supplement 1C . DOI: http://dx . doi . org/10 . 7554/eLife . 15550 . 00710 . 7554/eLife . 15550 . 008Figure 2—figure supplement 1 . XBP1-RLuc reporter dose response and Tg-normalized MGE profiling for 281 top ER proteostasis regulators .", "( A ) Activation of Renilla luciferase ( RLuc ) luminescence in HEK293T-Rex cells stably expressing XBP1-RLuc treated with the indicated concentration of thapsigargin ( Tg ) for 18 hr .", "Error bars represent standard deviation for n = 3 replicates .", "The Z’ score is for [Tg] = 500 nM .", "( B ) Activation of RLuc luminescence in HEK293T-Rex cells stably expressing XBP1-RLuc treated with the indicated concentration of tunicamycin ( Tm ) for 18 hr .", "Error bars represent standard deviation for n = 3 replicates .", "( C ) Heat map of normalized gene expression from MGE profiling experiment shown in Figure 2C .", "The expression of the ATF6 ( blue ) , XBP1s ( green ) and PERK ( orange ) target genes induced by the addition of the small molecule ER proteostasis regulators were normalized to the induction observed upon addition of Tg prior to plotting .", "The resulting heat map is shown with compounds clustered according to their activity .", "Molecules highlighted with green on the right are global UPR activators as they robustly induce genes regulated by the three arms of the UPR .", "Alternatively , molecules highlighted in pink preferentially activate ATF6 . DOI: http://dx . doi . org/10 . 7554/eLife . 15550 . 008 We compared the activation of this XBP1-RLuc reporter with that observed for the ERSE-FLuc reporter , both being normalized to the Tg control ( defined as 100% activation ) .", "Importantly , Tg ( 1 of the 281 screening hits ) robustly activates both the ERSE-FLuc and XBP1-Rluc reporters , confirming the ability of this approach to identify global ER stressors ( Figure 2B ) .", "In contrast , the majority of molecules identified in the primary screen ( 200/281 ) activate the ERSE-FLuc reporter >2-fold better than the XBP1s-Rluc reporter ( Figure 2B ) .", "This suggests that small molecules identified from the HTS preferentially activate the ATF6 transcriptional program independent of ER stress and/or global UPR activation .", "As a final filter , we performed multiplex gene expression ( MGE ) profiling in HEK293T-Rex cells treated with the 281 hits to directly monitor transcriptional changes and identify compounds that preferentially active the ATF6 transcriptional program ( Figure 1A ) .", "While the ATF6 and XBP1s luciferase reporters were indispensible for initial high-throughput screening , we wanted to ensure that any further prioritization was based on a comprehensive picture of the transcriptional remodeling induced by the compounds .", "For instance , we wanted to make certain that the compounds induce multiple ATF6 targets in addition to BiP , as well as monitor that no other stress-responsive signaling pathways are affected .", "For this approach , we defined 5–6 target genes that were previously identified as preferential targets of the ATF6 , XBP1s or PERK signaling pathway ( Lu et al . , 2004; Shoulders et al . , 2013 ) , as well as genes downstream of other stress responsive signaling pathways , including the heat shock response ( HSR ) , the oxidative stress response ( Nrf2 ) , and inflammatory signaling ( NFκB ) ( see Figure 2—source data 2 for a complete list of genes ) .", "The MGE analysis quantifies mRNA levels of these target genes in HEK293T-Rex cells treated with our prioritized 281 ER proteostasis regulators .", "This provides a medium-throughout approach to define transcriptional changes induced by our screening hits .", "We performed hierarchical clustering of the 281 screening hits based on the MGE data ( Figure 2C ) .", "This showed that compounds grouped into three distinct transcriptional profiles:", "1 ) global UPR activators that activate ATF6 , XBP1s and PERK target genes ( green ) ,", "2 ) preferential activators of ATF6 targets ( pink ) , and", "3 ) compounds with weak to no transcriptional effect .", "Apart from the UPR , no compounds significantly induced gene targets regulated by other stress-responsive signaling pathways ( Figure 2C ) .", "In order to compare the relative induction of UPR-associated transcriptional programs activated downstream of ATF6 , XBP1s and PERK , we normalized transcript changes to those observed in Tg-treated cells ( Figure 2—figure supplement 1C ) .", "This allows direct comparison of transcriptional reprogramming across the three arms of the UPR .", "Overall , eighteen molecules , including the known ER stressors Tg and Tm , were identified to induce genes regulated by all three arms of the UPR , indicating that these molecules induce ER stress and/or global UPR activation ( Figure 2C ) .", "In contrast , 79 compounds exhibited preferential activation of genes regulated in the ATF6 transcriptional program , relative to IRE1/XBP1s and PERK .", "We prioritized 8 compounds for further characterization ( Table 1 — highlighted in Figure 2C ) .", "At this prioritization stage , we primarily focused on the MGE data and less emphasis was placed on the ATF6 or XBP1s luciferase reporter activation , as the MGE results provided richer information .", "The prioritized compounds were selected based on their ability to preferentially activate the ATF6 transcriptional program with varying magnitude according to the MGE analysis ( Figure 2C ) .", "Furthermore , we focused on compounds containing a 2-amino-p-cresol moiety ( cluster F , Figure 1—figure supplement 1B–C ) , as these compounds were enriched within our top 79 preferential activators of the ATF6 transcriptional program ( Figure 2C ) , but we ensured that other representatives of diverse structural classes were included .", "Only catechols ( cluster A; Figure 1—figure supplement 1B–C ) were mostly excluded , as these are known to be associated with adverse pharmacology ( Baell and Holloway , 2010 ) . 10 . 7554/eLife . 15550 . 009Table 1 . Prioritized ER proteostasis regulators . DOI: http://dx . doi . org/10 . 7554/eLife . 15550 . 009NameStructureMWPolar Surface AreaAlogPStructural moieties ( Figure 1—figure supplement 1B–C ) % ERSE-FLuc activation*% XBP1-RLuc activation*Promiscuity**5 271 . 241 . 132 . 62-60 . 9 ± 6 . 09 . 1 ± 0 . 71 out of 31132283 . 358 . 563 . 27F140 . 1 ± 17 . 410 . 3 ± 7 . 92 out of 32145260 . 375 . 363 . 01F , H83 . 4 ± 6 . 324 . 5 ± 4 . 71 out of 31147255 . 349 . 333 . 35B , F63 . 2 ± 0 . 617 . 5 ± 1 . 91 out of 32148322 . 3149 . 122 . 00B , F52 . 5 ± 14 . 316 . 6 ± 2 . 43 out of 31238314 . 769 . 642 . 94B , L68 . 2 ± 5 . 761 . 1 ± 0 . 1 . 52 out of 31258393 . 291 . 153 . 76C , D28 . 6 ± 6 . 132 . 6 ± 1 . 01 out of 31263257 . 290 . 443 . 10C20 . 7 ± 2 . 32 . 4 ± 1 . 01 out of 31*% Tg activation from triplicate confirmation screen at 6 . 8 μM** Number of screening assays in which the compound was found active The toxicity of these 8 prioritized molecules was assessed in HEK293T-Rex cells .", "The compounds demonstrated maximum toxicity of <10–43% that of doxorubicin , with all of them exhibiting a cytotoxic concentration 50% ( CC50 ) of >17 μM ( the highest tested concentration ) ( Supplementary file 2 ) .", "These results suggest that our three-tiered screening strategy ( Figure 1A ) allowed identification of generally non-toxic small molecule ER proteostasis regulators that preferentially activate the ATF6 transcriptional program .", "Comprehensive transcriptome analysis in HEK293T-Rex cells was employed to further validate the capacity of three prioritized small molecule ER proteostasis regulators — 132 , 147 , and 263 — to preferentially activate the ATF6 transcriptional program .", "These molecules were selected because they were structurally diverse and exhibited variable levels of ATF6 activation ( measured by reporter assays and MGE ) ( Table 1 , Figure 2C ) .", "As a global UPR activation control , mRNA-seq analysis was also performed on HEK293T-Rex cells treated with Tg .", "This transcriptome analysis reveals that 132 , 147 , and 263 preferentially induce UPR-regulated genes involved in ER PN maintenance such as BiP , GRP94 and SEL1L ( Figure 3—figure supplement 1A–C , red ) .", "Compound 132 generated the most robust induction of these genes , relative to 147 and 263 , consistent with the levels of ERSE-FLuc activation afforded by these molecules ( Table 1 ) .", "Importantly , these small molecules induced three genes that were not upregulated by Tg-dependent global UPR activation — HSPA1A , HSPA1B , and HMOX1 ( Figure 3—figure supplement 1D–F ) .", "HSPA1A and HSPA1B are transcriptional targets of the heat-shock response ( HSR ) -associated transcription factor HSF1 ( Ryno et al . , 2014 ) and HMOX1 is a transcriptional target of the oxidative stress response ( Yang et al . , 2015 ) .", "Comparing the transcriptional profile previously observed for HSF1 activation ( Ryno et al . , 2014 ) to that observed for 132 , 147 , and 263 showed no significant correlation , indicating that these molecules do not induce the HSR ( Figure 3—figure supplement 1G–J ) .", "Similarly , we did not observe increased expression of oxidative stress markers , indicating that these molecules do not globally activate the oxidative stress response ( Figure 3—figure supplement 1K ) .", "These results confirm that our molecules selectively induce UPR-regulated ER proteostasis genes independent of other stress-responsive signaling pathways .", "The ability of 132 , 147 , and 263 to preferentially activate transcriptional programs induced downstream of the ATF6 , IRE1/XBP1s , and PERK arms of the UPR was next assessed from the mRNA-seq data .", "For this analysis , we defined genesets of >15 UPR target genes activated downstream of each of these three UPR signaling pathways .", "Previously published transcriptional profiles of chemical genetic activation of XBP1s , ATF6 or PERK defined these genesets ( Lu et al . , 2004; Shoulders et al . , 2013 ) .", "The induction of the selected genes by 132 , 147 , and 263 was then normalized to that observed with Tg , allowing direct comparisons across the three genesets ( Figure 3—source data 1 ) .", "The ability of this approach to define preferential ER proteostasis remodeling was confirmed by demonstrating that chemical genetic ATF6 activation in HEK293DAX cells selectively induces the ATF6 transcriptional program ( Figure 3A , B ) . 10 . 7554/eLife . 15550 . 010Figure 3 . Small molecule ER proteostasis regulators preferentially activate the ATF6 transcriptional program .", "( A ) Heat map showing the upregulation of ATF6 ( blue ) , XBP1s ( red ) or PERK ( green ) target genes in the mRNA-Seq analysis of HEK293T-Rex cells treated with Tg ( 1 µM ) , 132 ( 10 µM ) , 147 ( 10 µM ) or 263 ( 10 µM ) .", "Genes used in this analysis were selected from published reports indicating their selectivity for ATF6 , XBP1s or PERK ( Lu et al . , 2004; Shoulders et al . , 2013 ) .", "The induction of each gene was normalized to the respective induction observed with the global UPR activator Tg and reported as % Tg induction .", "The expression of these target genes in the mRNA-Seq analysis of TMP-dependent DHFR-ATF6 activation in HEK293DAX cells is also shown .", "The data used to prepare this heat map is included in Figure 3—source data 1 .", "( B–E )", "Box plots showing the relative activation of ATF6 , XBP1s , and PERK genesets in HEK293DAX cells following TMP-dependent DHFR-ATF6 activation ( B ) , or HEK293T-Rex cells treated with 132 ( C ) , 147 ( D ) or 263 ( E ) from the data shown in Figure 3A .", "Differences in activation of the ATF6 geneset relative to the XBP1s or PERK genesets were confirmed by one-way ANOVA and the p-values of unpaired t-tests are shown .", "( F–H )", "Box plots comparing the average fold change at the protein levels of ATF6 , XBP1s and PERK target sets in HEK293T-Rex cells treated with 10 µM 132 ( F ) , 147 ( G ) , or 263 ( H ) for 16 hr as observed in the proteomics analysis .", "The protein sets used here are the same as the genesets for the analysis of the RNA-Seq data ( A–E ) .", "Differences in activation of the ATF6 target protein set relative to the XBP1s or PERK target protein sets were confirmed by one-way ANOVA and the p-values of unpaired t-tests are shown .", "The data used to prepare these graphs can be found in Figure 3—source data 1 .", "( I–J )", "Plot showing the relative activation of the ERSE-FLuc ( blue ) and XBP1-RLuc ( red ) reporters in HEK293T-Rex cells treated with the indicated concentration of 147 ( I ) or 263 ( J ) for 15 hr .", "Error bars represent standard error for n = 3 replicates . DOI: http://dx . doi . org/10 . 7554/eLife . 15550 . 01010 . 7554/eLife . 15550 . 011Figure 3—source data 1 . Excel spreadsheet containing the RNA-seq and proteomic geneset data used to prepare Figure 3A–H . This spreadsheet contains 7 tabs including a Table of Contents , Geneset mRNA ATF6 , Geneset mRNA XBP1s , Geneset mRNA PERK , Geneset proteomics ATF6 , Geneset proteomics XBP1s , and Geneset proteomics PERK . DOI: http://dx . doi . org/10 . 7554/eLife . 15550 . 01110 . 7554/eLife . 15550 . 012Figure 3—figure supplement 1 . mRNA-Seq analysis shows that ER proteostasis regulators do not globally activate the Heat Shock Response ( HSR ) or oxidative stress response pathways .", "( A–C )", "Volcano plots showing log2 mRNA fold-change ( FC , relative to vehicle-treated controls ) versus -log10 Benjamini Hochberg ( BH ) adjusted p-value for mRNAs in HEK293T-Rex cells treated with small molecule ER proteostasis regulators 132 ( A ) , 263 ( B ) or 147 ( C ) ( all treated at 10 µM , 6 hr ) .", "The red symbols represent ER proteostasis factors as identified in the Source Data .", "The ATF6-regulated ER proteostasis genes BiP , GRP94 , and SEL1L are highlighted .", "( D–F )", "Correlation of log2 mRNA fold change ( FC ) with Tg ( 1 µM; 6 hr; relative to vehicle-treated control ) versus log2 mRNA FC with small molecule ER proteostasis regulators 132 ( D ) , 263 ( E ) or 147 ( F ) in HEK293T-Rex cells ( treated as indicated above ) .", "All genes displaying a significant change ( BH adjusted p-value < 0 . 1 ) for Tg-treatment or treatment with small molecule ER proteostasis regulators are shown .", "The red symbols represent ER proteostasis factors , as identified in the Source Data .", "The gray dashed line reflects levels of gene induction observed following Tg-induced ER stress ( slope = 1 ) .", "The red dashed line corresponds to a least squares linear regression that reflects the induction of ER proteostasis gene induction observed following treatment with the indicated small molecule ER proteostasis regulator .", "Treatment with 132 ( D ) shows a clear correlation with Tg treatment indicating global UPR activation , while 263 ( E ) and 147 ( F ) do not globally increase transcription of Tg-regulated genes and show lower levels of select ER proteostasis gene induction .", "The ATF6-regulated ER proteostasis genes BiP , GRP94 , and SEL1L are highlighted .", "G–J .", "Plot showing the log2 transcript fold change with Shield-1-dependent FKBP-HSF1 activation in HEK293T-Rex cells stably expressing FKBP-HSF1 ( mRNA-seq analysis ) ( Ryno et al . , 2014 ) versus log2 mRNA fold change with Tg ( G ) or small molecule ER proteostasis regulators 132 ( H ) , 263 ( I ) , or 147 ( J ) in HEK293T-Rex cells .", "The FKBP-HSF1 fusion protein is activated by the addition of the pharmacologic chaperone Shield-1 , which stabilizes the active FKBP-HSF1 protein and promotes HSF1 transcriptional activity ( Ryno et al . , 2014 ) .", "All gene expression data used in this analysis are included in the Source Data .", "Only genes whose expression is significantly altered ( FDR < 0 . 1 ) by FKBP-HSF1 activation in HEK293T-Rex cells are shown .", "( K ) Plot comparing the expression of oxidative stress target genes in HEK293T-Rex cells treated with vehicle or Tg ( 1 µM; grey ) , 132 ( 10 µM; blue ) , 147 ( 10 µM; red ) or 263 ( 10 µM; green ) .", "The data are presented as log reads per kilobase million ( RPKM ) of vehicle versus log RPKM for the indicated treatment using the data from our mRNA-seq analysis .", "Genes that fall on the diagonal are not induced by small molecule treatment .", "Oxidative stress genes were selected from the SABIOsciences Oxidative Stress PCR Array . DOI: http://dx . doi . org/10 . 7554/eLife . 15550 . 01210 . 7554/eLife . 15550 . 013Figure 3—figure supplement 1—source data 1 . Excel spreadsheet describing the RNA-seq data used to prepare Figure 3—figure supplement 1A–K . This spreadsheet contains 4 tabs including a Table of Contents , RNA-seq FC Tg 132 147 263 ATF6 , HSF1 genes , and oxidative stress genes . DOI: http://dx . doi . org/10 . 7554/eLife . 15550 . 01310 . 7554/eLife . 15550 . 014Figure 3—figure supplement 2 . ER proteostasis regulators induce ATF6 targets at the protein level and time-dependence of ATF6 and XBP1s target activation .", "( A–C )", "Correlation between mRNA fold changes ( FC ) and protein fold changes for treatment of HEK293T-Rex cells with compounds 132 ( A ) , 263 ( B ) , and 147 ( C ) .", "mRNA changes were measured by RNA-Seq after 10 µM compound treatment for 6 hr .", "Protein changes were measured by Tandem-Mass-Tag-based quantitative proteomics after 10 µM compound treatment for 16 hr .", "ER proteostasis genes are marked in red and select ATF6 targets are labeled .", "( D ) Representative immunoblot and quantification of select proteins regulated by the ATF6 transcriptional program assessed by quantitative Western blot .", "HEK293T-Rex cells were treated with ER proteostasis regulators ( 10 µM ) for 16 hr and whole cell lysates were separated by SDS-PAGE and immunoblotting was carried out using antibodies against the indicated proteins .", "Quantification was performed on an Odyssey Licor .", "Error bars represent standard error for n = 3 replicates .", "*p<0 . 05; **p<0 . 01; ***p<0 . 001 ( E–F ) Time course of induction of BiP ( a gene regulated by the ATF6 transcriptional program ) and SEC24D ( a gene regulated by the XBP1s transcriptional program ) mRNA as measured by qPCR in HEK293T cells treated for the indicated times with 10 µM of 263 ( E ) or 147 ( F ) .", "Error bars represent the standard error for n > 3 biological replicates . DOI: http://dx . doi . org/10 . 7554/eLife . 15550 . 01410 . 7554/eLife . 15550 . 015Figure 3—figure supplement 2—source data 2 . Excel spreadsheet describing the whole cell proteomic data used to prepare Figure 3—figure supplement 2A–C . RNA-seq data for genes identified by proteomics is also shown .", "This spreadsheet contains 4 tabs including a Table of Contents , 132 Proteomics RNA-Seq , 263 Proteomics RNA-seq , and 147 Proteomics RNA-seq . DOI: http://dx . doi . org/10 . 7554/eLife . 15550 . 015 Compound 132 significantly activates the ATF6 transcriptional program ( 75% relative to Tg ) , but also robustly induces expression of IRE1/XBP1s and PERK genesets ( Figure 3A , C ) .", "While modest preferential activation of the ATF6 transcriptional program is observed , this indicates that 132 is a global UPR activator .", "In contrast , compound 147 and to a lesser extent 263 show preferential activation of the ATF6 transcriptional program relative to IRE1/XBP1s and PERK albeit to lower levels , showing 45% and 50% activation relative to Tg , respectively ( Figure 3A , D , E ) .", "Reprogramming of the ER PN was next monitored using quantitative proteomics in HEK293T-Rex cells treated with 132 , 147 or 263 .", "Protein level changes induced by the addition of these small molecules correlated with transcript changes observed by mRNA-seq ( Figure 3—figure supplement 2A–C ) .", "Furthermore , the extent of protein expression changes followed the same trend as the transcript changes: induction was highest with compound 132 , relative to 263 and 147 .", "We further confirmed this trend by quantitative immunoblotting monitoring the expression of several proteins regulated by the ATF6 transcriptional program ( Figure 3—figure supplement 2D ) .", "Applying an analogous geneset analysis to that described above showed that 132 increased levels of proteins regulated downstream of ATF6 , IRE1/XBP1s and PERK to similar extents , further confirming that 132 is a global UPR activator ( Figure 3F ) .", "However , 147 and 263 preferentially increased levels of proteins regulated by the ATF6 transcriptional program , further confirming that these molecules reprogram the ER PN through a mechanism involving preferential activation of this transcriptional program ( Figure 3G , H ) .", "Preferential activation of the ATF6 transcriptional program by 147 and 263 could result from a mild level of ER stress that should increase upon addition of higher concentrations of these molecules or longer duration of treatment .", "When monitoring the activation of the ATF6-selective ERSE-FLuc reporter in HEK293 cells treated with increasing concentrations of 147 and 263 ( Figure 3I , J ) , neither compound showed significant increases in ERSE-FLuc activation at concentrations >10 µM – the concentration used for mRNA-seq .", "Similarly , increasing the concentration of these molecules did not significantly increase activation of the IRE1/XBP1s-selective XBP1-RLuc reporter .", "Furthermore , we observe a maximal BiP induction at 6 hr in cells treated with 147 or 263 over a 24 hr time course and no time-dependent increases in expression of the XBP1s target Sec24D ( Figure 3—figure supplement 2E–F ) .", "These results indicate that increasing the concentration or duration of treatment with small molecule ER proteostasis regulators does not alter their preferential activation of the ATF6 transcriptional program , suggesting that the compounds do not function by inducing a mild level of ER stress .", "We next evaluated whether the preferential activation of the ATF6 transcriptional program afforded by our small molecules requires endogenous ATF6 .", "Expression of the ATF6 target gene BiP was measured in ATF6+/+ mouse embryonic fibroblast ( MEF ) cells treated with the top 8 prioritized small molecule ER proteostasis regulators shown in Table 1 .", "All of these molecules induced BiP expression in these cells , albeit to varying extents ( Figure 4A ) .", "In contrast , BiP induction was significantly inhibited in the ATF6-/- MEFs , showing that small molecule-dependent upregulation of a gene included in the ATF6 transcriptional program requires endogenous ATF6 .", "Importantly , we did not observe significant levels of XBP1 splicing or eIF2α phosphorylation in cells treated with the ER proteostasis regulators , indicating that these molecules do not significantly activate the IRE1/XBP1s or PERK arms of the UPR ( Figure 4B , C ) . 10 . 7554/eLife . 15550 . 016Figure 4 . ER proteostasis regulators depend on endogenous ATF6 activation .", "( A ) BIP mRNA measured by qPCR in ATF6+/+ and ATF6-/- MEFs treated with the indicated small molecule ER proteostasis regulators ( 10 µM; 6 hr ) .", "Error bars show standard error for n > 3 .", "*p<0 . 05; **p<0 . 01; ***p<0 . 001 .", "( B ) Gel showing XBP1 splicing in HEK293T cells measured by RT-PCR .", "RNA was extracted from cells treated with global ER stressors Tg ( 500 nM ) or tunicamycin ( Tm; 1 µg/mL ) or 10 µM of the indicated small molecule ER proteostasis regulator for 3 hr .", "The small molecule BIX is included as a control .", "After generation of cDNA , primers flanking the XBP1 splicing site were used to amplify the longer unspliced ( XBP1u ) or 26-nt shorter spliced ( XBP1s ) segment .", "( C ) Immunoblot showing eIF2α phosphorylation in HEK293T cells treated with Tg ( 500 nM ) or 10 µM of the indicated small molecule ER proteostasis regulator for the indicated time .", "( D ) Activation of ATF6 as measured by nuclear localization of GFP-ATF6 .", "U2OS-GFP-ATF6 cells were treated with the top 8 small molecule ER proteostasis regulators ( 10 µM; 5 hr ) or Tg ( 100 nM; 5 hr ) and subcellular localization of GFP was assessed by confocal microscopy ( representative images are shown in Figure 4—figure supplement 1A ) .", "The nuclear:ER ratio of GFP signal corresponding to activation of GFP-ATF6 was calculated by comparing vehicle to Tg treatment .", "Error bars show standard error for n = 3 replicates .", "Dotted line shows mean plus three standard deviations from vehicle treated controls ( 28 . 65% ) .", "***p<0 . 001 using one way ANOVA followed by Tukey’s Multiple comparison test ( compared to vehicle ) .", "( E ) Plot showing ERSE-FLuc activation in HEK293T-Rex cells treated with the top 8 small molecule ER proteostasis regulators ( 10 µM; 18 hr ) in the presence ( red bars ) or absence ( grey bars ) of the S1P inhibitor PF-429242 ( 10 µM; 18 hr ) .", "Cells treated with Tg ( 500 nM; 18 hr ) are shown as a control .", "Error bars show standard error for n = 3 .", "***p<0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 15550 . 01610 . 7554/eLife . 15550 . 017Figure 4—figure supplement 1 . Select ER proteostasis regulators show proteolytic processing and nuclear translocation of ATF6 . ( A ) Representative images of U2OS-GFP-ATF6 cells incubated with Tg ( 100 nM ) , Tm ( 2 . 5 µg/mL ) , or the indicated ER proteostasis regulators ( 10 μM ) for 5 hr then imaged by confocal microscopy .", "GFP-ATF6 is shown in green in the upper merged images and in grayscale beneath .", "The ER is marked with GRP94 ( blue ) while nuclear DNA is marked with DAPI ( red ) .", "Yellow in the merged image indicates translocation of GFP-ATF6 to the nucleus .", "Quantification of nuclear ATF6 localization from images such as these are shown in Figure 4D .", "( B ) Immunoblot showing ATF6 processing from full-length ( ATF6α ( P ) ) to the cleaved ATF6α ( N ) ) in HEK293T cells treated with Tg ( 500 nM ) or 10 µM of the indicated small molecule ER proteostasis regulators for the indicated time .", "The small molecule BIX is included as a control .", "The red arrows show the active ATF6 transcription factor domain that results from S1P/S2P cleavage . DOI: http://dx . doi . org/10 . 7554/eLife . 15550 . 017 ATF6 activation involves trafficking of the full-length ATF6 protein to the Golgi .", "ATF6 is then processed by site-1 and site-2 proteases ( S1P/S2P ) to release the active cytosolic bZIP transcription factor , facilitating nuclear localization ( Ye et al . , 2000 ) .", "To evaluate the influence of the 8 ER proteostasis regulators on ATF6 processing , we measured ATF6 nuclear translocation using a fluorescence-based high-content imaging assay that quantifies nuclear-localized ATF6-GFP relative to ER-localized ATF6-GFP .", "Treatment with small molecules 132 and 263 increased the nuclear fraction of ATF6-GFP , reflecting increased ATF6 processing ( Figure 4D—figure supplement 1A ) .", "Compound 132 and 263 treatment also led to increased accumulation of the 50 kDa ATF6 fragment , resulting from S1P/S2P endoproteolysis in the Golgi , as shown by immunoblotting ( Figure 4—figure supplement 1B ) .", "Other molecules , including 147 , did not significantly increase ATF6-GFP nuclear localization or accumulation of the 50 kDa ATF6 fragment , although it cannot be excluded that they also induce ATF6 processing , albeit to a lower extent that falls outside the dynamic range or sensitivity of these assays .", "To more sensitively evaluate the involvement of ATF6 endoproteolytic processing in small molecule-dependent activation of the ATF6 transcriptional program , we utilized the S1P inhibitor PF-429242 , which prevents Tg-mediated ERSE-FLuc activation ( Figure 4E ) ( Hay et al . , 2007 ) .", "PF-429242 also inhibited the activation of ERSE-FLuc in cells treated with all 8 ER proteostasis regulators ( Figure 4E ) , reflecting the dependence of our small molecules on S1P proteolysis for ATF6 activation .", "Collectively , the ATF6 knockout , nuclear localization and S1P inhibition results indicate that the small molecule ER proteostasis regulators activate the ATF6 transcriptional program through a mechanism involving S1P-dependent processing of endogenous ATF6 .", "ER reprogramming afforded by stress-independent chemical genetic ATF6 activation selectively reduces secretion of destabilized , amyloidogenic TTR variants from liver-derived HepG2 cells ( Chen et al . , 2014; Shoulders et al . , 2013 ) .", "Thus , small molecule ER proteostasis regulators that preferentially activate the ATF6 transcriptional program , such as 147 and 263 , should similarly reduce secretion of destabilized , amyloidogenic TTR from HepG2 cells .", "We confirmed that 147 and 263 induced expression of the ATF6-target gene BiP in HepG2 cells by qPCR ( Figure 5—figure supplement 1A ) .", "We also showed that the treatment dose of these small molecules ( 10 µM ) does not influence HepG2 viability , although higher concentrations ( 100 µM ) do reduce viability ( Figure 5—figure supplement 1B ) .", "We used [35S] metabolic labeling to quantify secretion of FLAG-tagged destabilized A25T TTR ( FTTTRA25T ) or FLAG-tagged wild-type TTR ( FTTTRWT ) from HepG2 cells stably expressing DHFR-ATF6 ( HepG2ATF6 ) .", "Cells were pretreated with the global ER stressor Tg or small molecule ER proteostasis regulators 147 or 263 ( Figure 5A ) .", "Pretreatment with Tg reduced secretion of both FTTTRA25T and FTTTRWT ( Figure 5B—figure supplement 1C ) .", "However , pretreatment with 147 and 263 reduced FTTTRA25T secretion by 45% , but did not significantly influence secretion of FTTTRWT ( Figure 5B—figure supplement 1C ) .", "The reduction in FTTTRA25T secretion induced by these molecules correlates with increased degradation of FTTTRA25T ( Figure 5C ) .", "Notably , the selective reduction in secretion and increase in degradation for FTTTRA25T induced by 147 and 263 is strictly analogous to that observed after chemical genetic ATF6 activation in these cells ( Figure 5—figure supplement 1D , E ) ( Chen et al . , 2014 ) .", "This indicates that these small molecule ER proteostasis regulators phenocopy the capacity of chemical genetic ATF6 activation to selectively reduce secretion of destabilized , amyloidogenic TTR variants relative to the wild-type protein . 10 . 7554/eLife . 15550 . 018Figure 5 . Small molecule ER proteostasis regulators reduce secretion of destabilized amyloidogenic FTTTRA25T from HepG2 cells .", "( A ) Representative autoradiogram of [35S]-labeled FTTTRWT or FTTTRA25T in the lysate ( Lys ) and media ( Med ) of HepG2ATF6 cells pretreated for 15 hr with 10 µM small molecule 147 or 263 , or 1 µM Tg , incubated with [35S] for 30 min , and then incubated in fresh non-radioactive media for 0 or 4 hr .", "The experimental paradigm is shown above .", "( B ) Quantification of the normalized fraction of FTTTRWT ( open bars ) or FTTTRA25T ( colored bars ) secreted following a 4 hr chase in HepG2ATF6 cells pretreated for 15 hr with Tg ( 1 µM; blue ) or 10 µM small molecule 147 ( red ) or 263 ( green ) .", "Fraction secreted was calculated using the equation: fraction secreted = [extracellular [35S]-TTR signal at t=4h / ( extracellular [35S]-TTR signal at t=0h + intracellular [35S]-TTR signal at t=0h ) ] .", "Normalized fraction secreted was calculated relative to vehicle-treated controls using the equation: normalized fraction secreted = fraction secreted treatment / fraction secreted vehicle .", "Representative autoradiograms are shown in Figure 5A .", "Raw fraction secreted is shown in Figure 5—figure supplement 1C .", "Error bars show standard error for n = 4 replicates; *p<0 . 05 , ***p<0 . 001 .", "( C ) Quantification of the normalized fraction of FTTTRWT ( open bars ) or FTTTRA25T ( colored bars ) remaining ( lysate + media ) following a 4 hr chase in HepG2ATF6 cells pretreated for 15 hr with Tg ( 1 µM; blue ) or 10 µM small molecule 147 ( red ) or 263 ( green ) .", "Fraction remaining was calculated using the equation: [ ( extracellular [35S]-TTR signal at t=4h + intracellular [35S]-TTR signal at t=4h ) / ( extracellular [35S]-TTR signal at t=0h + intracellular [35S]-TTR signal at t=0h ) ] .", "Normalized fraction remaining was calculated relative to vehicle-treated controls using the equation: normalized fraction remaining = fraction remaining treatment / fraction remaining vehicle .", "Representative autoradiograms are shown in Figure 5A .", "Error bars show standard error for n = 4 replicates; *p<0 . 05 , **p<0 . 01 , ***p<0 . 001 .", "( D ) Quantification for normalized total [35S]-labeled proteins secreted from HepG2ATF6 cells following 15 hr pretreatment with 1 µM Tg ( blue ) , TMP ( 10 µM ) -dependent DHFR-ATF6 activation ( purple ) , or 10 µM small molecule ER proteostasis regulators 147 ( red ) or 263 ( green ) .", "The experimental protocol and a representative autoradiogram for this experiment is shown in Figure 5—figure supplement 1F .", "The recovery of [35S] labeled proteins is normalized to the vehicle-treated control .", "Error bars show standard error for n = 4 replicates .", "***indicates p<0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 15550 . 01810 . 7554/eLife . 15550 . 019Figure 5—figure supplement 1 . ER proteostasis regulators reduce secretion of destabilized amyloidogenic TTRA25T from HepG2 cells .", "( A ) Plot showing the fold increase of BiP mRNA in HepG2ATF6 cells treated with Tg ( 1 µM ) , TMP ( activates DHFR-ATF6; 10 µM ) or 10 µM of the indicated small molecule ER proteostasis regulators .", "Error bars show standard error for n = 3 .", "*p<0 . 05 , **p<0 . 01 .", "( B ) Toxicity from 48 hr incubation of HepG2ATF6 cells with Tg ( 1 µM ) or small molecules 147 or 263 at the indicated concentration as read by CellTiter-Glo .", "Error bars represent standard error from n = 6 replicates .", "( C ) Quantification of the fraction FTTTRWT ( open bars ) or FTTTRA25T ( colored bars ) secreted following a 4 hr chase in HepG2ATF6 cells pretreated for 15 hr with Tg ( 1 µM; blue ) or 10 µM small molecule 147 ( red ) or 263 ( green ) .", "Fraction secreted was calculated using the equation: fraction secreted = [extracellular [35S]-TTR signal at t / ( extracellular [35S]-TTR signal at t=0 + intracellular [35S]-TTR signal at t=0 ) ] .", "Representative autoradiograms are shown in Figure 5A .", "Error bars show standard error for n = 4 replicates .", "( D ) Quantification of normalized fraction secreted of FTTTRWT ( grey ) or FTTTRA25T ( red ) from HepG2ATF6 cells following 15 hr pretreatment with TMP ( 10 µM; activates DHFR-ATF6 ) .", "Fraction secreted was calculated using the equation: fraction secreted = [extracellular [35S]-TTR signal at t / ( extracellular [35S]-TTR signal at t=0 + intracellular [35S]-TTR signal at t=0 ) ] .", "Normalized fraction secreted was calculated relative to vehicle-treated controls using the equation: normalized fraction secreted = fraction secreted treatment / fraction secreted vehicle .", "Error bars show standard error for n = 9 .", "**p<0 . 01 .", "( E ) Quantification of normalized fraction remaining of FTTTRWT ( grey ) or FTTTRA25T ( red ) from HepG2ATF6 cells following 15 hr pretreatment with TMP ( 10 µM; activates DHFR-ATF6 ) .", "Fraction remaining was calculated using the equation: [ ( extracellular [35S]-TTR signal at t + intracellular [35S]-TTR signal at t ) / ( extracellular [35S]-TTR signal at t=0 + intracellular [35S]-TTR signal at t=0 ) ] .", "Normalized fraction remaining was calculated relative to vehicle-treated controls using the equation: normalized fraction remaining = fraction remaining treatment / fraction remaining vehicle .", "Error bars show standard error for n = 9 .", "*p<0 . 05 .", "( F ) Representative autoradiogram and Coomassie images of total [35S]-labeled protein secreted from HepG2ATF6 cells following 15 hr pretreatment with 1 µM Tg , TMP ( 10 µM ) -dependent DHFR-ATF6 activation , or 10 µM small molecule ER proteostasis regulators 147 or 263 .", "The experimental protocol used for this experiment is shown above .", "Quantification of this experiment is shown in Figure 5D . DOI: http://dx . doi . org/10 . 7554/eLife . 15550 . 019 We next evaluated whether pretreatment by 147 and 263 influences secretion of the endogenous secretory proteome from HepG2ATF6 cells using a [35S] metabolic labeling approach ( Figure 5—figure supplement 1F ) ( Chen et al . , 2014; Shoulders et al . , 2013 ) .", "Treatment with the ER stressor Tg significantly reduced the recovery of [35S] labeled proteins in conditioned media , consistent with the ability of ER stress to induce translational attenuation and globally disrupt protein secretion ( Figure 5D—figure supplement 1F ) .", "However , chemical genetic ATF6 activation , or pharmacologic activation of the ATF6 transcriptional program through addition of compound 147 or 263 , did not influence the levels of [35S] labeled proteins in media , demonstrating that preferential ATF6 activation does not globally disrupt secretion of the endogenous proteome .", "We next tested whether the ER proteostasis regulators reduce the secretion of a destabilized , amyloidogenic immunoglobulin LC ( ALLC ) that undergoes proteotoxic extracellular aggregation in association with AL ( Arendt et al . , 2008 ) .", "Chemical genetic ATF6 activation reduces secretion and extracellular aggregation of ALLC without notably affecting secretion of a stable , non-amyloidogenic LC ( Cooley et al . , 2014 ) .", "We evaluated whether our prioritized ER proteostasis regulators similarly reduce secretion of the same ALLC from an AL patient-derived plasma cell line ( ALMC-2 ) ( Arendt et al . , 2008 ) .", "Compound 147 induced expression of the ATF6 target gene BiP in ALMC-2 cells but unexpectedly , 263 did not ( Figure 6A ) .", "Treatment with compound 147 reduced ALLC secretion >50% from ALMC-2 cells , as measured by an ELISA while 263 did not significantly influence ALLC secretion ( Figure 6B ) .", "Dose-dependent treatment with 147 measuring ALLC secretion afforded an EC50 value of 1 . 1 µM ( Figure 6C ) .", "The attenuation in ALLC secretion saturated above 10 µM , producing a similar dose-response curve to 147-dependent ATF6 reporter activation ( Figure 3I ) .", "Conversely , compared to the reduction in ALLC secretion , these molecules only minimally affected the secretion of an energetically normal , fully assembled IgG from a control KAS-6/1 plasma cell line isolated from a multiple myeloma patient that did not present with AL ( Figure 6D ) ( Westendorf et al . , 1996 ) .", "These data indicate that small molecule ER proteostasis regulators that preferentially activate the ATF6 transcriptional program , e . g . , 147 , selectively reduce secretion of destabilized , amyloidogenic LCs , but not IgGs .", "Additional ER proteostasis regulator compounds tested also reduced secretion of amyloidogenic ALLC from ALMC-2 cells , while only minimally affecting IgG secretion from KAS-6/1 cells ( Figure 6—figure supplement 1A , B ) . 10 . 7554/eLife . 15550 . 020Figure 6 . Small molecule ER proteostasis regulators reduce secretion of destabilized , amyloidogenic immunoglobulin light chains from AL patient-derived plasma cells .", "( A ) qPCR analysis of BiP expression in ALMC-2 cells treated for 6 hr with 10 µM of the indicated small molecule ER proteostasis regulators .", "Error bars show standard error for n = 3 replicates .", "*p< 0 . 05 .", "( B ) Graph showing relative media levels of ALLC measured by ELISA ( red ) and cellular viability ( blue ) of ALMC-2 cells pretreated with 10 µM of the indicated small molecule ER proteostasis regulators .", "Cells treated with 500 nM Tg are shown as a control .", "Error bars show standard error for n > 6 replicates .", "***p<0 . 001 .", "( C ) Dose response curves of reduction in ALLC secretion from ALMC-2 cells after 147 treatment .", "ALMC-2 cells were pretreated for 15 hr , and subsequently the media was exchanged and conditioned on the cells for 2 hr .", "Secreted ALLC concentration was measured by ELISA using an antibody directed against free λ LC ( red ) .", "Cell viability was measured by CellTiter-Glo ( blue ) .", "Error bars represent standard error for n = 3 replicates .", "Data was fit to four parameter logistic functions to calculate EC50 values .", "( D ) Graph showing relative media levels of IgG measured by ELISA ( red ) and cellular viability ( blue ) of KAS-6/1 cells treated with 10 µM of the indicated small molecule ER proteostasis regulators .", "Cells treated with 500 nM Tg are shown as a control .", "Error bars show standard error for n = 3 replicates .", "**p<0 . 01 , ***p<0 . 001 .", "( E ) Quantification of ALLC immunopurifications from ALMC-2 cells after treatment with the indicated ER proteostasis regulator ( 10 µM; 16 h ) and in situ cross-linking .", "The relative recovery of GRP94 and BiP in each sample normalized to the recovered ALLC is shown .", "A representative immunoblot is shown in Figure 6—figure supplement 1D .", "Error bars show standard error for n = 4 replicates .", "*p<0 . 05 .", "( F ) Quantification of ALLC aggregates in conditioned media prepared on ALMC-2 cells pretreated with the indicated ER proteostasis regulator ( 10 µM; 16 hr ) .", "After pretreatment , media were exchanged and the volume adjusted to account for decreases in cell viability with several compounds .", "After conditioning for 8 hr , the media were collected and then heated to 55°C for 8 hr to induce ALLC aggregation .", "Quantification of the total ( red ) and aggregate ( blue ) ALLC amounts are shown .", "Error bars show standard error for n > 3 replicates .", "*p<0 . 05 , **p<0 . 01 , ***p<0 . 001 .", "Blue Native ( BN ) PAGE/Immunblots of the conditioned media are shown in Figure 6—figure supplement 1H .", "Quantification of ALLC aggregates in conditioned media pretreated with additional ER proteostasis regulator compounds is shown in Figure 6—figure supplement 1I . DOI: http://dx . doi . org/10 . 7554/eLife . 15550 . 02010 . 7554/eLife . 15550 . 021Figure 6—figure supplement 1 . ER proteostasis regulators reduce secretion and extracellular aggregation of a destabilized , amyloidogenic light chain from AL patient-derived plasma cells .", "( A ) Graph showing relative media levels of ALLC measured by ELISA ( red ) and cellular viability ( blue ) of ALMC-2 cells pretreated with 10 µM of the indicated small molecule ER proteostasis regulators .", "Results from treatment with Tg , 147 and 263 ( Figure 6B ) are shown for comparison .", "Cells treated with 500 nM Tg are shown as a control .", "Error bars show standard error for n > 6 replicates .", "*p<0 . 05 , **p<0 . 01 , ***p<0 . 001 .", "( B ) Graph showing relative media levels of IgG measured by ELISA ( red ) and cellular viability ( blue ) of KAS-6/1 cells treated with 10 µM of the indicated small molecule ER proteostasis regulators .", "Results from treatment with Tg , 147 and 263 ( Figure 6D ) are shown for comparison Cells treated with 500 nM Tg are shown as a control .", "Error bars show standard error for n = 3 replicates .", "*p<0 . 05 , **p<0 . 01 , ***p<0 . 001 .", "( C ) Plot comparing the reduction in ALLC secretion from ALMC-2 cells to BiP induction in the same cells .", "The data plotted here are taken directly from Figure 6A as well as respective measurements with additional prioritized ER proteostasis regulators ( Table 1 ) .", "( D ) Representative immunoblots of the ALLC co-immunopurifications from ALMC-2 cells after treatment with the indicated ER proteostasis regulator ( 10 µM; 16 hr ) and in situ cross-linking .", "Quantification is performed in Figure 6E .", "( E ) Plot showing the fraction secretion of ALLC from ALMC-2 cells during a cycloheximide ( CHX ) chase .", "Cells were treated for 15 hr with 500 nM Tg or 10 µM 147 , followed by media exchange and addition of 50 µg/mL CHX to inhibit new protein translation .", "ALLC concentrations in the lysate and media were quantified by ELISA at 0 , 3 or 6 hr after CHX addition .", "Fraction secreted was calculated as [ALLC]media , t/ ( [ALLC]media , t = 0 + [ALLC]lysate , t = 0 ) .", "Error bars represent standard error from n = 3 replicates .", "*p<0 . 05 , ***p< 0 . 001 .", "( F ) Plot showing the normalized intracellular fraction of ALLC at 6 hr after the CHX in the experiment described in ( E ) .", "The intracellular fraction ( 6 h ) was calculated as [ALLC]lysate , t = 6h / ( [ALLC]media , t = 0 + [ALLC]lysate , t = 0 ) and then normalized to the Vh treatment .", "Error bars represent standard error from n = 3 replicates .", "*p<0 . 05 , ***p<0 . 001 .", "( G ) Plot showing the fraction ALLC remaining from the cycloheximide chase experiment described in ( E ) .", "Fraction remaining was calculated as ( [ALLC]lysate , t + [ALLC]media , t ) / ( [ALLC]media , t = 0 + [ALLC]lysate , t = 0 ) .", "Error bars represent standard error from n = 3 replicates .", "( H ) Blue Native ( BN ) -PAGE/Immunoblot of conditioned media prepared on ALMC-2 cells pretreated with the indicated ER proteostasis regulator ( 10 µM; 16 hr ) .", "After pretreatment , media were exchanged and the volume adjusted to account for decreases in cell viability with several compounds .", "After conditioning for 8 hr , the media were collected and then heated to 55 ºC for 8 hr to induce ALLC aggregation .", "An SDS-PAGE/immunoblot of the same samples is shown as a control .", "( I ) Quantification of the total ( red ) and aggregate ( blue ) ALLC amounts in the immunoblots as shown in Figure 6—figure supplement 1H .", "Results from treatment with 147 and 263 ( Figure 6F ) are shown for comparison .", "Error bars show standard error for n > 3 replicates .", "*p< 0 . 05 , **p<0 . 01 , ***p<0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 15550 . 021 Tg treatment reduced viability of ALMC-2 and KAS-6/1 cells ( Figure 6B , D ) .", "Thus , reductions of ALLC secretion afforded by these treatments in part reflect cell death .", "However , the preferential activator of the ATF6 transcriptional program , 147 , only minimally influenced the viability of ALMC-2 or KAS-6/1 cells ( Figure 6B–D ) , suggesting that the reductions in ALLC secretion afforded by this molecule involve reprogramming of the ER PN .", "Consistent with this prediction , small molecule-dependent reductions in ALLC secretion from ALMC-2 cells strongly correlate with BiP induction in these cells ( Figure 6—figure supplement 1C ) .", "Furthermore , immunopurification of ALLC from ALMC-2 cells pretreated with 147 and subjected to in situ crosslinking showed increased interactions between ALLC and the ATF6-regulated chaperones BiP and GRP94 ( Figure 6E , Figure 6—figure supplement 1D ) .", "However , pretreatment with 263 , which did not induce BiP in ALMC-2 cells , did not increase the co-isolation of these chaperones .", "These results suggest that pharmacologic reductions in ALLC secretion are linked to reprogramming of the ER proteostasis environment .", "We previously showed that chemical genetic ATF6 activation reduces ALLC secretion through a mechanism involving prolonged ER retention ( Cooley et al . , 2014 ) .", "In contrast , genetic XBP1s activation reduces ALLC secretion by increasing its degradation .", "Therefore , evaluating the mechanism by which the small molecules reduce ALLC secretion provides insight into the specific type of ER reprogramming involved in this process .", "A cycloheximide chase assay demonstrated that pretreatment with 147 reduced ALLC secretion by 30% through a mechanism involving increased ER retention ( Figure 6—figure supplement 1E–G ) .", "This indicates that 147 reduces secretion of ALLC through a mechanism similar or identical to that previously observed with chemical genetic ATF6 activation .", "Finally , we evaluated whether pharmacologic ATF6-mediated reductions in ALLC secretion attenuated extracellular ALLC aggregation .", "Soluble ALLC aggregates were quantified in conditioned media from ALMC-2 cells after heating to 55°C for 8 hr using Blue-Native PAGE and immunoblotting ( Cooley et al . , 2014 ) .", "Total ALLC protein levels were also determined by SDS-PAGE and immunoblotting .", "Treatment with 147 decreased extracellular ALLC aggregates by >50% ( Figure 6F—figure supplement 1H ) .", "Similar reduction in aggregation was observed for additional ER proteostasis regulators tested that lowered ALLC secretion ( Figure 6F—figure supplement 1H , I ) .", "This indicates that the reduction in ALLC secretion afforded by our ER proteostasis regulators attenuates extracellular LC aggregation ." ], [ "Reprogramming of subcellular proteostasis environments through activation of stress-responsive signaling pathways is a promising strategy to restore mutant protein folding or enhance mutant protein degradation ( Calamini and Morimoto , 2012; Chen et al . , 2015; Das et al . , 2015 ) .", "Here , we employed high-throughput screening in combination with medium-throughput transcriptional profiling to identify small molecule ER proteostasis regulators that preferentially activate the ATF6 transcriptional program of the UPR .", "We show that pharmacologic activation of the ATF6 transcriptional program requires S1P-dependent processing of endogenous ATF6 .", "Furthermore , we show that our small molecule ER proteostasis regulators phenocopy the capacity of chemical genetic ATF6 activation to selectively reduce secretion and extracellular aggregation of destabilized amyloidogenic variants of proteins such as TTR and LC .", "These results reveal a new strategy to identify small molecules that reprogram the ER PN and correct imbalances in ER function through preferential activation of a specific sub-UPR transcriptional program .", "Our phenotypic screening approach sought to identify small molecule ER proteostasis regulators that activated the ATF6 transcriptional program , without placing a constraint for the compounds to act directly on ATF6 .", "Nonetheless , prioritized compounds such as 147 and 263 function through a mechanism that requires endogenous ATF6 .", "ATF6 is activated through a mechanism distinct to that utilized by the two other arms of the UPR .", "In response to ER stress , ATF6 traffics to the Golgi where it is endoproteolytically processed by S1P and S2P ( Ye et al . , 2000 ) .", "This releases the ATF6 bZIP transcription factor into the cytosol , facilitating its translocation to the nucleus where it promotes induction of the ATF6 transcriptional program .", "ATF6 is not known to contain any allosteric small molecule binding sites .", "Thus , it is likely that our molecules promote ATF6 activation through interactions with proteins that regulate its activity such as BiP and the PDIs .", "Both of these are predicted to be involved in initiating ATF6 trafficking to the Golgi , although their role in this process remains poorly defined ( Nadanaka et al . , 2007; Shen et al . , 2002 ) .", "Our small molecules could also preferentially induce ATF6 activity by targeting or mimicking metabolites required for activation of this pathway .", "Previous work shows that PERK and IRE1 can be activated independent of ATF6 in response to alterations in cellular lipid composition ( Volmer et al . , 2013 ) but no such mechanism has been reported for ATF6 to date .", "Interestingly , ER proteostasis regulators identified in our screen appear to preferentially induce ATF6 activation through distinct mechanisms .", "This is evident for molecules such as 263 , which demonstrates varying levels of BiP induction in distinct cell types ( e . g . , 263 induces BiP in HEK293T and HepG2 cells , but not in ALMC-2 cells ) .", "This is in contrast to compound 147 that activates the ATF6 pathway in all cell types tested .", "This capacity to activate ATF6 in a cell type specific fashion could be harnessed to define the underlying molecular mechanisms involved in ATF6 activation and develop compounds that induce tissue-selective ER PN remodeling .", "As we continue to optimize the potency and ATF6 selectivity of these small molecule ER proteostasis regulators , new insights into the molecular pathways that regulate ATF6 activity will likely emerge .", "One mechanism by which the ATF6 transcriptional program influences ER function is by increasing the stringency of ER protein quality control ( Chen et al . , 2015 ) .", "Stress-independent ATF6 activation selectively attenuates the secretion and subsequent proteotoxic aggregation of destabilized , disease-associated proteins without affecting the secretion of the endogenous proteome ( Chen et al . , 2014; Chiang et al . , 2012; Cooley et al . , 2014; Shoulders et al . , 2013; Smith et al . , 2011 ) .", "Our top small molecule ER proteostasis regulators ( e . g . , 147 ) phenocopy the ability of chemical genetic ATF6 activation to selectively reduce secretion of destabilized , disease-associated variants of amyloidogenic proteins such as TTR and immunoglobulin LC .", "This suggests that our small molecules could similarly phenocopy the ATF6-dependent reductions in the trafficking and intracellular aggregation of destabilized , aggregation-prone proteins associated with other gain-of-toxicity protein misfolding diseases ( e . g . , A1AT-Z aggregation in liver disease and rhodopsin aggregation in retinal degeneration ( Chiang et al . , 2012; Smith et al . , 2011 ) ) .", "Thus , small molecule ER proteostasis regulators identified herein have the potential to broadly ameliorate degenerative diseases associated with aggregation of secretory proteins .", "The long-term consequences of periodic pharmacologic activation of the ATF6 arm of the UPR are unclear .", "Mechanism-based toxicity associated with adapting ER proteostasis to ameliorate secretory protein misfolding and/or aggregation is possible .", "While some small molecule ER proteostasis regulators identified show mild toxicity , a subset of compounds , especially compounds that preferentially activate the ATF6 transcriptional program ( e . g . , 147 ) , show no significant toxicity across multiple cell models .", "This likely reflects the limited activation of the IRE1/XBP1s and PERK arms of the UPR afforded by these molecules , minimizing apoptosis signaling induced downstream of these two UPR signaling pathways ( Hetz , 2012; Tabas and Ron , 2011 ) .", "The moderate levels of ATF6 activation achieved using our small molecule ER proteostasis regulators likely also minimize pathologic imbalances in ER function .", "It is known that high levels of ATF6 activation induce hepatic steatosis in zebrafish , whereas lower levels do not ( Howarth et al . , 2014 ) .", "Finally , the low molecular weight and structural simplicity of the small molecules identified by our multi-tiered screening strategy ( e . g . , 147 ) provides a significant amount of chemical space that can be explored through structure-activity relationships to optimize potency , selectivity and dosing regimens , while minimizing toxicity .", "Several of our prioritized molecules include functional groups found in pan-assay interference compounds ( PAINS ) ( Baell and Walters , 2014 ) , but given that these ER proteostasis regulators showed activity in multiple complementary screening assays , they are unlikely to act through off-target pathways .", "Consistent with this , the molecules depend on ATF6 and its canonical activation pathway for the induction of ATF6 target genes .", "Furthermore , the RNA-Seq and proteomics profiling analysis for 147 and 263 confirmed that these molecules exert very little off-target transcriptome changes and proteome remodeling apart from preferential ER PN activation .", "Both 147 and 263 contain functional groups that after metabolic oxidation to a quinone methide could modify proteins covalently .", "This suggests that the covalent modification of ATF6 or a protein involved in the regulation of ATF6 activity ( e . g . , BiP or PDIs ) ( Nadanaka et al . , 2007; Shen et al . , 2002 ) by our small molecules could be involved in the activation mechanism .", "Future medicinal chemistry efforts will focus on exploring the mechanism of action of these compounds and the potential involvement of covalent protein modification for their activity .", "The establishment of ER proteostasis regulators , such as 147 , provides a unique opportunity to define the underlying molecular mechanism ( s ) of ATF6 activation and to probe the involvement of ATF6 in regulating ER PN function .", "These molecules also represent a valuable resource to define the therapeutic potential of ATF6 transcriptional program activation to correct pathologic imbalances in ER proteostasis in cellular and animal models of protein misfolding and aggregation diseases .", "As we continue to develop our top small molecule ER proteostasis regulators through medicinal chemistry , we will establish second generation molecules that optimize ER PN reprogramming to ameliorate pathologic imbalances in ER proteostasis associated with diverse protein aggregation diseases such as the systemic amyloidosis ." ], [ "All compounds were obtained from the commercial vendors listed in Supplementary file 3 and were used without further purification by dissolving in sterile dimethyl sulfoxide ( DMSO ) .", "The identity of screening compounds was confirmed by LC-MS analysis and all compounds were shown to be >90% pure .", "ERSE-Firefly luciferase reporter was cloned into a vector suitable for mammalian cell selection by transferring ERSE-FLuc from ERSE-FLuc . pGL3 ( Yoshida et al . , 1998 ) into a promotorless pcDNA3 . 1 vector using Xba1 and Not1 restriction sites to create ERSE . FLuc . pcDNA3 . 1 .", "XBP1s-Renilla luciferase was generated from a known XBP1s-GFP reporter ( Iwawaki et al . , 2004 ) .", "GFP was exchanged for Renilla luciferase by the Polymerase Incomplete Primer Extension ( PIPE ) method ( Klock and Lesley , 2009 ) using the following primers: vector , XBP1s-GFP . pCEFL , CTGAAGAACGAGCAGTAAGTGAGCAAGGGCGAGGAG and CGTACACCTTGGAAGCAGATCTTGAATCTGAAGAGTCAATACC; gene , CMV-Renilla , CGGTATTGACTCTTCAGATTCAAGATCTGCTTCCAAGGTGTACG and CTCCTCGCCCTTGCTCACTTACTGCTCGTTCTTCAG to create XBP1s-RLuc pCEFL .", "TTR point mutations were incorporated into the FLAG2-TTR pcDNA3 . 1 vector through site-directed mutagenesis ( Shoulders et al . , 2013 ) .", "HEK293T-Rex ( ATCC ) , HEK293T ( ATCC ) , HEK293DAX ( Shoulders et al . , 2013 ) , HepG2 ( ATCC ) , HepG2ATF6 ( Chen et al . , 2014 ) , and ATF6+/+ MEFs and ATF6-/- MEFs ( a kind gift from Randal Kaufman’s lab ) were cultured in High-Glucose Dulbecco’s Modified Eagle’s Medium ( DMEM ) supplemented with glutamine , penicillin/streptomycin and 10% fetal bovine serum .", "Cells were routinely tested for mycoplasma every 6 months .", "No further authentication of cell lines was performed by the authors .", "HEK293T-Rex cells containing the ERSE-FLuc or XBP1s-RLuc reporters were created by transfection with ERSE-FLuc pcDNA3 . 1 or XBP1s-RLuc pCEFL by calcium phosphate followed by culturing in geneticin sulfate ( G-418 , 500 μg/mL , ERSE ) or puromycin ( 20 μg/mL , XPB1s ) .", "Creation and maintenance of HEK293DAX cells has been described previously ( Shoulders et al . , 2013 ) .", "Creation and maintenance of HepG2ATF6 has been described previously ( Chen et al . , 2014 ) .", "U2OS cells stably expressing GFP-ATF6 were purchased from Thermo Fisher Scientific ( Waltham , MA ) ( 084_01 ) and cultured with 500 μg/mL G418 .", "Transient transfections of FTTTR variants into HepG2ATF6 cells were performed with Lipofectamine 3000 ( Life Technologies , Carlsbad , CA ) .", "ALMC-2 and KAS-6/1 plasma cells ( a kind gift from Diane Jelinak’s laboratory ) were cultured in Iscove’s Modified Dulbecco’s Medium ( IMDM ) GlutaMAX ( Life Technologies ) supplemented with penicillin/streptomycin , 5% fetal bovine serum and 2 ng/mL interleukin-6 ( IL-6 ) , as previously described ( Arendt et al . , 2008 ) .", "All cells were cultured under typical tissue culture conditions ( 37°C , 5% CO2 ) .", "96-well plates were seeded with HEK293T cells ( 20 , 000 cells/well ) and incubated at 37°C overnight .", "Cells were treated with compounds in media to give a final concentration of 10 μM and incubated for 6 hr .", "Media was removed , then cell pellets in the plates were frozen at -80°C .", "On the day of assay , cell pellets were thawed , lysed and analyzed for gene expression as previously described ( Calamini et al . , 2012 ) .", "RNA expression changes were log2 transformed and clustering was performed in R using a Euclidean distance function and Ward’s method for hierarchical clustering .", "HEK293T-Rex and HEK293DAX cells in 12-well plates were treated for 6 hr with vehicle , 1 μM Tg , 10 μM TMP ( in HEK293DAX ) , or 10 μM 132 , 147 or 263 in biological triplicate at 37°C .", "Cells were harvested , and RNA was extracted using the RNeasy Mini Kit ( Qiagen , Hilden , Germany ) .", "Total RNA was quantified using NanoDrop ( ND-1000 ) .", "Samples were run on the Illumina HiSeq system .", "Single end , 100 bp-long reads from RNA-Seq experiments were aligned to the GRCh37 . p13 human genome reference assembly using SeqMan NGen 11 . 2 . 1 ( DNAStar , Inc . , Madison , WI ) .", "The assembly data were then imported into ArrayStar with QSeq ( DNAStar , Inc . ) to quantify the gene expression levels .", "The sequence counts were normalized to reads per kilobase per million ( RPKM ) after filtering out non-mRNA sequence features .", "The statistical significance of the difference between the expression levels of a gene under different conditions was assessed using a Student’s t-test with the Benjamini-Hochberg multiple testing correction .", "ATF6- and XBP1s-selective target genes were identified from transcriptional profiles of HEK293DAX cells following stress-independent activation of TMP-dependent DHFR-ATF6 and/or doxycycline inducible XBP1s , as previously described ( Shoulders et al . , 2013 ) .", "PERK-selective target genes were identified from Lu et al . , 2004 .", "These genesets are described in Figure 3—source data 1 .", "Only ATF6- , XBP1s- or PERK-selective genes induced >1 . 5 fold in Tg-treated samples were used in this analysis .", "The log transformed fold-increase of these target genes in HEK293T-Rex cells treated with the respective ER proteostasis regulators was then normalized to the log transformed fold increase of these genes induced by Tg treatment .", "The plots shown in Figure 3B–E were prepared as box and whisker plots using Kaleidograph .", "Differential activation of the ATF6 , XBP1s , and PERK genesets was assessed by one-way ANOVA and significance of pairwise comparison confirmed by unpaired t-test .", "An analogous strategy was employed to prepare the heat map shown in Figure 2—figure supplement 1C using transcriptional data from the MGE analysis .", "HEK293T-Rex cells in 6-well plates were treated for 16 hr with vehicle , or 132 , 147 or 263 at 37°C .", "Lysates were prepared in radioimmunoprecipitation assay ( RIPA ) buffer ( 150 mM NaCl , 50 mM Tris pH 7 . 5 , 1% Triton X-100 , 0 . 5% sodium deoxycholate , and 0 . 1% SDS ) with fresh protease inhibitor cocktail ( Roche , Indianapolis , IN ) and centrifuged for 20 min at 10000 x g .", "Protein concentrations of supernatants were determined by BCA ( Thermo Fisher ) .", "For each sample , 100 μg of lysate was washed by chloroform/methanol precipitation .", "Air-dried pellets were resuspended in 1% RapiGest SF ( Waters ) and brought up in 100 mM HEPES ( pH 8 . 0 ) .", "Proteins were reduced with 5 mM Tris ( 2-carboxyethyl ) phosphine hydrochloride ( Thermo Fisher ) for 30 min and alkylated with 10 mM iodoacetamide ( Sigma Aldrich , St . Louis , MO ) for 30 min at ambient temperature and protected from light .", "Proteins were digested for 18 hr at 37°C with 2 μg trypsin ( Promega ) .", "After digestion , 20 μg of peptides from each sample were reacted for 1 hr with the appropriate TMT-NHS isobaric reagent ( Thermo Fisher ) in 40% ( v/v ) anhydrous acetonitrile and quenched with 0 . 4% NH4HCO3 for 1 hr .", "Samples with different TMT labels were pooled and acidified with 5% formic acid .", "Acetonitrile was evaporated on a SpeedVac and debris was removed by centrifugation for 30 min at 18 , 000 x g .", "MuDPIT microcolumns were prepared as described ( Ryno et al . , 2014 ) .", "LCMS/MS analysis was performed using a Q Exactive mass spectrometer equipped with an EASY nLC 1000 ( Thermo Fisher ) .", "MuDPIT experiments were performed by 5 min sequential injections of 0 , 10 , 20 , 30 , … , 100% buffer C ( 500 mM ammonium acetate in buffer A ) and a final step of 90% buffer C / 10% buffer B ( 20% water , 80% acetonitrile , 0 . 1% fomic acid , v/v/v ) and each step followed by a gradient from buffer A ( 95% water , 5% acetonitrile , 0 . 1% formic acid ) to buffer B . Electrospray was performed directly from the analytical column by applying a voltage of 2 . 5 kV with an inlet capillary temperature of 275°C .", "Data-dependent acquisition of MS/MS spectra was performed with the following settings: eluted peptides were scanned from 400 to 1800 m/z with a resolution of 30000 and the mass spectrometer in a data dependent acquisition mode .", "The top ten peaks for each full scan were fragmented by HCD using a normalized collision energy of 30% , a 100 ms activation time , a resolution of 7500 , and scanned from 100 to 1800 m/z .", "Dynamic exclusion parameters were 1 repeat count , 30 ms repeat duration , 500 exclusion list size , 120 s exclusion duration , and exclusion width between 0 . 51 and 1 . 51 .", "Peptide identification and protein quantification was performed using the Integrated Proteomics Pipeline Suite ( IP2 , Integrated Proteomics Applications , Inc . , San Diego , CA ) as described previously ( Ryno et al . , 2014 ) .", "The geneset analysis ( Figure 3F–H ) was performed analogous to the analysis of the RNA-seq data using the same proteins from the genesets for ATF6 , XBP1s and PERK and log transformed fold changes of protein expression .", "The plots shown in Figure 3F–H were prepared as box and whisker plots using Kaleidograph .", "Differential activation of the ATF6 , XBP1s , and PERK target protein sets was assessed by one-way ANOVA and significance of pairwise comparison confirmed by unpaired t-test .", "The comparison of RNA-seq and proteomics data ( Figure 3—figure supplement 2A–C ) and the data for the geneset analysis ( Figure 3F–H ) are included as Source Data .", "Unless otherwise noted , all p-values were calculated by performing a paired or unpaired ( noted ) t-test .", "HEK293T-Rex cells incorporating either the ERSE-FLuc or XBP1s-RLuc reporters were collected by trypsinization and resuspended at a density of 500 , 000 cells per mL .", "The assay was started by dispensing 5 μL of cell suspension into each well of white , solid-bottom 1536-well plates using a flying reagent dispenser ( FRD ) and placed into an online incubator for 3 hr .", "Cells were then treated for 48 hr with various concentrations of proteostasis regulator , DMSO ( 0% toxicity ) or 340 μM Doxorubicin as a positive control ( 100% toxicity ) .", "After 48 hr of incubation , toxicity was determined using an ATP detection method ( CellTiter-Glo , Promega ) .", "HepG2 cells were plated at 5000 cells/well in a translucent , flat-bottomed 96 well plate , and treated for 48 hr with vehicle , 10 μM Tg or 10 μM ER proteostasis regulator .", "ALMC-2 or KAS-6/1 cells were plated at 33 , 000 cells/well in 96-well plates , then treated with proteostasis regulators for 16 hr as described .", "Cell metabolic activity was measured using the CellTiter-Glo assay ( Promega ) .", "CellTiter-Glo reagent was added to cell culture media at a 1:1 ratio and incubated for 2 min on an orbital shaker to induce cell lysis .", "The plate was then incubated at room temperature for 10 min to stabilize the luminescent signal and read on a Tecan F200 Pro microplate reader .", "Cells were treated as described at 37°C , washed with Dulbecco’s phosphate-buffered saline , and then RNA was extracted using the RNeasy Mini Kit ( Qiagen ) .", "qPCR reactions were performed on cDNA prepared from 500 ng of total cellular RNA using the QuantiTect Reverse Transcription Kit ( Qiagen ) .", "The FastStart Universal SYBR Green Master Mix ( Roche ) , cDNA , and appropriate human primers ( Shoulders et al . , 2013 ) or mouse primers ( BiP; gtccaggctggtgtcctctc and gattatcggaagccgtggag ) purchased from Integrated DNA Technologies were used for amplifications ( 45 cycles of 1 min at 95°C , 10 s at 95°C , 30 sec at 60°C ) in an ABI 7900HT Fast Real Time PCR machine .", "Primer integrity was assessed by a thermal melt to confirm homogeneity and the absence of primer dimers .", "Transcripts were normalized to the housekeeping gene RPLP2 and all measurements were performed in triplicate .", "Data were analyzed using the RQ Manager and DataAssist 2 . 0 softwares ( ABI , Foster City , CA ) .", "XBP1 splicing was assessed by RT-PCR followed by gel electrophoresis on a 3% agarose gel using the following primers flanking the XBP1 splicing sites: CCTTGTAGTTGAGAACCAGG , GAGTCAATACCGCCAGAATC .", "300 μL of 1 . 375 x 104 U2OS-GFP-ATF6 cells per mL were plated per well in 96 well imaging plate ( ibidi 89626 , Madison , WI ) and sealed with breathable seals ( E&K Scientific T896100 , Santa Clara , CA ) two days prior to drug addition .", "Immediately prior to addition to cells , compounds were diluted to 6x in media from 500x DMSO stock and 60 μL 6x was added to cells for 1x final ( 0 . 2% DMSO ) .", "After 5 hr , media was removed and cells were fixed in 4% PFA ( Electron Microscopy Sciences 15714 , Hatfield , PA ) in PHEM buffer ( 60 mM PIPES , 25 mM HEPES , 10 mM EGTA , 2 mM MgCl2-hexahydrate , pH 6 . 9 ) for 15 min at RT .", "Cells were permeabilized with PHEM-Tx ( PHEM containing 0 . 1% Triton X-100 , two washes , 5 min at RT ) , washed twice in PHEM and blocked in PHEM containing 2% normal goat serum ( Jackson Immunoresearch Laboratories 005-000-121 , West Grove , PA ) for 1 hr at RT .", "Primary antibodies were incubated in blocking solution overnight at 4 ºC .", "Cells were washed three times in PHEM-Tx then incubated with secondary antibodies and nuclear stain ( DAPI , Thermo Fisher D-1306 , 5 μg/ml ) in blocking solution for 2 hr at RT protected from light .", "Cells were washed three times in PHEM-Tx , then twice in PHEM .", "Antibodies used were rat anti-GRP94 9G10 ( Abcam ab2791 , Cambridge , MA ) , mouse anti-GFP 3E6 ( Life Technologies A11120 ) , anti-rat-Alexa-555 ( Life Technologies A21434 ) , and anti-mouse-Alexa-488 ( Life Technologies A11029 ) , each at 1:1000 dilution .", "Plate was imaged on a spinning disk confocal with Yokogawa CSUX A1 scan head , Andor iXon EMCCD camera and 20x Plan Apo Objective NA 0 . 79 .", "Using the μManager high-content screening plugin 'HCS Site Generator' ( Edelstein et al . , 2014 ) 49 fields per well were acquired for a mean cell number per well of 368 ± 12 .", "Images were analyzed using CellProfiler ( Carpenter et al . , 2006 ) , MATLAB R2014a and GraphPad Prism 5 .", "Masks for the ER and nucleus of each cell were created using the GRP94 and DAPI staining , respectively .", "The ratio of the GFP intensity in the nucleus versus the ER was calculated for each cell and plotted as a histogram per well .", "A threshold for the minimum ratio of nuclear to ER signal corresponding to an activated ( i . e . , nuclear localized ATF6 ) cell was calculated as the minimum nuclear: ER ratio greater than 1 where the number of ER stressed cells ( Tg ) was greater than the corresponding unstressed control .", "Percent activation per well was calculated as the percentage of cells per well with a nuclear: ER ratio greater than the calculated threshold for that plate .", "Mean percent activation per well for a minimum of three replicate wells per treatment was plotted and error bars are standard error of the mean .", "Compounds are annotated as hits if they show percent activation more than three standard deviations away from the mean of vehicle treated control .", "HepG2ATF6 cells plated on poly-D-lysine coated dishes were metabolically labeled in DMEM-Cys/-Met ( Corning CellGro , Mediatech Inc . , Manassas , VA ) supplemented with glutamine , penicillin/streptomycin , dialyzed fetal bovine serum , and EasyTag EXPRESS [35S] Protein Labeling Mix ( Perkin Elmer ) for 30 min .", "Cells were washed twice with complete media and incubated in pre-warmed DMEM for the indicated times .", "Media or lysates were harvested at the indicated times .", "Lysates were prepared in RIPA buffer with fresh protease inhibitor cocktail ( Roche ) .", "FLAG-tagged TTR variants were immunopurified using M1 anti-FLAG agarose beads ( Sigma Aldrich ) and washed three times with RIPA buffer .", "The immunoisolates were then eluted by boiling in Laemmli buffer and separated on SDS-PAGE .", "For total secreted media , 15 µL aliquots from media incubated for 4 hrs on pulsed cells were run on a SDS-PAGE .", "The gels were then dried , exposed to phosphorimager plates ( GE Healthcare , Pittsburgh , PA ) , and imaged with a Typhoon imager .", "Band intensities were quantified by densitometry in ImageQuant .", "Fraction secreted was calculated using the equation: fraction secreted = [extracellular [35S]-TTR signal at t / ( extracellular [35S]-TTR signal at t=0 + intracellular [35S]-TTR signal at t=0 ) ] .", "Fraction remaining was calculated using the equation: [ ( extracellular [35S]-TTR signal at t + intracellular [35S]-TTR signal at t ) / ( extracellular [35S]-TTR signal at t=0 + intracellular [35S]-TTR signal at t=0 ) ] .", "For immunoblotting , cells were lysed in 50 mM Tris buffer , pH 7 . 5 containing 0 . 1% TritonX ( Fisher Scientific ) and supplemented with protease inhibitor cocktail ( Roche ) .", "For immunoblots of endogenous ATF6 , cells were washed in PBS containing protease inhibitor cocktail and 10 µM MG-132 and subsequently lysed in 1x Laemmli buffer , supplemented in the same way , by repeating cycles of boiling and vortexing .", "Protein lysate concentrations were normalized by Bradford ( Bio-Rad , Hercules , CA ) or BCA assay ( Thermo Fisher ) .", "Lysates or media were boiled for 5 min in Laemmli buffer + 100 mM DTT before loading onto SDS-PAGE gel .", "Proteins were transferred from gel slabs to nitrocellulose , and the Odyssey Infrared Imaging System ( Li-Cor Biosciences ) was used to detect proteins of interest .", "For immunoprecipitations , cells were washed with PBS then cross-linked with 0 . 5 mM Dithiobis ( succinimidiyl propionate ) ( DSP ) for 30 min at room temperature .", "The reaction was quenched by addition of 100 mM Tris pH 7 . 5 , then RIPA buffer was added to the cell pellets for lysis .", "Lysates were cleared by centrifugation at centrifuged at 10000 x g for 15 min .", "Proteins were immunopurified using sheep polyclonal free λ LC antibody ( Bethyl Laboratories A80-127A , Montgomery , TX ) that was covalently conjugated to CNBr-activated Sepharose 4B ( GE Healthcare ) .", "After four washes in RIPA buffer , proteins were eluted by boiling in Laemmli buffer + 100 mM DTT and samples were separated by SDS-PAGE and transferred to nitrocellulose membranes .", "Blots were probed with the following primary antibodies: monoclonal mouse M2 anti-FLAG ( 1:500 , Sigma Aldrich ) , polyclonal rabbit anti-TTR ( 1:1000 , Dako , Carpinteria , CA ) , rabbit polyclonal anti-human lambda light chain ( 1:1000 , Bethyl Laboratories A90-112A ) , mouse monoclonal anti-Grp78 ( 1:500 , Santa Cruz Biotechnology sc-166490 , Dallas , TX ) , rabbit polyclonal anti-Grp94 ( 1:1000 , GeneTex GTX103203 , Irvine , CA ) , rabbit polyclonal anti-PDIA4 ( 1:1000 , Proteintech Group 14712-1-AP , Rosemont , IL ) , mouse monoclonal anti-ATF6α ( 1–7 ) ( BioAcademia , 1:1000 , Osaka , Japan ) , rabbit anti-phospho eIF2α ( CellSignaling Technologies #9721 , 1:500 , Danvers , MA ) , mouse anti-eIF2α ( Abcam ab5369 , 1:1000 ) and mouse monoclonal anti β-actin ( 1:10000 , Sigma Aldrich ) .", "ALMC-2 or KAS-6/1 plasma cells were plated 100 , 000 cells/well in 150 µL of media in 96-well MultiScreenHTS filtration plates ( EMD Millipore MSBVS1210 , Billerica , MA ) .", "Triplicate wells were treated with DMSO or compounds at the indicated concentrations and incubated for 16 hr .", "Media was removed by filtration using a QIAvac 96 vaccuum manifold ( Qiagen ) and wells were washed two times with 150 µL media .", "Wells were then incubated with 150 µL of fresh media for 2 hr and the conditioned media was harvested into a 96-well plate using the vacuum manifold .", "Free LC and IgG concentrations were determined by ELISA in 96-well plates ( Immulon 4HBX , Thermo Fisher ) .", "Wells were coated overnight at 37 ºC with sheep polyclonal free λ LC antibody ( Bethyl Laboratories , A80-127A ) at a 1:500 dilution or human IgG-heavy and light chain antibody ( Bethyl Laboratories , A80-118A ) at a 1:2000 dilution in 50 mM sodium carbonate ( pH 9 . 6 ) .", "In between all incubation steps , the plates were rinsed extensively with Tris-buffered saline containing 0 . 05% Tween-20 ( TBST ) .", "Plates were blocked with 5% non-fat dry milk in TBST for 1 hr at 37ºC .", "Media analytes were diluted between 5 – 200 fold in 5% non-fat dry milk in TBST and 100 µL of each sample was added to individual wells .", "Light chain or IgG standards ranging from 3 – 300 ng/mL were prepared from purified human Bence Jones λ light chain or human reference serum ( Bethyl Laboratories , P80-127 and RS10-110 ) .", "Plates were incubated at 37 ºC for 1 . 5 hr while shaking .", "Finally , HRP-conjugated goat anti-human λ light chain antibody ( Bethyl Laboratories , A80-116P ) was added at a 1:5 , 000 dilution or HRP-conjugate IgG-Fc fragment cross-adsorbed antibody ( Bethyl Laboratories , A80-304P , 1:30 , 000 dilution ) was added in 5% non-fat dry milk in TBST , followed by a 1 . 5 hr incubation of the plates at 37 ºC .", "The detection was carried out with 2 , 2'-azinobis ( 3-ethylbenzothiazoline-6-sulfonic acid ) ( ABTS , 0 . 18 mg/mL ) and 0 . 03% hydrogen peroxide in 100 mM sodium citrate pH 4 . 0 .", "Detection solution ( 100 µL ) was added to each well and the plates were incubated at room temperature .", "The absorbance was recorded at 405 nm and the values for the LC standards were fitted to a 4-parameter logistic function .", "Light chain or IgG concentrations were averaged from at least 3 independent replicates under each treatment and then normalized to vehicle conditions .", "ALMC-2 plasma cells were plated 2 x 106 cells/well in 1 mL of media in 12-well plates .", "Wells were treated with DMSO or 10 µL of compounds and incubated for 16 hr .", "Cells were transferred to microcentrifuge tubes and washed two times in media .", "Small aliquots ( 10 µL ) of cells were removed for a cytotoxicity assay as described above .", "Cells were resuspended in media ( 1 mL for DMSO treatment ) and the volume was adjusted for each treatment based on cell viability .", "Cells were conditioned in the media for 8 hr and then were removed by centrifugation .", "The media samples were immunoprecipitated overnight at 4 ºC with recombinant Protein A – Sepharose 4B resin ( Life Technologies ) to remove fully assembled IgGs that interfere with LC aggregate detection .", "Cleared media samples were then heated to 55 ºC for 8 hr to induce LC aggregation , added to Blue-Native PAGE loading dye ( 10% glycerol , 0 . 5% Coomassie G-250 ) and then loaded onto 3–12% Bis-Tris gradient gels ( Life Technologies ) .", "The cathode buffer contained 50 mM Tricene and 15 mM Bis-Tris , pH 7 . 0 with 0 . 02% Coomassie G-250 .", "The anode buffer contained 50 mM Bis-Tris pH 7 . 0 .", "The gels were transferred onto PVDF membranes and LC was detected by rabbit anti-human λ light chain polyclonal antibody ( Bethyl Laboratories ) , followed by HRP-conjugated secondary antibodies .", "The blots were imaged using a chemiluminescence substrate ( Luminata Forte Western Luminescence Substrate , EMD Millipore ) and imaged with a ChemiDoc XRS+ scanner ( Bio-Rad ) ." ] ]

[ "Imbalances in endoplasmic reticulum ( ER ) proteostasis are associated with etiologically-diverse degenerative diseases linked to excessive extracellular protein misfolding and aggregation .", "Reprogramming of the ER proteostasis environment through genetic activation of the Unfolded Protein Response ( UPR ) -associated transcription factor ATF6 attenuates secretion and extracellular aggregation of amyloidogenic proteins .", "Here , we employed a screening approach that included complementary arm-specific UPR reporters and medium-throughput transcriptional profiling to identify non-toxic small molecules that phenocopy the ATF6-mediated reprogramming of the ER proteostasis environment .", "The ER reprogramming afforded by our molecules requires activation of endogenous ATF6 and occurs independent of global ER stress .", "Furthermore , our molecules phenocopy the ability of genetic ATF6 activation to selectively reduce secretion and extracellular aggregation of amyloidogenic proteins .", "These results show that small molecule-dependent ER reprogramming , achieved through preferential activation of the ATF6 transcriptional program , is a promising strategy to ameliorate imbalances in ER function associated with degenerative protein aggregation diseases ." ]

[ "Newly made proteins must be folded into specific three-dimensional shapes before they can perform their roles in cells .", "Many proteins are folded in a compartment called the endoplasmic reticulum before being transported to their final location .", "However , if a cell suddenly needs to make a large number of new proteins , it can overwhelm the endoplasmic reticulum and unfolded proteins may accumulate .", "The cell responds to this stress by activating the unfolded protein response , which increases the folding capacity of the endoplasmic reticulum to match the demand .", "However , if the stress persists , then the unfolded protein response instructs the cell to die to protect the rest of the body .", "A protein called ATF6 is involved in one branch of the unfolded protein response .", "Endoplasmic reticulum stress causes ATF6 to move from the endoplasmic reticulum to another cell compartment where certain enzymes are able to cut the protein .", "A fragment of ATF6 then moves to the nucleus of the cell to activate genes needed to increase the cell’s capacity to fold proteins .", "Errors in protein folding can cause serious diseases in humans and other animals .", "Drugs that target ATF6 might be able to regulate part of the unfolded protein response to treat these diseases .", "However , no drugs that act on ATF6 had been identified .", "Now , two groups of researchers have independently identified small molecules that specifically target ATF6 .", "Plate et al . used a new approach to screen over 600 , 000 small molecules and identified a small number that could activate ATF6-regulated genes without inducing global endoplasmic reticulum stress .", "Further experiments tested whether any of these ATF6 drug candidates could prevent the release of incorrectly folded versions of two particular proteins from cells that are associated with types of amyloid disease in humans .", "One of the small molecules tested effectively reduced the release of these proteins and prevented harmful deposits of the proteins forming in the spaces surrounding cells .", "In an independent study , Gallagher et al . identified a type of small molecule that can inhibit the activity of ATF6 .", "Together , these findings may lead to further development of new drugs for treating diseases associated with incorrect protein folding in the endoplasmic reticulum ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "biochemistry and chemical biology", "cancer biology" ]

[ [ "Doxorubicin ( Adriamycin ) is used widely to treat diverse types of cancer , yet its effectiveness is hampered by the existence of drug-resistant cancer cells .", "The reason for drug resistance is unclear mainly because the mechanism through which doxorubicin inhibits proliferation of cancer cells is not completely understood .", "Doxorubicin has been proposed to exert its cytostatic action through intercalation into DNA and production of free radicals ( Gewirtz , 1999 ) .", "However , these mechanisms are unlikely to be clinically relevant as the concentration of doxorubicin required to produce these effects is much higher than that achievable in patients ( Gewirtz , 1999 ) .", "Inhibition of topoisomerase II by doxorubicin at clinically achievable concentrations leads to DNA breaks , but a consistent relationship between DNA strand breaks and the cytostatic action of the drug has not been demonstrated ( Gewirtz , 1999 ) .", "Thus , the mechanism through which doxorubicin inhibits cell proliferation remains unclear .", "In addition to blocking cell proliferation , doxorubicin induces renal fibrosis in mice by stimulating production of collagen ( Lee and Harris , 2011 ) .", "The dual ability of doxorubicin to block cell proliferation and to induce collagen expression caught our attention inasmuch as we recently showed that both responses can be activated by a transcription factor called cAMP response element-binding protein 3-like 1 ( CREB3L1 , also known as OASIS ) ( Denard et al . , 2011 ) .", "CREB3L1 belongs to a family of transcription factors synthesized as transmembrane precursors ( Omori et al . , 2002 ) and activated through a process designated as Regulated Intramembrane Proteolysis ( RIP ) ( Brown et al . , 2000 ) .", "The transcription factor domain of CREB3L1 is located in the NH2-terminal 374-amino acids that project into the cytosol ( Figure 1A ) .", "The COOH-terminal domain of 124 amino acids projects into the lumen of the endoplasmic reticulum ( ER ) ( Figure 1A ) .", "Viral infection triggers the RIP of CREB3L1 , which undergoes two sequential cleavages mediated by Site-1 protease ( S1P ) and Site-2 protease ( S2P ) ( Denard et al . , 2011 ) .", "The S1P-catalyzed cleavage at the luminal side is a prerequisite for the S2P-catalyzed intramembrane cleavage that releases the NH2-terminal domain of the protein from membranes , allowing it to drive transcription of genes that suppress cell proliferation such as p21 ( Denard et al . , 2011 ) .", "Nuclear CREB3L1 also activates genes required for assembly of the collagen matrix , including collagen 1α1 ( Denard et al . , 2011 ) .", "These dual activities prompted us to hypothesize that doxorubicin functions by stimulating proteolytic activation of CREB3L1 . 10 . 7554/eLife . 00090 . 003Figure 1 . Doxorubicin stimulates RIP of CREB3L1 . ( A ) Schematic diagram of CREB3L1 .", "( B ) , ( D ) On Day 0 , Huh7 cells ( B ) or wild type and mutant CHO cells ( D ) were seeded at 4 × 105 cells per 60 mm dish .", "On day 1 , cells were treated with 500 nM doxorubicin .", "On day 2 , 24 hr after the treatment , the cells were separated into nuclear and membrane fractions , and analyzed by immunoblot with antibodies directed against CREB3L1 , calnexin and LSD1 .", "( C ) On day 0 , huh7 cells were seeded at 1 × 105 cells per 60 mm dish .", "On day 1 they were transfected with pCMV-CREB3L1 ( Δ381-519 ) ( 0 . 1 µg per dish ) as indicated .", "On day 2 , they were treated with 500 nM doxorubicin as indicated .", "On day 3 , 24 hr after the treatment , the cells were treated with 10 µM MG132 for 2 hr as indicated .", "Nuclear fraction of the cells was then analyzed by immunoblot analysis with antibody reacting against CREB3L1 and LSD1 .", "( E ) On day 0 , CHO-7 cells were seeded at 2 × 105 cells per 60 mm dish .", "On day 1 , some cells were changed into sterol-depleting medium ( medium A containing 50 µM compactin , 50 µM mevalonate , and 5% lipoprotein deficient serum [LPDS] ) with or without supplementation of sterols ( 1 µg/ml 25-hydroxycholesterol and 10 µg/ml cholesterol ) .", "Other cells were changed into normal medium ( medium A supplemented with 5% fetal calf serum [FCS] ) containing the indicated concentrations of doxorubicin .", "On day 2 , 24 hr after the treatment , the cells were separated into nuclear and membrane fractions , and analyzed by immunoblot with antibodies directed against SREBP2 , calnexin and LSD1 . DOI: http://dx . doi . org/10 . 7554/eLife . 00090 . 003 In the current study , we determine that doxorubicin induces proteolytic activation of CREB3L1 , and this cleavage is required for doxorubicin to inhibit proliferation of cancer cells .", "We further demonstrate that doxorubicin-stimulated production of ceramide is required for RIP of CREB3L1 .", "These results suggest that CREB3L1 expression may be a useful biomarker in identifying cancer cells sensitive to doxorubicin ." ], [ "To analyze proteolytic activation of CREB3L1 , we fractionated human hepatoma Huh7 cells ( Nakabayashi et al . , 1982 ) into membrane and nuclear fractions , and used an antibody reacting against the NH2-terminal domain of CREB3L1 ( Denard et al . , 2011 ) to examine the cleavage of CREB3L1 through immunoblot analysis .", "In the absence of doxorubicin , CREB3L1 existed as the full length precursor ( ∼80 kDa ) in membranes and the cleaved nuclear form of CREB3L1 ( ∼55 kDa ) was barely detectable ( Figure 1B , lane 1 ) .", "Treatment with doxorubicin markedly raised the amount of the nuclear form of CREB3L1 ( Figure 1B , lane 2 ) .", "The amount of membrane protein calnexin and nuclear protein lysine-specific demethylase 1 ( LSD1 ) was not altered by doxorubicin treatment ( Figure 1B ) .", "Doxorubicin may increase the amount of nuclear CREB3L1 through stimulation of CREB3L1 precursor cleavage or inhibition of nuclear CREB3L1 degradation , which was reported to be carried out by proteasomes ( Murakami et al . , 2009 ) .", "To determine whether doxorubicin inhibits degradation of nuclear CREB3L1 , we transfected Huh7 cells with a cDNA encoding NH2-terminal fragment of CREB3L1 resembling the cleaved nuclear form of the protein ( pCMV-CREB3L1 ( Δ381-519 ) ) ( Denard et al . , 2011 ) .", "The amount of transfected nuclear form of CREB3L1 was not affected by doxorubicin ( Figure 1C , lanes 2 and 3 ) .", "However , this amount was increased in cells treated with the proteasome inhibitor MG132 ( Figure 1C , lanes 5 and 6 ) , suggesting that overexpression of the transfected protein did not overwhelm the machinery that degrades nuclear CREB3L1 .", "These results suggest that doxorubicin does not stabilize nuclear CREB3L1 .", "Thus , doxorubicin appears to increase nuclear CREB3L1 by stimulating proteolysis of its precursor .", "To determine whether doxorubicin-stimulated cleavage of CREB3L1 was catalyzed by S1P and S2P , we analyzed the cleavage in mutant Chinese Hamster Ovary ( CHO ) cells deficient in S1P or S2P ( Rawson et al . , 1997 , 1998 ) .", "In wild type CHO cells , doxorubicin stimulated cleavage of CREB3L1 to produce the nuclear form ( Figure 1D , lane 2 ) .", "In contrast , doxorubicin failed to produce the nuclear form of CREB3L1 in mutant cells deficient in either S1P or S2P ( Figure 1D , lanes 4 and 6 ) .", "In wild type CHO cells , we also detected in the membrane fraction a cleaved fragment with a molecular weight similar to that of the nuclear form ( Figure 1D , lanes 1 and 2 ) .", "This fragment was absent in cells deficient in S1P ( Figure 1D , lanes 3 and 4 ) but dramatically elevated in cells deficient in S2P ( Figure 1D , lanes 5 and 6 ) .", "These findings suggest that this membrane-bound fragment is the intermediate form of CREB3L1 that was cleaved by S1P but not by S2P .", "Similar cleavage intermediates were observed in earlier studies of SREBP-2 , a prototypes of RIP substrates , in mutant CHO cells deficient in S2P ( Rawson et al . , 1997; Ye et al . , 2000 ) .", "SREBP-2 was cleaved in sterol-depleted CHO cells ( Figure 1E , lane 1 ) to activate genes required for cholesterol synthesis and uptake ( Brown and Goldstein , 2009 ) .", "However , this cleavage was not activated by doxorubicin ( Figure 1E , lanes 4 and 5 ) .", "Thus , doxorubicin appears to specifically induce proteolytic activation of CREB3L1 .", "An alternative approach to determine the effect of doxorubicin on proteolytic activation of CREB3L1 is to analyze the effect of the compound on expression of target genes activated by CREB3L1 .", "In Huh7 cells transfected with a control shRNA ( Huh7-shControl ) , doxorubicin induced the expression of collagen 1α1 and p21 ( Figure 2A , B ) , both of which were shown to be direct targets of CREB3L1 ( Murakami et al . , 2009; Denard et al . , 2011 ) .", "In Huh7 cells stably transfected with a shRNA targeting CREB3L1 ( Huh7-shCREB3L1 ) ( Denard et al . , 2011 ) in which expression of CREB3L1 was drastically reduced ( Figure 2C ) , induction of these genes was markedly blunted ( Figure 2A , B ) . 10 . 7554/eLife . 00090 . 004Figure 2 . Doxorubicin induces transcription of genes activated by CREB3L1 . ( A ) , ( B ) On day 0 , indicated cells were seeded at 3 × 105 cells per 60 mm dish .", "On day 1 , the cells were treated with the indicated concentration of doxorubicin .", "On day 2 , 24 hr after the treatment , some of the cells were harvested for quantification of p21 mRNA through RT-QPCR ( B ) .", "On day 4 , 72 hr after the treatment , the rest of the cells were harvested for quantification of collagen 1α1 ( COL1A1 ) mRNA through RT-QPCR ( A ) .", "( A ) , ( B ) The value of each mRNA in cells that were not treated with the drug is set to 1 .", "( C ) Immunoblot analysis of CREB3L1 in indicated cells . DOI: http://dx . doi . org/10 . 7554/eLife . 00090 . 004 Inasmuch as CREB3L1 was required for doxorubicin to induce expression of p21 , a well-characterized inhibitor of the cell cycle ( Sherr and Roberts , 1999 ) , we determined whether CREB3L1 was also required for doxorubicin to inhibit cell proliferation .", "For both untransfected Huh7 cells and those transfected with the control shRNA ( Huh7-shControl ) , doxorubicin completely blocked their proliferation at a concentration between 50 and 150 nM ( Figure 3A ) .", "This concentration of doxorubicin also resulted in maximal cleavage of CREB3L1 in Huh7 cells ( Figure 3B ) .", "For Huh7-shCREB3L1 cells , doxorubicin at concentrations up to 500 nM failed to block their proliferation ( Figure 3A ) .", "These concentrations of doxorubicin were not enough to trigger apoptosis of Huh7 cells , which became apparent only when the cells were treated with 5 µM of the compound ( Figure 3C ) .", "To rule out the off-target effects of the shRNA , we also transfected Huh7 cells with two distinct siRNA targeting regions of CREB3L1 that is different from that targeted by the shRNA .", "Transfection with these siRNA knocked down CREB3L1 mRNA by more than 90% ( Figure 3D ) , and the treatment also rendered Huh7 cells more resistant to doxorubicin ( Figure 3E ) . 10 . 7554/eLife . 00090 . 005Figure 3 . CREB3L1 is required for doxorubicin to suppress proliferation of Huh7 cells .", "( A ) On day 0 , indicated cells were seeded at 1 . 5 × 105 cells per 60 mm dish .", "On day 1 , they were treated with the indicated concentrations of doxorubicin .", "On day 3 , 48 hr after the treatment , the cells were quantified to determine cell proliferation .", "The number of cells just prior to the drug treatment and after treatment with no drug for 48 hr is set to 0% and 100% , respectively .", "( B ) Huh7 cells treated with the indicated concentrations of doxorubicin were analyzed as described in Figure 1B .", "( C ) On day 0 , Huh7 cells were seeded at 4 × 105 cells per 60 mm dish .", "On day 1 , cells were treated with the indicated concentrations of doxorubicin .", "On day 3 , 48 hr after the treatment , cells were harvested to determine the percentage of the cells that underwent apoptosis through TUNEL assay .", "( D ) , ( E ) On day 0 , Huh7 cells were seeded at 1 × 105 cells per 60 mm dish .", "On day 1 , the cells were transfected with indicted siRNAs .", "On day 2 , the cells were treated with indicated concentrations of doxorubicin .", "On day 4 , 48 hr after the treatment , some of the cells were harvested for quantification of CREB3L1 mRNA by RT-QPCR ( D ) , while the others were used for determination of cell proliferation as described in Figure 3A ( E ) .", "( A ) , ( C ) , ( D ) , ( E ) Results are reported as mean ± S . E . M . of three independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 00090 . 005 If proteolytic activation of CREB3L1 is required for doxorubicin to inhibit cell proliferation , then the amount of CREB3L1 expressed in cancer cells may determine their sensitivity to doxorubicin .", "To test this hypothesis , we analyzed SV589 cells , an immortalized line of human fibroblasts ( Yamamoto et al . , 1984 ) , and MCF-7 cells , a line of human breast cancer cells ( Soule et al . , 1973 ) .", "Compared to Huh7 cells , expression of CREB3L1 was higher in SV589 cells and lower in MCF-7 cells ( Figure 4A ) .", "The sensitivity of the cells to growth inhibition by doxorubicin followed the order of CREB3L1 expression ( Figure 4B ) .", "Similar to Huh7 cells , knockdown of CREB3L1 by two duplexes of siRNA targeting different regions of CREB3L1 in SV589 cells ( Figure 4C ) made them more resistant to doxorubicin ( Figure 4D ) .", "Since MCF-7 cells expressed very little CREB3L1 , we used these cells to study the effect of CREB3L1 overexpression on sensitivity to doxorubicin .", "We stably transfected MCF-7 cells with a plasmid encoding CREB3L1 and selected one clone of the cells with relatively low expression ( MCF7/pCREB3L1 ( L ) ; eightfold above parental cells ) and another clone with high expression of CREB3L1 ( MCF7/pCREB3L1 ( H ) ; 300-fold above parental cells ) ( Figure 4E ) .", "The eightfold overexpression of CREB3L1 in MCF7/pCREB3L1 ( L ) cells lowered the IC50 for doxorubicin from 500 nM to 10 nM , and the 300-fold overexpression of CREB3L1 in MCF7/pCREB3L1 ( H ) cells further reduced the IC50 to ∼1 nM ( Figure 4F ) .", "In this experiment , cells were treated with doxorubicin for 2 days .", "To determine the effect of CREB3L1 expression on proliferation of the cells treated with doxorubicin for a longer period of time , we incubated MCF-7 and MCF7/pCREB3L1 ( H ) cells with 15 nM doxorubicin for 6 days .", "This treatment did not affect proliferation of MCF-7 cells , but markedly blocked proliferation of MCF7/pCREB3L1 ( H ) cells , as determined by direct cell counting ( Figure 4G ) and by measurement of cellular DNA content ( Figure 4H ) .", "Thus , CREB3L1 expression level is a key determinant of cellular sensitivity to doxorubicin . 10 . 7554/eLife . 00090 . 006Figure 4 . Sensitivity of cancer cells to doxorubicin is correlated to their expression of CREB3L1 . ( A ) , ( E ) RT-QPCR quantification of CREB3L1 mRNA in indicated cells with its value in Huh7 ( A ) or MCF-7 cells ( E ) set to 1 .", "( B ) , ( F ) Effect of doxorubicin on proliferation of the indicated cells was determined as described in Figure 3A .", "( C ) , ( D ) SV-589 cells were treated and analyzed as described in Figure 3D , E .", "( G ) , ( H ) On day 0 , indicated cells were seeded at 1 . 5 × 105 cells per 60 mm dish .", "On day 1 , the cells were treated with or without 15 nM doxorubicin .", "After incubation for the indicated period of time , cell proliferation was determined by direct counting of the cells ( G ) or by measurement of the amount of cellular DNA ( H ) .", "( G ) , ( H ) The number of cells just before doxorubicin treatment at time 0 is set to one .", "( A–H )", "Results are reported as mean ± S . E . M . of three independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 00090 . 006 We then determined the relationship between doxorubicin-induced cleavage of CREB3L1 and DNA breaks caused by inhibition of topoisomerase .", "Doxorubicin induced appearance of histone γH2AX , a marker for DNA breaks ( Figure 5A , lane 2 ) .", "However , this effect was unaffected by knockdown of CREB3L1 expression ( Figure 5A , lane 5 ) .", "This result suggests that cleavage of CREB3L1 does not lead to doxorubicin-induced DNA breaks .", "To investigate whether DNA breaks may lead to cleavage of CREB3L1 , we examined etoposide , another chemotherapeutic drug that inhibits topoisomerase ( Stähelin and von Wartburg , 1991 ) .", "Unlike doxorubicin , etoposide failed to induce cleavage of CREB3L1 ( Figure 5B , lane 3 ) , even though etoposide was as effective as doxorubicin in causing DNA breaks ( Figure 5A , lanes 2 and 3 ) .", "Accordingly , knockdown of CREB3L1 in Huh7 cells did not increase their resistance to etoposide ( Figure 5C ) , and overexpression of CREB3L1 in MCF7 cells also did not increase their sensitivity to the compound ( Figure 5D ) .", "These results suggest that induction of CREB3L1 cleavage by doxorubicin is not related to its inhibitory activity towards topoisomerase .", "Besides etoposide , CREB3L1 was also not required for bleomycin or paclitaxel to inhibit cell growth , an observation suggesting that CREB3L1 may be specifically involved in doxorubicin-induced suppression of cell proliferation ( Figure 5E–G ) . 10 . 7554/eLife . 00090 . 007Figure 5 . CREB3L1 activation is independent from DNA breaks .", "( A ) On day 0 , indicated cells were seeded at 4 × 105 cells per 60 mm dish .", "On day 1 , cells were treated with 500 nM doxorubicin or 500 nM etoposide .", "On day 2 , 24 hr after the treatment , the cells were harvested for immunoblot analysis with antibodies reacting against γH2AX or actin .", "( B ) Huh7 cells were seeded and treated as described in ( A ) .", "On day 2 , cells were separated into nuclear and membrane fractions and analyzed by immunoblot analysis as described in Figure 1B .", "( C ) The effect of etoposide on proliferation of the indicated cells was determined as described in Figure 3A .", "( D ) – ( G ) On day 0 , indicated cells were seeded at 1 . 5 × 105 cells per 60 mm dish .", "On day 1 , cells were treated with indicated concentrations of etoposide ( D ) , doxorubicin ( E ) , bleomycin ( F ) , or paclitaxel ( G ) .", "On day 3 , 48 hr after the treatment , proliferation of the cells was determined by measurement of cellular DNA .", "The amount of DNA just prior to the drug treatment and after treatment with no drug for 48 hr is set to 0% and 100% , respectively .", "( C ) – ( G ) Results are reported as mean ± S . E . M . of three independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 00090 . 007 RIP of membrane-bound transcription factors is known to be a signal transduction pathway that transfers signals from the ER to nucleus ( Brown et al . , 2000 ) .", "Since ER is the site where most lipids are synthesized , we wondered whether doxorubicin may alter homeostasis of certain lipids that may result in cleavage of CREB3L1 .", "Doxorubicin and daunorubicin , a chemotherapeutic drug derived from doxorubicin , were reported to induce de novo synthesis of ceramide ( Bose et al . , 1995; Liu et al . , 2008 ) , a class of lipid known to inhibit cell proliferation ( Ogretmen and Hannun , 2004 ) .", "De novo synthesis of ceramide is initiated with the condensation of palmitate and serine ( Gault et al . , 2010 ) .", "This rate-limiting step in de novo synthesis of ceramide is catalyzed by serine palmitoyltransferase ( SPT ) ( Linn et al . , 2001 ) .", "Ceramide synthesis also requires a reaction catalyzed by ceramide synthase ( Mullen et al . , 2012 ) .", "We confirmed that doxorubicin stimulated ceramide synthesis by showing that treatment with the compound increased the amount of [14C]palmitate incorporated into ceramide in Huh7 cells ( Figure 6A ) .", "Mass spectroscopy analysis revealed that doxorubicin primarily increased the amount of ceramide containing palmitate ( 16:0 ) as the amide-linked fatty acid ( Figure 6B ) .", "In contrast to doxorubicin , etoposide failed to induce ceramide synthesis at a concentration at which cell proliferation was inhibited ( Figure 6C ) . 10 . 7554/eLife . 00090 . 008Figure 6 . Doxorubicin stimulates synthesis of ceramide .", "( A ) On day 0 , Huh7 cells were seeded at 2 × 105 per 60-mm dish .", "On day 1 , the cells were treated with or without 500 nM doxorubicin .", "On day 2 , 20 hr after the treatment , the cells were labeled with indicated concentrations of [14C]palmitate for additional 4 hr .", "Cell lipids were then extracted to determine the amount of [14C]palmitate incorporated into ceramide .", "*p=0 . 003; **p=0 . 02 .", "( B ) On day 0 , Huh7 cells were seeded at 1 . 5 × 105 per 60-mm dish .", "On day 1 , the cells were treated with or without 500 nM doxorubicin .", "On day 2 , 24 hr after the treatment , the cells were harvested for ceramide analysis via LC-MS as described in ‘Materials and methods’ .", "The amount of ceramide with indicated amide-linked fatty acids was presented .", "( C ) Huh7 cells were treated with 500 nM doxorubicin or 1 µM etoposide , labeled with 3 µM [14C]palmitate , and analyzed as described in Figure 6A .", "( A ) – ( C ) Results are reported as mean ± S . E . M . of triplicate incubations from a representative experiment . DOI: http://dx . doi . org/10 . 7554/eLife . 00090 . 008 To determine whether doxorubicin-induced ceramide synthesis is required to stimulate cleavage of CREB3L1 , we treated Huh7 cells with myriocin , an inhibitor of SPT ( Miyake et al . , 1995 ) .", "This treatment inhibited doxorubicin-induced cleavage of CREB3L1 ( Figure 7A ) .", "Co-treatment with myriocin rendered the cells more resistant to doxorubicin ( Figure 7B ) .", "Fumonisin B1 , an inhibitor of ceramide synthase ( Wang et al . , 1991 ) , also blocked doxorubicin-induced cleavage of CREB3L1 ( Figure 7C ) .", "The observations that inhibition of two different enzymes involved in ceramide synthesis were both effective in blocking doxorubicin-induced cleavage of CREB3L1 strongly suggest that this cleavage is caused by increased synthesis of ceramide . 10 . 7554/eLife . 00090 . 009Figure 7 . Doxorubicin-induced synthesis of ceramide stimulates cleavage of CREB3L1 . ( A ) , ( C ) On day 0 , Huh7 cells were seeded at 4 × 105 per 60-mm dish .", "On day 1 , the cells were treated with indicated concentrations of myriocin ( A ) or fumonisin B1 ( C ) for 2 hr , followed by co-incubation with 200 nM doxorubicin .", "On day 2 , 24 hr after the doxorubicin treatment , cells were analyzed for cleavage of CREB3L1 by immunoblot analysis as described in Figure 1B .", "( B ) Huh7 cells treated with or without 30 µM myriocin for 2 hr followed by co-treatment with doxorubicin were analyzed as described in Figure 3A .", "( D ) Huh7 cells treated with 10 µM C6-ceramide for 3 hr were analyzed as described in Figure 6B .", "Results are reported as mean ± S . E . M . of triplicate incubations from a representative experiment .", "( E ) Huh7 cells treated with indicated concentration of C6-ceramide for 24 hr were analyzed as described in Figure 1B .", "( F ) Indicated cells treated with indicated concentration of C6-ceramide for 48 hr were analyzed as described in Figure 3A .", "( B ) , ( F ) Results are reported as mean ± S . E . M . of three independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 00090 . 009 To more directly determine the effect of ceramide on cleavage of CREB3L1 , we treated Huh7 cells with C6-ceramide , a cell-permeable analogue of ceramide that contains a short acyl chain .", "It was reported previously that C6-ceramide was converted to naturally-existing ceramide in cells through the ceramide salvage pathway ( Kitatani et al . , 2008 ) .", "Indeed , our mass spectroscopy analysis confirmed that treatment with C6-ceramide increased nearly all species of ceramide in Huh7 cells ( Figure 7D ) .", "This treatment stimulated CREB3L1 cleavage even in the absence of doxorubicin ( Figure 7E ) .", "These results suggest that doxorubicin-induced synthesis of ceramide leads to cleavage of CREB3L1 .", "Thus , CREB3L1 appears to suppress cell proliferation in response to accumulation of ceramide .", "This conclusion was further supported by the observation that knockdown of CREB3L1 in Huh7 cells completely abolished the ability of C6-ceramide to inhibit cell proliferation ( Figure 7F ) ." ], [ "The current study establishes a crucial role for CREB3L1 in inhibiting cell proliferation in response to doxorubicin .", "We show that the sensitivity of cellular response to doxorubicin is positively correlated to CREB3L1 expression in cancer cells .", "Importantly , the concentration of doxorubicin required to proteolytically activate CREB3L1 is within clinically relevant concentration ranges found in the serum of patients treated with the drug ( <1 µM ) ( Gewirtz , 1999 ) .", "These findings raise the possibility that the clinical response to doxorubicin may be determined by the level of CREB3L1 produced in tumor cells .", "Thus , measuring CREB3L1 expression in tumor cells may be useful in identifying cancer patients who are most likely to benefit from doxorubicin treatment .", "However , this hypothesis is difficult to test with the currently available clinical data .", "This is because most cancer patients are treated with chemotherapy regime containing doxorubicin but not doxorubicin alone .", "Thus , even if the patients respond to the treatment , it is difficult to discern whether they respond to doxorubicin or other anti-cancer drugs in the regime .", "A clinical study using doxorubicin alone to treat tumors that express high amount of CREB3L1 will be required to determine whether CREB3L1 expression can be used as a biomarker to predict treatment outcome of doxorubicin .", "An important finding in the current study is that doxorubicin-induced accumulation of ceramide is required for cleavage of CREB3L1 .", "This is the second example of a transcription factor whose proteolytic activation is regulated by a lipid synthesized in the ER .", "The first such example is SREBP-2 , a transcription factor that regulates cholesterol metabolism ( Brown and Goldstein , 2009 ) .", "When ER cholesterol content is less than 4% of total lipid , SREBP-2 are transported from the ER to Golgi complex where it is cleaved by S1P and S2P ( DeBose-Boyd et al . , 1999; Radhakrishnan et al . , 2008 ) .", "These cleavages liberate the NH2-terminal domain of SREBP-2 from membranes , allowing it to enter the nucleus where it activates all genes required for cholesterol synthesis and uptake ( Horton et al . , 2003 ) .", "When ER cholesterol content exceeds 8% of total lipid , SREBP-2 is retained in the ER so that it is separated from S1P and S2P that are localized in the Golgi complex .", "Consequently , cleavage of SREBP-2 is inhibited ( Nohturfft et al . , 2000; Radhakrishnan et al . , 2008 ) .", "If the mechanism through which ceramide regulates cleavage of CREB3L1 is similar to that employed by cholesterol to regulate cleavage of SREBP-2 , then excessive ceramide is predicted to trigger the transportation of CREB3L1 from the ER to Golgi complex .", "Such similarity might also explain why a twofold increase in ceramide is sufficient to induce cleavage of CREB3L1 , as ceramide may also function through the same switch-like mechanism used by cholesterol to regulate SREBP-2 cleavage .", "Our current study demonstrates that proteolytic activation of CREB3L1 is required for doxorubicin to induce expression of p21 .", "However , expression of p21 alone may not be sufficient to suppress cell proliferation .", "We have shown previously that CREB3L1 induces transcription of multiple genes that suppress cell proliferation ( Denard et al . , 2011 ) .", "Thus , CREB3L1 may function similar to p53 as a master regulator of cell proliferation .", "It was reported previously that doxorubicin inhibited cell proliferation through both p53 dependent and independent pathways ( Lupi et al . , 2007 ) .", "Since CREB3L1 is able to inhibit proliferation of doxorubicin-treated Huh7 cells in which p53 is inactivated by mutations ( Hsu et al . , 1993 ) , CREB3L1-mediated pathway is likely to be p53-independent .", "Most cancer cells are thought to originate from genome damage .", "Since p53 is activated in response to genome damage to inhibit cell proliferation , the protein is frequently inactivated by mutations in human cancer cells ( Levine et al . , 1991 ) .", "Unlike p53 , CREB3L1 is activated by ceramide or ER stress ( Murakami et al . , 2006 , 2009 ) but not genome damage .", "Owing to the lack of selection pressure against expression of CREB3L1 , most cancer cells may still express functional CREB3L1 .", "This may be the reason why doxorubicin is effective against many varieties of cancers .", "Thus , more effective chemotherapeutic reagents against cancers may be generated by development of compounds that specifically activate CREB3L1 ." ], [ "We obtained rabbit anti-LSD1 from Cell Signaling ( Boston , MA ) ; mouse anti-calnexin from Enzo Life Sciences ( Farmingdale , NY ) ; mouse anti-γH2AX from Millipore ( Billerica , MA ) ; rabbit anti-Actin and anti-p21 from Abcam ( Cambridge , MA ) ; peroxidase-conjugated secondary antibodies from Jackson ImmunoResearch ( West Grove , PA ) ; Doxorubicin ( Cat# D1515-10MG ) , bleomycin , etoposide , paclitaxel and N-Hexanoyl-D-sphingosine ( C6-Ceramide ) from Sigma-Aldrich ( St . Louis , MO ) ; Myriocin and fumonisin B1 from EMD Biosciences ( Darmstadt , Germany ) ; and [14C]palmitate ( 55 mCi/mmol ) from ARC ( St . Louis , MO ) .", "A rabbit polyclonal antibody against human CREB3L1 was generated as previously described ( Denard et al . , 2011 ) .", "Doxorubicin stock solution ( 2 . 5 mg/ml ) was made by adding nuclease-free water ( Ambion , Carlsbad , CA ) directly to the vial , and was stored at 4°C for no more than 2 weeks .", "SRD-12B and M19 cells are mutant CHO cells deficient in S1P and S2P , respectively ( Rawson et al . , 1997 , 1998 ) .", "These cells were maintained in medium A ( 1:1 mixture of Ham's F12 medium and Dulbecco's modified Eagle's medium containing 100 U/ml penicillin and 100 µg/ml streptomycin sulfate ) supplemented with 5% ( vol/vol ) fetal calf serum ( FCS ) , 5 µg/ml cholesterol , 1 mM sodium mevalonate , and 20 µM sodium oleate .", "Their parental CHO-7 cells are a clone of CHO-K1 cells selected for growth in lipoprotein-deficient serum ( Metherall et al . , 1989 ) and were maintained in medium A supplemented with 5% ( vol/vol ) newborn calf lipoprotein-deficient serum .", "Huh7 and SV589 cells were maintained in medium B ( Dulbecco's modified Eagle's medium with 4 . 5 g/l glucose , 100 U/ml penicillin , 100 mg/ml streptomycin sulfate , and 10% [vol/vol] FCS ) .", "Single cell clones of Huh7-shControl and Huh7-shCREB3L1 cells were generated by stably transfecting Huh7 cells with a control shRNA or shRNA targeting CREB3L1 , respectively , as previously described ( Denard et al . , 2011 ) .", "These cells were maintained in medium B supplemented with 10 µg/ml puromycin .", "MCF-7 cells were maintained in medium C ( RPMI-40 media with 100 U/ml penicillin , 100 mg/ml streptomycin sulfate , and 10% [vol/vol] FCS ) .", "MCF7/pCREB3L1 ( L ) and MCF7/pCREB3L1 ( H ) were generated by stably transfecting MCF-7 cells with pTK-CREB3L1 encoding human CREB3L1 driven by the thymidine kinase promoter .", "These cells were maintained in medium C supplemented with 700 µg/ml G418 .", "All cells were incubated in monolayers at 37°C in 5% CO2 except for CHO and MCF-7-derived cells that were cultured at 37°C in 8% CO2 .", "None of the cells were allowed to reach more than 80% confluence during maintenance .", "Cells were transfected with indicated plasmids using Fugene 6 reagent ( Promega ) as described by the manufacturer , after which the cells were used for experiments as described in the ‘Figure legends’ .", "Cell homogenates were separated into nuclear and membrane fractions ( Sakai et al . , 1996 ) , and analyzed by SDS-PAGE ( 15% for γH2AX , and 10% for the rest of the proteins ) followed by immunoblot analysis with the indicated antibodies ( 1:1000 dilution for anti-CREB3L1 and anti-LSD1 , 1:3000 dilution for anti-calnexin , 1:2000 dilution for anti-γH2AX and 1:10 , 000 dilution for anti-Actin ) .", "Bound antibodies were visualized with a peroxidase-conjugated secondary antibody using the SuperSignal ECL-HRP substrate system ( Pierce ) .", "RT-QPCR was performed as previously described ( Liang et al . , 2002 ) .", "Each measurement was made in triplicate from cell extracts pooled from duplicate dishes .", "The relative amounts of RNAs were calculated through the comparative cycle threshold method by using human 36B4 mRNA as the invariant control .", "The number of cells was determined by direct counting or measurement of cellular DNA content with Quant-iT dsDNA Assay Kit ( Life Technologies ) .", "Results from each experiment were reported as the mean value from triplicate incubations .", "Duplexes of siRNA were synthesized by Dharmacon Research .", "The siRNA sequences targeting human CREB3L1 and the control siRNA targeting GFP was reported previously ( Adams et al . , 2004; Denard et al . , 2011 ) .", "Cells were transfected with siRNA using Lipofectamine RNAiMAX reagent ( Invitrogen ) as described by the manufacturer , after which the cells were used for experiments as described in the figure legends .", "TUNEL assay was performed with the APO-BrdU TUNEL Assay kit ( Invitrogen ) as described in the manufacturer's directions .", "Cells were subjected to flow cytometry on a FACSCaliber Flow Cytometer ( Becton Dickinson ) to determine percent of apoptotic cells .", "At least 5000 cells were collected for each measurement .", "Results from each experiment were reported as mean of triplicate measurements .", "Ceramide synthesis measured by radiolabeled analysis was performed by incubating cells with [14C]palmitate followed by homogenizing the cells in buffer A ( 10 mM HEPES pH 7 . 6 , 1 . 5 mM MgCl2 , and 10 mM KCl ) .", "Lipids in the homogenate were extracted by 0 . 5 ml of chloroform/methanol ( 2:1; vol/vol ) , dried , and dissolved in 70 µl of chloroform/methanol ( 1:1; vol/vol ) .", "Lipid extracts were mixed with 50 µg of non-radioactive ceramide standard ( Avanti Polar Lipids ) and analyzed by Thin Layer Chromatography ( TLC ) on POLYGRAM SIL G plates in a solvent system of chloroform/acetate ( 90:10; vol/vol ) for ceramide separation .", "Following visualization by exposing the TLC plates to I2 vapor , bands containing ceramide were excised , and the amount of radioactivity in it was determined by scintillation counting .", "The activity of ceramide synthesis was determined by radioactivity found in the ceramide band normalized by the amount of cellular protein .", "The statistical analysis was performed with one tailed paired t-test .", "LC-MS analyses of ceramide were performed by UPLC-MS/MS at UT Southwestern Medical Center Mouse Metabolic Phenotyping Core .", "The equipment consisted of a Shimadzu Prominence UPLC system equipped with a CBM-20A controller , a DGU-A3 degasser , three UPLC solvent delivery modules LC-ADXR , a CTO-20AC column oven/chiller maintained at 30°C , a SIL-20ACTHT autosampler .", "The UPLC system is attached to an API 5000 LC-MS/MS system ( Applied Biosystems/MDS SCIEX , Concord ON , Canada ) .", "The mass spectrometer is equipped with a Turbo V ion source operating the TurboIonSpray probe in positive mode .", "Quantitative analysis of sphingolipids was achieved using selective reaction monitoring scan mode .", "Chromatographic separations were obtained by reverse phase LC on a 2 . 1 ( i . d . ) × 150 mm Kinetex C8 ( Phenomenex , Torrance , CA ) column under a complex gradient elution , using three different mobile phases: eluent A consisting of CH3OH/H2O/HCOOH , 58/41/1 , vol/vol/vol with 5 mM ammonium formate , eluent B consisting of CH3OH/HCOOH , 99/1 , vol/vol with 5 mM ammonium formate , and eluent C consisting of CH3OH/CH2Cl2 35/65 with 5 mM ammonium formate .", "The amount of ceramide measured was normalized against the amount of cellular protein ." ] ]

[ "Doxorubicin is used extensively for chemotherapy of diverse types of cancer , yet the mechanism through which it inhibits proliferation of cancer cells remains unclear .", "Here we report that doxorubicin stimulates de novo synthesis of ceramide , which in turn activates CREB3L1 , a transcription factor synthesized as a membrane-bound precursor .", "Doxorubicin stimulates proteolytic cleavage of CREB3L1 by Site-1 Protease and Site-2 Protease , allowing the NH2-terminal domain of CREB3L1 to enter the nucleus where it activates transcription of genes encoding inhibitors of the cell cycle , including p21 .", "Knockdown of CREB3L1 mRNA in human hepatoma Huh7 cells and immortalized human fibroblast SV589 cells conferred increased resistance to doxorubicin , whereas overexpression of CREB3L1 in human breast cancer MCF-7 cells markedly enhanced the sensitivity of these cells to doxorubicin .", "These results suggest that measurement of CREB3L1 expression may be a useful biomarker in identifying cancer cells sensitive to doxorubicin ." ]

[ "Cancer is a broad term to describe over 200 diseases that are caused by cells proliferating in an out-of-control manner .", "Cell replication and division are normally very tightly regulated , and as cells become old , damaged or mutated , they are either repaired or undergo programmed cell death ( apoptosis ) .", "However , if defective cells continue to replicate , the resulting clusters of abnormal cells can become cancerous .", "With so many different types of cancer , there is no ‘magic bullet’ to cure all of them .", "Many cancer therapies are targeted , relying on drugs that block the spread of cancer by interfering with specific molecules involved in the growth and progression of certain tumors .", "However , the fact that diseased cells replicate faster than normal cells in many forms of cancer makes it possible to use non-specific drugs , such as doxorubicin , to treat tumors when targeted therapies are not available .", "Doxorubicin can induce DNA breaks in a variety of different cancers by inhibiting the activity of topoisomerase II but a consistent relationship between the inhibition of this enzyme and the blocking of cell proliferation has not been established .", "This lack of understanding of the mechanism through which doxorubicin inhibits cell proliferation makes it difficult to identify cancer patients who are most likely to benefit from doxorubicin treatment .", "Denard et al . have now shown that doxorubicin blocks cell replication by cleaving a transcription factor called CREB3L1 .", "This latest work builds on previous work in which they showed that cleavage of this transcription factor can inhibit the replication of cells infected with hepatitis C virus .", "It has been known since 2000 that CREB3L1 is a membrane protein with one end inside the lumen of the endoplasmic reticulum , and the other end ( which is terminated with an NH2 group ) in the cytosol of the cell .", "When CREB3L1 is cleaved , the NH2-terminal domain travels into the nucleus of the cell , where it drives the transcription of genes that suppress the cell cycle .", "Denard et al . clearly show that doxorubicin triggers the cleavage of CREB3L1 by stimulating the production of ceramide molecules .", "Thus , It might be possible , with further research , to use CREB3L1 as a biomarker to identify tumors that are suitable for treatment by doxorubicin ." ]

[ "Introduction", "Results", "Discussion", "Material and methods" ]

[ "ecology" ]

[ [ "In nature , plants are attacked by a multitude of herbivore species .", "By removing plant tissues and interrupting essential physiological processes , herbivory can negatively affect plant fitness , impose selection pressure , and thus drive the evolution of plant defenses ( Züst et al . , 2012; Agrawal et al . , 2012; Huber et al . , 2016a; 2016b ) .", "Herbivory does not necessarily reduce plant survival and reproduction , however ( Bruelheide and Scheidel , 1999; Morris et al . , 2007; Corbett et al . , 2011 ) .", "For example , minor insect herbivory that occurs early in the growing season can induce plant defenses , and thereby ensure enhanced protection during times of greater herbivore abundance ( Agrawal , 1998; Kessler and Baldwin , 2004; Poelman et al . , 2008 ) .", "Likewise , herbivory that reduces competitor abundance may benefit a given individual ( Agrawal et al . , 2012 ) .", "Accordingly , measuring the impact of herbivory on plant reproductive fitness in an ecologically relevant setting ( e . g . , one that includes the full suite of native herbivores ) , is essential for identifying the selective forces underlying the evolution of plant defenses .", "Most plant defenses are regulated by jasmonates ( Howe and Jander , 2008 ) .", "Interrupting the biosynthesis of these hormones results in decreased constitutive and induced production of volatile and non-volatile secondary metabolites , defensive proteins and structural defenses ( Thaler et al . , 2002; Li et al . , 2004; Paschold et al . , 2007; Howe and Jander , 2008; Zhou et al . , 2009 ) .", "Surprisingly , however , despite the availability of many different jasmonate deficient mutants ( McConn et al . , 1997; Thaler et al . , 2002; Zhou et al . , 2009; Kallenbach et al . , 2012 ) , fitness benefits of endogenous jasmonate production in herbivore-attacked plants in natural environments have not been demonstrated so far .", "Exogenous applications of jasmonic acid ( JA ) or methyl jasmonate ( MeJA ) are known to enhance plant defenses and fitness ( Baldwin , 1998; Thaler , 1999; Heil et al . , 2001 ) , but these treatments remain difficult to interpret because they may not simulate dosages , localizations , and timings of endogenous jasmonate responses .", "Apart from inducing defenses , jasmonates can also reduce plant growth ( Baldwin , 1998; Hanley , 1998; Redman et al . , 2001; Zavala and Baldwin , 2006; Bodenhausen and Reymond , 2007; Machado et al . , 2013 ) , by interfering , for example , with the gibberellin and auxin signaling cascades ( Onkokesung et al . , 2010; Yang et al . , 2012 ) .", "This suggests that the evolution of jasmonate defense signaling is accompanied by growth suppression that may reduce fitness benefits under low herbivore pressure .", "Our understanding of jasmonates in plant-herbivore interactions is also wanting because most research has focused on leaf-feeding arthropods ( Mafli et al . , 2012; Falk et al . , 2014 ) , and much less is known with respect to vertebrate herbivores , even though vertebrates are often the primary consumers in plant communities ( Paige and Whitham , 1987; Hodgson and Illius , 1996 ) .", "What we do know is that vertebrate herbivores can exert strong selective pressure on plants ( Collins et al . , 1998; Becerra , 2015 ) by influencing growth ( Paige and Whitham , 1987; Bergman , 2002; Liu et al . , 2012; Ishihama et al . , 2014 ) , structural defenses ( Abrahamson , 1975; White , 1988; Young and Okello , 1998; Takada et al . , 2001; Wilson and Kerley , 2003; Young et al . , 2003; Kato et al . , 2008 ) , reproductive timing ( Zamora et al . , 2001 ) and mortality ( Veblen et al . , 1989; Gill , 1992; Vila and Guibal , 2001; Saint-Andrieux et al . , 2009 ) .", "We also know that vertebrates tend to avoid plants that are rich in secondary metabolites , including condensed tannins and phenolics ( Cooper and Owen-Smith , 1985; Owen Smith , 1993; Furstenburg and van Hoven , 1994; O'reilly-Wapstra et al . , 2004; Jansen et al . , 2007; DeGabriel et al . , 2009; Rosenthal and Berenbaum , 2012 ) .", "Furthermore , it has been shown that silencing the production of the nervous toxin nicotine can engender increased leaf damage by vertebrate browsers in the field ( Steppuhn et al . , 2008 ) .", "Despite these advances , it remains unclear to what extent endogenous jasmonates actually help plants to maintain their fitness when facing herbivore communities that include vertebrates .", "To assess the importance of jasmonates in protecting plants from native arthropod and vertebrate herbivores , we studied three experimental N . attenuata populations in their native environment in the Great Basin Desert ( United States ) .", "Each population consisted of a mix of jasmonate deficient and wild type plants .", "For each population , we characterized the damage that was caused by vertebrates and arthropods and then correlated damage patterns with plant flower production as a measure of the plant’s reproductive potential .", "We then assessed the impact of simulated herbivory on jasmonate-dependent flower production and defoliation tolerance under glasshouse conditions .", "As part of this glasshouse work , we quantified primary and secondary metabolites in the specific plant parts that experienced herbivory and damage from the different kinds of herbivores in the field .", "Based on our findings we conducted a controlled feeding experiment to assess the impact of jasmonate deficiency on consumption rates by cottontail rabbits that resided in our study area .", "Finally , we conducted a complementation experiment with the same rabbits to assess whether nicotine was the main jasmonate-dependent deterrent affecting consumption rates ." ], [ "To evaluate the impact of jasmonates on herbivory-dependent plant fitness , we established three experimental N . attenuata populations ( henceforth called 'Lytle' , 'Poplar' and 'Snow' ) across the field station of the Lytle Ranch Preserve ( St . George , UT , USA; Figure 1—figure supplement 1 ) .", "In each plot , at least 12 jasmonate-deficient inverted repeat allene-oxide cyclase ( irAOC ) plants and empty vector controls ( EV , 'wild type' ) were planted .", "The irAOC line has been characterized previously ( Kallenbach et al . , 2012 ) .", "Its herbivory-induced jasmonate levels are reduced by more than 95% ( Kallenbach et al . , 2012; Machado et al . , 2013 ) while flower production is similar to WT plants in the absence of herbivore attack ( Machado et al . , 2013 ) .", "Five to seven weeks after the establishment of the populations , we recorded herbivore damage and counted the number of flowers on each plant as a strong predictor of Darwinian fitness ( Van Dam and Baldwin , 2001; Glawe et al . , 2003; Baldwin , 2003 ) .", "Across all three plots , we observed four main herbivore damage types: leaf removal , stem peeling , leaf chewing and leaf sucking/piercing .", "The different damage types are characteristic for different herbivores including deer , rabbits , wood rats , gophers , caterpillars , ants , mirid bugs and leafhoppers ( Meldau et al . , 2009; Stitz et al . , 2011; Kallenbach et al . , 2012; Schuman et al . , 2012; Ðinh et al . , 2013; Schäfer et al . , 2013 ) .", "Across all plots , jasmonate deficiency significantly increased herbivore damage and decreased flower production ( Figure 1 ) .", "In the Lytle plot , jasmonate-deficient plants suffered more stem peeling and leaf removal by vertebrates , but similar arthropod damage compared to wild type plants .", "Jasmonate-deficient plants also produced fewer flowers .", "Likelihood ratio tests based on Generalized Linear Models ( GLMs ) showed that this effect was associated with the higher occurrence of vertebrate stem peeling in irAOC plants .", "In the Snow plot , irAOC plants suffered more vertebrate leaf removal and damage from leaf chewing and leaf sucking/piercing insects than the wild type plants , but only leaf removal by vertebrates was associated with a reduction in flower production .", "In the Poplar plot , no vertebrate damage was observed , and jasmonate-deficiency increased damage by leaf sucking/piercing arthropods , but did not decrease flower production ( Figure 1 , Figure 1—figure supplement 2 ) .", "Overall , jasmonate deficiency increased vertebrate damage more strongly than arthropod damage , and only jasmonate-dependent changes in vertebrate damage translated into a decrease in flower production . 10 . 7554/eLife . 13720 . 003Figure 1 . Jasmonate-deficiency reduces flower production by increasing vertebrate damage in nature . Effects of jasmonate deficiency on vertebrate and invertebrate damage by damage type and effect of damage type on flower production across three experimental plots ( 'Lytle' , 'Poplar' and 'Snow' ) are shown ( n = 12–19 ) .", "Solid lines indicate significant effects of jasmonate-deficiency on herbivore damaage patterns and flower production .", "The herbivores responsible for the different damage types were identified based on field observations and characteristic feeding patterns .", "Jasmonate-deficiency increases damage by vertebrate and arthropod herbivores , but only vertebrate damage leads to a reduction of flower production as a strong predictor of plant reproductive potential . DOI: http://dx . doi . org/10 . 7554/eLife . 13720 . 00310 . 7554/eLife . 13720 . 004Figure 1—source data 1 . Parameters used to determine the presence of different herbivores in the three experimental populations . DOI: http://dx . doi . org/10 . 7554/eLife . 13720 . 00410 . 7554/eLife . 13720 . 005Figure 1—source data 2 . Quantification and herbivore association of the different damage types observed in the three experimental populations . DOI: http://dx . doi . org/10 . 7554/eLife . 13720 . 00510 . 7554/eLife . 13720 . 006Figure 1—source data 3 . Leaf-herbivore damage screen and fitness measurements . DOI: http://dx . doi . org/10 . 7554/eLife . 13720 . 00610 . 7554/eLife . 13720 . 007Figure 1—figure supplement 1 . Overview of the different experimental N . attenuata populations used in the present study . Satellite picture from googlemaps . com . DOI: http://dx . doi . org/10 . 7554/eLife . 13720 . 00710 . 7554/eLife . 13720 . 008Figure 1—figure supplement 2 . Detailed results of the analysis of the overall effect of jasmonate signaling and herbivore damage on N . attenuata flower production in the field . Mean herbivore damage in the Lyttle", "( a ) , Poplar", "( b ) and Snow", "( c ) plot .", "Effect of type of damage on plant fitness", "( d ) .", "Mean flower production in the different plots", "( e ) .", "Asterisks indicate significant differences ( *p<0 . 05; **p<0 . 01; ***p<0 . 001 ) .", "N . S: not significant . DOI: http://dx . doi . org/10 . 7554/eLife . 13720 . 00810 . 7554/eLife . 13720 . 009Figure 1—figure supplement 3 . Photographic evidence of herbivore damage and the associated herbivores in the Lytle plot . Mule deer and cottontail rabbits were regularly encountered in the vicinity of the Lytle plot ( Top , white arrows ) .", "Bottom left: Typical EV plant in the Lytle plot , with partial defoliation .", "Bottom right: IrAOC plant that was completely defoliated and stem-peeled by vertebrate browsing .", "Stem peeling is typically caused by mountain cottontail rabbits ( Sylvilagus nuttallii ) , black-tailed jackrabbits ( Lepus californicus ) and woodrats ( Neotoma spp . ) .", "Cottontail rabbit picture by Pavan Kumar . DOI: http://dx . doi . org/10 . 7554/eLife . 13720 . 00910 . 7554/eLife . 13720 . 010Figure 1—figure supplement 4 . Photographic evidence of herbivore damage and the associated herbivores in the Snow plot . Several gophers were observed within the snow plot ( Top , white arrow ) .", "Bottom left: Typical EV plant in the snow plot .", "Bottom right: IrAOC plant that was partially defoliated by animal browsing .", "Gopher picture by Arne Weinhold . DOI: http://dx . doi . org/10 . 7554/eLife . 13720 . 010 To understand the impact of herbivore damage patterns and jasmonate-deficiency on plant flower production in more detail , we mimicked the different types of damage that we observed in the field in a controlled glasshouse experiment and quantified flower production over the entire flowering period ( Figure 2 ) .", "Attack by chewing invertebrates was mimicked by wounding ( W ) the plants with a pattern wheel and treating the wounds with Manduca sexta oral secretions ( OS ) ( W+OS treatments ) ( Machado et al . , 2013 ) .", "Vertebrate damage was mimicked by removing either the rosette leaves ( rosette defoliation ) , the rosette and stem leaves ( full defoliation ) or the rosette and stem leaves plus the stem bark ( stem peeling ) .", "The artificially peeled stems looked similar to the vertebrate-damaged stems in the field , and histological staining revealed that stem peeling resulted in the removal of the epidermis and cortex from the stems ( Figure 2—figure supplement 1 ) .", "In wild type plants , W+OS induction did not significantly reduce peak flower production ( Figure 2a ) .", "All other treatments led to significant reductions in flower production: rosette defoliation reduced flower production by 24% , rosette and stem leaf removal reduced flower production by 53% , and stem peeling resulted in a marked 78% reduction in flower numbers .", "Full defoliation and stem peeling also delayed the onset of flowering by more than two weeks .", "Overall , the same flowering patterns were observed in EV and irAOC plants ( Figure 2b ) .", "However , the total number of flowers was significantly higher in defoliated and stem-peeled irAOC plants than in similarly treated EV plants . 10 . 7554/eLife . 13720 . 011Figure 2 . Simulated vertebrate attack strongly reduces plant flower production . Average ( ±SE ) flower production of wild type", "( a ) and jasmonate deficient irAOC plants", "( b ) following different simulated herbivory treatments ( n =10–12 ) .", "Different letters indicate significant differences between treatments within genotypes ( p<0 . 05 ) .", "Asterisks indicate significant differences between genotypes within treatments ( *p<0 . 05 ) .", "W+OS: Wounding + application of Manduca sexta oral secretions . DOI: http://dx . doi . org/10 . 7554/eLife . 13720 . 01110 . 7554/eLife . 13720 . 012Figure 2—source data 1 . Fitness costs of induction and defoliation in the glasshouse . DOI: http://dx . doi . org/10 . 7554/eLife . 13720 . 01210 . 7554/eLife . 13720 . 013Figure 2—figure supplement 1 . Toluene-blue staining of stem-peeled EV plants at the early bolting stage . Experimental stem peeling as a mimick of feeding patterns observed in the Lytle plot removed epidermis , cortex and phloem cells from the plant .", "The remaining tissue consisted of xylem and pith cells . DOI: http://dx . doi . org/10 . 7554/eLife . 13720 . 013 As a first step to understand the mechanism behind the significant differences in vertebrate damage between EV and irAOC plants in the field , we profiled primary and secondary metabolites in the stems and leaves of glasshouse-grown plants .", "Redundancy analyses ( RDAs ) showed that simulated herbivory led to dramatic changes in both leaf- and stem carbohydrate profiles and secondary metabolite levels in a jasmonate-dependent manner ( permutation test for the effect of the genotype x treatment interaction: p<0 . 001 in both tissues ) ( Figure 3 and Figure 3—figure supplement 1 ) .", "The RDAs further revealed that diterpene glycosides ( DTGs ) , rutin , nicotine , glucose and fructose in the leaves and nicotine , glucose and fructose in the stems explained most of the metabolic differences between induced EV and irAOC plants ( Figure 3 ) .", "Closer inspection of these metabolites showed that under control conditions , irAOC plants have lower nicotine and DTG concentrations in the leaves and lower nicotine levels in the stems than EV plants .", "Upon induction , these differences became even more pronounced ( Figure 3 ) .", "Constitutive rutin , glucose and fructose concentrations on the other hand did not differ between genotypes , but were more strongly depleted in induced EV plants ( Figure 3 ) . 10 . 7554/eLife . 13720 . 014Figure 3 . Herbivory-induced jasmonates increase secondary metabolites and decrease sugars in leaves and stems . Results of a redundancy analysis of the metabolic profiles in the leaves", "( a ) and in the stems", "( b ) of wild type ( EV ) and jasmonate-deficient ( irAOC ) plants are shown ( n = 3–5 ) .", "Insets depict average ( ±SE ) concentrations of metabolites explaining most of the variation between induced EV plants and the other treatment*genotype combinations ( correlation coefficients > |0 . 8| ) .", "Letters indicate significant differences between treatments within genotypes ( p<0 . 05 ) .", "Asterisks indicate significant differences between genotypes within treatments ( p<0 . 05 ) .", "Control: intact plants; W+W: wounded and water-treated plants; W+OS: wounded and M . sexta oral secretions-treated plants; DTGs: diterpene glycosides . DOI: http://dx . doi . org/10 . 7554/eLife . 13720 . 01410 . 7554/eLife . 13720 . 015Figure 3—source data 1 . Plant primary and secondary metabolite concentrations . DOI: http://dx . doi . org/10 . 7554/eLife . 13720 . 01510 . 7554/eLife . 13720 . 016Figure 3—figure supplement 1 . Secondary metabolite profiles in leaves and stems of N . attenuata in response to simulated herbivore attack . Mean ( ±SE ) glucose", "( a ) , fructose", "( b ) , sucrose", "( c ) , starch", "( d ) nicotine", "( e ) , chlorogenic acid", "( f ) , rutin", "( g ) and diterpene glycosides ( DTGs )", "( h ) ( n=5 ) .", "Different letters indicate significant differences between treatments within genotype and tissue ( p<0 . 05 ) .", "n . s: not significant .", "Asterisks indicate significant differences between genotypes within treatment ( *p<0 . 05 ) .", "L . O . D: limit of detection .", "Control: intact plants; W+W: wounded and water treated plants; W+OS: wounded and M . sexta-oral secretions treated plants . DOI: http://dx . doi . org/10 . 7554/eLife . 13720 . 016 Feeding preferences by vertebrate herbivores are influenced by plant chemistry .", "Toxic secondary metabolites in particular can reduce food uptake ( Bryant et al . , 1991; Iason , 2005; Rosenthal and Berenbaum , 2012; Camp et al . , 2015 ) .", "To determine whether the nicotine deficiency observed in irAOC plants could explain the higher stem and leaf-removal by vertebrates in the field , we developed a biotechnology-driven in vitro feeding assay for cottontail rabbits ( Sylvilagus nuttallii ) ( Figure 4—figure supplement 1 ) .", "Cottontail rabbits can feed on N . attenuata in nature ( Karban , 1997; Baldwin , 2003 ) and were present in our field experiment ( Figure 1—figure supplement 3 ) .", "In a first step , we produced food pellets by drying N . attenuata shoots of wild type and irAOC plants and pressing them into pellets .", "To determine whether drying affects N . attenuata defenses , we measured secondary metabolites in plants before and after drying ( Figure 4—figure supplement 2 ) .", "The drying process did not affect the water-loss corrected abundance of DTGs .", "The abundances of nicotine , chlorogenic acid and rutin were reduced , but jasmonate-dependent differences were conserved .", "The most abundant phenol amides dicaffeoylspermidine , dicoumarylspermidine , ferolylputrescine and caffeoylputrescine were degraded during the drying process , while less abundant phenol amides increased in concentration .", "From these data , we concluded that the dried plant material could be used to assess the impact of jasmonate-dependent alkaloids , DTGs and phenolic compounds , but not phenol amides , on vertebrate feeding preferences .", "In a first set of experiments , we presented eight individual cottontail rabbits with three types of food pellets: one consisting of pure dried alfalfa leaves as a rabbit maintenance food , and two others consisting of", "1 ) a mixture of dried alfalfa leaves and EV or", "2 ) dried alfalfa leaves and irAOC plant material at a ratio of 5:1 to account for typical food mixing by the rabbits .", "We then monitored food consumption as a response variable for feeding preference over 24 hr ( Figure 4 ) .", "Comparisons of adjusted confidence intervals of asymptotes of logistic models fitted for each treatment showed that cottontails rejected EV pellets and strongly preferred to feed from both irAOC pellets and pure alfalfa ( Figure 4a , Figure 4—figure supplement 3 ) .", "Similar feeding preferences were observed in two independent experiments ( Figure 4a , Figure 4—figure supplement 4a ) .", "From our metabolite profiling , we had identified nicotine as a prominent candidate that might drive feeding preference of vertebrates .", "We therefore tested the hypothesis that nicotine may be responsible for the observed feeding patterns by complementing nicotine levels of irAOC pellets to match those observed in EV pellets ( Figure 4b , inset ) .", "Strikingly , rabbits refused both nicotine complemented-irAOC and EV pellets and preferentially fed on alfalfa pellets ( Figure 4b ) .", "Similar results were obtained in two independent experiments ( Figure 4b , Figure 4—figure supplement 4b ) . 10 . 7554/eLife . 13720 . 017Figure 4 . Jasmonate-dependent nicotine determines feeding preferences of a native vertebrate herbivore . Average amounts of food consumption by cottontail rabbits in a choice experiment .", "Choice experiments were carried out with EV , irAOC and alfalfa pellets", "( a ) ( n = 8 ) or with EV , irAOC+nicotine and alfalfa pellets", "( b ) ( n = 9 ) .", "Letters indicate significant differences in curve asymptotes tested based on asymptote confidence intervals of logistic models ( p<0 . 001 ) .", "Insets: nicotine contents of the different pellets .", "Letters indicate significant differences ( p<0 . 05 ) .", "N . D: not detected . DOI: http://dx . doi . org/10 . 7554/eLife . 13720 . 01710 . 7554/eLife . 13720 . 018Figure 4—source data 1 . Impact of drying on secondary metabolite profiles . DOI: http://dx . doi . org/10 . 7554/eLife . 13720 . 01810 . 7554/eLife . 13720 . 019Figure 4—source data 2 . Nicotine concentrations in pellets . DOI: http://dx . doi . org/10 . 7554/eLife . 13720 . 01910 . 7554/eLife . 13720 . 020Figure 4—source data 3 . Results of choice experiment ( repetition ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 13720 . 02010 . 7554/eLife . 13720 . 021Figure 4—source data 4 . Results of nicotine complementation experiment ( repetition ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 13720 . 02110 . 7554/eLife . 13720 . 022Figure 4—figure supplement 1 . Experimental setup for feeding preference assays for cottontail rabbits ( Sylvilagus nuttallii ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 13720 . 02210 . 7554/eLife . 13720 . 023Figure 4—figure supplement 2 . Secondary metabolite profiles in the leaves of N . attenuata before and after drying . Mean concentrations ( ±SE ) of nicotine", "( a ) , diterpeneglycosides ( DTGs )", "( b ) , rutin", "( c ) , chlorogenic acid", "( d ) dicaffeoylspermidine", "( e ) , dicoumarylspermidine", "( f ) , ferolylputrescine", "( g ) , caffeoylputrescine", "( h ) , caffeoylspermidine", "( i ) , diferuloylspermidine", "( j ) , coumarylputrescine", "( k ) and ferolylspermidine", "( l ) ( n = 5 ) .", "Asterisks indicate significant differences between genotypes within water content status ( *p<0 . 05; **p<0 . 01; ***p<0 . 001 ) .", "N . S: not significant .", "L . O . D: limit of detection . DOI: http://dx . doi . org/10 . 7554/eLife . 13720 . 02310 . 7554/eLife . 13720 . 024Figure 4—figure supplement 3 . Asymptotes and confidence intervals of the feeding preference assays for cottontail rabbits ( Sylvilagus nuttallii ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 13720 . 02410 . 7554/eLife . 13720 . 025Figure 4—figure supplement 4 . Results of the repetition of the feeding preference assays for cottontail rabbits ( Sylvilagus nuttallii ) .", "Choice experiments were carried out with EV , irAOC and alfalfa pellets", "( a ) ( n = 8 ) or with EV , irAOC+nicotine and alfalfa pellets", "( b ) ( n = 9 ) .", "Letters indicate significant differences in curve asymptotes tested based on asymptote confidence intervals of logistic models ( p<0 . 001 ) .", "Insets: nicotine contents of the different pellets .", "Letters indicate significant differences ( p<0 . 05 ) .", "N . D: not detected . DOI: http://dx . doi . org/10 . 7554/eLife . 13720 . 025" ], [ "Since their discovery 25 years ago ( Johnson et al . , 1990 ) , jasmonates have emerged as key signals that govern plant responses and resistance to herbivores ( Howe and Jander , 2008 ) .", "Early experiments demonstrated that the application of jasmonates strongly reduced herbivore damage in the field and increased seed production under high herbivore pressure ( Baldwin , 1998; Agrawal , 1999 ) .", "Experiments with jasmonate biosynthesis and perception mutants subsequently revealed that a reduction in endogenous jasmonate signaling increased plant susceptibility to a large number of organisms including detritivorous crustaceans ( Farmer and Dubugnon , 2009 ) , caterpillars of noctuid moths ( Bodenhausen and Reymond , 2007 ) , spider mites ( Li et al . , 2002 ) , beetles ( Kessler et al . , 2004 ) slugs ( Falk et al . , 2014 ) and tortoises ( Mafli et al . , 2012 ) .", "However , despite the increasing number of available jasmonate mutants in different plant species , it has remained unclear whether endogenous jasmonate production actually benefits plant fitness in nature .", "Filling this gap of knowledge is critical to understand the evolution and natural variation of defense signaling ( Machado et al . , 2013; Li et al . , 2015; Xu et al . , 2015b; Xu et al . , 2015a ) .", "Our experiments show that in the field , jasmonate-deficient plants were more strongly damaged by vertebrate and invertebrate herbivores .", "However , only in populations that experienced vertebrate herbivory did this effect translate into a reduction in flower production .", "Stem peeling in particular was associated with a jasmonate-dependent reduction in flower producion .", "Mimicking vertebrate and arthropod herbivory in the greenhouse confirmed that the type of damage caused by vertebrates has a strong negative impact on the plant’s reproductive potential .", "Plant damage at later developmental stages was not assessed in the present study .", "Furthermore , the outcrossing success of the different genotypes was not determined due to safety precautions related to the use of transgenic plants .", "Earlier studies demonstrate however that leaf damage at later developmental stages has little impact on plant fitness ( Zavala and Baldwin , 2006 ) and that flower production in the absence of future damage is a valid and robust approximation for plant fitness in the predominantly self-pollinating N . attenuata ( Baldwin et al . , 1997; Baldwin , 2003; Hettenhausen et al . , 2012; Schuman et al . , 2012 ) .", "Our results therefore highlight the importance of jasmonates in safeguarding the plant’s reproductive potential under vertebrate attack .", "Vertebrate herbivores can have a profound impact on plant fitness and the distribution of plant species in nature ( Hulme , 1994; 1996; Shimoda et al . , 1994; Palmisano and Fox , 1997; Waller and Alverson , 1997; Gómez and Zamora , 2000; Sessions and Kelly , 2001; Warner and Cushman , 2002; Maron and Crone , 2006; Maron and Kauffman , 2006; Nisi et al . , 2015 ) .", "For instance , Paliurus ramosissimus trees that are intensively bark peeled by sika deer ( Cervus nippon ) will take nearly 30 years to recover ( Ishihama et al . , 2014 ) .", "Similarly , strong growth delays and increased tree mortality have been observed in other species ( Veblen et al . , 1989; Gill , 1992; Palmisano and Fox , 1997; Vila and Guibal , 2001; Saint-Andrieux et al . , 2009 ) .", "Surprisingly however , vertebrate herbivores have rarely been considered in the context of plant defense signaling ( Dyer , 1980; Smith et al . , 1991; Liu et al . , 2012 ) .", "Instead , plant defense regulation has mostly been investigated in the context of arthropod herbivores and pathogens ( Dixon et al . , 1994; Chen et al . , 1995; Yang et al . , 1997; Rojo et al . , 2003; Wu and Baldwin , 2009 , Wu and Baldwin , 2010; Schmelz , 2015 ) .", "Although arthropod damage in the field is generally low ( Steppuhn et al . , 2004; Rayapuram and Baldwin , 2007; Pandey and Baldwin , 2008; Rayapuram et al . , 2008; Johnson et al . , 2009; Meldau et al . , 2009; Stitz et al . , 2011; Fischer et al . , 2012; Kallenbach et al . , 2012; Schuman et al . , 2012; Schäfer et al . , 2013 ) , local outbreaks can lead to substantial plant damage ( Van Bael et al . , 1999; Cease et al . , 2012; DeRose and Long , 2012; Ðinh et al . , 2013 ) , and several studies show that arthropods influence plant fitness ( Baldwin , 1998; Maron , 1998; Kessler and Baldwin , 2004; Machado et al . , 2013 ) and may drive the evolution of plant defenses ( Huber et al . , 2016; Poelman and Kessler , 2016; Züst and Agrawal , 2016 ) including plant defense signaling ( Durrant et al . , 2015; Xu et al . , 2015a ) .", "Given the context-dependent potential of both arthropod and vertebrate herbivores to act as agents of natural selection , it seems crucial to include both groups when studying the ecology and evolution of plant defense regulation ( Strauss , 1991; Hulme , 1994; 1996; Palmisano and Fox , 1997; Sessions and Kelly , 2001; Oduor et al . , 2010 ) .", "Numerous studies have found that plant secondary metabolites can influence vertebrate foraging patterns ( Freeland and Janzen , 1974; McArthur et al . , 1995; Mithen et al . , 1995; Dearing et al . , 2005; Scogings et al . , 2011; Mkhize et al . , 2015 ) .", "Vertebrates tend to avoid plants that are rich in condensed tannins , phenolics and alkaloids ( Cooper and Owen-Smith , 1985; Owen Smith , 1993; Furstenburg and van Hoven , 1994; O'reilly-Wapstra et al . , 2004; Jansen et al . , 2007; Steppuhn et al . , 2008; DeGabriel et al . , 2009; Rosenthal and Berenbaum , 2012 ) : Quinolizidine alkaloids for instance seem to protect lupin ( Lupinus spp ) plants from rabbits ( Oryctolagus spp . ) ( Wink , 1985; 1988; Fattorusso and Taglialatela-Scafati , 2008 ) .", "However , apart from toxic secondary metabolites , primary metabolites such as carbohydrates can also affect mammalian feeding preferences ( Mayland et al . , 2000 ) .", "In this study , leaves and stems of jasmonate-deficient plants consistently contained lower levels of nicotine and higher levels of glucose and fructose compared to wild type plants .", "While both processes may have influenced vertebrate feeding preferences , we hypothesized that nicotine as a strong nervous toxin may have played a dominant role in increasing vertebrate feeding in jasmonate-deficient plants .", "The experiments with captive cottontails support this inference: Cottontails refused to feed on pellets containing 20% wild type N . attenuata shoots , but readily fed on pellets containing jasmonate-deficient , and thus nicotine-deficient , irAOC shoots .", "When irAOC pellets were complemented with nicotine to match the concentration in wild-type plants , rabbits avoided the two pellet types and only consumed alfalfa .", "This finding suggests that the amount of nicotine produced via jasmonate signaling is sufficient to dictate cottontail feeding preferences .", "From the plant’s perspective , these results illustrate that nicotine effectively protects N . attenuata against sympatric vertebrates in a similar manner as it does against leaf feeding arthropods ( Steppuhn and Baldwin , 2007 ) .", "They further suggest that the increased damage and reduced flower production in jasmonate-deficient plants in the field can be attributed to reduced nicotine concentrations .", "Nicotine is a nervous system toxin that can bind to acetylcholine receptors of synaptic cells leading to neuromuscular blockade ( Albuquerque et al . , 2009; Prochaska and Benowitz , 2016 ) .", "Its median lethal dose ( LD50 ) varies between 3–50 mg*kg-1 for small rodents ( Wink , 1993; Karačonji , 2005 ) .", "As nicotine is produced at concentrations of up to 3 mg*g FW-1 in N . attenuata shoots , the consumption of a single wild-type plant can be lethal to a small vertebrate herbivore .", "Interestingly , certain vertebrates such as the desert woodrat ( Neotoma lepida ) seem to excise N . attenuata leaves , dry them and transfer them to their nests ( Baldwin , 2003 ) .", "It has been proposed that this behavior may allow woodrats to use nicotine as pharmaceutical means to reduce ectoparasite loads ( Baldwin , 2003 ) .", "Whether nicotine can provide benefits to vertebrates in this way remains to be tested .", "In conclusion , our study demonstrates the importance of jasmonates as regulatory signals that boost nicotine production and thereby protect the leaves and stems of N . attenuata from a diverse range of herbivores .", "By studying the interaction between plants and their natural herbivore communities , we show that jasmonates can provide fitness benefits in nature , and that these fitness benefits are derived from protecting vital plant tissues from vertebrates ." ], [ "In spring 2012 , we established three experimental N . attenuata populations ( 'Lytle' , 'Poplar' and 'Snow' ) across the field station of the Lytle Ranch Preserve ( St . George , UT , USA; Figure 1—figure supplement 1 ) .", "Each population consisted of at least twelve jasmonate-deficient inverted repeat allene-oxide cyclase plants ( irAOC , line 457 ) and empty vector controls ( EV , line A-03-9-1 , 'wild type' ) that were planted in alternation approximately 1 m apart .", "The irAOC line has been characterized previously ( Kallenbach et al . , 2012 ) and was chosen for its strongly reduced jasmonate levels ( Kallenbach et al . , 2012; Machado et al . , 2013 ) .", "Plants were germinated and planted as described ( Krügel et al . , 2002; Schuman et al . , 2012; Machado et al . , 2013 ) .", "Seeds of the transformed N . attenuata lines were imported under APHIS notification number 07-341-101n and experiments were conducted under notification number 06-242-02r .", "In the Snow population , half of the plants were induced by wounding the leaves with a pattern wheel and applying Manduca sexta oral secretions ( W+OS ) as described ( 23 ) .", "As the induction treatment had no significant effect on herbivore damage or flower production , the parameter was not included in the final statistical models .", "Wild type ( EV ) and jasmonate-deficient irAOC plants from all three populations were screened for herbivore damage five weeks after transplantation .", "The type of damage was classified as leaf removal , stem peeling , leaf chewing and leaf sucking/piercing and was attributed to different herbivores by using knowledge from previous studies ( Baldwin , 2003; Kessler et al . , 2004; Steppuhn et al . , 2004; Meldau et al . , 2009; Stitz et al . , 2011; Kallenbach et al . , 2012; Schuman et al . , 2012; Ðinh et al . , 2013; Schäfer et al . , 2013 ) .", "The parameters used to determine the presence of herbivores and details about the quantification of the different damage types are summarized in Figure 1—source datas 1 , 2 .", "Photographic evidence of some herbivores is provided in Figure 1—figure supplements 3 , 4 .", "As a measure of reproductive potential , the numbers of flowers were counted for each plant in the different populations immediately following the herbivore impact assessments ( Van Dam and Baldwin , 2001; Glawe et al . , 2003; Sime and Baldwin , 2003 ) .", "Flowers were removed after counting according to APHIS regulations for the use of transgenic plants in the field .", "In the plot surrounding the Snow population , we observed a spike in gopher activity in the week after the flower count .", "To be able to include this attack into our statistical model , we reassessed herbivore damage and counted the number of regrowing flowers two weeks later .", "This second flower count was then used for statistical analysis .", "To determine the relative value of leaves and stem bark for plant reproduction , we evaluated reproductive output in plants that were subjected to four different regimes of simulated herbivory: simulated insect attack , stem peeling , and full and partial defoliation ( n =10–12 ) .", "Wild type ( EV ) and irAOC plants were grown as described elsewhere ( Krügel et al . , 2002 ) .", "When plants reached the bolting stage , insect attack by chewing herbivores was mimicked by wounding 3 leaves and applying M . sexta oral secretions as described ( W+OS ) ( Machado et al . , 2015 ) .", "In another batch of plants , vertebrate herbivory was simulated by removing either the rosette leaves , or by removing both rosette and stem-leaves ( i . e , full defoliation ) .", "Finally , we mimicked the vertebrate damage patterns observed in the Lytle plot population by removing all leaves and peeling off the bark .", "To remove the bark , a small incision was made into the epidermis at the stem base .", "The epidermis and bark were then pulled off with forceps from the bottom to the top , leaving only xylem and pith of the stems ( See below 'Histological staining' ) ( Figure 2—figure supplement 1 ) .", "Intact plants were used as controls .", "Following the different treatments , all plants were left to grow in a fully randomized setup , and the number of flowers was counted every 3–4 days until the end of the flowering period .", "As a first step to understand the mechanism behind the significant differences in vertebrate grazing patterns between wild type ( EV ) and jasmonate-deficient irAOC plants in the field , we profiled primary and secondary metabolites in the stems and leaves for several plant treatments .", "Prolonged leaf-wounding and simulated insect attack treatments were carried out as described ( Machado et al . , 2013; 2015 ) ( n = 3–5 ) .", "Nicotine , chlorogenic acid , rutin and total 17-hydroxygeranyllinalool diterpene glycosides were determined by HPLC-DAD as described ( Keinänen et al . , 2001 ) .", "Sugars and starch were measured as described ( Velterop and Vos , 2001; Smith and Zeeman , 2006; Machado et al . , 2013; 2015 ) .", "To determine which of the jasmonate-dependent changes in host plant chemistry may be responsible for the preference for irAOC plants observed in the field , we developed an in vitro feeding assay for cottontail rabbits ( Sylvilagus nuttallii ) as follows .", "To investigate which tissues were removed from the stems by vertebrates , we simulated this type of herbivory by experimentally removing the epidermal tissue from bolting wild type plants as described above .", "Peeled and non-peeled stems where then stained with toluene blue for 15 s and photographed using a stereomicroscope equipped with a digital CCD camera ( SteREO Discovery . V8 , 14 Carl Zeiss Microimaging ) and processed with AxioVision LE software ( Carl Zeiss 15 Microimaging ) .", "All statistical analyses were performed using the R software ( R Development Core Team , 2015 ) unless otherwise stated:" ] ]

[ "Endogenous jasmonates are important regulators of plant defenses .", "If and how they enable plants to maintain their reproductive output when facing community-level herbivory under natural conditions , however , remains unknown .", "We demonstrate that jasmonate-deficient Nicotiana attenuata plants suffer more damage by arthropod and vertebrate herbivores than jasmonate-producing plants in nature .", "However , only damage by vertebrate herbivores translates into a significant reduction in flower production .", "Vertebrate stem peeling has the strongest negative impact on plant flower production .", "Stems are defended by jasmonate-dependent nicotine , and the native cottontail rabbit Sylvilagus nuttallii avoids jasmonate-producing N . attenuata shoots because of their high levels of nicotine .", "Thus , endogenous jasmonates enable plants to resist different types of herbivores in nature , and jasmonate-dependent defenses are important for plants to maintain their reproductive potential when facing vertebrate herbivory .", "Ecological and evolutionary models on plant defense signaling should aim at integrating arthropod and vertebrate herbivory at the community level ." ]

[ "Plants are attacked by many different herbivores , including insects and mammals , and often produce toxins in response to protect themselves .", "Toxin production is regulated by plant hormones called jasmonates .", "It is commonly assumed this ability helps plants to survive and reproduce in nature .", "However , proof that a plant's own jasmonates ( also known as \"endogenous jasmonates\" ) can increase a plant's fitness in the wild is lacking , especially in the context of attack by mammals .", "Machado et al . have now asked whether endogenous jasmonates increase the fitness of coyote tobacco plants that were under attack by herbivores in their natural habitats in Southwestern Utah .", "Plants that lacked jasmonates were attacked more strongly by various herbivores , yet unexpectedly only the damage by mammals – including gophers , deer and rabbits – caused the plants to produce fewer flowers .", "Since plants with more flowers tend to produce more offspring , the number of flowers is a measure of a plant’s fitness .", "Damage by insects , which are often seen as major enemies of plants , did not result in a significant impact on the number of flowers .", "Laboratory experiments then revealed that damaging plants in a similar way to mammalian herbivores strongly reduced the plants’ fitness .", "However mimicking insect damage did not have such a large effect .", "Finally , feeding experiments with cottontail rabbits revealed that jasmonate-producing plants are protected by higher levels of the nicotine toxin , which can explain why these plants fare better when attacked by mammals in nature .", "Jasmonates are well known to regulate plant defenses and provide protection against a wide variety of herbivores .", "However , these new findings show that this only translates into fitness benefits for the plants against a subset of herbivores .", "A major challenge in the future will be to study how diverse communities of herbivores shape the evolution of plant defense signaling .", "Including larger herbivores , like mammals , into such experiments will be challenging but necessary to understand how plants survive in nature ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "stem cells and regenerative medicine" ]

[ [ "The intestinal epithelium is one of the most rapidly renewing tissues , undergoing complete turnover in approximately 3 days ( Leblond and Walker , 1956 ) .", "This rapid turnover protects against insults from bacterial toxins and metabolites , dietary antigens , mutagens , and exposure to DNA damaging agents including irradiation .", "Upon insult , the rapid intestinal regeneration is particularly important as impaired regeneration can result in epithelial barrier defects that can lead to rapid dehydration and translocation of intestinal microbiota into the bloodstream .", "The processes of normal tissue turnover and intestinal regeneration are driven by intestinal stem cells ( ISCs ) that reside at the bottom of crypt and generate the precursors for the specialized differentiated cells ( Barker , 2014; Li and Clevers , 2010 ) .", "It has been extensively reported that ISC compartment includes two functionally and molecularly distinct stem cell populations ( Barker , 2014; Li and Clevers , 2010; Gehart and Clevers , 2015 ) : The active crypt base columnar ( CBC ) stem cells ( Sato et al . , 2011 ) , ( Barker et al . , 2007 ) and a more dormant , reserve ISC population that reside above the crypt base and exhibit no Wnt pathway activity , also referred as +4 cells due to their position at the crypt ( Montgomery et al . , 2011; Sangiorgi and Capecchi , 2008; Tian et al . , 2011; Takeda et al . , 2011; Li et al . , 2014; Yan et al . , 2012 ) .", "The CBCs often identified and isolated based on the expression of Lgr5 , a Wnt target gene ( Barker et al . , 2007 ) .", "During homeostasis , steady-state proliferation of CBCs is driven by extrinsic niche signals – high canonical Wnt activity promotes CBC self-renewal and proliferation ( Barker et al . , 2007; Miyoshi , 2017 ) while BMP signals antagonize it ( Kosinski et al . , 2007 ) .", "In contrast to the active CBCs , the reserve ISCs represent a slow-cycling population of stem cells that are resistant to high doses of ionizing radiation and appear dispensable for homeostasis ( Sangiorgi and Capecchi , 2008; Yousefi et al . , 2016 ) .", "These reserve ISCs are identified through CreERT knockin reporter alleles at the Bmi1 and Hopx loci , as well as by an Tert-CreERT transgene ( Montgomery et al . , 2011; Sangiorgi and Capecchi , 2008; Tian et al . , 2011; Takeda et al . , 2011; Li et al . , 2014 ) .", "Reserve ISCs do not have an active Wnt signaling pathway and are refractory to Wnt signals in their resting state ( Takeda et al . , 2011; Li et al . , 2014; Li et al . , 2016 ) .", "Although the activity of the BMP pathway has never been directly examined specifically in reserve ISCs , indirect evidence suggests that it may help to promote their dormancy ( Reynolds et al . , 2014; He et al . , 2004; Kishimoto et al . , 2015 ) .", "During epithelial regeneration upon stresses , reserve ISCs give rise to Wnthigh Lgr5+ CBCs that generate the precursor cells of the specialized differentiated cells ( Tian et al . , 2011; Takeda et al . , 2011; Li et al . , 2014 ) .", "In addition , it has been documented that Lgr5-CreERT- or Bmi1-CreERT-marked cells can act as the cells of origin of intestinal cancer in mice ( Sangiorgi and Capecchi , 2008; Barker et al . , 2009 ) .", "However , it remains unclear how ISCs differentially sense and respond to multiple signals under both physiological and pathological conditions , and whether these signals contribute to intestinal tumorigenesis .", "MicroRNAs represent a broad class of 18–22 nucleotide noncoding RNAs that negatively regulate the stability and translation of target mRNAs .", "Mounting evidence indicates that microRNAs play important roles in stress-activated pathways ( Leung and Sharp , 2010; Mendell and Olson , 2012; Emde and Hornstein , 2014 ) and in control of somatic stem cell fate and tumorigenesis ( Gangaraju and Lin , 2009; Sun and Lai , 2013; Yi and Fuchs , 2011 ) .", "Hundreds of microRNAs have been identified in the intestinal epithelium ( McKenna et al . , 2010 ) .", "Global ablation of microRNA activity through genetic deletion of the microRNA processing enzyme Dicer demonstrated that microRNAs are critical for homeostasis of intestinal epithelium ( McKenna et al . , 2010 ) .", "Recently , numerous reports demonstrate that specific microRNAs play important roles in the complex intestinal immune system and in the epithelium during homeostasis including miR-155 , miR-29 , miR-122 , miR-21 , miR-146a and miR-143/145 ( Runtsch et al . , 2014 ) .", "Particularly , miR-143/145 are essential for intestinal epithelial regeneration after injury , acting non cell-autonomously in sub-epithelial myofibroblasts ( Chivukula et al . , 2014 ) , indicating potential importance of microRNA activity in intestinal regeneration .", "In the ISC compartment , the function of miR-31 is of a particular interest , as it becomes overexpressed in colorectal cancer ( Bandrés et al . , 2006; Cottonham et al . , 2010; Wang et al . , 2009; Yang et al . , 2013 ) and increases during the progression of inflammation-associated intestinal neoplasia ( Olaru et al . , 2011 ) .", "In addition , it has been reported that miR-31 is enriched in mammary stem/progenitor cells , suggesting a potential role in somatic stem cells ( Ibarra et al . , 2007 ) .", "Here we utilized gain- and loss-of-function mouse models to show that a damage-responsive microRNA , miR-31 drives proliferative expansion of both active and dormant ISCs , and acts as an oncogene promoting intestinal tumorigenesis in different models .", "Our findings implicated miR-31 as a potential high-value therapeutic target for a broad range of intestinal regenerative disorders and cancers ." ], [ "Elevated miR-31 expression has been previously observed in colorectal cancers ( Bandrés et al . , 2006; Cottonham et al . , 2010; Wang et al . , 2009; Yang et al . , 2013 ) , however its expression in normal intestinal epithelium , particularly in ISCs , remains unclear .", "To begin addressing a potential role for miR-31 in the intestinal epithelium and ISCs , first we examined its expression pattern in intestine .", "MiR-31 expression levels are the highest in the Lgr5-GFPhighcrypt base columnar stem cells , intermediate in Lgr5-GFPlow transit-amplifying cell population and the lowest in Lgr5-GFPneg populations ( Figure 1A ) .", "Higher level of miR-31 was also found in Hopx+ reserve ISCs than that in bulk epithelial cells ( Figure 1A ) , based on isolation with Hopx-CreERT;mTmG alleles from mice 15 hr after tamoxifen injection .", "Consistently , in situ hybridization revealed that miR-31 expression levels are generally higher in the crypts than villi .", "MiR-31 is predominantly expressed in the epithelial cells of intestinal crypt , including stem cells and transit amplifying cells ( Figure 1B ) .", "Next , we examined miR-31 expression in response to intestinal injury .", "Mice were exposed to 12 Gy γ-IR and then miR-31 expression was examined at various timepoints during the recovery phase .", "MiR-31 levels transiently and markedly drop by 24 hours ( coincident with full proliferative arrest/DNA damage response ) , and then sharply upregulated 48 hours post-γ-IR ( during initiation of regenerative proliferation from the radioresistant ISCs ) , and then return to baseline levels within one week ( after full recovery ) ( Figure 1C ) .", "In situ hybridization reveals miR-31 expressing cells to be located in the regenerative foci known to exhibit high Lgr5 expression and Wnt pathway activity ( Figure 1D ) .", "Together , these data suggest a role for this microRNA in ISC-driven regeneration .", "To determine the function of miR-31 in the mouse intestine , we generated both gain- and loss- of-function mouse models .", "MiR-31 gain-of-function was achieved with a targeted , inducible Rosa26-rtTA;TRE-miR-31 mouse model ( TRE-miR31 ) and doxycycline ( Dox ) -mediated induction of miR-31 in the intestinal epithelium was validated by qRT-PCR ( Figure 1—figure supplement 1A , B ) .", "For the loss-of-function , we generated constitutive miR-31 null mice using RNA-guided CRISPR/Cas9 nucleases ( Figure 1—figure supplement 1C ) .", "The 402 bp DNA fragment containing miR-31 was deleted in the knockout ( KO ) allele ( Figure 1—figure supplement 1D ) , which was validated by sequencing and qRT-PCR ( Figure 1—figure supplement 1E ) .", "We also generated a Villin-Cre-mediated intestine-specific conditional miR-31 null mice ( cKO ) using traditional homology-directed gene targeting ( Figure 1—figure supplement 1F ) .", "The expression of miR-31 was markedly reduced in the cKO intestinal epithelium ( Figure 1—figure supplement 1G ) .", "The induction of miR-31 in TRE-miR31 intestine and deletion of miR-31 in KO intestine were also confirmed by in situ hybridization ( Figure 1—figure supplement 1H ) .", "MiR-31 induction in response to Dox administration in TRE-miR31 mice resulted in a significant reduction in body weight after 2 weeks ( Figure 1E ) and intestinal lengths were moderately , but significantly shorter than controls ( Figure 1E ) .", "Dox treatment of TRE-miR31 mice for 2 weeks resulted in expansion of intestinal crypts ( Figure 1F ) .", "Unexpectedly villus lengths were mildly shortened , and thus the total length of the crypt-villus was not significantly altered in TRE-miR31 mice ( Figure 1—figure supplement 2A ) .", "The expanded crypts were also found in the TRE-miR31 duodenum and ileum ( Figure 1—figure supplement 2B ) .", "The length of intestinal crypts in the control M2rtTA mice was not significantly altered at different time points in response to Dox treatment ( Figure 1—figure supplement 2C , D ) .", "In contrast , crypts were significantly expanded in TRE-miR31 mice after 10 days of Dox treatment , this crypt expansion remained stable for up to 1 year with continuous Dox induction ( Figure 1—figure supplement 2C–E ) .", "Given that crypt elongation reached maximal levels within 2 weeks of Dox induction , we conducted most of the subsequent assays at this time point .", "More mitotic cells were found in the TRE-miR31 crypts ( Figure 1G and Figure 1—figure supplement 3A , B ) , while more apoptotic cells were detected at the top of TRE-miR31 villi ( Figure 1H and Figure 1—figure supplement 3A , B ) .", "The number of Lgr5+ ISCs increased in TRE-miR31 mice after 10 day Dox treatment , while no significant difference was found between them after 7 days of Dox induction ( Figure 1—figure supplement 3C , D ) .", "In addition , there were fewer differentiated cells including enteroendocrine , goblet and Paneth cells in TRE-miR31 intestine than the controls ( Figure 1—figure supplement 4A , B ) , indicating an impaired cell differentiation .", "These results suggest that miR-31 induction accelerates the conveyer-belt movement of proliferative cells exiting the cell cycle and progressing into the villi to ultimately be shed into the lumen , which could comprise the differentiation of specialized intestinal cell types .", "Next , we examined the consequence of miR-31 loss in both miR-31 germline knockout ( KO ) and Villin-Cre-driven intestinal epithelial conditional KO ( cKO ) mice .", "We followed these mice up to six months .", "Both miR-31 KO and cKO mice were viable and fertile with no apparent gross phenotypes observed .", "No differences in the body weight and intestinal length were found between control and miR-31 KO mice ( Figure 1—figure supplement 5A ) , and the transmission of miR-31 knockout alleles generally followed Mendelian ratios ( Figure 1—figure supplement 5B ) .", "Despite this , loss of miR-31 led to a significant reduction in crypt height with fewer proliferative cells ( Figure 1I and Figure 1—figure supplement 5C , D ) .", "Interestingly , loss of miR-31 gave rise to a certain number of apoptotic cells throughout the crypt-villus axis , while apoptotic cells are predominantly presented at the tip of control villi and very rare apoptotic cells are presented in crypt-villus axis ( Figure 1—figure supplement 5C , D ) .", "Deletion of miR-31 also led to increased numbers of enteroendocrine and Paneth cells , while the number of goblet cells remained unaltered in miR-31 KO intestines ( Figure 1—figure supplement 6A , B ) .", "Moreover , the phenotype of shortened crypts with fewer proliferative cells was also found in cKO intestine ( Figure 1—figure supplement 7A , B ) .", "Loss of miR-31 gave rise to more apoptotic cells in cKO intestinal epithelium , including in cKO crypts , while cleaved-caspase3+ apoptotic cells were nearly entirely absent from control crypts ( Figure 1—figure supplement 7C , D ) .", "These results suggest that miR-31 loss functions within intestinal epithelium .", "We further analyzed DNA synthesis and migration of epithelial cells along the crypt-villus axis after a single pulse of BrdU .", "Upward movement of BrdU+ cells from crypts to villi was enhanced in TRE-miR31 mice , and this movement was impaired in miR-31−/− mice ( Figure 1—figure supplement 8 ) .", "Taken together , these data indicate that miR-31 functions within the intestinal epithelium to maintain a proper balance between stem cell proliferation , differentiation , and epithelial cell death for optimal intestinal homeostasis .", "Higher expression levels of miR-31 in Lgr5+ CBCs prompted us to examine its effect on their renewal .", "Lgr5+ ISC frequency was markedly increased in TRE-miR31 , and significantly reduced in miR-31−/− and cKO intestine ( Figure 2A–C and Figure 2—figure supplement 1A ) .", "A 1 . 5 hr pulse of EdU incorporation demonstrated that the frequency of actively proliferating Lgr5-GFP+/EdU+ cells is higher in TRE-miR31 mice and conversely lower in miR-31−/− mice ( Figure 2D ) .", "In line with these in vivo findings , miR-31 induction increased the frequency of budding organoids in vitro , and caused more buds per organoid and more elongated crypts ( Figure 2E and Figure 2—figure supplement 1B ) .", "Furthermore , lineage-tracing assay reveals that miR-31 induction in the intestine increases the height of traced lineages derived from Lgr5-CreERT-marked ISCs ( Figure 2F , G and Figure 2—figure supplement 1C ) .", "Interestingly , miR-31 induction significantly repressed Hopx expression , while deletion of miR-31 increased it ( Figure 2H ) .", "Consistently , miR-31 induction in the intestine repressed lineage tracing from Hopx-CreERT-marked reserve ISCs ( Figure 2I , and Figure 2—figure supplement 1D , E ) .", "In contrast to miR-31 overexpression , deletion of miR-31 within intestinal epithelium induced quiescence ( residence in G0 ) in Lgr5-GFP+ cells concomitant to an increase in apoptosis and a decrease in cycling ( G1/S/G2/M ) ( Figure 2J and Figure 2—figure supplement 1F ) .", "In agreement , higher frequency of apoptotic organoids and compromised budding was found in the cKO crypts ( Figure 2K ) , and more apoptotic cells were found inside of the cKO organoids ( Figure 2—figure supplement 1G ) .", "Taken together , these data strongly indicate that miR-31 promotes proliferative expansion of Lgr5+ CBCs , and concomitantly prevents their apoptosis .", "The dynamic changes of miR-31 expression in response to irradiation prompted us to investigate its function during intestinal epithelial injury repair .", "Intestinal histology of cKO and control Vil-Cre mice was comparable two hours after 12 Gy γ-IR ( Figure 3A ) .", "However , by 4 days post-γ-IR , there were significantly fewer regenerative foci and fewer proliferative cells per regenerative focus in cKO mice ( Figure 3A ) .", "Consistently , intestinal regeneration in response to γ-IR was significantly impaired in miR-31−/− mice ( Figure 3—figure supplement 1A , B ) .", "Conversely , in the intestine of TRE-miR31 mice pre-treated for 2 weeks with Dox , there were more regenerative foci with higher numbers of proliferative cells than in the control mice ( Figure 3—figure supplement 1A , B ) .", "These data suggest that miR-31 is important for intestinal epithelial regeneration in response to irradiation .", "To understand the phenotype resulting from miR-31 modulation , we assayed for apoptotic cells in cKO mice at early stages after irradiation .", "Loss of miR-31 increased apoptosis in the crypts 2 and 4 hours post-irradiation prior to any overt histological changes ( Figure 3B ) .", "Quantification of apoptotic cell position analysis reveals that apoptotic events occur with the highest frequently in CBC cells , but are still found in transit-amplifying and +4 zones of cKO crypts , compared to control mice ( Figure 3B ) .", "Further , flow cytometry for live cell and apoptotic markers within the Lgr5-GFP+ population confirmed higher frequency of late apoptotic Lgr5+ cells ( AnnexinV+/7AAD+ ) and lower frequency of early apoptotic Lgr5+ cells ( AnnexinV+/7AAD− ) and live Lgr5+ cells ( AnnexinV-/7AAD- ) in cKO mice , relative to controls ( Figure 3—figure supplement 1C ) .", "These data suggest that loss of miR-31 increases apoptosis of Lgr5+ cells in response to irradiation .", "Next , we examined its effect on cell proliferation .", "Cell cycle analysis indicates that more Lgr5-GFP+ cells resided in G0 relative to G1/S/G2/M in cKO mice 2 hours after γ-IR ( Figure 3—figure supplement 1D ) .", "In agreement , expression levels of Lgr5 were dramatically up-regulated in TRE-miR31 mice and prominently down-regulated in miR-31−/− mice at multiple time points after irradiation ( Figure 3C ) , and consequently miR-31 induction promoted lineage regeneration from Lgr5+ cells in response to irradiation ( Figure 3D , E ) .", "Reserve ISCs , marked either by Bmi1-CreER or Hopx-CreER reporters , have been reported to resist high dose of radiation , being able to replenish the depleted CBC compartment and regenerate the epithelium after irradiation ( Sangiorgi and Capecchi , 2008; Tian et al . , 2011; Takeda et al . , 2011; Yan et al . , 2012 ) , ( Yousefi et al . , 2016 ) .", "Thus , we examined the response of Hopx-CreER-marked reserve ISCs to 12 Gy γ-IR upon miR-31 induction and deletion .", "Lineage-tracing assay revealed that miR-31 induction promoted epithelial regeneration from the Hopx+ reserve stem cells ( Figure 3F and Figure 3—figure supplement 1E ) .", "Conversely , the number and the size of regenerative foci originating from Hopx-CreER;Rosa26-LoxP-Stop-LoxP-LacZ-marked cells were markedly reduced in miR-31−/− mice ( Figure 3G ) .", "In line with this , the frequency of LacZ+/Ki67+ cells was significantly lower in miR-31−/− mutants compared to controls ( Figure 3H ) .", "Taken together , miR-31 deficiency-mediated the reduction in proliferation and increase in apoptosis within both CBC and reserve ISC compartments can account for the impaired regeneration of miR-31 null intestine .", "Canonical Wnt pathway activity is a major driving force for self-renewal of CBCs and epithelial regeneration after injury ( Clevers et al . , 2014 ) , and , thus we examined the effect of miR-31 on Wnt activity .", "We utilized Axin2-LacZ Wnt reporter mice , which act as a broad readout for canonical Wnt activity , and normally showed its activity to be restricted to the base of crypts in control mice , as expected ( Figure 4A ) ( Davies et al . , 2008 ) .", "In contrast , Wnt pathway activity was strikingly absent from CBCs of miR-31−/− crypts , appearing only faintly above the crypt base in the early TA zones ( Figure 4A ) .", "Conversely , Wnt activity was expanded in TRE-miR31 crypts ( Figure 4A , B ) .", "In agreement , the number of nuclear β-Catenin-positive cells was significantly reduced in miR-31−/− intestinal crypts at 2 and 4 months of age ( Figure 4—figure supplement 1A ) .", "Conversely , they increase in TRE-miR31 crypts 14 days and 2 months after Dox induction ( Figure 4—figure supplement 1B ) .", "Consistently , the expression levels of Ctnnb1 ( encoding β-Catenin ) and the Wnt targets , Ccnd1 ( encoding Cyclin D1 ) , Myc and Axin2 were significantly reduced in miR-31−/− intestine both at the RNA and protein levels ( Figure 4C , D ) .", "In contrast , expression levels of the above genes were enhanced in TRE-miR31 intestinal epithelium following 2 weeks of Dox induction ( Figure 4E , F ) .", "The reduction in Ctnnb1 and Wnt targets was further confirmed in conditional miR-31 KO intestine ( Figure 4G ) .", "To test whether Wnt activity is directly impacted by miR-31 , we analyzed the effects of gain- and loss-of-function of miR-31 on expression of Wnt target genes in HCT116 human colorectal carcinoma cells .", "Ccnd1 , Ctnnb1 , Myc and Axin2 were markedly increased in miR-31 over-expressing cells , relative to controls ( Figure 4H ) .", "Conversely , these genes were downregulated upon miR-31 inhibition ( Figure 4H ) .", "Considering that HCT116 cells are heterozygous for a β-Catenin gain-of-function mutation at the Gsk3b target site S45 ( Ctnnb1+/S45mt ) ( Ilyas et al . , 1997 ) , ( Kaler et al . , 2012 ) , we examined β-Catenin protein levels .", "Consistently , β-Catenin was up-regulated in the presence of miR-31 mimics , and down-regulated upon miR-31 inhibition ( Figure 4—figure supplement 1C ) .", "The Wnt reporter ( Topflash/Fopflash ) assay using HCT116 cells further confirmed that miR-31 induction enhanced Wnt activity , while inhibition of miR-31 repressed it ( Figure 4I ) .", "To test the functional relevance of miR-31 potentiation of canonical Wnt activity , we cultured organoids with varying combinations of miR-31 induction and R-spondin , the Lgr5 ligand .", "Wnt activation by R-spondin is critical for normal organoid growth and budding ( Sato et al . , 2011 ) .", "Interestingly , we observed that miR-31 induction via TRE-miR31 was sufficient to maintain crypt organoid growth and budding in the absence of R-spondin ( Figure 4J , K ) and that the Dox-treated TRE-miR31 organoids can be normally passaged at least five times , similar to the organoids cultured with R-spondin ( Figure 4L ) .", "Together , these findings demonstrate that miR-31 activates the canonical Wnt signaling in the crypts of small intestine .", "BMP and TGFβ pathways are known to inhibit the canonical Wnt pathway , inhibiting proliferation and promoting intestinal progenitor differentiation ( Reynolds et al . , 2014; He et al . , 2004; Furukawa et al . , 2011 ) .", "We thus examined the effects of miR-31 on BMP and TGFβ signals .", "BMP-specific Smad1/5/8 and TGFβ-specific Smad2/3 phosphorylation were significantly increased in miR-31−/− intestine ( Figure 5A and Figure 5—figure supplement 1A ) , and downregulated in TRE-miR31 intestine ( Figure 5A and Figure 5—figure supplement 1B ) , suggesting an inhibitory effect of miR-31 on BMP and TGFβ signaling pathways .", "Consistently , we observed a significant increase on the expression of BMP target genes including Id1 , Id2 , Id3 , Msx1 , Msx2 and Junb and TGFβ target genes Cdkn1c ( p57 ) , Cdkn1a ( p21 ) , Cdkn2a ( p16 ) , and Cdkn2b ( p15 ) in miR-31−/− intestine ( Figure 5B ) .", "Conversely , BMP and TGFβ targets were repressed upon forced expression of miR-31 in TRE-miR31 intestine following 2 weeks of Dox induction ( Figure 5C ) .", "The upregulation of BMP and TGFβ targets was further confirmed upon conditional miR-31 deletion in cKO intestine ( Figure 5D , E ) .", "BMP-specific Smad1/5/8 and TGFβ-specific Smad2/3 phosphorylation were also increased in miR-31 cKO cultured organoids ( Figure 5—figure supplement 1C ) .", "Further , we examined the effect of miR-31 on BMP and TGFβ signaling in HCT116 colorectal cancer cells .", "These cells carry biallelic mutations in the Tgfbr2 gene , but still express functional TGFBR2 proteins and respond to TGFβ ( de Miranda et al . , 2015 ) .", "In line with the in vivo findings , we found down-regulation of p-Smad2/3 and p-Smad1/5/8 in HCT116 cells treated with miR-31 mimics , and their up-regulation in cells treated with miR-31 inhibitor ( Figure 5—figure supplement 1D ) .", "Luciferase assays using BMP- and TGFβ-responsive luciferase reporters , BRE-Luc and CAGA-Luc , respectively , revealed that inhibition of miR-31 resulted in significant increases in luciferase activities , and that miR-31 mimics decreased them ( Figure 5F , G ) .", "More importantly , increasing concentrations of the BMP inhibitor Noggin in organoid culture was able to rescue the budding defect in miR-31 cKO organoids in a dose-dependent manner ( Figure 5H , I ) .", "Together , these data suggest that miR-31 promotes ISC proliferation possibly through repressing BMP and TGFβ signaling pathways in a cell-autonomous manner .", "To understand how miR-31 regulates Wnt , BMP and TGFβ pathways , we analyzed miR-31 binding sites in 3’UTRs of transcripts encoding for regulators of these pathways .", "Genes containing miR-31 binding sites include Wnt antagonists Axin1 , Gsk3b , and Dkk1 , along with transcripts containing BMP/TGFβ signaling pathway components such as Smad3 , Smad4 , Bmpr1a and Tgfbr2 ( Figure 6—figure supplement 1A ) .", "The expression of Axin1 , Gsk3b , Dkk1 , Smad3 , Smad4 , Bmpr1a and Tgfbr2 was significantly upregulated in miR-31−/− intestine ( Figure 6A ) and remarkably downregulated in TRE-miR31 intestine following Dox induction ( Figure 6B ) , suggesting that they are negatively regulated by miR-31 .", "The upregulation of these putative target genes was further confirmed in conditional miR-31 KO intestine ( Figure 6C ) .", "Axin1 , Gsk3b , Dkk1 , Bmpr1a and Smad4 were selected for further validation at protein level ( Figure 6D , E and Figure 6—figure supplement 2A–C ) and in organoids cultured from miR-31 cKO mice ( Figure 6—figure supplement 3A ) .", "This effect was further confirmed in HCT116 cells with miR-31 modulation ( Figure 6—figure supplement 3B ) .", "Next , we validated the direct repression of target transcripts by miR-31 activity using WT-3’UTR-luciferase constructs for Axin1 , Gsk3b , Dkk1 , Bmpr1a , Smad3 and Smad4 .", "Mutation of the miR-31 3’UTR binding site in these constructs abrogated this repression ( Figure 6F and Figure 6—figure supplement 1B ) .", "Furthermore , RNA crosslinking , immunoprecipitation , and RT-PCR ( CLIP-PCR ) assays with Ago2 antibodies confirmed that transcripts of Axin1 , Dkk1 , Gsk3b , Smad3 , Smad4 and Bmpr1a were highly enriched in Ago2 immunoprecipitates , and that increasing miR-31 activity augmented their enrichment ( Figure 6G ) , providing evidence that miR-31 directly binds to these transcripts .", "Taken together , these findings indicate that Axin1 , Gsk3b , Dkk1 , Smad3 , Smad4 , and Bmpr1a transcripts are the direct targets of miR-31 .", "Next , we asked whether these targets functionally contribute to impaired regeneration in miR-31−/− mice .", "Derepression of these target transcripts was observed in miR-31−/− intestine after irradiation ( Figure 6H , I ) .", "As a consequence , Wnt activity was reduced , while the BMP and TGFβ activities were increased in miR-31−/− intestine , evidenced by β-Catenin , p-Smad1/5/8 and p-Smad2/3 immunohistochemistry assays ( Figure 6J ) .", "Considering that intestinal regeneration following irradiation requires Wnt hyperactivity ( Davies et al . , 2008 ) , and that BMP activity counterbalances Wnt signaling ( He et al . , 2004 ) , our findings suggest that miR-31 is an important amplifier of Wnt signaling during intestinal regeneration .", "Given that miR-31 promotes proliferation and inhibits apoptosis in the ISCs , it is plausible that miR-31 may function in intestinal tumorigenesis .", "Supporting this notion , miR-31 has been found to be upregulated in human colorectal cancers and in colitis ( Bandrés et al . , 2006; Cottonham et al . , 2010; Wang et al . , 2009; Yang et al . , 2013 ) .", "We tested the role of miR-31 in intestinal tumorigenesis and observed that miR-31 mimics promoted proliferation of HCT116 , SW480 and LOVO colon cancer cells in vitro ( Figure 7—figure supplement 1A ) .", "Conversely , inhibition of miR-31 with anti-miR-31 abrogated growth of these cells ( Figure 7—figure supplement 1A ) .", "We further performed xenograft assays using miR-31 mimics- and inhibitor-treated HCT116 cells .", "Thirty days after grafting , tumor volume and weight were increased in miR-31 mimic-treated tumors , and markedly reduced in miR-31 knockdown tumors ( Figure 7A ) .", "The decrease in tumor size from miR-31 inhibition coincided with the reduction in Ki67+ and Cyclin D1+ proliferating cells ( Figure 7B and Figure 7—figure supplement 1B ) , and correlated with reduced Wnt activity and increased BMP and TGFβ activities ( Figure 7—figure supplement 1B ) .", "To verify these findings in more physiologically relevant settings , we examined tumor formation in the AOM-DSS ( Azoxymethane-Dextran Sodium Sulfate ) model of the inflammation-driven colorectal adenocarcinoma ( De Robertis et al . , 2011 ) .", "In comparison with the controls , we observed a marked decrease in both tumor size and number in miR-31−/− mice ( Figure 7C ) , along with a concomitant reduction in proliferating cells ( Figure 7D , E ) , and reduced Wnt pathway and increased BMP and TGFβ activity ( Figure 7D , F ) .", "This tumor-promoting effect of miR-31 in mice became even more evident when miR-31 was deleted in Vil-Cre;Apcflox/+ mice .", "Intestinal adenomas form in this mouse model upon loss of heterozygosity at the Apc locus , which is relevant to human disease in that spontaneous loss of Apc is found in the vast majority of human colorectal cancer ( Kinzler et al . , 1991; Nagase et al . , 1992 ) .", "Loss of miR-31 in this animal model remarkably reduced tumor burden ( Figure 7G ) , which was associated with decreased Wnt activity , enhanced BMP and TGFβ signaling , and decreased proliferating cells ( Figure 7H–J and Figure 7—figure supplement 1C ) .", "Correspondingly , the miR-31 targets Axin1 , Dkk1 , Gsk3β , Smad4 and Bmpr1a were up-regulated in the miR-31 null tumors ( Figure 7—figure supplement 1D ) .", "Together , these data demonstrate that miR-31 plays an oncogenic role in intestinal and colorectal tumorigenesis by mediating activation of Wnt and repression of BMP and TGFβ signaling pathways .", "Lastly , we asked how radiation injury induces miR-31 expression .", "We analyzed a 2 kb region upstream of the transcription start site of the miR-31 gene locus for the potential binding sites of transcription factors using the JASPAR database and identified one STAT3 and two NF-κB binding sites ( Figure 8A ) .", "Interestingly , the STAT3 and NF-κB signaling pathways were shown to be activated in response to γ-IR , evidenced by p-STAT3 and p65 levels , respectively ( Figure 8B , C ) .", "The activation of the STAT3 pathway occurred mainly in the regenerative foci where miR-31 is highly induced , while NF-κB was more prominently activated in villi where little miR-31 is present and not in the regenerative foci ( Figure 8D ) .", "This suggested a link between STAT3 activity and miR-31 upon irradiation .", "To verify whether active STAT3 signaling could induce miR-31 expression , mICc12 intestinal epithelial cells were treated with IL-6 , a known activator of the STAT3 signaling .", "Indeed , miR-31 expression was significantly induced upon IL-6 treatment ( Figure 8E ) , concomitant with the activation of the STAT3 pathway ( Figure 8F ) .", "In contrast , inhibition of STAT3 signaling with Stattic prominently dampened miR-31 induction response to IL-6 treatment ( Figure 8G ) , and reduced STAT3 signaling ( Figure 8H ) .", "This inhibitory effect on miR-31 expression was further validated using Stat3 siRNA ( Figure 8I , J ) .", "Importantly , miR-31 was induced by IL-6 in the organoid cultures , indicating that this is an epithelial cell-autonomous mechanism ( Figure 8K ) .", "Luciferase reporter assays reveal that IL-6 is able to induce its activity , while mutation of the p-STAT3 binding site blocked it ( Figure 8L ) .", "Furthermore , Chromatin Immunoprecipitation ( ChIP ) assays show that p-STAT3 is recruited to its binding site on the miR-31 promoter ( Figure 8M ) .", "Thus , our data strongly suggest that STAT3 activity potentiates miR-31 induction to promote crypt regeneration in response to radiation injury ." ], [ "The intestinal epithelium is one of the most rapidly renewing tissues ( Leblond and Walker , 1956 ) .", "Those Lgr5+ CBC stem cells residing at the base of crypts maintain the proliferative capacity necessary to meet this demands of high-turnover tissue , which is driven by activation of the canonical Wnt pathway , as well as repression of BMP signaling ( Li and Clevers , 2010 ) , ( Li et al . , 2014 ) , ( Kosinski et al . , 2007 ) .", "Wnt pathway activity and BMP inhibition are believed to be the niche for cycling CBCs .", "However , it is largely unknown how those Lgr5+ CBCs integrate the signals of Wnt antagonists and activators of BMP and TGFβ .", "Here we show that the miR-31 activates Wnt signaling by directly repressing a cohort of Wnt antagonists Dkk1 , Axin1 and Gsk3b , and represses BMP/TGFβ signaling by directly inhibiting activators of the pathways , Smad3 , Smad4 and Bmpr1a , pointing to an important role of miR-31 acting as a rheostat to integrating niche signals sensed by cycling CBCs .", "In agreement with this point , our in vivo analysis demonstrated that miR-31 induction increases the number of Lgr5+ CBCs whereas miR-31 deletion reduces CBC frequency .", "Niche Wnt signals likely originate from sub-epithelial telocytes whose presence is required for CBC activity , and possibly to a lesser extent from Paneth cells , who secrete Wnt ligands but are dispensable for CBC activity ( Durand et al . , 2012; Aoki et al . , 2016; Sato et al . , 2011; Kim et al . , 2012; San Roman et al . , 2014; Kabiri et al . , 2014 ) .", "BMP antagonists noggin and gremlin are similarly secreted by sub-mucosal tissues below the crypts ( Kosinski et al . , 2007 ) , repressing the BMP signaling in CBCs .", "Thus , sub-epithelial mesenchyme constitutes an extrinsic niche for cycling ISCs .", "In contrast to secretory signals from an extrinsic niche , miR-31 appears to be an intrinsic coordinator of these extrinsic niche signals , supporting canonical Wnt and represses BMP/TGFβ signals within CBCs .", "Thus , we identify miR-31 as a cell-autonomous post-transcriptional regulator of the ISC niche , maintaining proliferative capacity of cycling CBC cells .", "In addition , we also noticed that miR-31 loss resulted in an increased apoptosis in CBC cells , suggesting the importance of miR-31 in maintaining cell survival .", "The molecular mechanism by which miR-31 protects against apoptosis warrants future study .", "The response to high dose of γ-IR can be separated into two distinct stages .", "First , within 24 hours , the majority of CBCs die via apoptosis and subsequent mitotic death , caused by residual misrepaired and unrepaired of DNA double-strand breaks ( Hua et al . , 2012 ) .", "Next , between 24 hours and 4 days after γ-IR , rare surviving CBCs and quiescent reserve ISCs enter the cell cycle and form regenerative foci that produce mitotically active Lgr5+ cells that repair lost epithelium ( Yousefi et al . , 2016; Hua et al . , 2012 ) .", "We assume that reserve ISCs also undergo the same process , although lack of direct evidence .", "In line with this , miR-31 is dramatically reduced within the first 24 hours post γ-IR , most likely due to loss of CBCs .", "Loss of miR-31 led to an marked increase in apoptosis in both CBCs and +4 cells 2 hours post-γ-IR .", "Based on our data , we conclude that during the first stage miR-31 acts as an anti-apoptotic factor , protecting CBCs and reserve ISCs against apoptosis .", "During the second stage , the surviving stem cells start proliferating to repopulate the depleted intestinal epithelium .", "The surviving stem cells are relatively damage-resistant ( Tian et al . , 2011; Takeda et al . , 2011; Li et al . , 2014; Yousefi et al . , 2016; Ritsma et al . , 2014 ) , a property attributed to their quiescence , a state likely maintained by BMP/TGFβ signaling and inactivation of Wnt signaling ( Li et al . , 2014; Yousefi et al . , 2016; He et al . , 2004 ) .", "We show that miR-31 is prominently induced at the regenerative foci 36 hr post-γ-IR and that miR-31 activates Wnt , and represses BMP/TGFβ activities .", "This points to the potential importance of miR-31 in activating the surviving ISCs .", "Given BMP/TGFβ inhibiting ability of miR-31 , we speculate that the homeostatic insensitivity of reserve ISCs to Wnt ligands ( Yan et al . , 2012 ) results from their having active BMP and TGFβ pathways , that must be suppressed for cells to become competent to respond to Wnt ligands .", "Our findings suggest that miR-31 functions as an activator of dormant reserve ISCs .", "We also want to mention that the expression patterns of Bmi1 and Hopx are not specific to +4 position , as both of these transcripts are found non-specifically throughout the crypt base ( Li et al . , 2014; Muñoz et al . , 2012; Itzkovitz et al . , 2011 ) .", "This means that miR-31-activated stem cells represent a complex population including +4 cells , surviving Lgr5+ cells , and those TA cells dedifferentiated in response to irradiation .", "Taken together , our findings suggest that miR-31 functions as the anti-apoptotic factor in ISCs during the early post-γ-IR stage , and , potentially , serves as the cell-intrinsic activator of surviving ISCs regenerative foci promoting regeneration .", "Future studies will be needed to comprehensively test this idea .", "Many reports have showed that miR-31 is overexpressed in CRC tissues ( Bandrés et al . , 2006; Cottonham et al . , 2010; Wang et al . , 2009 ) and increases in progressively during progression from normal to inflammatory bowl disease ( IBD ) to IBD-related neoplasia ( Olaru et al . , 2011 ) .", "We demonstrate that miR-31 promotes tumor development using several models , including cancer cells xenografting , AOM- and DSS- induced inflammation-driven tumors , and Apc-loss driven tumors , characterized by activated Wnt , and repressed BMP/TGFβ signalings .", "Indeed , several reports showed that miR-31 is overexpressed in colorectal cancer ( CRC ) tissues ( Bandrés et al . , 2006; Cottonham et al . , 2010; Wang et al . , 2009 ) .", "Wnt signaling is aberrantly up-regulated in CRCs , which due primarily to mutations in the Wnt antagonist APC ( Novellasdemunt et al . , 2015 ) .", "Our current study suggests that miR-31 up-regulation might also contribute to Wnt activation in CRCs .", "In addition , decreased BMP and TGFβ signaling is also often found in CRCs ( Bellam and Pasche , 2010; Hardwick et al . , 2008 ) , and can be the consequence of miR-31 upregulation .", "As such , our data suggests that miR-31 acts as the oncogenic microRNA in CRCs .", "Moreover , tight association between miR-31 induction and STAT3 pathway activation in intestinal tissues is worth noting .", "Our molecular data suggest direct activation of miR-31 expression by STAT3 signaling pathway .", "Indeed , many reports showed that constitutive activation of STAT3 is frequently detected in primary human colorectal carcinoma ( Kusaba et al . , 2005; Corvinus et al . , 2005 ) and contributes to invasion , survival , and growth of colorectal cancer cells ( Tsareva et al . , 2007; Lin et al . , 2005 ) .", "Therefore , our current study suggests a signaling pathway involving STAT3 , miR-31 and WNT/BMP/TGFβ that promotes colorectal tumorigenesis .", "In summary , we propose a model in which miR-31 functions as a cell-intrinsic master modulator of the intestinal stem cell niche signaling during normal homeostasis , regeneration and tumorigenesis ( Figure 9 ) .", "During homeostasis , miR-31 functions to integrate niche signals , supporting canonical Wnt activity and represses BMP/TGFβ signaling pathways within cycling CBC stem cells .", "MiR-31 is stress inducible and plays an important role in epithelial regeneration .", "In response to high dose of γ-IR , miR-31 is markedly induced via STAT3 signaling pathway , and appears capable of regulating the activation state of a population of dormant , radiation resistant reserve ISCs during regeneration .", "Further , we demonstrate that miR-31 acts as an oncomiR in promoting tumor growth ." ], [ "All mouse experiment procedures and protocols were evaluated and authorized by the Regulations of Beijing Laboratory Animal Management and strictly followed the guidelines under the Institutional Animal Care and Use Committee of China Agricultural University ( approval number: SKLAB-2011-04-03 ) .", "To generate TRE-miR-31 transgenic mice , the mmu-miR-31 sequence was amplified using the following primers: Forward 5’-CTCGGATCCTGTGCATAACTGCCTTCA-3’ ( BamHI site was added ) , and Reverse 5’-CACAAGCTTGAAGTCAGGGCGAGACAGAC-3’ ( HindIII site was added ) , and was inserted into pTRE2 vector ( Clontech ) to generate a pTRE2-miR31 construct .", "TRE-miR31 transgenic mice were produced using standard protocols and crossed with Rosa26-rtTA mice which harboring the modified reverse tetracycline transactivator ( M2rtTA ) targeted to and under transcriptional control of the Rosa26 locus .", "Constitutive miR-31−/− mice were generated using CRISPR/Cas9 approach at the Nanjing Animal Center , and 402 bp DNA fragment containing miR-31 was deleted to produce the null allele .", "Conditional miR-31 KO allele was generated at the Shanghai Model Animal Center , the first exon ( 14806–15522 ) of miR-31 was targeted with flanking LoxP sites resulting in the 2 LoxP locus .", "Villin-Cre ( Vil-Cre ) mice were purchased from the National Resource Center of Model Mice ( stock number:T000142 ) .", "mTmG , Lgr5-eGFP-CreERT , Apc floxed , and Rosa26-LSL-lacZ mice were obtained from Jackson Laboratories ( stock number: 007576 , 008875 , 009045 and 009427 ) .", "Hopx-CreERT mice were obtained from John Epstein laboratory .", "Axin2-LacZ mice were obtained from Yi Zeng laboratory .", "HCT116 , SW480 and LOVO human colorectal cancer cell lines are purchased from American Type Culture Collection ( ATCC ) and the mouse mICc12 intestinal epithelial cell line was obtained from the Institute of Interdisciplinary Research ( Fudan University , Shanghai , China ) who originally obtained them from Dr A Vandervalle ( Institut National de la Santé et de la Recherche Médicale , Faculté X , Paris , France ) .", "They were confirmed to come from a mouse cell line by Beijing Microread Genetics Co . , Ltd using STR profiling .", "No cell lines are on the list of commonly misidentified cell lines .", "We have tested for mycoplasma contamination using a Mycoplasma Detection Kit , and no mycoplasma contamination was detected in any of the cultures .", "These cell lines were cultured in DMEM/F12 medium .", "The sequence of miR-31 inhibitor is 5’-AGCUAUGCCAGCAUCUUGCCU-3’ .", "The sequence of Scramble RNA is 5’-CAGUACUUUUGUGUAGUACAA-3’ .", "The Sequence of miR-31 mimics: 5’-AGGCAAGAUGCUGGCAUAGCU-3’ 3’-CUAUGCCAGCAUCUUGCCUUU-5’ The sequence of negative control for miR-31 mimics: 5’-UUCUCCGAACGUGUCACGUUU-3’ 3’-ACGUGACACGUUCGGAGAAUU-5’ .", "For the induction , 2 mg/mL Dox ( Doxycycline hyclate , Sigma ) was added to the drinking water along with 1% w/v sucrose .", "Mice were induced at 8 weeks of age .", "To isolate intestinal epithelial cells , mouse intestine was dissected longitudinally and rinsed three times with ice-cold 1x DPBS , then cut into 2–4 mm long pieces , incubated in 1x DPBS containing 2 mM EDTA and 0 . 2 mM DTT for 30 min at 4°C on a rotating platform .", "Suspended cells were then collected folowing gentle vortexing .", "To isolate intestinal crypts , rinsed small intestine was cut-opened and and villi were scraped using coverslip glass , the technique which left the crypts attached .", "Crypts were then detached after tissue incubation in 1x DPBS with 2 mM EDTA for 30 min at 4°C with gentle vortexing .", "Isolated crypts were counted and pelleted as previously described ( Sato et al . , 2009 ) .", "Dissected intestine was incubated with 5 mM EDTA and 1 . 5 mM DTT in HBSS for 30 min at 4°C .", "Single cell suspension was produced following Dispase ( BD Biosciences ) treatment and passing cells through 40 μm cell strainer .", "Flow cytometry analysis was performed using BD LSR Fortessa cell analyzer ( BD Biosciences ) .", "PI-negative cells were selected , then gated for single cells based on the forward-scatter height vs . forward-scatter width ( FSC-H vs . FSC-W ) and side-scatter height vs . side-scatter width ( SSC-H vs . SSC-W ) profiles .", "The size of the nozzle for all sorting runs was 100 μm ( 20 psi ) .", "Lgr5-eGFP+ cells were quantified by flow cytometry in TRE-miR31;Lgr5-eGFP-CreERT and M2rtTA;Lgr5-eGFP-CreERT mice after two weeks of Dox treatment .", "Lgr5-eGFP+ cells in miR-31+/−;Lgr5-eGFP-CreERT and miR-31−/−;Lgr5-eGFP-CreERT mice were quantified using the same method .", "Crypt culture was performed as previously described in Sato et al . ( 2009 ) .", "A total of 500 isolated crypts from TRE-miR31 , M2rtTA , Vil-Cre and Vil-Cre;miR31fl/fl ( cKO ) mice were mixed with 80 μL of matrigel ( BD Bioscience ) and plated in 24-well plates .", "After matrigel polymerization , 500 μL of crypt culture medium [advanced DMEM/F12 ( Gibco ) , 2 mM Glutamax ( Invitrogen ) , 100 U/mL penicillin , 100 μg/mL streptomycin ( Sigma ) , 1 mM N-acetyl cysteine ( Sigma ) , B27 supplement ( Invitrogen ) , N2 supplement ( Invitrogen ) , 50 ng/mL mouse , EGF ( Peprotech ) , 100 ng/mL mouse Noggin ( Peprotech ) and 10% human R-spondin-1 ( Peprotech ) ] was added to M2rtTA , Vil-Cre and Vil-Cre;miR-31fl/fl small intestine crypt cultures .", "For TRE-miR31 culture , human R-spondin-1 was removed from the medium , and instead 2 μg/mL of Dox was added .", "For miR-31 in situ hybridizations , digoxigenin ( DIG ) -labeled probes ( Exiqon ) were used following the manufacturer’s protocol .", "Both DIG-labeled miR-31 and scrambled probes ( Exiqon ) were hybridized at 61°C .", "U6 probe was used as the positive control .", "In situ signals were detected by staining with Anti-DIG-AP antibody ( Roche ) and developed using BM purple substrate ( Roche ) .", "Total RNA was isolated from total mouse small intestinal epithelial cells using TRIzol reagent ( Life Technologies ) according to the manufacturer’s instructions .", "Each RNA sample was reverse transcribed with the M-MLV Reverse Transcriptase ( Sigma ) using Oligo ( dT ) primers .", "Real-time PCR was performed using the LightCycler 480 SYBR Green I master mix on a LightCycler 480 real-time PCR system ( Roche ) .", "qRT-PCR primers were follows: Axin1-forward: 5’- TTCTGGGTTGAGGAAGCAGC −3’; Axin1-reverse: 5’- GATTAGGGGCTGGATTGGGT-3’; Axin2-forward: 5’- GGCTAGCTGAGGTGTCGAAG −3’; Axin2 -reverse: 5’- GCCAGTTTCTTTGGCTCTTT −3’; Ctnnb1-forward: 5’- TCCTAGCTCGGGATGTTCAC −3’; Ctnnb1 -reverse: 5’- TTCTGCAGCTTCCTTGTCCT −3’; Bmpr1a-forward: 5’- GCTGTCATCATCTGTTGTCCTGG −3’; Bmpr1a-reverse: 5’- CATTACCACAAGGGCTACACCACC −3’; Myc-forward: 5’- CTACTCGTCGGAGGAAAG −3’; Myc-reverse: 5’- ACTAGACAGCATGGGTAAG −3’; Ccnd1-forward: 5’- TGGTGAACAAGCTCAAGTGG −3’; Ccnd1-reverse: 5’- GGCGGATTGGAAATGAACT −3’; Dkk1-forward: 5’- TCCGAGGAGAAATTGAGGAA −3’; Dkk1-reverse: 5’- CCTGAGGCACAGTCTGATGA −3’; Gsk3b-forward: 5’- CCAACAAGGGAGCAAATTAGAGA −3’; Gsk3b-reverse: 5’- GGTCCCGCAATTCATCGAAA −3’; Id1-forward: 5’- ACCCTGAACGGCGAGATC −3’; Id1-reverse: 5’- GCGGTAGTGTCTTTCCCAGA −3’; Id2-forward: 5’- CTACTCGTCGGAGGAAAG −3’; Id2 -reverse: 5’- ACTAGACAGCATGGGTAAG −3’; Id3-forward: 5’- TCCGGAACTTGTGATCTCCA −3’; Id3-reverse: 5’- GTAAGTGAAGAGGGCTGGGT −3’; Junb-forward: 5’- CGGATGTGCACGAAAATGGA −3’; Junb-reverse: 5’- GACCCTTGAGACCCCGATAG −3’; Msx1-forward: 5’- CAGAGTCCCCGCTTCTCC −3’; Msx1-reverse: 5’- CTGAGCGAGCTGGAGAATTC −3’; Msx2-forward: 5’- TTCACCACATCCCAGCTTCT −3’; Msx2-reverse: 5’- TTCAGCTTTTCCAGTTCCGC −3’; Cdkn2b-forward: 5’- GCCCAATCCAGGTCATGATG −3’; Cdkn2b-reverse: 5’- TCACACACATCCAGCCGC −3’; Cdkn2a-forward: 5’- AGAGCTAAATCCGGCCTCAG −3’; Cdkn2a -reverse: 5’- CTCCCTCCCTCCTTCTGCT −3’; Cdkn1a-forward: 5’- ATCACCAGGATTGGACATGG −3’; Cdkn1a -reverse: 5’- CGGTGTCAGAGTCTAGGGGA −3’; Cdkn1b-forward: 5’- GGGGAACCGTCTGAAACATT −3’; Cdkn1b -reverse: 5’- AGTGTCCAGGGATGAGGAAG −3’; Cdkn1c-forward: 5’- GTTCTCCTGCGCAGTTCTCT −3’; Cdkn1c -reverse: 5’- GAGCTGAAGGACCAGCCTC −3’; Smad3-forward: 5’- ACAGGCGGCAGTAGATAACG −3’; Smad3-reverse: 5’- AACGTGAACACCAAGTGCAT −3’; Smad4-forward: 5’- GGCTGTCCTTCAAAGTCGTG −3’; Smad4-reverse: 5’- GGTTGTCTCACCTGGAATTGA −3’; Tgfbr2-forward: 5’- TTGTTGAGACATCAAAGCGG −3’; Tgfbr2-reverse: 5’- ATAAAATCGACATGCCGTCC −3’; Rela-forward: 5’- agataccaccaagacccacc-3’; Rela-reverse: 5’- ggtgaccagggagattcgaa −3’; Ikkb-forward: 5’-agaagtacaccgtgaccgtt-3’;Ikkb-reverse: 5’-gggaagggtagcgaacttga-3’; IL-1-forward: 5’- tacctgtgtctttcccgtgg-3’; IL-1-reverse: 5’- ttgttcatctcggagcctgt-3’; IL-6-forward: 5’- gccagagtccttcagagaga-3’; IL-6-reverse: 5’-ggtcttggtccttagccact-3’; IL-18-forward: 5’- gtctaccctctcctgtaagaaca-3’; IL-18-reverse: 5’- tggcaagcaagaaagtgtcc-3’; Tnf-forward: 5’- aatggcctccctctcatcag-3’; Tnf-reverse: 5’- cccttgaagagaacctggga-3’; Stat3-forward: 5’- tgacatggatctgacctcgg-3’; Stat3-reverse: 5’- tgcccagattgcccaaagat −3’; For quantification of microRNA expression , mature miR-31 was quantified using TaqMan microRNA assays according to the manufacturer’s instructions .", "U6 snRNA was used as the internal control ( Applied Biosystems ) .", "Intestines were rinsed with 1x DPBS , fixed in 10% formalin , paraffin-embedded and sectioned at 5 μm .", "Sections were stained with hematoxylin and eosin ( H&E ) .", "For immunohistochemistry , antigen retrieval was performed by heating slides in 0 . 01 M citrate buffer ( pH 6 . 0 ) in a microwave .", "Sections were then immunostained using ABC peroxidase method ( Vector labs ) with diaminobenzidine ( DAB ) as the enzyme substrate and hematoxylin as the counterstain .", "For immunofluorescence staining , paraffin sections were microwave pretreated in 0 . 01 M citrate buffer ( pH 6 . 0 ) , and incubated with primary antibodies , then incubated with secondary antibodies ( Invitrogen ) and counterstained with DAPI in the mounting medium ( Vector labs ) .", "The following antibodies were used: anti-Ki67 ( 1:150 , Leica ) , anti-GFP ( 1:200 , Abcam ) , anti-Axin1 ( 1:100 , Cell Signaling ) , anti-Gsk3β ( 1:2000 , Abcam ) , anti-Dkk1 ( 1:50 , Santa Cruz ) , anti-β-Catenin ( 1:500 , Sigma ) , anti-BrdU ( 1:50 , Abcam ) , anti-cleaved Caspase3 ( 1:100 , Cell Signaling ) , anti-p-Smad1/5/8 ( 1:200 , Cell Signaling ) , anti-p-Smad2/3 ( 1:200 , Cell Signaling ) , anti-CyclinD1 ( 1:50 , Abcam ) , anti-p65 ( 1:400 , Cell Signaling ) , anti-p-STAT3 ( 1:800 , Cell Signaling ) .", "To generate reporter constructs for luciferase assays , 300–600 bp fragments in length containing predicted miR-31 target site in the 3’UTRs of Axin1 , Dkk1 , Bmpr1a , Gsk3b , Smad3 and Smad4 were cloned into the psiCHECK-2 vector ( Promega ) between the XhoI and NotI sites immediately downstream of the Renilla luciferase gene .", "To generate reporters with mutant 3’UTRs , nucleotides in the target site complementary to the sequence of the miR-31 seed region sequence were mutated using QuikChange Site-Directed Mutagenesis kit according to the manufacturer’s protocol ( Stratagene ) .", "293T cells were seeded in 96-well plates one day before transfection .", "10 ng of each reporter construct was co-transfected with miR-31 mimics or scramble RNA at a final concentration of 50 nM into 293T cells using Lipofectamine 2000 according to the manufacturer’s protocol ( Invitrogen ) .", "After 24 hr , firefly and renilla luciferase activities were measured with the Dual-Glo luciferase assay system according to the manufacturer’s instructions ( Promega ) and then be calculated using this formula ( WT-mimics/WT-mimics NC ) / ( MUT-mimics/MUT-mimic NC ) .", "The primers used for amplifying 3’-UTRs of candidate target genes of miR-31 were as follows: Dkk1-forward: 5’-GCGCTCGAGTGGGCTTGAATTTGGTAT-3’; Dkk1-reverse:5’-TTAGCGGCCGCGTCCCGACTATCCTGTGA-3’; Smad3-forward: 5’-CCGCTCGAGCACCACACCGAATGAATG-3’; Smad3-reverse: 5’-ATAAGAATGCGGCCGCTGGCAATCCTTTACCATAGC-3’; Gsk3b-forward: 5’-TTAGCGGCCGCTCAGTTTCACAGGGTTAT-3’; Gsk3b-reverse: 5’-GCGCTCGAGACAAAGGCATTCAAGTAG-3’; Axin1-forward: GCCTCGAGTCAGTCAGGTGGACAGCC; Axin1-reverse:TAGCGGCCGCACACGGACACTTGGAAGG; Bmpr1a-forward: GCCTCGAGAATTAAACAATTTTGAGGGAG; Bmpr1a-reverse: TTGCGGCCGCCTACAGTTACAAGGTGGAT; Smad4-forward: 5’- TTACTCCTAGCAGCACCC −3’; Smad4-reverse: 5’-CAGTTGTCGTCTTCCCTC-3’; For western blotting assay , intestinal epithelial tissues were lysed in lysis buffer ( Beyotime , China ) with 1% PMSF ( Phenylmethylsulfonyl fluoride ) .", "After quantification using a BCA protein assay kit ( Beyotime , China ) , 30 μg of total protein was separated by 10% SDS-PAGE under denaturing conditions and transferred to PVDF membranes ( GE Healthcare ) .", "Membranes were blocked in 5% nonfat dry milk in incubation buffer and incubated with primary antibodies , followed by incubation with the secondary antibody and chemiluminescent detection system ( Pierce ) .", "The primary antibodies were: anti-GAPDH ( Sigma ) , anti-β-Tubulin ( Sigma ) , anti-CyclinD1 ( Santa Cruz ) , anti-c-Myc ( Santa Cruz ) , anti-β-Catenin ( Sigma ) , anti-Dkk1 ( Santa Cruz ) , anti-Gsk3β ( Abcam ) , anti-Axin1 ( Cell Signaling ) , anti-p-Smad2/3 ( Cell Signaling ) , anti-p21 ( Santa Cruz ) , anti-Smad4 ( Santa Cruz ) , anti-p-Smad1/5/8 ( Cell Signaling ) , anti-Bmpr1a ( Abcam ) , anti-p65 ( Cell Signaling ) , anti-STAT3 ( Cell Signaling ) , anti-p-p65 ( Cell Signaling ) , anti-p-STAT3 ( Cell Signaling ) .", "For irradiation , 2-month-old adult mice were subjected to 12 Gy γ-IR and executed at appointed time .", "Seven week-old control and miR-31−/− mice were intraperitoneally injected with AOM ( Sigma-Aldrich , ) at 10 mg/kg body weight .", "One week after AOM injection , mice were treated with the so-called DSS cycle , comprised of two steps in which mice were fed with 2 . 5% ( w/v ) DSS ( molecular weight 36 , 000–50 , 000 , MP Biomedicals ) for 7 days followed by 14 days of normal water feeding .", "Mice were subjected to a total of three DSS cycles .", "After treatment , mice were sacrificed and distal colon tissues were collected and tumor number and volume were evaluated .", "The transcript of primary miR-31 is located at Chromosome 4 , NC_000070 . 6 ( 88910557 . . 88910662 , complement ) in the mouse genome .", "The upstream 2 kb region of transcript start site ( TSS ) was identified as the miR-31 promoter in this study , which is located at Chromosome 4 , NC_000070 . 6 ( 88910663 . . 88912663 ) and was cloned into the pGL3-Basic reporter constructs .", "The binding site of STAT3 is located at 88911572–88911582 .", "The binding site 1 and 2 of p65 are located at 88912038–88912048 and 88912409–88912419 , respectively .", "ChIP assay was performed according to the manufacturer’s protocol with minor modifications , using Simple-ChIP enzymatic chromatin immunoprecipitation kit ( Cell Signaling Technology ) .", "The sonicated nuclear fractions were divided for input control and for overnight incubated at 4°C with p-STAT3 or the positive control with H3 , negative control with IgG .", "The recruited genomic DNA from the ChIP assays was quantified by qPCR with primers specific to p-Stat3 binding elements of the miR-31 promoter regions .", "Primers were as follows: p-STAT3-binding site forward: 5’-TCCAGGCAAGAAAGTGAGGG −3’; p-STAT3- binding site reverse: 5’- TGAGTAACAGTGCAACAGAGC-3’ .", "The 21nt oligonucleotide miR-31 inhibitor ( 5-AGCUAUGCCAGCAUCUUGCCU-3 ) or negative control Scramble RNA ( 5-CAGUACUUUUGUGUAGUACAA-3 ) were transfected into HCT116 cells with or without CHIR99021 ( GSK3β inhibitor ) .", "The apoptotic cells were evaluated by FITC-Annexin V/PI staining ( BD PharMingen ) and analyzed by FACS ( Becton , Dickinson ) .", "CLIP-PCR assay performed as previously described with modification ( Wang et al . , 2015 ) .", "Cells were treated with scramble RNA or miR-31 inhibitor , and then harvested after being irradiated at 400 mJ/cm2 twice .", "They were then re-suspended in PXL buffer with RNAsin ( Promega ) and RQ1 DNAse ( Promega ) , and spun at 15000 rpm for 30 min .", "Supernatant was collected .", "Protein A Dynabeads ( Dynal , 100 . 02 , Thermo Fisher ) and goat anti-rabbit IgG ( Jackson ImmunoResearch , ) or Ago2 antibody were incubated for 4 hr at 4°C with rotation .", "The supernatant was added to the beads for 2–4 hr at 4°C .", "Beads were then washed twice and digested with Proteinase K ( 4 mg/ml ) for 20 min at 37°C .", "RNA was then extracted using Trizol Reagent ( Invitrogen ) and quantified by qRT-PCR .", "All analyses were performed in triplicate or greater and the means obtained were used for independent t-tests .", "Asterisks denote statistical significance ( *p<0 . 05; **p<0 . 01; ***p<0 . 001 ) .", "All data are reported as mean ±SD .", "Means and standard deviations from at least three independent experiments are presented in all graphs ." ] ]

[ "Intestinal regeneration and tumorigenesis are believed to be driven by intestinal stem cells ( ISCs ) .", "Elucidating mechanisms underlying ISC activation during regeneration and tumorigenesis can help uncover the underlying principles of intestinal homeostasis and disease including colorectal cancer .", "Here we show that miR-31 drives ISC proliferation , and protects ISCs against apoptosis , both during homeostasis and regeneration in response to ionizing radiation injury .", "Furthermore , miR-31 has oncogenic properties , promoting intestinal tumorigenesis .", "Mechanistically , miR-31 acts to balance input from Wnt , BMP , TGFβ signals to coordinate control of intestinal homeostasis , regeneration and tumorigenesis .", "We further find that miR-31 is regulated by the STAT3 signaling pathway in response to radiation injury .", "These findings identify miR-31 as a critical modulator of ISC biology , and a potential therapeutic target for a broad range of intestinal regenerative disorders and cancers ." ]

[ "Cells lining the inner wall of the gut help to absorb nutrients and to protect the body against harmful microbes and substances .", "Being on the front line of defense means that these cells often sustain injuries .", "Specialized cells called intestinal stem cells keep the tissues healthy by replacing the damaged and dying cells .", "The intestinal stem cells can produce copies of themselves and generate precursors of the gut cells .", "They also have specific mechanism to protect themselves from cell death .", "These processes are regulated by different signals that are generated by the cell themselves or the neighboring cells .", "If these processes get out of control , cells can easily be depleted or develop into cancer cells .", "Until now , it remained unclear how intestinal stem cells can differentiate between and respond to multiple and simultaneous signals .", "It is known that short RNA molecules called microRNA play an important role in the signaling pathways of damaged cells and during cancer development .", "In the gut , different microRNAs , including microRNA-31 , help to keep the gut lining intact .", "However , previous research has shown that bowel cancer cells also contain high amounts of microRNA-31 .", "To see whether microRNA-31 plays a role in controlling the signaling systems in intestinal stem cells , Tian , Ma , Lv et al . looked at genetically modified mice that either had too much microRNA-31 or none .", "Mice with too much microRNA-31 produced more intestinal stem cells and were able to better repair any cell damage .", "Mice without microRNA-31 gave rise to fewer intestinal stem cellsand had no damage repair , but were able to stop cancer cells in the gut from growing .", "The results showed that microRNA-31 in intestinal stem cells helps the cells to divide and to protect themselves from cell death .", "It controlled and balanced the different types of cell signaling by either repressing or activating various signals .", "When Tian et al . damaged the stem cells using radiation , the cells increased their microRNA-31 levels as a defense mechanism .", "This helped the cells to survive and to activate repair mechanisms .", "Furthermore , Tian et al . discovered that microRNA-31 can enhance the growth of tumors .", "These results indicate that microRNA-31 plays an important role both in repairing gut linings and furthering tumor development .", "A next step will be to see whether cancer cells use microRNA-31 to protect themselves from chemo- and radiation therapy .", "This could help scientists find new ways to render cancerous cells more susceptible to existing cancer therapies ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "neuroscience" ]

[ [ "Assessment of responses to nociceptive stimuli in either animals or humans is a cornerstone of pain research upon which many inferences from experimental , pharmacological and clinical investigations are based ( Kruger and Light , 2010; Szallasi , 2010 ) .", "However , our appreciation of the full range of behavioral and physiological parameters that comprise a ‘nociceptive response’ remains incomplete , as does our appreciation of the integration of this information at the spinal level .", "This in turn affects the ability to interpret the results of interventions and manipulations of nociceptive circuits and participating molecular circuits .", "Time and magnitude are two , of many , domains in which human and animal behavioral responses are quantified following application of noxious stimuli and from which endpoints can be derived .", "However , when considering acute and chronic pain , responses in the temporal and magnitude domains occupy an extraordinarily broad range .", "Among the earliest , are action potentials from primary afferent nociceptive fibers and reflex withdrawal responses ( Creed and Sherrington , 1926; Jensen et al . , 2015; Levinsson et al . , 2002; Lundberg , 1979; Morgan , 1998 ) followed by limb withdrawal reactions ( Burke et al . , 1971; Lundberg , 1979; Morgan , 1998 ) .", "Over the longer-term , more integrated behavioral endpoints are measurable such as limb guarding , changes in operant assay responding ( Neubert et al . , 2005; Thut et al . , 2007; Murphy et al . , 2014 ) , modification of activity levels ( Cobos et al . , 2012; Dolan et al . , 2010 ) , interference with activities of daily living ( Jirkof et al . , 2013; Rock et al . , 2014 ) , alterations in experimental conditioned place preference ( Wagner et al . , 2014; Xie et al . , 2014 ) , and the production of facial grimacing ( Langford et al . , 2010; Sotocinal et al . , 2011; Matsumiya et al . , 2012 ) .", "Additionally , the plasticity of several of these behaviorally assessed endpoints is evident when testing is performed during ongoing appetitive or attentional behaviors ( Foo and Mason , 2005 , Foo and Mason , 2009; Foo et al . , 2009 ) .", "The fact that a noxious stimulus can be received anywhere on the body suggests that the noxious sensory integrator system is present at all levels of the spinal cord and trigeminal system .", "Ideally , nociception should be integrated with higher order neural circuits related to formulating an effective escape strategy , threat assessment and reference to previous experience and threats .", "Lastly , this system likely overlaps or accesses a generalized motor control circuit geared to generate rapid motor responses to abrupt environmental perturbations that might include slipping or tripping while in motion , and responses to abrupt auditory or visual stimuli and , therefore , can incorporate input from corticospinal , tectospinal , cerebello-spinal , and rubrospinal descending tracts .", "Appropriately integrated motor reactions to these stimuli have strong adaptive value .", "Many of the tests for evaluating baseline pain , sensitization during inflammation , tissue damage , or assessing the actions of analgesic drugs or genetic manipulations , employ acute , episodic stimuli ( Mitchell et al . , 2014 ) .", "Acute stimuli generally involve application of mechanical force , noxious thermal heat , or noxious cold ( Simone and Kajander , 1997; Cain et al . , 2001 ) .", "Even here , the designation of ‘acute’ occupies a broad temporal range , from very brief noxious stimuli such as pin prick , an electrical pulse , or von Frey hairs ( Mitchell et al . , 2010; Chaplan et al . , 1994 ) , to a 100 ms laser pulse , and much longer duration tests employing progressively increasing ramp-style stimuli like the Randall-Sellito paw pressure test ( Randall et al . , 1957 ) , hot plate responses ( Caterina et al . , 1997; Davis et al . , 2000 ) , or the ‘Hargreaves’ thermal withdrawal test ( Hargreaves et al . , 1988; Iadarola et al . , 1988 ) .", "However , the exact behavioral components that comprise , and even precede , the withdrawal endpoint have not been adequately explored .", "We posit that a fine-grained analysis of the sensory-motor integrative responses to nociceptive stimuli is a critical prerequisite for defining the neural circuits that generate noci-responsive behaviors and identifying the underlying molecular , cellular , and circuit-level processes that comprise these neural programs .", "In this report , we show that , in addition to the spinal segmental responses to noxious stimuli , there is a corresponding rapid and simultaneous multi-segmental , multi-limb response .", "Furthermore , we observe that this response can be inhibited by the animal’s posture , providing evidence for a potential endogenous analgesia system that gates access of nociceptive information to the ventral horn motor neuronal withdrawal circuits .", "We show that this gate operates via opioid- and non-opioid-dependent mechanisms .", "Our observations provide several new , largely unexplored behavioral responses that can be used for assessment of acute nociceptive stimuli , effects of pharmacological treatments , modification of circuits by genetic manipulations , and how more chronic manipulations or models affect spinal excitability and the integrated sensory-motor response to noxious test stimuli .", "The system can be viewed as a second gate control for nociception that is permissive to the appropriate flow of information from noxious afferent input to motor output ." ], [ "The results comprise three behavioral endpoints: latency to first movement , pattern of movement for the four limbs and head , and whether the stimulus caused limb withdrawal .", "These endpoints were measured following two stimuli: noxious heat ( laser ) or pinprick ( sharp ) , and under two postural conditions: standing on all four limbs ( quadrupedal stance ) or when standing only on two hindlimbs ( bipedal stance ) ( Figure 1 ) .", "In the latter stance , we also determined the behavioral endpoints in the presence or absence of naloxone . 10 . 7554/eLife . 23584 . 003Figure 1 . Experimental design and protocol .", "( A ) Adult unrestrained rats were placed in a Plexiglas chamber which allows them to move freely .", "The chamber was placed on an elevated glass or a wire mesh surface depending on the stimulus type ( i . e . laser; sharp or blunt ) .", "Stimulus location is indicated by the yellow lightning symbol and the limbs and head are color coded .", "Selective thermal activation was achieved using a brief ( 100 ms ) , small-diameter ( 1 . 6 mm ) , and high-energy ( 5000 mA ) infrared diode laser pulse .", "Pinprick stimuli were applied by pushing a sharp pin against the foot pad until the skin was dimpled but not penetrated and a rounded wooden stick was used for non-noxious blunt stimulus .", "A high-speed camera captured the animal’s postural changes via a mirror under the platform surface .", "The collected data were transferred and stored on a computer for off-line analysis .", "( B ) The experimental protocol had three phases: habituation– allowing the rat to get used to the chamber and the required posture ( on four or two limbs ) ; stimulus – sensory stimulation was delivered to the target limb; response– a 500–800 ms window where any postural changes or movements were documented .", "( C ) A typical response to a thermal Aδ afferent activation .", "A sequence of video frames ( a–d ) demonstrating the response to a laser pulse applied to the left hind limb of rat standing on four limbs ( Video 1 ) .", "For simplicity , only the stimulated limb ( blue ) and the head ( red ) are marked .", "Immediately after the stimulus ( a– 0 ms ) , all limbs are in place and no movement has occurred .", "Bottom left insert: zoom in to the stimulated hindpaw where the black dot represents the laser target .", "The head was the first body part to move", "( b ) , followed by the movement of the stimulated limb", "( c ) while the head continued moving toward the stimulated area .", "Red and blue arrows demonstrate the overall trajectory of the head and left hindlimb movement , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 23584 . 003 The bipedal posture suppresses or delays the withdrawal response to the two noxious stimuli until the animal shifts from a bipedal stance to a quadrupedal stance .", "The withdrawal reaction , when it does occur , is inhibited until the forepaws are placed on the test platform or are about to contact the platform .", "This is evident in the delay to hindlimb withdrawal seen with the sharp stimulus where withdrawal occurs after ~50 ms in the quadrupedal stance , whereas , when the animal is standing on its hindlimbs , the withdrawal of the stimulated hindlimb occurs after ~90 ms . The additional 40 ms allows the animal’s posture to be stabilized ( i . e . all four limbs are in contact with the platform prior to withdrawal ) .", "Further analysis of the trials , revealed three types or levels of withdrawal inhibition when in the bipedal stance: complete , delayed , and partial ( see bar plots in Figure 5Cb and Cd ) .", "Complete inhibition means that no movement of the stimulated limb was seen after nociceptive stimulation .", "Delayed inhibition was characterized by an initial twitch followed by full withdrawal of the limb .", "Partial inhibition was characterized by a twitch , usually of a toe on the stimulated paw , but no withdrawal of the limb .", "At least one of the three types of inhibition occurred in ~50% of all trials administered to rats in the bipedal stance for both thermal laser and sharp stimuli .", "We hypothesized that the postural inhibition and slowing of the behavioral responses to noxious stimuli might result from modulation of an endogenous opioid peptide-mediated suppression of the withdrawal reflex and orienting movements .", "To test this idea , we injected animals systemically with naloxone , an opioid receptor antagonist ( Videos 9 and 10 ) .", "Contrary to our expectation , the response rate to noxious stimuli was unchanged in the presence of naloxone ( laser: 63% , n = 19; sharp 55% , n = 20 , Figure 6A ) when the rat was standing on its hindlimbs , compared to pre-drug control conditions ( laser no drug vs . naloxone , p=0 . 4026; sharp no drug vs . naloxone , p>0 . 4; laser naloxone vs . sharp naloxone , p>0 . 7 ) .", "The presence of naloxone also did not change the average withdrawal latency to thermal laser stimulation when comparing three conditions ( quadrupedal , bipedal , and bipedal + naloxone ) ( Figure 6B ) .", "However , naloxone significantly reduced the latency to the first movement in response to the sharp stimulus ( no drug , quadrupedal: 46 . 9 ± 4 . 0 ms; no drug bipedal: 86 . 3 ± 11 . 0 ms; bipedal with naloxone: 55 . 1 ± 7 . 0 ms , p<0 . 05; Figure 6B ) . 10 . 7554/eLife . 23584 . 020Video 9 . Hindlimb laser stimulus when the rat is rearing up in the presence of naloxone . DOI: http://dx . doi . org/10 . 7554/eLife . 23584 . 02010 . 7554/eLife . 23584 . 021Video 10 . Hindlimb sharp stimulus when the rat is rearing up in the presence of naloxone . DOI: http://dx . doi . org/10 . 7554/eLife . 23584 . 021 One limitation of these studies is that we did not use a vehicle injection as a control for the naloxone injection .", "Injections are anxiogenic and could influence the timing of withdrawal responses to noxious stimuli ( Lapin , 1995 ) .", "We note , however , that the latency of the first response to a thermal stimulus was unchanged following naloxone injection , suggesting that the effects of the injection per se did not alter the responses to all noxious stimuli .", "Despite this observation , we cannot eliminate the possibility that the injection might have influenced the responses to the pin prick stimulus and we consider this potential confound in more detail in the discussion ." ], [ "Systematic evaluation of behaviors evoked by brief thermal and mechanical nociceptive stimuli reveals a coordinated interaction between noxious sensory input and the evoked multisegmental motor responses .", "A new inhibitory gating mechanism was also identified which we refer to as the nociceptive-motor gate control circuit .", "While it is possible to characterize the gate as an analgesic system , its actions are not strictly to suppress ‘pain’ as is commonly conceptualized , but rather to achieve a specific motor goal and , as exemplified by early orientation of the head , to perform a threat assessment .", "The overall function is to enable escape from a noxious stimulus while preserving balance and coordinates the elaboration of a full withdrawal response and , if necessary , additional avoidance behaviors .", "We show that the nociceptive-motor gate control circuit has an opioid component but also requires activation of non-opioid inhibitory elements .", "We enumerate the properties such a nociceptive-motor gate must possess and identify a known spinal integrator system , as fulfilling many of these requirements .", "It is also recognized that further elements of integration exist in the medial dorsal spinal cord such as the spino-cerebellar projections of Clark’s column and projections to lateral reticular nucleus ( Pivetta et al . , 2014 ) .", "The inhibitory component of the nociceptive-motor gate exerts potent control over nociceptive inputs and is activated in a context-dependent fashion .", "Whether the analgesic properties of the nociceptive-motor gate can be adapted for therapeutic purposes is a question open to further investigation ." ], [ "Male Sprague-Dawley rats ( 250–350 g; N = 9 ) were housed under a 12/12 hr light-dark cycle and allowed access to food and water ad libitum .", "The ambient temperature of the holding and testing rooms was 21–22°C .", "Procedures were performed in accordance with the National Institutes of Health ( NIH ) Guidelines for the Care and Use of Laboratory Animals , and approved by the institutional Animal Care and Use Committee .", "All efforts were made to minimize animal numbers and distress .", "Unrestrained rats were placed under plastic enclosures on an elevated glass ( thermal stimulus trials ) or wire mesh platform ( pinprick and blunt stimulus trials ) .", "The enclosures ( 23 × 13 × 13 cm ) were large enough for the rats to move or to stand freely ( Figure 1A ) .", "All animals were habituated to the testing environment for 3 consecutive days prior to commencement of behavioral testing by placing them on the glass test plate under the enclosure for 10 min .", "On test days , stimuli were delivered following a 10-min habituation period .", "Thermal or mechanical stimuli were delivered with the rat's body supported either by all four limbs or only their hindlimbs for a period of at least 3–5 s before administering the stimulus ( Figure 1B ) .", "Responses to both fore- and hindlimb stimulations were explored when the rat was resting on all four limbs .", "When the rat was on its hindlimbs , only its hindlimbs were stimulated .", "In the bipedal stance , behavioral responses to noxious stimuli were also tested after intraperitoneal injection of the opioid antagonist , naloxone hydrochloride dehydrate ( 5 mg/kg ) ( Sigma-Aldrich , USA ) ( van der Kooy and Nagy , 1985; Hylden et al . , 1991; McNally et al . , 2000 ) .", "Naloxone was administered using a 30-g needle attached to a zero-dead volume insulin syringe ( Terumo , Elkton , MD ) , 30 min prior to the experiment to minimize any stress-induced analgesia related to handling and the injection procedure .", "A control , vehicle injection was not performed .", "For thermal stimulation , the laser collimator , which is designed to maintain a constant beam diameter over 2 cm , was attached to a movable support and positioned below the glass , with the 1 . 6 mm diameter beam directed at the tip of a toe .", "The laser device is supplied with a red aiming beam to precisely position the infrared beam on the skin .", "Great care was taken to keep the glass surface dry and free of debris or excrement so that the skin could be stimulated directly by the laser .", "As reported previously ( Mitchell et al . , 2010 , 2014 ) , the response to an infrared laser stimulus that preferentially excites Aδ fibers in normal rats is characterized by rapid paw withdrawal and , with the intensities used , was frequently accompanied by orientation of the head to the stimulated paw followed by paw shaking and licking ( see Video 3 ) .", "To further quantify and characterize this sequence of events , we used a high-speed 12-bit monochrome camera ( AOS Technologies AG , Switzerland ) to record limb and head movements before , during , and after the stimulus .", "A rectangular mirror , positioned at a 45° angle beneath the platform ( Figure 1A ) , was used to direct the image of the animal onto the camera lens .", "Initially , images were acquired at 500 frames per second ( fps ) but additional experiments showed that 130–170 fps could capture the time course of the behavioral sequence with sufficient temporal resolution .", "A recording of 1 to 2 s encompassed the period before stimulation , delivery of the stimulus , and the animal’s response and this recording was then transferred to a computer for subsequent analysis ( Figure 1C ) .", "Recordings commenced when the rats were standing still just prior to the moment of stimulation , irrespective of their initial posture ( e . g . standing on two or four legs ) .", "To quantify the timing of the limb and head movements , frames were counted from the end of the stimulus ( laser , pinprick , or blunt ) to the onset of the first-observable movement or muscle twitch using commercially available software ( AOS Imaging studio V3 . 7 , AOS Technologies AG , Switzerland ) .", "In addition , we determined the time of movement onset and offset for all four limbs and the head .", "The behavioral responses of the stimulated limb were further analyzed and divided into four types of response ( withdrawal , twitch transitioning to withdrawal , twitch only , or no response ) .", "This categorization was based on the effects of the different postural states on the response .", "To represent the temporal relationship of the different limb movements , we constructed limb movement histograms comprised of all assays in which animals responded with a limb lifting from the platform .", "We set bins of 50 ms each with 0 ms corresponding to the onset of the stimulus for ‘Sharp’ and ‘Blunt’ and to the end of the 100 ms laser pulse for ‘Laser’ stimulus .", "A limb was counted as moving in all the bins from the beginning of movement until the time the movement ended or reached the set end time of 500 ms . We also calculated the mean first response latency ( Mean ± SEM ) for each limb and the probability of limb movement in response to the stimulus by counting the number of the trials that each limb demonstrated any type of movement divided by the total number of trials .", "We also broke down the type of movement ( full withdrawal; twitch to withdrawal; twitch only; no movement ) and its probability for the stimulated limb in response to a stimulus .", "Data analysis and plots were done in Matlab ( Mathworks , MA , RRID:SCR_001622 ) and Prism6 ( GraphPad Software , Inc . , CA , RRID:SCR_002798 ) .", "The statistics are listed in the Results section and in the figure legends .", "A post-hoc power analysis was done using G-Power 3 . 1 . 9 . 2 .", "The two-way ANOVA analysis had a power of 0 . 92 for the results presented in Figure 3 and 0 . 71 for the results presented in Figure 6B .", "Despite the low power , the p values for Figure 6B indicated statistical significance ." ] ]

[ "Physiological responses to nociceptive stimuli are initiated within tens of milliseconds , but the corresponding sub-second behavioral responses have not been adequately explored in awake , unrestrained animals .", "A detailed understanding of these responses is crucial for progress in pain neurobiology .", "Here , high-speed videography during nociceptive Aδ fiber stimulation demonstrated engagement of a multi-segmental motor program coincident with , or even preceding , withdrawal of the stimulated paw .", "The motor program included early head orientation and adjustments of the torso and un-stimulated paws .", "Moreover , we observed a remarkably potent gating mechanism when the animal was standing on its hindlimbs and which was partially dependent on the endogenous opioid system .", "These data reveal a profound , immediate and precise integration of nociceptive inputs with ongoing motor activities leading to the initiation of complex , yet behaviorally appropriate , response patterns and the mobilization of a new type of analgesic mechanism within this early temporal nociceptive window ." ]

[ "A bee sting or a pinprick are examples of painful experiences that trigger an immediate response in humans and other animals .", "Scientists have begun mapping how different parts of the nervous system control how the body reacts to pain .", "But there are still many questions about what happens in the very first moments after pain .", "For example , does the response depend on what the body is doing when the painful event occurs ?", "Examining how animals move in response to pain may help answer these questions and possibly point to new strategies for treating pain .", "Now , Blivis et al . show that the nervous system orchestrates a sequence of movements in the whole body in the first 500 milliseconds after a painful event .", "In the experiments , a high-speed video camera recorded what happened when rats experience a pinprick or brief burst from a hot laser on one paw .", "When a rat is on all four paws , it first moves it head and then picks up its foot after one of these painful experiences .", "In fact , the position of the rat’s entire body moves to enable the head to turn towards the source of the pain .", "This may help the rat assess the threat and decide what to do about it .", "When a rat is standing on two hind legs , however , the animal’s pain reaction is delayed until the animal attains a more stable footing .", "The rat puts its front paws down , before moving its foot from the source of the pain .", "Future studies are needed to identify which parts of the brain and spinal cord are active during these early , rapid movements and if something similar happens in humans .", "If a similar process occurs in humans , scientists might be able to develop new pain medications that take advantage of the system that temporarily suppresses the body’s immediate reaction to pain .", "These medications could , in future , be used to treat the heightened sensitivity to pain that can occur after an injury , or the intense “breakthrough” pain experienced by cancer patients that cannot be controlled by their usual pain medication ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "ecology", "microbiology and infectious disease" ]

[ [ "Multispecies microbial communities ( microbiomes ) influence animal biology in diverse ways ( McFall-Ngai et al . , 2013 ) : microbiomes modulate disease ( van Nood et al . , 2013 ) , metabolize nutrients ( Zhu et al . , 2011 ) , synthesize vitamins ( Degnan et al . , 2014 ) , and modify behavior ( Bravo et al . , 2011 ) .", "A central goal in host-microbiome studies is to understand the molecular mechanisms underpinning these diverse microbiome functions .", "Some aspects of microbial community function are the product of inter-species interactions ( Rath and Dorrestein , 2012; Manor et al . , 2014; Gerber , 2014; Gonzalez et al . , 2012 ) .", "For example , microorganisms modulate the metabolomes of neighboring species ( Derewacz et al . , 2015; Jarosz et al . , 2014 ) and microbial metabolites ( e . g . , antibiotics ) alter bacterial transcriptional responses ( Goh et al . , 2002 ) .", "Despite current understanding of microbial inter-species interactions in vitro , some of which has been elucidated in exquisite detail , the consequences of microbial interspecies interactions within host-associated microbiomes are just beginning to be explored experimentally .", "Insight into host-associated microbiome function has stemmed mostly from whole-microbiome [e . g . , re-association of germ-free hosts with whole microbiomes ( Ridaura et al . , 2013 ) and modeling microbiome function based on gene annotation ( Costello et al . , 2009 ) ] or single-microorganism [e . g . , re-association of germ-free hosts with a single microorganism ( Ivanov et al . , 2009 ) ] studies .", "However , these approaches tend to reveal only limited insight into inter-species microbial interactions , which can provide hosts with essential services .", "For example , termite symbionts carry genes necessary for metabolism of different parts of complex carbohydrates ( Poulsen et al . , 2014 ) , yet their function has not been demonstrated in vivo; co-occurring human gut symbionts share polysaccharide breakdown products cooperatively ( Rakoff-Nahoum et al . , 2014 , 2016 ) , but the consequences of such interactions for the host are unknown; inter-species bacterial interactions protect Hydra from fungal infection ( Fraune et al . , 2015 ) , but the mechanism of host protection is unclear .", "The need to understand the effects of inter-species microbiome interactions motivated our current work .", "Attractive model systems in which to study the outcomes of inter-species microbial interactions for host biology would include a tractable host that harbors a simple multispecies microbiome .", "Here , we report the use of Drosophila melanogaster to study interactions in a simple microbiome and their consequences for host behavior .", "The Drosophila microbiome consists largely of yeasts , acetic acid bacteria , and lactic acid bacteria ( Chandler et al . , 2011 , 2012; Broderick and Lemaitre , 2012; Camargo and Phaff , 1957; Staubach et al . , 2013 ) .", "Drosophila ingests microbiome members from the environment ( e . g . , fermenting fruit , [Camargo and Phaff , 1957; Barata et al . , 2012; Erkosar et al . , 2013; Blum et al . , 2013; Broderick et al . , 2014] ) , a behavior posited as a mechanism for Drosophila to select , acquire , and maintain its microbiome ( Broderick and Lemaitre , 2012; Blum et al . , 2013 ) .", "Drosophila behavior toward environmental microorganisms has focused on yeasts ( Becher et al . , 2012; Christiaens et al . , 2014; Schiabor et al . , 2014; Palanca et al . , 2013; Venu et al . , 2014 ) .", "Yeasts attract Drosophila via ester production ( Christiaens et al . , 2014; Schiabor et al . , 2014 ) , induce Drosophila egg-laying behavior ( Becher et al . , 2012 ) , and are vital for larval development ( Becher et al . , 2012 ) .", "Lactic and acetic acid bacteria produce metabolites ( e . g . , acids ) that may repel Drosophila at high acid concentrations , while also inducing egg-laying preference for sites containing acetic acid ( Ai et al . , 2010; Joseph et al . , 2009 ) .", "One motivation of our study was to analyze Drosophila behavior toward the yeast and bacteria that dominate the Drosophila microbiome .", "Yeast and bacteria are largely studied within separate Drosophila sub-disciplines , despite their shared habitat ( Broderick and Lemaitre , 2012 ) .", "Yeasts serve as food , providing Drosophila vitamins , sterols , and amino acids ( Broderick and Lemaitre , 2012 ) .", "Lactic and acetic acid bacteria are gut microbiome members ( Wong et al . , 2011 ) promoting larval development ( Shin et al . , 2011; Storelli et al . , 2011 ) , increasing resistance to pathogens ( Blum et al . , 2013 ) , inducing intestinal stem cell proliferation ( Buchon et al . , 2009 ) , and reducing adult sugar and lipid levels ( Newell and Douglas , 2014; Wong et al . , 2014 ) .", "Since microorganisms that are traditionally considered ‘food’ co-exist with those considered ‘microbiome’ in fruit fermentations and the two groups provide Drosophila with different resources , we hypothesized that Drosophila might detect a beneficial community via metabolites that are produced cooperatively by the desirable symbionts .", "Alternatively , Drosophila might detect a different metabolite as the signal for each symbiont .", "Fruit undergoes a well-characterized ripening process in which cell-wall degrading enzymes and amylases convert the firm , starchy tissue into soft , sugar-rich fruit ( El-Zoghbi , 1994; Abu-Goukh and Bashir , 2003; Mao and Kinsella , 1981 ) .", "The high sugar content supports microbial colonization and fermentation by Drosophila-associated microorganisms , including yeasts , lactic acid bacteria , and acetic acid bacteria ( Barata et al . , 2012; Barbe et al . , 2001 ) .", "Drosophila avoids ‘green’ fruit and is attracted to ‘overripe’ fruit ( Turner and Ray , 2009 ) , yet it is unclear how Drosophila behavior is influenced by the dynamic multispecies fruit microbiome and its metabolic properties .", "To this end , we developed a model fruit fermentation system that afforded measurement of microbial populations , microbial metabolites , and Drosophila behavior .", "Here we demonstrate the importance of emergent microbiome metabolism—quantitatively different or unique metabolites produced by the microbiome , but not by any of its members in isolation—on behavior , suggesting that Drosophila larvae and adults benefit by behaviorally selecting a multispecies , interactive microbiome ." ], [ "To determine whether Drosophila responds to emergent microbial community metabolites , we used the T-maze olfactory assay to analyze Drosophila behavioral responses to several Drosophila microbiome members grown individually or in communities ( Figure 1A , Supplementary file 2 , Figure 1—figure supplement 1 ) .", "When the strains were grown individually , Drosophila was strongly attracted to yeasts , moderately attracted to acetic acid bacteria , and neutral or slightly repelled by lactic acid bacteria ( Figure 1B , C ) .", "Because strains within a microbial group attracted Drosophila similarly , a representative yeast , acetic acid bacterium , and lactic acid bacterium were used to test the effect of interactions between microbiome members on Drosophila behavior .", "Drosophila preferred microbial communities grown together to microorganisms grown individually and then mixed prior to analysis ( defined throughout as a separate-culture mixture , Figure 1D ) .", "Focusing on a model Saccharomyces cerevisiae and Acetobacter malorum community , we found that when tested against apple juice medium ( AJM ) , Drosophila attraction to the community was stronger than to the separate-culture mixture or individual members ( Figure 1E ) .", "In sum , Drosophila detects , and prefers , microorganisms growing together to a mixture of the same strains combined after they had completed growth . 10 . 7554/eLife . 18855 . 003Figure 1 . Drosophila detection of microbe-microbe metabolite exchange .", "( A ) T-maze setup and quantification .", "( B ) Drosophila behavior toward yeasts ( blue ) , acetic acid bacteria ( red ) , and lactic acid bacteria ( brown ) ( Supplementary file 2 ) .", "Mean ± SEM of 12–36 replicates ( n = 2–6 experiments ) .", "Each T-maze replicate uses a technical replicate of a microbial culture and one cohort of Drosophila maintained in separate vials for 3–5 days .", "Mock ( two empty tubes ) , ACV ( 25% apple cider vinegar versus water ) , and benzaldehyde ( 1% versus paraffin oil [PO] ) .", "The one-sample t-test was used to assess the mean deviance from 0 .", "Symbols: NS p>0 . 05; *p≤0 . 05; **p≤0 . 01; ***p≤0 . 001; ****p≤0 . 0001 .", "( C ) Mean Drosophila behavior toward each microorganism was graphed according to microbial group .", "The means were compared by one-way ANOVA with Tukey’s post-hoc comparison .", "( D ) Drosophila behavior toward community combinations of a representative yeast , acetic acid bacterium , and lactic acid bacterium in relation to their separate-culture mixture ( grown individually and mixed; Sc = S . cerevisiae; Am= A . malorum; Lp = L . plantarum cs ) grown for 96 hr; Drosophila preference for the three- versus two-membered community is the last column .", "Mean ± SEM of 12–18 replicates ( n = 2–3 experiments ) .", "The one-sample t-test assessed the mean deviance from 0 .", "( E ) Drosophila olfactory behavior toward the S . cerevisiae and A . malorum community and its constituent parts relative to media grown for 48–60 hr .", "Mean ± SEM of 18–30 replicates ( n = 5 experiments ) .", "A one-way ANOVA followed by post-hoc Tukey’s multiple comparison correction test evaluated whether the means of the experimental groups were different from one another . DOI: http://dx . doi . org/10 . 7554/eLife . 18855 . 00310 . 7554/eLife . 18855 . 004Figure 1—source data 1 . Raw Drosophila preference data for Figure 1B , C . DOI: http://dx . doi . org/10 . 7554/eLife . 18855 . 00410 . 7554/eLife . 18855 . 005Figure 1—source data 2 . Raw Drosophila preference data for Figure 1D . DOI: http://dx . doi . org/10 . 7554/eLife . 18855 . 00510 . 7554/eLife . 18855 . 006Figure 1—source data 3 . Raw Drosophila preference data for Figure 1E . DOI: http://dx . doi . org/10 . 7554/eLife . 18855 . 00610 . 7554/eLife . 18855 . 007Figure 1—figure supplement 1 . Drosophila melanogaster olfactory behavior toward different culture volumes of Saccharomyces cerevisiae and Acetobacter malorum . The top three experimental groups are controls: Mock ( empty tube versus empty tube ) recapitulates alternating of test and control arms , as in all experimental groups; apple cider vinegar ( ACV [25% in water] ) is the positive control and tested against water only; Benzaldehyde ( 1% ) is the negative control and is a 100-fold dilution of benzaldehyde in paraffin oil ( PO ) tested against paraffin oil only .", "In all experimental groups , 10 µl of total volume was used; the culture amount is specified , when appropriate , on the right-hand portion of the plot .", "The remaining volume in the microbial groups is water .", "Media control ( AJM ) is always 5 µl of AJM mixed with 5 µl of water .", "Data points represent the mean +/- SEM combined from three experiments ( n = 12 per experimental group ) .", "A one-sample t-test assessed the mean deviance from 0 .", "NS p>0 . 05; *p≤0 . 05; **p≤0 . 01; ***p≤0 . 001; ****p≤0 . 0001 .", "Based on this analysis , we used a total microbial culture volume of 5 µl throughout the manuscript , unless otherwise noted . DOI: http://dx . doi . org/10 . 7554/eLife . 18855 . 00710 . 7554/eLife . 18855 . 008Figure 1—figure supplement 1—source data 1 . Raw Drosophila preference data for Figure 1—figuresupplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 18855 . 008 We next measured the attractiveness and other properties of the co-culture over time .", "When grown alone , the microorganisms had similar growth profiles ( Figure 2A ) .", "However , when grown with A . malorum , S . cerevisiae populations first increased , then decreased between 60 and 72 hr , and were undetectable by 96 hr ( Figure 2A ) .", "The decrease in S . cerevisiae viable counts mirrored a decrease in pH ( Figure 2—figure supplement 1A ) .", "Drosophila did not prefer the co-culture relative to the separate-culture mixture at 34 hr; however , Drosophila was more attracted to the co-culture from 48–127 hr ( Figure 2B ) .", "Moreover , the Drosophila attraction to the 96 hr co-culture was stronger than its preference for the 48 , 54 , or 60 hr co-cultures ( Figure 2B ) .", "Drosophila preference for the co-culture correlated with lower pH and S . cerevisiae population density , despite Drosophila olfactory avoidance of acid ( Ai et al . , 2010 ) and reliance on yeast for nutrition ( Anagnostou et al . , 2010 ) ( Figure 2C , D ) .", "Drosophila preference did not correlate with viable A . malorum populations ( Figure 2—figure supplement 1B ) .", "Drosophila preference for the co-culture increased relative to sterile media during 34–96 hr of growth , which is consistent with the increase in Drosophila attraction being due to a property of the co-culture rather than to a decrease in attraction to the separate-culture mixture ( Figure 2—figure supplement 1C ) .", "Moreover , Drosophila was more attracted to the 72 hr co-culture than individual cultures or the separate-culture mixture at any other growth stage ( i . e . 24 , 34 , 72 hr; Figure 2—figure supplement 1D , E ) .", "In sum , several properties of the microbial community ( e . g . S . cerevisiae density , pH ) parallel Drosophila detection of , and preference for , the co-culture . 10 . 7554/eLife . 18855 . 009Figure 2 . Drosophila temporal preference for metabolite exchange .", "( A ) S . cerevisiae and A . malorum viable populations .", "Mean ± SEM of 2–3 experiments with one pooled replicate ( 2–3 cultures from the same colony ) per experiment .", "Limit of detection is 20 CFU/mL .", "A curve was fitted to the data with 40 values .", "Subsequently , an exponential plateau equation was compared between the individual cultures from 0 to 72 hr .", "The null hypothesis that the k values are the same was not rejected ( p>0 . 05 ) .", "A separate analysis compared a slope of 0 between S . cerevisiae alone and S . cerevisiae with A . malorum from 48–127 hr .", "The null hypothesis that the slopes were the same was rejected ( p=0 . 0205 ) .", "( B ) Drosophila olfactory behavior toward co-cultured S . cerevisiae and A . malorum versus its separate-culture mixture as a function of culture age .", "Mean ± SEM of 16–18 replicates from three experiments .", "Two statistical tests were run .", "First , a one-sample t-test assessed whether Drosophila was attracted , neutral , or repelled by the test arm by evaluating mean deviance from 0 .", "Symbols: NS p>0 . 05; *p≤0 . 05; **p≤0 . 01; ***p≤0 . 001; ****p≤0 . 0001 .", "Second , a one-way ANOVA followed by Dunnet’s post-hoc multiple comparison test evaluated whether Drosophila was attracted to the co-culture aged 96 hr differently than other aged co-cultures .", "The results are shown in pink; unique letters indicate difference ( p<0 . 05 ) from 96 hr . ( C ) Relationship between pH and Drosophila preference for the S . cerevisiae and A . malorum co-culture versus the separate-culture mixture .", "Each data point represents the pH of a co-culture and the mean RI of Drosophila toward the same co-culture .", "A linear standard curve with an unconstrained slope was generated and compared to a null model with slope = 0 .", "The data fit to an unconstrained slope better than to the null model ( p<0 . 0001 , slope = −0 . 3295 ) .", "( D ) Relationship between S . cerevisiae populations and Drosophila preference for the co-culture versus the separate-culture mixture .", "Each data point represents viable S . cerevisiae populations of the culture along with the mean RI value toward the co-culture containing S . cerevisiae .", "A semilog standard curve with an unconstrained slope was generated and compared to a null model with slope = 0 .", "The data fit to an unconstrained slope better than to the null model ( p<0 . 0001 , slope = −0 . 0349 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 18855 . 00910 . 7554/eLife . 18855 . 010Figure 2—source data 1 . Raw Drosophila preference data for Figure 2B & Figure 2—figure supplement 1C . DOI: http://dx . doi . org/10 . 7554/eLife . 18855 . 01010 . 7554/eLife . 18855 . 011Figure 2—source data 2 . Raw Drosophila preference data , microbial population data , and pH data for Figure 2A , C , D & Figure 2—figure supplement 1A , B . DOI: http://dx . doi . org/10 . 7554/eLife . 18855 . 01110 . 7554/eLife . 18855 . 012Figure 2—figure supplement 1 . Properties of the co-culture and its relationship to Drosophila preference .", "( A ) pH of experimental groups as a function of microbial growth time .", "Mean pH ± SEM of three experiments with one pooled replicate per experiment .", "( B ) Relationship between A . malorum populations and Drosophila preference for the co-culture versus the separate-culture mixture .", "Each data point represents viable A . malorum populations in the co-culture along with the mean RI behavioral value toward the co-culture containing A . malorum .", "A semi-log standard curve with an unconstrained slope was generated and compared to a null model with slope = 0 .", "The data do not fit to an unconstrained slope better than to the null slope = 0 model ( p=0 . 1132 ) .", "( C ) Drosophila attraction to the co-culture versus sterile media as a function of co-culture age ( grown 34 hr – 127 hr ) .", "Mean RI ± SEM of three experiments with 16–18 total replicates .", "A one-sample t-test assessed whether the group means were different from 0 .", "NS p>0 . 05; *p≤0 . 05; **p≤0 . 01; ***p≤0 . 001; ****p≤0 . 0001 Significance is denoted beside or within bars of each experimental group .", "ACV = apple cider vinegar ( 25% in water ) ; PO = paraffin oil .", "( D ) Behavior of Drosophila toward the co-culture grown for 72 hr versus S . cerevisiae alone , A . malorum alone , or the separate-culture mixture grown for different periods of time that correspond to different stages of growth ( e . g . late log , stationary; see [E] ) .", "Mean ± SEM of 11–12 replicates in two experiments .", "A one-sample t-test compared the experimental group means to 0 .", "( E ) Viable populations of conditions in ( D ) Mean ± SEM of 2 pooled replicates where each replicate contains two replicate cultures . DOI: http://dx . doi . org/10 . 7554/eLife . 18855 . 01210 . 7554/eLife . 18855 . 013Figure 2—figure supplement 1—source data 1 . Raw Drosophila preference data and microbial population data for Figure 2—figure supplement 1D , E . DOI: http://dx . doi . org/10 . 7554/eLife . 18855 . 013 Mutants in broadly and narrowly tuned ionotropic and olfactory receptors [Irs and Ors , respectively , ( Abuin et al . , 2011; Hallem and Carlson , 2006 ) ] were used to evaluate the role of Drosophila olfactory reception in discriminating the co-culture from the separate-culture mixture during and immediately following peak attraction ( Figure 3—figure supplement 1 ) .", "During the most attractive phase of the co-culture ( e . g . 67–115 hr ) , homozygous mutants of Drosophila ORCO and Or42b showed a significant reduction in attraction to the co-culture , whereas no role was detected for Drosophila homozygous mutants in several Irs or Or35a ( Figure 3A ) .", "As co-culture growth proceeded ( e . g . 139–163 hr ) , attraction decreased and the role of Or42b and ORCO waned ( Figure 3 ) .", "An independent homozygous mutant of ORCO also showed reduced attraction to the co-culture , whereas the heterozygotes ORCO/+ and Or42/+ were attracted to the co-culture similarly to wild-type flies ( Figure 3B ) . 10 . 7554/eLife . 18855 . 014Figure 3 . Role of olfactory receptor mutants in Drosophila detection of inter-species microbial interactions .", "( A ) The mean rank of the response index of the various Drosophila mutants toward the co-culture was compared with the mean rank of wild-type fly behavior toward the co-culture using the Kruskal-Wallis test followed by Dunn’s post-hoc multiple comparisons testing .", "Symbols: *p≤0 . 05; **p≤0 . 01; ***p≤0 . 001; ****p≤0 . 0001 .", "A lack of symbol indicates no difference when comparing each mutant group to the wild-type group .", "The behavioral responses of all Drosophila ( wild-type and each mutant ) toward the co-culture was greater than 0 ( using the non-parametric Wilcoxon signed rank test in which the medians were compared to 0 , p<0 . 05 , no symbols shown ) .", "Mean +/- SEM of 12–24 replicates per time point per fly condition ( n = 2–4 experiments per time point ) .", "( B ) The mean rank of mutant fly behavior toward the co-culture was compared between wild-type and the specified conditions using the Kruskal-Wallis test followed by Dunn’s post-hoc host multiple comparisons testing .", "Mean +/- SEM of 11–12 replicates ( n = 2 experiments ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 18855 . 01410 . 7554/eLife . 18855 . 015Figure 3—source data 1 . Raw Drosophila preference data and microbial population data for Figure 3A and Figure 3—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 18855 . 01510 . 7554/eLife . 18855 . 016Figure 3—source data 2 . Raw Drosophila preference data for Figure 3B . DOI: http://dx . doi . org/10 . 7554/eLife . 18855 . 01610 . 7554/eLife . 18855 . 017Figure 3—figure supplement 1 . Effect of co-culture age on Drosophila attraction and microbial density .", "( A ) Attraction of wild-type Drosophila to different aged co-cultures ( grown 67–163 hr , S . cerevisiae and A . malorum ) .", "Mean ± SEM of 12–24 replicates per group ( n = 2–4 experiments ) .", "A one-way ANOVA with Tukey’s post-hoc multiple comparisons assessed the difference between all experimental groups .", "B ) Corresponding viable counts at different times of microbial growth .", "Mean ± SEM of 2–3 replicates . DOI: http://dx . doi . org/10 . 7554/eLife . 18855 . 017 ORCO is a required co-receptor for all other Or gene products ( Larsson et al . , 2004 ) and Or42b , one of the most conserved olfactory receptors , detects esters and 1 , 1-diethoxyethane ( Mathew et al . , 2013; Asahina et al . , 2009; Stökl et al . , 2010 ) .", "These results suggest that Or42b enables Drosophila to distinguish the co-culture from the separate-culture mixture .", "Moreover , a non-ORCO factor explains ~40% of Drosophila co-culture preference ( Figure 3A , B ) .", "Previous work found that ORCO is fully responsible for the Drosophila attraction to apple cider vinegar ( Semmelhack and Wang , 2009 ) , suggesting that the behavioral circuit activated by inter-species interactions between S . cerevisiae and A . malorum is distinct from the circuit activated by apple cider vinegar .", "We speculated that the emergent property of co-culture attractiveness might arise from a distinct metabolic profile of the co-culture .", "Using gas chromatography-mass spectrometry ( GC-MS ) , we identified volatiles unique to or differentially produced in the co-culture compared to the separate-culture mixture .", "Five co-culture volatiles ( ethanol , isobutanol , isoamyl alcohol , acetic acid , isoamyl acetate ) were confirmed with standards ( Table 1—source data 1 and 2 ) and quantified with standard curves ( Table 1—source data 3 and 4 ) .", "The alcohol concentrations were lower , and acetic acid and isoamyl acetate were unique in the co-culture relative to the other experimental groups ( Table 1 ) .", "The molecular profile was reminiscent of ethanol catabolism as the unique co-culture metabolic process .", "We therefore hypothesized that ethanol catabolism was the emergent metabolic process . 10 . 7554/eLife . 18855 . 018Table 1 . Summary of volatiles detected using GC-MS .", "Relative abundance of volatiles in the co-culture ( S . cerevisiae and A . malorum grown together ) compared to the separate-culture mixture ( S . cerevisiae and A . malorum grown separately , and their quantities added in during analysis ) .", "GC-MS captured volatiles with XAD-4 beads suspended above the cultures during growth; subsequently , beads were methanol-extracted ( n = 6 experiments , Table 1—source data 1–2 ) .", "Quantification is based on two experiments in which a linear regression was computed with standards ( Table 1—source data 3–4 ) .", "Quantification is based on beads suspended above the cultures between 84 and 96 hr of culture growth . DOI: http://dx . doi . org/10 . 7554/eLife . 18855 . 01810 . 7554/eLife . 18855 . 019Table 1—source data 1 . Extracted ion chromatograms of five metabolites detected by gas chromatography-mass spectrometry ( GC-MS ) in Table 1 . Extracted ion chromatograms of the five metabolites detected by gas chromatography- mass spectrometry ( GC-MS ) .", "( A ) Schematic depicting the experimental setup ( B-F ) Representative extracted ion chromatograms from one replicate ( out of three total ) of one experiment ( out of 3–4 total ) of m/z values corresponding to major metabolites identified in the experimental conditions along with appropriate standards .", "Acetic acid ( B ) , isoamyl alcohol ( C ) , isoamyl acetate ( D ) isobutanol ( E ) , and ethanol ( F ) were identified as the five major metabolites in the co-culture ( S . cerevisiae and A . malorum ) .", "Isoamyl alcohol ( C ) , ethanol ( E ) , and isobutanol ( F ) were identified as the major metabolites in S . cerevisiae grown alone .", "Extracted ion chromatograms were constructed using the m/z value in the title of each graph .", "For acetic acid and isobutanol , the m/z value used corresponds to the molecular weight of the molecule .", "For ethanol , the m/z used corresponds to the molecular weight minus one ( hydrogen ) .", "For isoamyl alcohol , the m/z used corresponds to the loss of the hydroxyl group ( depicted ) , which may have picked up hydrogen and been lost as water .", "For isoamyl acetate , the m/z value corresponds to the molecule shown within the graph .", "In all cases , figures showing the complete mass spectra between the metabolite and standard are found in Table 1—source data 2 .", "Microorganisms were grown 72–96 hr . DOI: http://dx . doi . org/10 . 7554/eLife . 18855 . 01910 . 7554/eLife . 18855 . 020Table 1—source data 2 . Representative spectra of metabolites in Table 1 . Representative spectra of acetic acid ( A-B ) , isoamyl alcohol ( C-E ) , isoamyl acetate ( F-G ) , ethanol ( H-J ) and isobutanol ( K-L ) in standard and experimental samples .", "Standard concentrations are denoted on individual graphs .", "All mass spectra are one replicate ( out of 3–4 experiments with three replicates per experiment ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 18855 . 02010 . 7554/eLife . 18855 . 021Table 1—source data 3 . Linear regression of metabolites using GC-MS in Table 1 . Estimation of volatile quantity using GC-MS .", "Separate experiments are graphed in panels ( A-E ) and ( F-J ) .", "( A-E )", "Data points represent the value of a single replicate per concentration for each standard .", "The abundance of a single m/z value at a specific retention time was chosen for each standard .", "The values were fitted with a linear regression and the equation was used to estimate the concentration of the five metabolites in the experimental samples from the same experiment .", "( F-J )", "Data points represent the mean ± SEM of three replicates for a given concentration for each standard .", "The abundance of a single m/z value at a specific retention time was chosen for each standard .", "The values were fitted with a linear regression .", "The equation was used to estimate the concentration of the five metabolites in the experimental samples from the same experiment .", "When applicable an equation was calculated when the line was forced to go through X , Y = 0 , 0; these equations were used to calculate the concentrations of isoamyl alcohol , isoamyl acetate , and isobutanol . DOI: http://dx . doi . org/10 . 7554/eLife . 18855 . 02110 . 7554/eLife . 18855 . 022Table 1—source data 4 . Raw spectral abundance data as a function of concentration used for linear regressions in Table 1—source data 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 18855 . 022IdentityStandard confirmationRelative quantification ( co-culture: separate-culture mixture ) EthanolY5 . 0–12 . 6-fold reducedIsobutanolY7 . 3–24 . 7-fold reducedIsoamyl acetateYunique to co-cultureIsoamyl alcoholY3 . 6–6 . 4-fold reducedAcetic acidYunique to co-culture We next measured ethanol and acetic acid levels over time ( 24–156 hr ) and compared the chemical dynamics to Drosophila preference .", "Consistent with a relationship between ethanol catabolism , acetic acid anabolism , and Drosophila attraction , the dynamics of Drosophila co-culture preference mirrored ethanol depression and acetic acid accumulation in the co-culture ( Figure 4A ) .", "Furthermore , as ethanol catabolism and acetic acid anabolism proceeded ( 36–96 hr ) , Drosophila attraction toward the co-culture increased until 96 hr , at which point it decreased , consistent with lower turnover of ethanol at the end of ethanol catabolism ( Figure 4A , black line ) . 10 . 7554/eLife . 18855 . 023Figure 4 . Drosophila behavior and ethanol catabolism .", "( A ) Dynamics of ethanol , acetic acid , and Drosophila co-culture preference .", "Acetic acid was only detected in the co-culture .", "The abundance was derived from a linear regression calculated from standards ( Table 1—source data 3 ) .", "Chemical data is the mean ± SEM of two values calculated from two experiments with three replicates per experiment ( except acetic acid and ethanol concentrations at 144 and 156 hr , which are from one experiment with three replicates ) .", "Drosophila co-culture preference is the mean value of the preference shown in Figure 2B .", "The estimated ethanol concentrations in the co-culture and S . cerevisiae culture were compared with multiple t-tests and multiple comparisons correction by the Holm-Sidak method .", "Symbols: NS p>0 . 05; *p≤0 . 05; **p≤0 . 01; ***p≤0 . 001; ****p≤0 . 0001 .", "( B ) Drosophila preference for stages of ethanol catabolism .", "72 hr is ‘mid’ stage; 36 hr is ‘early’ stage and 144 is ‘late’ stage .", "The co-culture contains S . cerevisiae and A . malorum grown for the time indicated .", "AJM= apple juice media .", "Data points represent the mean ± SEM of the combined results of two experiments with 8–10 total replicates per group .", "The one-sample t-test was used to assess the mean deviance from 0 .", "( C ) Drosophila olfactory behavior toward specified conditions .", "Mean ± SEM of 2–7 experiments with 10–42 total replicates .", "Two statistical tests were used to evaluate the behavior .", "First , a one-sample t-test assessed the mean deviance from 0 .", "Symbols: NS p>0 . 05; *p≤0 . 05; **p≤0 . 01; ***p≤0 . 001; ****p≤0 . 0001 .", "Second , a one-way ANOVA with Tukey’s post-hoc comparison assessed whether the means of the bottom three experimental groups were different from one another ( differences are denoted by unique pink letters ) .", "Esters include ethyl acetate , isoamyl acetate , 2-phenethyl acetate , isobutyl acetate , 2-methylbutyl acetate , and methyl acetate; acid is acetic acid .", "Amounts added are based on physiological amounts in co-cultures and are found in Table 2 .", "The co-culture contains S . cerevisiae and the specified A . pomorum strain .", "acid= acetic acid . DOI: http://dx . doi . org/10 . 7554/eLife . 18855 . 02310 . 7554/eLife . 18855 . 024Figure 4—source data 1 . Raw spectral abundance data associated with metabolites graphed in Figure 4A . DOI: http://dx . doi . org/10 . 7554/eLife . 18855 . 02410 . 7554/eLife . 18855 . 025Figure 4—source data 2 . Raw Drosophila preference data for Figure 4B . DOI: http://dx . doi . org/10 . 7554/eLife . 18855 . 02510 . 7554/eLife . 18855 . 026Figure 4—source data 3 . Raw Drosophila preference data for Figure 4C . DOI: http://dx . doi . org/10 . 7554/eLife . 18855 . 02610 . 7554/eLife . 18855 . 027Figure 4—figure supplement 1 . Drosophila behavior toward the co-culture using A . malorum or A . pomorum . Drosophila behavior toward co-cultures grown for 96 hr using A . malorum or A . pomorum versus a media control ( AJM = apple juice medium ) .", "Result of two experiments with six replicates each .", "Data points represent mean ± SEM .", "An unpaired two-tailed t-test assessed the difference between the co-cultures grown with A . malorum or A . pomorum . DOI: http://dx . doi . org/10 . 7554/eLife . 18855 . 02710 . 7554/eLife . 18855 . 028Figure 4—figure supplement 1—source data 1 . Raw Drosophila preference data for Figure 2—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 18855 . 028 We hypothesized that Drosophila preferred the community during peak ethanol turnover ( e . g . co-cultures at ~72 hr of growth ) compared with the community during pre-ethanol catabolism ( e . g . co-culture at ~36 hr of growth ) or during late-stage ethanol catabolism , in which ethanol turnover is low ( e . g . co-culture at ~144 hr of growth; Figure 4A ) .", "Consistent with our hypothesis , Drosophila preferred the co-culture in the middle stage of ethanol catabolism to earlier or later stages of ethanol catabolism ( Figure 4B ) .", "To test directly whether ethanol catabolism underpinned Drosophila co-culture preference , we evaluated Drosophila preference for the co-culture harboring a mutant in adhA , which encodes pyrroloquinoline quinone-dependent alcohol dehydrogenase ( PQQ-ADH-I ) , the enzyme that converts yeast-derived ethanol into acetaldehyde on path to acetic acid ( Shin et al . , 2011 ) .", "Co-cultures using either A . malorum or A . pomorum wild-type ( WT ) along with S . cerevisiae were equally attractive to Drosophila ( Figure 4—figure supplement 1 ) .", "Drosophila preferred the co-culture containing A . pomorum WT versus a separate-culture mixture; however , Drosophila did not prefer the co-culture containing A . pomorum adhA versus a separate-culture mixture ( Figure 4C ) .", "Moreover , Drosophila preferred the co-culture containing A . pomorum WT to the co-culture containing A . pomorum adhA ( Figure 4C ) .", "In sum , ethanol catabolism is necessary for Drosophila to discriminate between the co-culture and the separate-culture mixture .", "We next identified additional metabolites unique to the co-culture using solid-phase microextraction gas chromatography-mass spectrometry ( SPME GC-MS ) .", "Acetic acid , six acetate esters , an acetaldehyde metabolic derivative ( acetoin ) , a putative acetaldehyde metabolic derivative ( 2 , 4 , 5-trimethyl-1 , 3-dioxolane ) , and two unknown metabolites were more abundant in the co-culture relative to the separate-culture mixture or co-culture with A . pomorum adhA ( Table 2 , Table 2—source data 1–6 ) .", "To determine the molecular basis for Drosophila co-culture preference , select metabolites were added to the co-culture containing A . pomorum adhA .", "Esters and acetic acid , but not esters alone , were sufficient to fully restore the attractiveness of the co-culture containing A . pomorum adhA to the co-culture containing A . pomorum WT levels ( Figure 4C ) . 10 . 7554/eLife . 18855 . 029Table 2 . Estimated concentrations of key metabolites in the co-culture using SPME GC-MS .", "Estimated concentrations of differentially concentrated or unique metabolites in the co-culture .", "Linear regression equations ( Lin . reg . eqs . 1 and 2 ) were estimated from individual experiments in which peak areas of different concentrations of metabolites were fitted with a linear regression ( Table 2—source data 2 , 3 , 5 and 6 ) .", "Normalized peak areas correspond to the specified metabolites in co-cultures containing S . cerevisiae and A . malorum .", "Separate estimates were derived from a normalized peak area estimated from a single experiment ( co-culture and standard samples were from a run with similar internal standard signal ) or from the mean normalized peak area estimated from all experiments ( co-cultures were run over four days , standards were run on two days ) .", "The final estimated concentration was an average of all estimated concentrations ( n = 4 estimates ( two from each standard regression equation times two estimates of the normalized peak area ) , except for methyl acetate , n = 2 estimates ) .", "The estimated concentrations ( except acetoin ) were added to the co-culture containing A . pomorum adhA ( Figure 4C ) .", "*Ethyl acetate , acetic acid , and acetoin concentrations were estimated from standards ( Table 2—source data 1 and 4 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 18855 . 02910 . 7554/eLife . 18855 . 030Table 2—source data 1 . Extracted ion chromatograms of differentially emitted or unique metabolites in the co-culture in Table 2 . Extracted ion chromatograms of differentially emitted or unique metabolites in the co-culture according to solid phase microextraction gas chromatography-mass spectrometry ( SPME GC-MS ) .", "Specific metabolites are displayed above each panel .", "For each panel , the left-most plot compares the co-culture containing S . cerevisiae and A . malorum to S . cerevisiae grown alone , A . malorum grown alone , or media ( AJM [apple juice medium] ) ; the right-most plot compares the co-culture containing S . cerevisiae and A . pomorum wild-type to the co-culture containing S . cerevisiae and A . pomorum adhA , since A . pomorum adhA is required for Drosophila co-culture preference ( Figure 5A ) .", "The two plots within the same panel contain the same standard .", "The y-axis for each plot is the ion current for a m/z value that discriminates the metabolite of interest over a specific retention time window .", "The following m/z values were chosen for each metabolite based on standards or , in the cases of putative and unknown metabolites ( I and J ) were chosen from the experimental groups: ( A ) m/z 74 . 04 ( B ) m/z 88 . 08 ( C ) m/z 73 . 03 ( D ) 87 . 05 ( E ) 74 . 02 ( F ) 104 . 04 ( G ) 60 . 05 ( H ) 88 . 05 ( I ) 101 . 06 ( J ) 101 . 06 .", "Each panel is one representative replicate of 1 experiment ( out of 3–5 total replicates in three experiments ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 18855 . 03010 . 7554/eLife . 18855 . 031Table 2—source data 2 . Linear regression of metabolites in defined metabolite mixtures in Table 2 . Normalized peak areas corresponding to metabolites in a defined metabolite mixture ( from SPME GC-MS ) .", "A linear regression was calculated to quantify the metabolites in the co-culture .", "Each concentration is from one replicate .", "A-E and F-I are two separate experiments .", "Linear regression was used to estimate the concentration of the metabolites in the co-culture containing S . cerevisiae and A . malorum ( Table 2 ) and to complement the co-culture containing A . pomorum adhA ( Figure 4C ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 18855 . 03110 . 7554/eLife . 18855 . 032Table 2—source data 3 . Peak area as a function of concentration used to estimate metabolite concentrations in co-cultures in Table 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 18855 . 03210 . 7554/eLife . 18855 . 033Table 2—source data 4 . Extracted ion chromatograms of various m/z values used in . DOI: http://dx . doi . org/10 . 7554/eLife . 18855 . 03310 . 7554/eLife . 18855 . 034Table 2—source data 5 . Peak areas as a function of metabolite concentration used in linear regression in Table 2—source data 2A–E . DOI: http://dx . doi . org/10 . 7554/eLife . 18855 . 03410 . 7554/eLife . 18855 . 035Table 2—source data 6 . Peak areas as a function of metabolite concentration used in Table 2—source data 2F–I . DOI: http://dx . doi . org/10 . 7554/eLife . 18855 . 035MetaboliteLin .", "Reg .", "eq .", "1 Lin .", "Reg .", "eq .", "2 Normalized peak area ( single experiment ) Normalized peak area ( Average , All experiments ) Estimated concentration ( % ) Isobutyl acetateY = 4151X − 0 . 1319Y = 3252X − 0 . 072510 . 291 . 160 . 00023Isoamyl acetatey = 8158XY = 7800X0 . 783 . 80 . 000262-Phenethyl acetateY = 5129X −0 . 04011 Y = 6972X −0 . 2013 1 . 21 . 90 . 000282-Methylbutyl acetate acetateY = 8995X − 0 . 05042Y = 8087X−0 . 13070 . 563 . 10 . 00023Methyl acetateY = 75 . 22X+0 . 004457 NA0 . 0180 . 0400 . 00033Ethyl acetateNANANANA~0 . 02*Acetic acidNANANANA~3 . 0*AcetoinNANANANA~0 . 01* Although acetate and its metabolic derivatives were sufficient for Drosophila co-culture preference , acetaldehyde is a reactive intermediate during ethanol catabolism whose metabolic derivatives might be increased in microbial communities compared with individual microbial cultures .", "Consistent with this idea , acetoin was moderately increased in the co-culture compared with the separate-culture mixture ( Table 2—source data 1 ) ; strikingly , acetoin was increased ~27 fold in the tri-culture ( S . cerevisiae , A . malorum , and L . plantarum ) compared to the co-culture ( Figure 5A , B , Figure 5—figure supplement 1 ) and was attractive to Drosophila ( Figure 5C ) .", "In sum , emergent metabolites from two- and three-membered communities , including acetaldehyde metabolic derivatives , attract Drosophila . 10 . 7554/eLife . 18855 . 036Figure 5 . Acetaldehyde metabolic derivatives as attractive microbial community generated metabolites .", "( A ) Representative chromatogram of m/z 88 . 05 in the tri-culture ( S . cerevisiae-A . malorum-L . plantarum ) compared to the co-culture ( S . cerevisiae and A . malorum ) .", "( B ) Estimated quantification is based on a linear regression of acetoin ( Figure 6—figure supplement 1 ) .", "Relative quantification of acetoin in the tri-culture ( one replicate with A . malorum and one replicate with A . pomorum from separate days ) and the co-culture ( one replicate with A . malorum and two replicates with A . pomorum from separate days ) .", "Difference in peak areas was assessed by an unpaired two-tailed t-test ( **p≤0 . 01 ) .", "( C ) Mean ± SEM of three experiments with 16–18 total replicates .", "A one-way ANOVA with Tukey’s post-hoc multiple comparisons correction assessed the differences between Drosophila behavior toward the co-culture with A . pomorum adhA and esters to various groups in which individual molecular groups were removed or added ( p>0 . 05; *p≤0 . 05; **p≤0 . 01; ***p≤0 . 001; ****p≤0 . 0001 ) .", "Esters include ethyl acetate , isoamyl acetate , 2-phenethyl acetate , isobutyl acetate , 2-methylbutyl acetate , and methyl acetate .", "Esters added are based on physiological amounts in co-cultures and are calculated in Table 2 and Table 2—source data 2 ) .", "Acetoin is added in a similar amount as the tri-culture .", "Sc = S . cerevisiae , Ap = A . pomorum . DOI: http://dx . doi . org/10 . 7554/eLife . 18855 . 03610 . 7554/eLife . 18855 . 037Figure 5—source data 1 . Extracted ion current for m/z 88 . 05 in Figure 5A . DOI: http://dx . doi . org/10 . 7554/eLife . 18855 . 03710 . 7554/eLife . 18855 . 038Figure 5—source data 2 . Peak areas associated with acetoin for Figure 5B . DOI: http://dx . doi . org/10 . 7554/eLife . 18855 . 03810 . 7554/eLife . 18855 . 039Figure 5—source data 3 . Raw Drosophila preference data for Figure 5C . DOI: http://dx . doi . org/10 . 7554/eLife . 18855 . 03910 . 7554/eLife . 18855 . 040Figure 5—figure supplement 1 . Acetoin linear regression . The curve is based on maximum m/z values ( 88 . 05 ) of three concentrations of acetoin .", "One replicate per concentration ( n = 1 experiment ) .", "The linear regression was used to estimate acetoin concentrations in the tri-culture and the co-culture ( Figure 5B ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 18855 . 04010 . 7554/eLife . 18855 . 041Figure 5—figure supplement 1—source data 1 . Extracted ion current for Figure 5—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 18855 . 041 To further investigate the potential role of acetaldehyde and its metabolic derivatives in Drosophila behavior , we performed a dose response in which acetaldehyde was added to the separate-culture mixture ( Figure 6—figure supplement 1A ) to evaluate its ability to induce attractiveness to co-culture levels .", "Even at the lowest tested levels , acetaldehyde supplementation stimulated the separate-culture mixture to attractiveness levels equal to the co-culture ( Figure 6—figure supplement 1A ) .", "Three acetaldehyde metabolic derivatives—acetoin , 1 , 1-diethoxyethane ( an acetal ) , and 2 , 3-butanedione—were sufficient to induce the attractiveness of the separate-culture mixture to levels equivalent to the co-culture containing A . pomorum WT using concentrations of each metabolite at or below the physiological concentration of acetoin found in the tri-culture ( Figure 6—figure supplement 1B ) .", "A pure metabolite mixture comprised of key metabolic groups produced by microbial communities and identified in this study ( esters , acetaldehyde metabolic derivatives , alcohols , acid ) attracted Drosophila similarly to the co-culture ( Figure 6A , B ) .", "Interestingly , the acetaldehyde metabolic derivatives alone were sufficient to attract Drosophila similarly to the co-culture ( Figure 6C ) .", "Moreover , removal of the acetaldehyde metabolic derivatives group alone reduced Drosophila attraction ( Figure 6D ) .", "In sum , acetaldehyde metabolic derivatives are potent Drosophila attractants . 10 . 7554/eLife . 18855 . 042Figure 6 . Drosophila behavior toward 21 metabolite mixtures .", "( A ) Supplementary file 5 contains the concentrations of all mixtures ( in 50% AJM ) .", "The co-culture was grown for 96 hr .", "Mean ± SEM of 4–6 replicates per experimental group .", "Groups were tested over five days .", "( B ) Drosophila attraction to a co-culture grown for 96 hr and metabolite mixture #21 .", "Mean ± SEM of three experiments with 17–18 replicates per group .", "A Mann-Whitney test compared the median values of the co-culture and metabolite mixture #21; the Wilcoxon Signed-Rank test compared the median value of fly behavior toward the co-culture relative to metabolite mixture #21 to 0 .", "( C ) Sufficiency of metabolite groups to attract Drosophila .", "The individual groups are: acetaldaldehyde metabolic derivatives ( 1 , 1-diethoxyethane; acetoin; 2 , 3-butanedione ) ; alcohols ( ethanol; isobutanol; isoamyl alcohol; 2-methyl , 1-butanol; benzeneethanol ) ; esters ( isoamyl acetate; ethyl acetate; isobutyl acetate; 2-phenethyl acetate; butyl acetate; 2-methylbutyl acetate; methyl acetate; phenethyl benzoate; propyl acetate; ethyl isobutyrate; ethyl hexanoate; isovaleric acid; butyl ester; ethyl octanoate; ethyl decanoate; ethyl laurate ) ; and acetic acid ( acetic acid ) .", "Mean ± SEM of 6 replicates of 1 experiment ( except the acetaldehyde metabolic derivative group which is 12 replicates from two experiments ) .", "A one-way ANOVA followed by Dunnet’s post-hoc comparison assessed the difference between the co-culture and all experimental groups .", "NS p>0 . 05; *p≤0 . 05; **p≤0 . 01; ***p≤0 . 001; ****p≤0 . 0001 ( D ) The same groups used in C were used and removed from metabolite mix #21 .", "The difference between the co-culture ( Sc-Am ) and each group was assessed in the same manner as in C . Mean +/- SEM of 6 replicates from one experiment . DOI: http://dx . doi . org/10 . 7554/eLife . 18855 . 04210 . 7554/eLife . 18855 . 043Figure 6—source data 1 . Concentrations of mixtures and raw Drosophila preference data for Figure 6 . DOI: http://dx . doi . org/10 . 7554/eLife . 18855 . 04310 . 7554/eLife . 18855 . 044Figure 6—figure supplement 1 . Acetaldehyde metabolic derivatives can complement the co-culture containing A . pomorum adhA , although their physiological concentrations are unknown .", "( A ) Dose response of acetaldehyde was given to the co-culture containing A . pomorum adhA ( along with a constant dose of 3 . 0% acetic acid ) .", "Metabolite additions were added to the culture in the noted percentages , allowed to sit 35 min at room temperature , mixed 1:1 in water , and assessed for Drosophila attractiveness .", "Mean ± SEM of 1–7 experiments with 12–42 total replicates per group .", "The mean rank of fly behavior toward all experimental conditions was compared using the Kruskal-Wallis test followed by Dunn’s post-hoc host multiple comparisons testing .", "Unique letters indicate difference ( p<0 . 05 ) ( B ) Role of acetic acid , acetaldehyde , or specified acetaldehyde metabolic derivatives in complementing the co-culture containing A . pomorum adhA .", "Acetic acid ( 3 . 0% ) and acetaldehyde ( 0 . 75% ) were added and allowed to sit at room temperature for 35 min , mixed 1:1 in water , and Drosophila attraction was assayed .", "2 , 3-butanedione ( 0 . 15% ) , 1 , 1-diethoxyethane ( 0 . 01% ) , and acetoin ( 0 . 15% ) were added to the culture , mixed 1:1 with water , and Drosophila behavior was assayed .", "Mean ± SEM of 2–7 experiments with 5–6 replicates per experiment .", "The mean rank of fly behavior toward all experimental conditions was compared using the Kruskal-Wallis test followed by Dunn’s post-hoc host multiple comparisons testing .", "Unique letters indicate difference ( p<0 . 05 ) .", "Sc = S . cerevisiae , Ap = A . pomorum . DOI: http://dx . doi . org/10 . 7554/eLife . 18855 . 04410 . 7554/eLife . 18855 . 045Figure 6—figure supplement 1—source data 1 . Raw Drosophila preference data for Figure 6—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 18855 . 04510 . 7554/eLife . 18855 . 046Figure 6—figure supplement 2 . Drosophila behavior toward water amended with nine metabolites ( 9-metabolite mixture ) versus three different apple cider vinegars ( ACV ) , a co-culture ( Sc-Am = S . cerevisiae and A . malorum ) , or tri-culture ( Sc-Am-Lp = S . cerevisiae , A . malorum , L . plantarum cs ) .", "Cultures were grown for 72 hr and mixed 1:1 with water , as in all other experiments .", "Data points represent the Mean ± SEM of two experiments with twelve total replicates .", "A one-sample t-test assessed whether the mean values of the experimental groups were different from 0 .", "NS > 0 . 05; *p≤0 . 05; **p≤0 . 01; ***p≤0 . 001; ****p≤0 . 0001 .", "The acetoin concentration was similar to that calculated from the tri-culture ( Figure 5A , 0 . 3% ) .", "The concentrations of all nine metabolites can be found in the materials and methods . DOI: http://dx . doi . org/10 . 7554/eLife . 18855 . 04610 . 7554/eLife . 18855 . 047Figure 6—figure supplement 2—source data 1 . Raw Drosophila preference data for Figure 6—figure supplement 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 18855 . 047 Overall , our results suggest that both esters and acetaldehyde metabolic derivatives are keystone microbial community metabolites that attract Drosophila .", "We next created a simple 9-metabolite mixture in water ( containing only one acid , four esters , and four acetaldehyde metabolic derivatives ) and measured Drosophila preference toward this mixture in relation to the yeast-acetic acid bacteria co-culture , the yeast-acetic acid bacteria-lactic acid bacteria microbial community , or apple cider vinegar ( ACV ) .", "The defined mixture used concentrations for each acetaldehyde metabolic derivative similar to the concentration of acetoin in the tri-culture and ester and acid concentrations that were in the range detected in the co-culture .", "The defined 9-metabolite mixture was more attractive than all other conditions ( Figure 6—figure supplement 2 ) .", "In sum , acetaldehyde metabolic derivatives and esters are potent Drosophila attractants whose detection may signal the presence of actively metabolizing , multispecies microbial communities .", "We hypothesized that Drosophila preference for communities during peak ethanol turnover reflected fitness benefits derived from ingesting metabolites associated with different staged communities .", "To test this hypothesis , we measured adult Drosophila survival when given ethanol and acetic acid concentrations characteristic of microbial cultures at different stages of ethanol catabolism .", "Adult Drosophila survival was highest when given metabolites associated with middle-staged ethanol catabolism compared with pre- or end-stage ethanol catabolism ( Figure 7—figure supplement 1A ) .", "In sum , Drosophila preference provides benefits associated with consumption of microbial community-generated metabolites .", "Next , we explored the relationship between Drosophila attraction and egg-laying preference .", "Drosophila preferred to lay eggs in the co-culture containing A . pomorum WT to the co-culture containing A . pomorum adhA ( Figure 7A ) .", "Therefore , we predicted that Drosophila larvae would develop more quickly in the wild-type condition than the adhA condition .", "In contrast , we found that larvae develop more slowly when consuming the co-culture containing A . pomorum WT compared with the co-culture containing A . pomomrum adhA ( Figure 7B ) .", "This result may be explained by the fact that A . pomorum WT kills the nutritious yeast cells , whereas the A . pomorum adhA mutant does not ( Figure 7—figure supplement 1B ) .", "Given the role of yeast in Drosophila development ( Becher et al . , 2012 ) the co-culture containing A . pomorum adhA , which supports yeast populations , may be more nutritive for developing Drosophila larvae than the co-culture containing A . pomorum WT . 10 . 7554/eLife . 18855 . 048Figure 7 . Drosophila egg-laying preference , nutrition , and pathogen protection .", "( A ) Drosophila was given a choice to lay eggs in a co-culture containing S . cerevisiae and A . pomorum wild-type ( WT ) or S . cerevisiae and A . pomorum adhA .", "The co-cultures were grown for 96 hr and mixed 1:1 with a 1 . 6% agarose solution .", "Drosophila was allowed to lay eggs for eight hours .", "The Wilcoxon signed-rank test evaluated whether the median value of each experimental group was different from 0 .", "Mean ± SEM of 16–18 replicates from two experiments .", "( B ) Drosophila ( 40 females and 15 males ) deposited eggs in fly vials for 4 hr containing the co-culture of S . cerevisiae and A . pomorum WT ( WT ) or the co-culture of S . cerevisiae and A . pomorum adhA ( adhA ) .", "Subsequently the number of pupae in each condition was monitored over time .", "Mean ± SEM of 5 replicates of 1 experiment .", "Between 12–16 d , larvae pupated in 3/5 WT replicates .", "Multiple unpaired t-tests were used to compare means at each time point .", "*p<0 . 05 .", "( C ) Drosophila ( 40 females and 15 males ) deposited eggs for 4 hr after which the total number of eggs were counted in the two experimental groups .", "Mean +/- SEM of 12 replicates of 1 of 2 representative experiments .", "A Mann-Whitney test compared the medians of each group .", "NS p>0 . 05; *p≤0 . 05; **p≤0 . 01; ***p≤0 . 001; ****p≤0 . 0001 .", "( D ) three days after egg-laying the plates containing eggs quantified in ( C ) were exposed to the open environment and the consequence of exposure was the growth of unidentified fungi , as pictured .", "Control plates that were not exposed to the environment did not harbor any fungi .", "In experiment 1 , 12/12 of the adhA plates harbored fungi and 0/12 plates of WT plates harbored fungi .", "In the second experiment 4/6 adhA plates harbored fungi and 0/6 of WT plates harbored fungi .", "( E , F )", "Following environmental exposure , the eggs were followed through pupation ( E ) and adulthood ( F ) .", "Mean +/- SEM of 12 replicates of 1 of 2 representative experiments .", "The median values in E and F were compared the same way as in C . DOI: http://dx . doi . org/10 . 7554/eLife . 18855 . 04810 . 7554/eLife . 18855 . 049Figure 7—source data 1 . Raw Drosophila egg-laying preference data for Figure 7A . DOI: http://dx . doi . org/10 . 7554/eLife . 18855 . 04910 . 7554/eLife . 18855 . 050Figure 7—source data 2 . Raw developmental data for Figure 7B , C , E , F . DOI: http://dx . doi . org/10 . 7554/eLife . 18855 . 05010 . 7554/eLife . 18855 . 051Figure 7—figure supplement 1 . Impact of co-culture metabolites on adult survival and yeast populations .", "( A ) Drosophila survival in the presence of acetic acid ( AA ) , ethanol ( EtOH ) or the combination of the two in water .", "Groupings were based on concentrations of metabolites estimated from pre-ethanol catabolism ( 9 . 4% ethanol , High EtOH ) , middle-staged ethanol catabolism ( 1 . 4% ethanol , 2 . 8% acetic acid , Mod . EtOH , Mod . AA ) and post ethanol catabolism ( 3 . 42% acetic acid , High AA ) .", "Data represent mean +/- SEM of 5–6 replicates of 1 representative experiment of 2 .", "Mod .", "EtOH , Mod .", "AA was different from High EtOH condition from 108 hr onward and High AA condition from 72 hr onward ( assessed by two-way ANOVA comparing time and condition , p≤0 . 05 , Tukey’s correction ) .", "Negative Cntrl is water and Positive Cntrl is Shield’s and Sang M3 Insect Medium .", "( B ) Photograph of yeast populations of a co-culture containing S . cerevisiae and A . malorum adhA ( left ) and S . cerevisiae and A . malorum WT ( right ) after growing for 72 hr . 50 ul of the culture was spread onto MRS plates containing a cocktail of antibiotics .", "Source Data Titles . DOI: http://dx . doi . org/10 . 7554/eLife . 18855 . 05110 . 7554/eLife . 18855 . 052Figure 7—figure supplement 1—source data 1 . Raw survival proportions for Figure 7-figuresupplement1A . DOI: http://dx . doi . org/10 . 7554/eLife . 18855 . 052 Another potential selective pressure on the choice of egg-laying sites is the presence of pathogens and parasites .", "The presence of parasitoid wasps increases Drosophila egg deposition in high ethanol concentration sites , which are protective to larvae ( Rollero et al . , 2015 ) .", "Drosophila also avoids laying eggs in habitats containing pathogenic molds by detecting geosmin ( Stensmyr et al . , 2012 ) .", "Additionally , acetic acid , a unique metabolite in the co-culture containing A . pomorum WT , inhibits phytopathogenic fungi ( Kang et al . , 2003 ) .", "To test whether the co-culture containing A . pomorum WT protects developing larvae from environmental fungi , we allowed Drosophila to lay eggs in co-culture containing S . cerevisiae and either A . pomorum WT or A . pomorum adhA and quantified the total number of eggs , pupae , and adults .", "We found that Drosophila laid significantly more eggs in the co-culture containing A . pomorum WT than the co-culture containing A . pomorum adhA ( Figure 7C ) .", "Following open-air exposure to environmental microbes , unidentified fungi grew on the co-cultures containing A . pomorum adhA , but did not grow on the co-cultures containing A . pomorum WT ( Figure 7D ) .", "Furthermore , more pupae and adults survived in the co-culture containing A . pomorum WT compared to the co-culture containing A . pomorum adhA ( Figure 7E , F ) .", "In sum , Drosophila egg-laying preference in the co-culture containing A . pomorum WT may reflect an underlying benefit in fungal pathogen defense ." ], [ "Here , we have demonstrated how emergent properties of a microbial community—volatile profile , population dynamics , and pH—influence Drosophila attraction , survival , and egg-laying behaviors .", "Our study is the first to identify the consequences of microbe-microbe metabolic exchange on animal behavior and discovers additional microbial interactions that attract Drosophila for further mechanistic study ( Figure 1D ) .", "Microbe-microbe metabolic exchange generates unique and quantitatively different volatiles from those resulting from individual microbial metabolism ( Table 1 and 2 , Figure 8 ) .", "Acetobacter-generated acetate coupled to Saccharomyces-derived alcohols spawn diverse acetate esters ( Table 1 and 2 ) .", "We hypothesize that more complex and diverse communities , comprising alcohol-producing yeasts , acetate-producing Acetobacter , and lactate-producing Lactobacillus , will generate a wider array of attractive esters ( Figure 8 ) .", "The community of S . cerevisiae , A . malorum , and L . plantarum emitted higher levels of acetoin and attracted Drosophila more strongly than the co-culture of S . cerevisiae and A . malorum ( Figure 1D , Figure 5 ) .", "Acetoin and 2 , 3-butanedione are formed by an α-acetolactate intermediate in bacteria and directly from acetaldehyde in yeast ( Chuang et al . , 1968 ) .", "We therefore hypothesize that communities of yeasts and bacteria may emit high levels of attractive acetaldehyde metabolic derivatives ( Figure 8 ) . 10 . 7554/eLife . 18855 . 053Figure 8 . Model of microbe-microbe metabolite exchange . Bolded are metabolites increased due to microbe-microbe interactions . DOI: http://dx . doi . org/10 . 7554/eLife . 18855 . 053 Previous studies have found that yeasts alone can produce esters in high concentrations ( Becher et al . , 2012; Christiaens et al . , 2014; Schiabor et al . , 2014 ) .", "In this study , we found that S . cerevisiae produced low quantities of esters when grown alone .", "One explanation for the low ester production is that in contrast to previous studies that have used more complex media , we used an apple juice medium that is much lower in nitrogen content .", "Nitrogen content positively correlates with the yeast ester production ( Becher et al . , 2012; Rollero et al . , 2015 ) .", "Our results suggest that environmental nitrogen availability might predict microbial ester production and Drosophila attraction .", "In high nitrogen environments , yeasts likely produce ester compounds and strongly attract Drosophila .", "However , in low nitrogen environments Acetobacter may be responsible for ester production and Drosophila attraction; Acetobacter may be capable of producing esters in low nitrogen conditions or may generate locally high nitrogen environments by assimilating nitrogen from yeast killed by its production of acetic acid .", "Future work should determine the relationship in wild fruit fermentations between nitrogen content and ester production by yeasts and bacteria .", "Drosophila behavioral studies have mostly focused on yeasts .", "Yeasts attract Drosophila and are the preferred substrate for Drosophila to lay eggs ( Becher et al . , 2012 ) .", "However , we find that Drosophila attraction toward the co-culture increases as yeast viability declines ( Figure 2 ) .", "One reason why Drosophila might be attracted to the co-culture as yeast populations decline is that yeasts provide essential nutrients .", "As such , the lysis of viable yeast by Acetobacter may benefit Drosophila through the liberation of nutrients .", "An alternative explanation is that in their interaction with Drosophila , Acetobacter may have benefited by evolving to produce esters that in other contexts ( e . g . high nitrogen environments ) are produced by yeasts .", "The contribution of Drosophila-associated bacteria to Drosophila behavior is not as well understood as yeasts ( Venu et al . , 2014 ) .", "Our results suggest that non-yeast microorganisms , especially when grown in microbial communities , affect Drosophila behaviors .", "We reason that additional studies that couple chemical microbial ecology with Drosophila behavior will herald the discovery of additional microbe-influenced behaviors and microbial community-generated metabolites .", "This study demonstrates the coordination of ethanol synthesis and catabolism by S . cerevisiae and Acetobacter , respectively , and the role of ethanol in Drosophila behavior and survival .", "Non-Saccharomyces Drosophila microbiome members also make ethanol ( Ruyters et al . , 2015 ) and diverse acetic acid bacteria catabolize ethanol , generalizing our findings to other microbial community combinations .", "Ethanol can have deleterious or beneficial fitness consequences for Drosophila depending on concentration ( Ranganathan et al . , 1987; Azanchi et al . , 2013 ) and ecological context ( Kacsoh et al . , 2013 ) .", "Our results are consistent with Drosophila using products of inter-species microbiome metabolism to detect a community that titrates ethanol concentration optimally for the host .", "Work that further dissects the consequences of acetic acid and ethanol concentrations on Drosophila biology and investigates other community-level metabolic profiles will be of interest to enrich the chemical and ecological portrait of the Drosophila microbiome .", "Drosophila egg-laying preference for the co-culture containing A . pomorum WT may provide a fitness tradeoff for the host .", "On the one hand , we observed that juice agar plates inoculated with the co-culture containing A . pomorum WT had fewer viable yeast cells and larvae developed more slowly , likely due to the lower vital nutrients ( e . g . protein , vitamins ) than would be available in the co-culture containing A . pomorum adhA .", "On the other hand , when exposed to environmental microbes , juice agar plates inoculated with the co-culture containing A . pomorum WT were not invaded by fungi , whereas the co-culture containing A . pomorum adhA was susceptible to fungal growth .", "This suggests that in more natural conditions the catabolism of ethanol into acetic acid , which delays larval development in the microbial community studied here ( e . g . in a community with S . cerevisiae ) , ultimately has a protective effect .", "Whether this is due to a direct elimination of pathogens or instead indirectly limits fungal competition , as has been shown for dietary yeasts and Aspergillus sp .", "( Rohlfs and Kürschner , 2010 ) is unknown .", "Future work that more thoroughly dissects the Drosophila fitness tradeoffs that result from its association with different microbiomes is of interest .", "Our work raises questions about the consequences of the observed behavior on microbiome assembly and stability in the Drosophila intestine .", "Drosophila possesses specific and regionalized gut immune responses to the microbiome ( Lhocine et al . , 2008; Ryu et al . , 2008; Paredes et al . , 2011; Costechareyre et al . , 2016 ) implying a tolerant environment in which privileged microbiome members are maintained and reproduce in the Drosophila intestine .", "Other work suggests that Drosophila acquires its adult microbiome from exogenous sources , that adult microbiome abundance drops without continuous ingestion of exogenous microorganisms , and that the microbiome can be shaped by diet ( Chandler et al . , 2011; Blum et al . , 2013; Broderick et al . , 2014 ) .", "As such , a combination of internal mechanisms , exogenous factors , and host behavior likely sculpt the microbiome; determining the relative contribution of each will be important moving forward .", "Complicating our understanding of the contribution of these factors is the opaque distinction between ‘microbiome’ and ‘food’ , since both are ingested from the environment ( Broderick , 2016 ) .", "To dissect the formation and stability of the Drosophila microbiome , the fate of ingested microorganisms needs to be monitored and microbial intestinal replication needs to be surveyed as a function of Drosophila behavior , age , immune status , microbiome membership , and nutritional state [e . g . using synthetic diets without yeast; ( Shin et al . , 2011; Piper et al . , 2014 ) ] .", "In sum , our results support a model in which the Drosophila olfactory system is tuned to fruity ( e . g . , esters ) and buttery ( several acetaldehyde metabolic derivatives , such as 2 , 3-butanedione ) smelling metabolites promoted by microbe-microbe interactions .", "We anticipate that accounting for microbial interactions in diverse host-microbe studies will lead to new insights into diverse aspects of microbial-animal symbioses ." ], [ "Fly stocks , genotypes , and sources are listed in Supplementary file", "1 . Drosophila melanogaster was reared at 25°C on a 12 hr:12 hr light: dark cycle on autoclaved food ( 5% yeast , 10% dextrose , 7% cornmeal , 0 . 6% propionic acid , 0 . 7% agar ) .", "Microorganisms used in this study are listed and described in Supplementary file", "2 . Microorganisms were streaked onto yeast-peptone dextrose ( YPD; 1% yeast extract ( Becton Dickinson , and Company , Franklin Lakes , NJ , USA ) , 2% peptone ( Becton Dickinson , and Company , Franklin Lakes , NJ , USA ) , and 2% dextrose [Avantor Performance Materials , Center Valley , PA , USA] ) or Man , de Rosa , Sharpe ( MRS , Fisher Scientific , Waltham , MA , USA ) plates from a freezer stock .", "The T-maze apparatus was a kind gift of the Carlson Laboratory .", "Flies were wet-starved for 15–26 hr prior to T-maze olfactory experiments by placing flies into vials containing Kimwipes ( Kimberly Clark , Dallas , TX , USA ) soaked with 2 mL of milliQ water .", "Flies were collected within four days ( <65 flies per vial ) of emergence and matured on autoclaved food .", "Flies between 3 and 10 days-old were used in experiments .", "Single microbial colonies were picked from rich media ( MRS and YPD ) plates and grown overnight .", "Cultures were washed 1X in PBS , diluted 100-fold , and 10 µl was aliquoted into 3 mL of apple juice media ( AJM , apple juice ( Martinelli’s Gold Medal , Watsonville , CA , USA ) , pH adjusted to 5 . 3 with 5M NaOH , with 0 . 5% yeast extract ) .", "Media was filtered with a 0 . 22 µM-size pore attached to a 250 mL polystyrene bottle ( Corning , NY , USA ) .", "For co-culture experiments , 1e3-1e5 CFU of each microorganism was placed simultaneously into AJM .", "Microorganisms were grown in 14 mL round bottom polypropylene tubes ( Corning Science , Tamaulipas , Mexico ) at 28°C , 200 rpm for the time noted in individual experiments .", "The microbial culture was diluted 1:1 with sterile milliQ water ( 0 . 22 µM filter [Millipore , Billerica , MA , USA] ) and placed directly onto autoclaved 10 mm round Whatman filter paper ( GE Healthcare Life Science , Pittsburgh , PA , USA ) placed near the bottom of 15 mL CentriStar centrifuge tubes ( Corning , NY ) .", "A total volume of 10 µl was used for all experiments .", "Tubes containing 10 µl of total volume ( 1:1 microbial culture: water ) placed onto 10 mM filter paper and Drosophila were placed into the behavioral room ( 20–25°C , 50–70% humidity maintained by a humidifier ( Sunbeam Tower Humidifier , Boca Raton , FL , USA ) and equilibrated for 10 min prior to the beginning of the experiment .", "Flies ( ~40–130 ) were knocked into the T-maze apparatus and rested for ~1 min .", "Subsequently , the two arms of the T-maze were twisted into the T-maze apparatus and the flies were allowed to choose from the test and control arms for 2 min in the dark .", "No airflow was used in the T-maze assay .", "Troubleshooting experiments in which red light was used to observe Drosophila behavior suggested that Drosophila stopped short of reaching the culture placed on the filter paper at the end of the tube .", "The test arm was alternated from one side of the apparatus to the other every experimental replicate .", "A Response Index ( RI ) was computed to analyze preference for the test arm ( flies in test arm-flies in control arm ) / ( total flies ) .", "Chemicals can be found in Supplementary file", "3 . Selective plates were used to distinguish S . cerevisiae from A . malorum .", "MRS containing 50 µg/mL cycloheximide selected for A . malorum while MRS containing 10 µg/mL chloramphenicol and 20 µg/mL tetracycline selected for S . cerevisiae .", "pH of filtered cultures ( 0 . 22 µM ) was measured using a Beckman Coulter pH meter ( Model Phi510 , Fullerton , CA , USA ) .", "Microbial samples were grown in AJM for a specified amount of time in 14 mL round bottom tubes fitted with an autoclaved tissue strainer ( 250 µM nylon mesh ( Thermo Scientific Pierce , Grand Island , NY ) holding between 0 . 03 and 0 . 05 grams of autoclaved Amberlite XAD-4 resin ( Sigma-Aldrich , St Louis , MO , USA ) prewashed in water and methanol .", "After microbial growth , XAD-4 from two cultures was dumped into an autoclaved glass vial .", "XAD-4 was swirled with 900 µl methanol for 30 s .", "500–750 µl of methanol was removed for GC-MS analysis .", "Quantification for Table 1 was derived from beads suspended above the cultures from 84–96 hr of growth .", "Quantification for Figure 4A was derived from beads suspended above the culture every 12 hr; time points on the graph refer to the end point of the 12 hr span ( e . g . 84 hr corresponds to beads suspended from 72–84 hr of growth ) .", "Samples ( 5 µl of methanol-extracted samples ) were injected into the GC-MS ( Agilent 7890A/5975C ) at 250°C using helium as the carrier gas at a flow rate of 1 . 1 mL per minute ( column head pressure 13 psi ) .", "The following chromatography temperature program was used for experiments to initially identify metabolites in the co-culture and individually grown microorganisms: 40°C for 3 min ramped at 1 . 7°C per minute to 200°C ( held for 3 min ) then to 220°C at 3°C per min and held for a further 5 min .", "The total run time was 111 . 78 min .", "For experiments focused on the five major metabolites , a shorter program was used that maintained the same first 10 min of the previous method ( all five volatiles eluted within 9 min ) .", "The chromatography temperature program was 40°C for 3 min , ramped at 1 . 7°C per min to 46 . 8°C and held for 3 min , then ramped at 60°C per min until 220°C and held for 5 min .", "The total run time was 17 . 9 min .", "The mass spectrometer was run in electron-impact ( EI ) mode at 70 eV .", "The temperatures of the transfer line , quadrupole , and ionization source were 220°C , 180°C , and 230°C respectively .", "The ionization was off during the first 4 min to avoid solvent overloading with a source temperature of 230°C .", "Mass spectra were recorded in the range of 35–300 m/z .", "Full scan mode was used at a scan rate of 6 scans/sec .", "The electron multiplier voltage was set in the relative mode to autotune procedure .", "In the initial experiments peaks were manually picked using Agilent Chemstation Software .", "Volatiles associated with peaks were searched against the National Institute of Standards ( NIST ) 11 database .", "Subsequent experiments focused on the five major volatiles identified in the initial experiments by performing extracted ion chromatograms using an ion that successfully identified a standard at a specific retention time .", "Quantification was performed by tabulating the maximum abundance of the ion at a characteristic retention time and using a linear regression equation from a dose-response of the standards ( Table 1—source data 3 and 4 ) .", "A Waters GCT Premier gas chromatography time of flight mass spectrometer ( Milford , MA ) with a DB-5MS column ( 30m x 0 . 25 mm ID x 0 . 25 μm film thickness; Agilent ) was used .", "Live cultures were transferred to autoclaved glass vials ( 20 mL , 23×75 mm , Supelco , Bellefonte , PA , USA ) with screw caps ( 18 mm , 35 Shore A , Supelco , Bellefonte , PA , USA ) after growing for 72 hr .", "The glass vials containing live microbial cultures were analyzed via a 50/30 μm carboxen/divinylbenzene/polydimethylsiloxane Stableflex solid-phase micro-extraction ( SPME ) fiber .", "The extraction methodology was based on previous studies using SPME to extract volatiles form vinegars ( Callejón et al . , 2008; Xiao et al . , 2011 ) .", "The syringe was inserted through the membrane of the caps and sampled the volatiles for 30 min at 45°C; subsequently , metabolites were desorbed for 30 s at 240C and baked for an additional 4 . 5 min in the injection port .", "The gas chromatograph was fitted with a microchannel plate ( MCP ) detector .", "The temperature program of the column was as follows: 40°C for 5 min , 2 °C a min for 17 . 5 min followed by 25 °C a min for 10 min .", "A final hold time of 5 min at 325°C was used .", "The carrier gas was helium .", "A split ratio of 250 was used based on better peak resolution .", "An internal standard of cineole ( Sigma-Aldrich , St . Louis , MO , USA ) was run with each sample and used to compute relative abundances .", "The mass detector was in the range of 40 to 650 m/z .", "To analyze the data , MassLynx software was used .", "The response threshold was set to an absolute area of 10 . 00 .", "The software automatically picked out peaks and computed peak areas .", "To obtain a relative quantification , peaks were compared across samples and normalized to the internal standard .", "Peaks were first searched against the NIST5 database to identify potential hits .", "Most potential metabolites were confirmed by a standard mixture in 50% AJM .", "The standard mixtures are in Supplementary file", "4 . A co-culture containing S . cerevisiae and A . pomorum WT or A . pomorum adhA was grown for 72 hr before use in the T-maze .", "For the physiological concentrations of acetate-derived metabolites , concentrations were added as in Table 2 and then mixed 1:1 with water prior to behavioral analysis .", "For the acetaldehyde metabolic derivatives chemical complementation group , a 1:1 mixture of the mutant co-culture: water was supplemented with 1 , 1-diethoxyethane , 2 , 3-butanedione , and acetoin at final concentrations of 0 . 01% , 0 . 15% and 0 . 15% , respectively and used immediately in the T-maze .", "Acetic acid and/or acetaldehyde were added to the culture , allowed to sit at RT for 35 min , mixed 1:1 with water and then placed into the T-maze vials .", "Standard curves were used to calculate the concentrations of individual metabolites ( Table 2—source data 2 , 3 , 5 and 6 ) .", "The standard curves were generated on two separate experiments in which 3 concentrations of each standard was used .", "The concentrations of the metabolites were independently calculated from the standard curve equations generated on the two separate days .", "Estimated concentrations from each standard curve equation were averaged ( Table 2 ) .", "The experimental data are based on the peak areas of the S . cerevisiae-A .", "malorum co-culture .", "The 9-metabolite mixture contains 1 . 5% acetic acid , 0 . 0003% isoamyl acetate , 0 . 0003% 2-phenethyl acetate , 0 . 01% ethyl acetate , 0 . 002% ethyl lactate , 0 . 3% 1 , 1-diethoxyethane , 0 . 3% 2 , 3-butanediol , 0 . 3% 2 , 3-butanedione , and 0 . 3% acetoin in filtered milliQ water .", "Adult male flies ( 0–3 d-old ) were collected and matured for one day on fly food .", "Flies were then placed into vials containing kimwipes with 5 mL of either Shields and Sang Insect Medium ( Sigma , St . Louis , MO; positive control ) , MilliQ water ( negative control ) , or MilliQ water with ethanol ( 9 . 4% ) , acetic acid ( 3 . 42% ) , or ethanol and acetic acid ( 1 . 4% and 2 . 8% respectively ) .", "Survival was assessed every 12 hr for 7 d .", "For each condition 5 mL was given at 0 and 12 hr and every 24 hr thereafter .", "Experimental replicates were considered separate vials ( 5–6 per group ) .", "Each replicate contained 8–31 flies .", "Egg-preference assay was adapted from Joseph et al 2009 ( Joseph et al . , 2009 ) .", "Microbial cultures grown for 96 hr were heated to 65°C for 10 min , mixed 1:1 with 1 . 6% agarose and poured into a 35×10 mm polystyrene tissue culture dish ( Fisher Scientific , PA , USA ) separated in two by a straight-edge razor blade .", "Flies were starved for ~18 hr prior to the experiment .", "The 35 mm petri dish was placed within clear flat top boxes with dimensions 2 5/16” X 2 5/16” X 5 1/16” ( TAP plastics , San Leandro , CA , USA ) .", "The test and control sides were alternated for each replicate .", "Drosophila aged 4–10 days ( n = 50–100 ) was allowed to lay eggs for 8 hr .", "After the assay , the number of eggs on deposited on each choice was tabulated and an egg-laying index was computed analogously to the olfactory response index .", "0–3 d-old Drosophila were collected ( 40 females and 15 males per tube ) .", "After three days , each tube of flies was placed into six oz .", "polypropylene square bottom Drosophila bottles ( Dot Scientific Inc . , MI , USA ) in which a 35×10 mm polystyrene tissue culture dish ( Fisher Scientific , PA , USA ) was fitted inside the opening hole .", "The culture dish contained either the co-culture with A . pomorum wild-type and S . cerevisiae or the co-culture with A . pomorum adhA and S . cerevisiae .", "Drosophila was allowed to lay eggs for 4 hr .", "The co-cultures were grown for 72 hr at 28 C , 200 rpm .", "The cultures were mixed 1:1 with 1 . 6% agarose and 4 mL was poured into each 35 mm culture dish .", "The eggs were counted manually immediately following the 4 hr time window of egg-laying .", "The plates were placed into an incubator at 60% humidity and 25C on a 12:12 hr light dark cycle .", "After three days , the plates were exposed to the environment by placing them on the floor with their lids off for 10 min .", "Subsequently , total pupae and adults were counted daily .", "In the case of no open environmental exposure ( Figure 7B ) , the co-culture was mixed with 1 . 6% agarose 1:1 .", "8 mL was distributed into narrow polypropylene fly vials ( 28 . 5×95 mm , VWR , PA , USA ) .", "After 4 hr , adults were removed and the eggs were placed in an incubator at 60% humidity and 25C on a 12:12 hr light dark cycle .", "Data analysis was performed in Prismv6 . 0b .", "Specific statistical tests are noted for individual experiments .", "In behavioral experiments , a Shapiro-Wilk normality test determined whether the underlying data were consistent or inconsistent with a normal distribution .", "If consistent , a parametric test was used to evaluate differences; if inconsistent , a non-parametric test was used ." ] ]

[ "Animals host multi-species microbial communities ( microbiomes ) whose properties may result from inter-species interactions; however , current understanding of host-microbiome interactions derives mostly from studies in which elucidation of microbe-microbe interactions is difficult .", "In exploring how Drosophila melanogaster acquires its microbiome , we found that a microbial community influences Drosophila olfactory and egg-laying behaviors differently than individual members .", "Drosophila prefers a Saccharomyces-Acetobacter co-culture to the same microorganisms grown individually and then mixed , a response mainly due to the conserved olfactory receptor , Or42b .", "Acetobacter metabolism of Saccharomyces-derived ethanol was necessary , and acetate and its metabolic derivatives were sufficient , for co-culture preference .", "Preference correlated with three emergent co-culture properties: ethanol catabolism , a distinct volatile profile , and yeast population decline .", "Egg-laying preference provided a context-dependent fitness benefit to larvae .", "We describe a molecular mechanism by which a microbial community affects animal behavior .", "Our results support a model whereby emergent metabolites signal a beneficial multispecies microbiome ." ]

[ "Animals associate with communities of microorganisms , also known as their microbiome , that live in or on their bodies .", "Within these communities , microbes – such as yeast and bacteria – interact by producing chemical compounds called metabolites that can influence the activity of other members of the community .", "These metabolites can also affect the host , helping with nutrition or causing disease .", "The behavior of an animal may help it to acquire its microbiome , although this has not been properly explored experimentally .", "For example , the fruit fly Drosophila melanogaster acquires members of its microbiome from the microbes found on the fermented fruit that it eats .", "It is possible that the flies – and other animals – respond to microbial metabolites , which act as signals or cues that cause the animal to avoid or seek the microbial community .", "The fruit fly microbiome is commonly studied in the laboratory because it has a much simpler composition than mammalian microbiomes .", "Previous studies have explored how the flies respond to odors produced by individual types of microbes , but none have explored how the behavior of the flies changes in response to the odors produced by a mixed microbial community .", "Fischer et al . now show that fruit flies are preferentially attracted to microbiome members that are interacting with each other .", "The flies detected members of the microbiome by responding to chemicals that are only produced when community members grew together .", "For example , one member of the microbial community produces ethanol that is then converted to acetate by another community member .", "Neither ethanol nor acetate alone attracted flies as strongly .", "Fischer et al . also discovered that both adult fruit flies and their larvae benefit from acquiring a mixture of different microbes at the same time .", "Adult flies benefit by avoiding harmful concentrations of either ethanol or acetic acid , and larvae benefit from developing in an environment that reduces how quickly disease-causing microbes can grow .", "Overall , the results presented by Fischer et al . detail how flies select a beneficial , interactive microbiome from an external reservoir of microorganisms .", "Flies also have internal mechanisms , like their immune system , that help them to select their microbiome .", "Therefore a future challenge will be to integrate the behavioral and internal selection mechanisms into a single model of microbiome acquisition ." ]

[ "Introduction", "Results and discussion", "Materials and methods" ]

[ "developmental biology", "cell biology" ]

[ [ "Embryogenesis requires dynamic interaction between tissues to create changing three-dimensional configurations , culminating in the completion of the body .", "In parallel to the amniote vertebrates ( Calvin and Oyen , 2007 ) , the insects have evolved extraembryonic ( EE ) tissues that arise in early embryogenesis to envelop the embryo ( Panfilio , 2008 ) .", "These are the amnion and the serosa , which are both simple , squamous epithelia .", "Like its vertebrate namesake , in most insect species the amnion encloses a fluid-filled cavity around the embryo .", "As the outermost cellular layer , the serosa provides mechanical and physiological protection ( Farnesi et al . , 2015; Jacobs et al . , 2013; 2014; Rezende et al . , 2008 ) .", "This protective configuration is not permanent , though , and a major reorganization of the EE tissues is essential for embryos to correctly close their backs in late development .", "Reorganization involves whole tissue eversion , contraction , and final apoptosis of both EE tissues ( Panfilio et al . , 2013 ) .", "For these events , perhaps the nearest morphogenetic equivalent in the model system Drosophila is eversion of the wing imaginal disc during metamorphosis , where the squamous peripodial epithelium also exhibits these behaviors ( Aldaz et al . , 2010 ) .", "However , research on Drosophila cannot address the morphogenesis of the two EE epithelia directly , due to the secondarily derived nature of its single EE tissue , the amnioserosa , which does not surround the embryo ( Rafiqi et al . , 2012; Schmidt-Ott , 2000 ) .", "Insect EE withdrawal – the active process whereby the EE tissues withdraw from the embryo and leave it uncovered – has been addressed at the level of gross morphology in classical descriptions for many species ( reviewed in Panfilio , 2008 ) .", "However , a major open question has been the organization and role of the amnion .", "This is primarily because it is difficult to visualize in its native topography with respect to other tissues .", "A lack of amnion-specific molecular markers ( discussed in Koelzer et al . , 2014 ) and the histological similarity and proximity of the serosa ( Panfilio and Roth , 2010; van der Zee et al . , 2005 ) have been particular challenges .", "Here , we present the first clear determination of the relative topography and role of the amnion in late development in a holometabolous insect , the red flour beetle , Tribolium castaneum .", "We characterize an enhancer trap line that labels the amnion and use this in conjunction with recently characterized serosal lines ( Koelzer et al . , 2014 ) to morphogenetically dissect which tissue is responsible for which aspects of EE tissue withdrawal .", "The topographical arrangement of the tissues differs strikingly from what was previously known in hemimetabolous insects and what had previously been hypothesized for Tribolium .", "To better appreciate the implications of this arrangement for morphogenesis , we situate these observations in the larger context of EE development at preceding and following stages .", "This global , mesoscopic approach to evaluating tissue interactions significantly improves our understanding the entire withdrawal process , including the first detailed examination of EE rupture in any insect .", "Furthermore , we provide evidence that while the serosa strongly drives the contraction and folding of the tissues , the amnion initiates EE rupture ." ], [ "To augment the toolkit for tissue-specific visualization in Tribolium , we identified and characterized an enhancer trap line with amniotic EGFP expression ( Figure 1 , see also Figure 1—figure supplement 1 , Video 1 ) .", "Prior to withdrawal morphogenesis , the EGFP-labeled tissue fully envelops the embryo but does not cover the yolk ( Figure 1A–B ) .", "To confirm that this tissue is indeed the amnion , and not a specialized region of the serosa , we examined EGFP expression after RNAi for Tc-zen1 , thereby eliminating serosal tissue identity ( van der Zee et al . , 2005 ) .", "In the absence of the serosa , the amnion occupies a dorsal position over the yolk , and indeed this tissue expresses EGFP ( Figure 1C–D ) . 10 . 7554/eLife . 13834 . 003Figure 1 . The enhancer trap line HC079 is an autonomous amniotic tissue marker . Images are lateral , with anterior left and dorsal up , shown as maximum intensity projections or a mid-sagittal schematic .", "Visualization reagents are indicated .", "( A–B )", "In wild type ( WT ) , EGFP expression is extraembryonic ( EE ) in a ventral domain that fully covers the embryo but not the yolk prior to rupture .", "See also Figure 1—figure supplement 1 , Video 1 .", "( C–D )", "Consistent with the WT EGFP domain being amniotic , the entire EE tissue expresses EGFP when serosal identity is eliminated after Tc-zen1RNAi .", "Here , the EE tissue does cover the yolk ( dorsal to the cardioblast cell row: double-headed arrow ) , and , during the mid-withdrawal stage shown here , acquires a diagnostic ‘crease’ ( arrow ) ( Panfilio et al . , 2013 ) .", "Scale bars are 100 µm .", "Abbreviations: H , head; T3 , third thoracic segment . DOI: http://dx . doi . org/10 . 7554/eLife . 13834 . 00310 . 7554/eLife . 13834 . 004Figure 1—figure supplement 1 . Developmental time course of EGFP expression in the enhancer trap line HC079 . ( A–B ) Here we provide a first characterization of the new enhancer trap line HC079 .", "See Koelzer et al . ( 2014 ) for mapping technique , original staging definitions , acquisition parameters , and analysis method .", "HC079 was mapped to a gene-poor , intergenic region on chromosome 3 .", "EGFP signal is first detected in the amnion during germband retraction , becoming increasingly bright throughout the tissue up to the time of serosal rupture , which heralds the onset of withdrawal morphogenesis .", "Relative EGFP signal quantification is plotted as the mean ± standard deviation during a 48-hr time-lapse recording , with corresponding values for landmark developmental stages ( GBE , germband extension; GBR , germband retraction; SR , serosal rupture; MT , muscle twitches ) .", "Note that 'serosal rupture' is the staging term but in fact refers to first discernible rupture of both EE tissues at the beginning of withdrawal morphogenesis .", "Landmark stage values are also based on nGFP embryos that were recorded in parallel for calibration ( combined n = 7–23 embryos per stage ) .", "Vertical red lines indicate the mean SR age .", "In A , relative EGFP signal is the mean grey value ( n = 10 ) , shown for minimum age ( from a four-hr egg collection ) relative to serosal rupture .", "In B , signal is the integrated density of the posterior and anterior egg halves ( 0–50% vs . 50–100% egg length ) for embryos recorded in both lateral and ventral aspect ( n = 5 ) , with standard deviation shown on only one side of each plot for clarity .", "Here , the x-axis shows minimum absolute age in hours after egg lay ( hours AEL ) .", "Vertical black lines and asterisks indicate the first time points at which the anterior and posterior EGFP values differ significantly ( *: p<0 . 05 , **: p<0 . 01 for paired , two-tailed t-tests ) .", "Although this difference in part reflects the relative area of visible amnion in lateral aspect ( greater anteriorly ) , the trend still holds when only embryos in ventral aspect are considered ( uniform relative amnion surface area ) .", "( C ) In our examination of rupture initiation in the amnion ( HC079 ) × heart ( G04609 ) cross compared to the serosa ( G12424 ) × heart ( G04609 ) cross , we found that the amnion cross develops at a faster rate , shown here as the minimum age at the time of serosal rupture ( x-axis; amnion cross: 3007 ( median ) ± 81 ( MAD ) min , n = 28; serosal cross: 3168 ( median ) ± 85 ( MAD ) min , n = 30; p = 4 . 942e-06 , Mann-Whitney U test ) , shown in relation to the duration of rupture initiation ( y-axis: same as main text Figure 4F ) .", "However , even when a normalization factor was introduced to adjust for developmental rate , rupture initiation is longer in the amnion background .", "( D–I )", "Selected still images of an example embryo in lateral aspect ( maximum intensity projections of deconvolved data; anterior left , dorsal up ) , showing EGFP signal predominantly in the amnion as well as in the eye ( E ) and multiple ringed domains within the legs ( arrows ) .", "After dorsal closure ( I ) , weak EGFP signal is also seen segmentally and in the usual nervous system tissues in which the core 3xP3 promoter drives expression ( Koelzer et al . , 2014 ) .", "Scale bar ( shown in D ) is 100 µm .", "The double-headed arrow in D marks the posterior tip of the abdomen , indicating that the embryo is midway through germband retraction at this time .", "See also Video 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 13834 . 00410 . 7554/eLife . 13834 . 005Video 1 . Time course of EGFP expression in the Tribolium enhancer trap line HC079 . The embryo is shown in lateral aspect with anterior left and dorsal up .", "Strengthening EGFP signal is specific to the amnion during germband retraction and up to the point of EE tissue rupture and withdrawal morphogenesis .", "In the later stages , additional embryonic expression domains occur in the eyes and legs .", "Throughout the movie , wandering yolk globules also exhibit low levels of EGFP , a feature observed for other enhancer trap lines from this screen ( Koelzer et al . , 2014 ) .", "The time stamp specifies minimum age from a four-hour egg collection , in hours after egg lay ( h AEL ) .", "Maximum intensity projections from a deconvolved z-stack ( 5 µm step size for a 60 µm stack ) recorded every ten minutes over a 48-hr time-lapse at 30ºC , acquired with an inverted DeltaVision RT microscope ( Applied Precision ) .", "Scale bar is 100 µm .", "Selected still images are shown and further described in Figure 1—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 13834 . 005 We then used the tissue-specific EE imaging lines to address the arrangement of the amnion and serosa during late development .", "Initially the two EE tissues are physically separate ( Handel et al . , 2005 ) , but they progressively come together during early germband retraction until the only clearly discernible amniotic region is a rim of tissue at the embryo’s dorsal margin ( Figure 2A-A2 ) .", "Previous histological studies in Tribolium and other holometabolous insects concluded that the region of overlap throughout the ventral half of the egg comprised a single EE cell layer , the EE tissues having intercalated or otherwise 'fused' ( e . g . , Kobayashi and Ando , 1990; Patten , 1884; van der Zee et al . , 2005 ) .", "Alternatively , this structure was interpreted as only serosa ( Panfilio et al . , 2013 ) , as the underlying amniotic region undergoes apoptosis in a hemimetabolous insect , the milkweed bug Oncopeltus fasciatus ( Panfilio and Roth , 2010 ) .", "In fact , we find that both tissues persist as apposed and very thin but distinct squamous epithelial layers that are evident in both sectioned and whole mount material ( Figure 2B–E , Figure 2—figure supplement 1 ) .", "Apposition occurs over the entire surface area of the amnion except its dorsal rim , such that the serosa and amnion form an extensive epithelial bilayer .", "Several hours after the completion of germband retraction , the EE membranes rupture and withdraw from the embryo ( Koelzer et al . , 2014 ) .", "Interestingly , the amnion-serosa bilayer is maintained throughout withdrawal morphogenesis , with all major folds and minor bends involving both EE tissue sheets ( Figure 3 ) .", "The new amnion EGFP line thus sheds light on EE tissue structure during withdrawal: rather than a single , pseudostratified tissue , each EE tissue persists as a monolayer , representing a novel EE tissue organization compared to what is known for other species .", "Given that the amnion persists as a discrete tissue throughout withdrawal morphogenesis , we then investigated its role in this process , starting with its structure at the onset of rupture . 10 . 7554/eLife . 13834 . 006Figure 2 . The amnion and serosa form a bilayer during the germband retraction stage . Images are lateral ( A–C , E ) or ventral-lateral ( D ) , with anterior left and dorsal up .", "( A ) During germband retraction , the amnion progressively comes together with the serosa , shown sagittally at an intermediate stage when the tissues are apposed anteriorly ( A1 ) but still separate posteriorly ( A2 ) .", "Fuchsin preparation causes embryo shrinkage ( Wigand et al . , 1998 ) , amplifying apparent amniotic cavity volume , but without altering tissue topography , which is consistent across dozens of stage-matched specimens .", "( B–D )", "Before rupture , the ventral EE tissue under the eggshell ( autofluorescent vitelline membrane ) is comprised of distinct serosal ( outer ) and amniotic ( inner ) layers .", "In sagittal sections ( B , C ) , tissue-specific EGFP labels continuous tissue sheets , while nuclei ( DAPI stain ) of the apposed EE tissue remain EGFP-negative and in a separate layer ( inset schematic ) .", "Maximum intensity projections ( D ) also show two epithelial layers , which can be distinguished by tissue-specific cellular morphologies .", "The box in the first panel indicates the magnified region in D1-D3 .", "Amnion-specific EGFP illuminates the nuclei and cell boundaries in this tissue ( D1 , D1′ ) .", "A phalloidin counterstain shows a complex network of F-Actin filaments , including weak signal for amniotic cell boundaries ( orange arrowheads ) , and a distinct pattern of thicker filaments in a double-walled , polygonal arrangement that corresponds to serosal cell boundaries ( D2 , D2′ , see Figure 2—figure supplement 1 for serosal EGFP labeled specimens and additional details ) .", "Finally , comparison of a nuclear counterstain ( DAPI ) with the EGFP nuclear signal distinguishes the nuclei of the two EE tissues ( here , EGFP-negative nuclei are serosal , shown in D2′ ) , providing the information for a schematic overlay of these distinct tissues ( D3 , D3′: outlined cells are the same as those indicated by arrows in the previous panels ) .", "( E ) The EE bilayer is illustrated in mid-sagittal view of the anterior , according to the color scheme in Figure 1B and with the serosa shown in blue .", "Scale bars are 100 µm ( A ) , 50 µm ( A1-A2 , D ) , and 10 µm ( B , C , D1-D3 ) .", "Abbreviations: A7 , seventh abdominal segment; Am , amnion; GBR , germband retraction; M , mandible; Ser , serosa; Vm , vitelline membrane; and as defined in Figure 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 13834 . 00610 . 7554/eLife . 13834 . 007Figure 2—figure supplement 1 . The amnion and serosa form a persistent bilayer comprised of two morphologically distinct epithelia , including in the anterior-ventral rupture competence zone . Although EGFP intensity varies between cells , EGFP signal for a single EE tissue type labels all cells within a continuous epithelial sheet .", "Moreover , the amnion and serosa have strikingly different cellular morphologies , enabling cells of both tissues to be distinguished , with the serosa overlying the amnion .", "( A–C )", "Both EE tissues can be discerned as continuous epithelia within the rupture competence zone ( shown after the completion of germband retraction , at 44–48 hr after egg lay ) .", "Shown are two examples with serosal EGFP ( A–B ) and one with amniotic EGFP ( C , reproduced from main text Figure 2D , for comparison ) .", "'Overview' images show lateral , ventral , and ventral-lateral views of maximum intensity projections ( A1 , B1 , C1 , respectively ) with anterior left and the region of interest around the head appendages indicated by the blue boxed region ( due to the curvature of the egg , only the regions actually visible in high-magnification projections of thinner z-stacks are indicated ) .", "'Merge' images ( A2 , B2 , C2 ) show maximum intensity projections of the indicated region , labeled for F-Actin ( red , phalloidin ) , nuclei ( blue , DAPI ) , and the specified EE tissue ( green , EGFP ) .", "Note that embryonic-specific signal ( F-Actin and DAPI signal for small cells not in contact with the tissue at the egg surface ) was manually deleted from deeper optical sections with the freehand selection tool in ImageJ before rendering the projected images shown here .", "'Schematic' images ( A3 , B3 , C3 ) show cell and nuclear outlines .", "Cell outlines were determined by EGFP signal ( for A3 , B3 , C3 ) and F-Actin signal ( for C3 , for serosal outlines only ) .", "Nuclear outlines were determined by EGFP signal or DAPI ( for all EGFP-negative nuclei only ) .", "Serosal cells are shown in cyan or blue; amniotic nuclei and cells are shown in orange or white .", "Dashed outlines highlight individual serosal ( red ) and amniotic ( orange ) cells , which are also labeled by arrows of the same color in the single-channel micrographs for EGFP ( A4 , B4 , C4 ) and F-Actin ( A5 , B5 , C5 ) .", "Strong F-Actin signal correlates with serosal cell shape ( compare A4 , A5 and B4 , B5 ) , such that F-Actin signal can be used to infer serosal cell shape even when that tissue is not labeled with EGFP ( C3 , C5 ) .", "Furthermore , the F-Actin-inferred serosal cell outlines correspond to EGFP-negative nuclei in the amnion enhancer trap line ( C3 ) .", "Note that in order to preserve anterior-ventral EE tissue structure and topography , the specimens presented here ( A–C ) are still within the vitelline membrane .", "After fixation , embryos were cut in half ( transversely ) with a razor blade to allow the phalloidin and DAPI staining reagents to penetrate into the sample .", "( The EGFP is the endogenous signal . ) ( D–E ) .", "For over a day prior to rupture , no cell mixing or change in cell number occurs in either the serosa ( D ) or the amnion ( E ) .", "Selected stills from time-lapse movies are shown at the times indicated relative to rupture ( at 30°C , z-stacks acquired every 2 min ) , in anterior aspect and oriented with the ventral region uppermost , with light sheet illumination coming from above .", "Individual nuclei were tracked hourly ( uniquely colored tracks ) , showing the static nature of serosal cells until the time of rupture , while amniotic nuclei move much more within the cells and the amniotic tissue itself shifts slightly anterior-dorsally .", "The initial opening at the time of rupture is indicated with an asterisk ( D4 , E4 ) .", "Axes were determined based on weak embryonic signal ( D5 , E5; the withdrawing edge of the amnion is still visible in E5 ) .", "Images shown here are maximum intensity projections with gamma corrections 0 . 5 ( serosa ) and 0 . 7 ( amnion ) as well as brightness correction to accommodate for changes in signal intensity of these lines during development ( see Figure 1—figure supplement 1 and Koelzer et al . , 2014 ) .", "For these specimens , cellular details at the time of rupture are shown in main text Figure 4D–E .", "Acquisition details are specified in Supplementary file 1 .", "( F ) The bilayer arrangement of the amnion and serosa can also be seen in transmission electron micrographs of sagittal sections ( examined after the completion of germband retraction , at 44–48 hr after egg lay; see also main text Figure 2B–C ) .", "The red box on the semithin section ( 1 µm thick , stained with toluidine blue ) approximately indicates the anterior-ventral region shown in F2–F3 .", "The arrow highlights an intercellular junction within the serosa ( F2 ) , and false coloring highlights the continuity of the two distinct epithelial layers ( F3 ) .", "Both EE tissues have very flat cells , with an apical-basal thickness of only ~200 nm in the amnion and ~450 nm in the serosa , in the aponuclear region shown here .", "Ultrathin sections ( 50–100 nm thick ) were cut with an 'Ultra' diamond knife on a Leica Ultracut UCT microtome , and examined with a Zeiss EM 109 electron microscope .", "Scale bars are 50 µm ( A1 , B1 , C1 , D1–D5 , E1–E5 ) , 20 µm ( A2–A5 , B2–B5 , C2–C5 ) , and 1 µm ( F2–F3 ) .", "Abbreviations: An , antenna; Am , amnion; D , dorsal; H , head; L , left; Lr , labrum; M , mandible; Mx , maxilla; R , right; Ser , serosa; T2 , second thoracic segment; V , ventral; Vm , vitelline membrane . DOI: http://dx . doi . org/10 . 7554/eLife . 13834 . 00710 . 7554/eLife . 13834 . 008Figure 3 . The extraembryonic bilayer moves as a single unit throughout late morphogenetic remodeling . Images are lateral , with anterior left and dorsal up , shown as maximum intensity projections ( A , D ) , sagittal optical sections ( B , C , E , F ) , or as a mid-sagittal schematic ( G ) .", "( A–G )", "The bilayered EE structure is maintained during early serosal compaction .", "The embryos shown here are at a stage when the serosa folds medially , such that mid-sagittal sections show anterior and posterior arms to the folding tissue ( B , E , G ) .", "Higher magnification images of cellular structure focus on the posterior arms ( C , F ) .", "Panels A–C and D–F each show a single embryo .", "Annotations: arrow , ruptured edge of both tissues; double-headed arrow , double bend in the tissues; dashed green lines delimit EGFP domains .", "Scale bars are 100 µm ( A , D ) , 50 µm ( B , E ) , and 10 µm ( C , C′ , F , F′ ) .", "Abbreviations and schematic color scheme as in previous figures . DOI: http://dx . doi . org/10 . 7554/eLife . 13834 . 008 While serosal cells overlying the amnion are fairly uniform in size and shape ( Figure 4A-A2 ) , close inspection of the amnion EGFP signal shows that an anterior-ventral cap of amnion cells is morphologically distinct ( Figure 4B ) .", "These large cells are rounder and have sharper cell outlines compared to cells elsewhere in the tissue , which are striated and elongated along the dorsal-ventral axis .", "The anterior-ventral amniotic cells also have brighter EGFP signal ( Figure 1—figure supplement 1 ) .", "Moreover , we consistently observe that EE rupture begins within this territory ( Figure 4C , see also Video 2 ) .", "Definitive opening only directly affects a handful of amniotic cells , and the exact location of opening within the anterior-ventral cap varies from lateral ( Figure 4C ) to more medial and anterior sites ( e . g . , Figure 2—figure supplement 1D–E ) .", "This suggests that the entire anterior-ventral cap constitutes a rupture competence zone . 10 . 7554/eLife . 13834 . 009Figure 4 . Rupture dynamics: amniotic initiation . Images are maximum intensity projections in lateral ( A–C ) , anterior ( D–E ) , or anterior-ventral ( G ) views .", "( A ) The serosa maintains a homogeneous cellular morphology throughout the region apposed to the amnion ( below the dashed white line , compare with Figure 1A′ ) , shown in detail for anterior ( Ant . , A1 ) and posterior ( A2 ) regions .", "( B–C )", "In contrast , amnion-EGFP signal before ( B ) and during ( C ) rupture highlights an anterior-ventral cap of morphologically distinct cells ( arrows ) ; the asterisk marks the tissue opening ( see also Video 2 ) .", "( D–E )", "Cell shape changes around the site of opening ( asterisk , red cells ) differ markedly between amniotic cell contortions ( D ) and more regular serosal cell shapes ( E ) .", "Colors mark unique amniotic cells or groups of 3–4 neighboring serosal cells .", "The nature of opening and cellular connections is further described in the main text , and long-term cell tracking is shown in Figure 2—figure supplement 1 .", "Time stamps are in minutes:seconds at 30°C , relative to rupture at 0:00 .", "The entire interval from initial opening until the EE tissue fully cleared the head was 8 min ( D ) and 4 min ( E ) .", "( F ) Box plot showing that the rupture initiation interval , as defined in the Materials and methods section , is longer in the amnion ( values are median ± median absolute deviation , at 27 . 5–28ºC ) .", "( G ) Tissue opening in a heterozygote embryo permits tracking of the ruptured EE tissue relative to embryonic anatomical landmarks ( 'T1' labels the proximal region of the first leg pair ) .", "The colored time scale shows duration of track segments over 36 . 7 min at 19 . 5 ± 1°C .", "Along the time scale and superimposed on the embryo are the site of rupture ( white asterisk ) and the withdrawing EE tissue edge ( white lines , line with arrowhead marks time point shown ) .", "Tracks are shown for selected nuclei .", "For comparison , rupture is also indicated along the time scale for a morphologically stage matched embryo with serosal EGFP ( blue asterisk; see also Figure 4—figure supplement 1 , Video 3 ) , which shows a shorter rupture initiation interval ( interval from the asterisk to the right end of the colored time scale ) , consistent with the data in panel F . Scale bars are 100 µm ( A , C ) and 50 µm ( A1–A2 , B , D1–D2 , E1–E2 , G ) .", "Panel B shows the same embryo as in Figure 1A . DOI: http://dx . doi . org/10 . 7554/eLife . 13834 . 00910 . 7554/eLife . 13834 . 010Figure 4—figure supplement 1 . Comparison of early tissue opening in the amnion and serosa .", "( A–B )", "The embryos are heterozygotes expressing EGFP in the specified extraembryonic tissue and in the embryonic landmark domains of the 'heart' enhancer trap line .", "They are shown in anterior-ventral views as maximum intensity projections of single time points taken from z-stacks acquired every 20 s with a light sheet microscope .", "Light sheet illumination is coming from above in both recordings ( acquisition details are specified in Supplementary file 1 ) .", "Image A is reproduced from main text Figure 4G for side-by-side comparison with image B . The colored time scale shows the duration of track segments over 36 . 7 min at 19 . 5 ± 1°C .", "The embryos are stage matched for the degree of EE tissue withdrawal by the end of the 36 . 7-min interval , when the EE tissue has cleared the proximal portion of the first leg pair ( T1 ) .", "Along the time scale and superimposed on the embryo are the site of rupture ( white asterisk ) and the withdrawing EE tissue edge ( white lines , line with arrowhead marks time point shown ) .", "In the main text , statistical analysis of the duration of 'rupture initiation' was based on embryos viewed in lateral aspect and was defined as the time from first discernible opening to when the EE tissues cleared the embryo’s head dorsally ( see Materials and methods , main text Figure 4F ) .", "Since dorsal head structures are not visible in the anterior-ventral movies shown here , the approximate equivalent duration is through the time when the maxillae are cleared ( Mx ) : the interval from 9:00 to 20:20 in the amnion ( 11:20 duration ) and from 20:00 to 27:20 in the serosa ( 7:20 duration ) .", "Tracks are shown for selected nuclei .", "Scale bars are 50 µm .", "See also Video 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 13834 . 01010 . 7554/eLife . 13834 . 011Video 2 . Tribolium extraembryonic tissue rupture and withdrawal shown with amnion-specific EGFP ( enhancer trap line HC079 ) .", "The embryo is shown in lateral aspect with anterior left and dorsal up , with amnion-specific EGFP expression , as well as restricted late embryonic expression domains in the legs and body segments ( see also Figure 1—figure supplement 1 ) .", "The movie spans late amnion morphogenesis , from 3 . 3 hr before rupture through late dorsal closure .", "Of particular note are the brighter , rounder amniotic cells in an anterior-ventral cap , in which rupture occurs .", "During withdrawal , the amnion everts ( turns inside out ) , such that the surface that had faced inward toward the embryo is flipped outward to face the vitelline membrane: this is particularly apparent from 35 to 49 min after rupture as the ruptured tissue edge folds over .", "Time is shown relative to tissue rupture at 0 min .", "Images are maximum intensity projections with a gamma correction of 0 . 7 from a z-stack ( 7 µm step size for a 77 µm stack ) recorded every seven minutes over a 9 . 9-hr time-lapse at 24°C , acquired with an Axio Imager . Z2 with ApoTome . 2 structured illumination ( Zeiss ) .", "Scale bar is 100 µm .", "Figure 4C shows a still at 21 min after rupture . DOI: http://dx . doi . org/10 . 7554/eLife . 13834 . 011 The amniotic cells at the opening exhibit impressive plasticity in both cell and nuclear shape on very short time scales as they are stretched in multiple directions .", "Remarkably , definitive rupture initiates not only by intercellular separation but in some cases by intracellular hole formation ( e . g . , Figure 4D1-D2: red cell ) .", "Among recordings with sufficient spatial and temporal resolution , we followed intracellular opening in three specimens .", "In the example shown here , thin cytoplasmic remnants of the affected cell that were distant from the nucleus and main cell body could be tracked initially , but these may fragment as the opening enlarges to the circumference of the egg .", "However , the nuclei ( and main cell body ) of cells at the site of rupture can be followed during withdrawal and appear to contribute to the persistent edge of the tissue ( also in Video 2 ) .", "These cellular contortions do not occur in the serosa , where cells at the site of rupture separate from their neighbors by spatial translation without substantial deviation from regular , polygonal cell shapes with centrally positioned nuclei ( Figure 4E1-E2 ) .", "Meanwhile , at no time prior to or during rupture did we observe cell loss in the amnion ( see also Figure 2—figure supplement 1E ) .", "There is also no discernible change in apical area or EGFP brightness of amniotic cells directly involved in rupture compared to cells elsewhere in the amniotic cap , such that the onset of rupture appears very abrupt .", "Thus , rupture in Tribolium within a specialized competence zone of the amnion is fundamentally different to the strategy in Oncopeltus .", "In the latter species , gradual apoptosis of the amnion at the rupture site begins over a day ( 16% development ) before rupture occurs but is in itself insufficient as a proximate trigger , which remains unknown ( Panfilio and Roth , 2010 ) .", "In addition to cell shape changes , we also quantified the rate of EE tissue rupture .", "We find that rupture initiation in Tribolium – defined as the short interval of initial opening of the EE membranes – is significantly longer in the amnion than in the serosa ( Figure 4F-G , [p<0 . 001 , Mann-Whitney U test; see Materials and methods for details of the landmarks that delimit this interval ) .", "This is also true even when slight differences in developmental rate between genetic backgrounds are considered ( see also Figure 1—figure supplement 1 ) .", "This result , supported by the distinct morphological appearance of the amnion at the site of rupture ( Figure 4B ) , suggests that the amnion initiates rupture .", "The serosa then ruptures after a slight delay of a few minutes .", "Indeed , the difference in initiation duration corresponds morphologically to only a slight opening of the amnion , generally no larger than the area of the anterior-ventral cap region ( 82% , n = 28 , data set from Figure 4F ) , before the serosa would also perceptibly rupture and then catch up with the amnion ( Figure 4G , Figure 4—figure supplement 1 , Video 3 ) .", "We therefore hypothesize that the amniotic rupture competence zone may differ from the rest of the tissue in the nature of its attachment to the serosa ( also based on tracking data in Figure 2—figure supplement 1D–E ) , given that the edges of the two tissues then retain apposition throughout subsequent withdrawal ( Figure 3C: arrow ) .", "Before evaluating the progression of withdrawal in detail ( below , Figure 5 ) , we then completed our assessment of EE rupture by analyzing how preceding events in early development restrict the source of upstream signals to determine the site of rupture . 10 . 7554/eLife . 13834 . 012Video 3 . Extraembryonic rupture filmed at high temporal resolution . Embryos are shown in anterior-ventral aspect with anterior left , and labeled with EGFP from a heterozygote cross of enhancer trap lines labeling the amnion ( line HC079 ) or serosa ( line G12424 ) with selected embryonic domains in the head , segments , and legs ( line G04609 , 'heart' ) .", "During the movie , the EE tissues rupture and withdraw to the extent that the head and first leg pair are exposed .", "Selected EE nuclei were tracked , where track color segments correspond to fixed units of time ( red , orange , yellow , and green: 8 . 3 min; blue: final 3 . 3 min ) .", "Elapsed time is shown .", "Images are maximum intensity projections from a z-stack ( 2 . 58 µm step size for a 175 or 200 µm stack ) recorded every 20 s at 19 . 5 ± 1°C and shown for a 36 . 7-minute period ( see Supplementary file 1 for further acquisition details ) .", "Single-sided light sheet illumination is from the top , resulting in deterioration of the signal in the lower part of the image due to scattering .", "The final frame shows time point 36:40 with the approximate position of mouthparts labeled for orientation: An , antennae; Lr , labrum; Mx , maxillae; Lb , labium; T1 , first thoracic segment .", "Scale bar is 50 µm .", "See also Figure 4—figure supplement 1 for a visual summary . DOI: http://dx . doi . org/10 . 7554/eLife . 13834 . 01210 . 7554/eLife . 13834 . 013Figure 5 . Withdrawal dynamics: serosa-driven progression . Images are lateral ( A–E , H ) , dorsal-lateral ( F , I ) or ventral ( G ) , with anterior left and dorsal up , shown as sagittal optical sections ( A–B , D–E ) or maximum intensity projections ( C , F–I ) .", "( A−E )", "Tracking and histological staining of withdrawing EE tissue edges .", "The WT EE tissue edge squeezes , then rapidly clears , the abdomen .", "In A , the blue bracket marks a 1-minute interval within a 6 . 7-hr total interval for the entire red/anterior and cyan/posterior tracks , at 21°C .", "The distance of the bracketed track segment from the vitelline membrane ( white outline ) reflects the degree of squeezing of the abdomen .", "After Tc-zen1RNAi ( z1 ) , an amnion-only posterior edge contracts slowly over an uncompressed abdomen .", "In B , the entire track represents a 3 . 9-hr interval at 21°C .", "Note that the track is close to the vitelline membrane outline .", "Slow amnion-only progression is in part due to ruffling of the tissue .", "The red track segment in B corresponds to the period of tissue ruffling shown morphologically in the region of the red bracket in C ( same staining reagents as in D ) .", "Furthermore , there is no apical F-actin enrichment in the folding tissue ( E , compare with D and Figure 3E–F ) .", "( F–G )", "While WT serosal contraction leads to a dorsally condensed tissue ( F ) , in strong Tc-zen2RNAi ( z2 ) phenotypes , the serosa condenses ventrally over the unopened amnion and the confined embryo ( G ) .", "The double arrowhead labels the cardioblasts; 'Y' marks the opaque yolk .", "( H–I )", "In weaker Tc-zen2RNAi ( z2 ) phenotypes the EE tissues can rupture and tear ectopically , leaving a 'belt' of EE tissue that squeezes the embryo ( arrows ) .", "Scale bars are 100 µm ( A , F–I ) and 50 µm ( B–E ) .", "Abbreviations as in previous figures . DOI: http://dx . doi . org/10 . 7554/eLife . 13834 . 013 Long-term examination shows that , in contrast to amniotic rupture within specialized cap cells , the relative position of rupture in the serosa is highly variable .", "At the blastoderm stage , the amnion and serosa initially share a lateral tissue boundary within the plane of the blastoderm epithelium ( van der Zee et al . , 2005 ) .", "As the EE tissues envelop the embryo , they maintain that boundary until they separate into discrete membranes at the serosal window closure stage ( Figure 6A; Benton et al . , 2013; Handel et al . , 2000 ) .", "As the EE tissues separate , serosal cells rapidly acquire fixed positions under the vitelline membrane ( Koelzer et al . , 2014 ) , while internally the embryo and amnion are not so tethered .", "Indeed , we find that over half of all embryos examined ( 59% , n = 69 ) rotate longitudinally during early germband extension relative to the serosa: up to 90º about the anterior-posterior axis , with a tendency to rest laterally ( Figure 6B , Figure 6—figure supplement 1 ) .", "More generally , rotation occurs irrespective of whether the embryo’s long axis is orthogonal ( this study ) or parallel ( data in Strobl and Stelzer , 2014 ) to gravity , suggesting an inherent anisotropy of the early egg .", "However , there is no counterpart rotation in later development ( n = 118 ) , including for 15 embryos filmed continuously and which exhibited typical frequencies of early rotation .", "In other words , 9 of 15 embryos rotated longitudinally such that the site of serosal window closure ( Figure 6A: boxed region ) did not correspond to the site of later EE tissue rupture ( Figure 6D: starburst ) .", "The time window for rotation ends shortly before the onset of amnion-serosa adhesion to form the bilayer during germband retraction ( Figure 2A , Figure 6—figure supplement 1 ) .", "Thus , early rotation without any later reversal is a feature of wild type Tribolium embryogenesis .", "This physically uncouples apposition of the mature amnion and serosa from the tissues’ relative orientation when they had initially detached from one another . 10 . 7554/eLife . 13834 . 014Figure 6 . Changing amnion-serosa interactions during Tribolium extraembryonic morphogenesis . Schematics are sagittal , with anterior left and dorsal up ( A , C–E ) , or transverse with dorsal up ( B ) , illustrating: initial separation during formation of the amnion and serosa as distinct epithelial covers ( A ) , relative rotation in many embryos ( B: shown at the position of the dashed line in A , see also Figure 6—figure supplement 1 ) , EE apposition at the retracted germband stage ( C ) , the successive stages of EE tissue withdrawal ( D ) , and final tissue structure during degeneration at the dorsal organ stage ( E , see also Figure 6—figure supplement 2 ) .", "Amnion-serosa apposition persists during withdrawal morphogenesis ( D ) , which involves EE tissue rupture ( starburst ) , curling over of the resulting tissue edge as it everts ( shown for the anterior-dorsal edge ) , and early serosal compaction as the EE edges fold medially .", "Abbreviations as in previous figures . DOI: http://dx . doi . org/10 . 7554/eLife . 13834 . 01410 . 7554/eLife . 13834 . 015Figure 6—figure supplement 1 . Frequency of embryonic longitudinal rotation during development .", "( A ) Sample sizes and film durations ( blue bars ) relative to landmark developmental stages ( yellow bars: SW , serosal window closure; GBE , germband extension complete; SR , serosal rupture; staging as in Koelzer et al . ( 2014 ) ) .", "Embryos were analyzed from multiple time-lapse data sets of at least 15 . 75-hr duration at 30°C , recorded with an inverted DeltaVision RT ( Applied Precision ) microscope .", "Pooled data included embryos in the nGFP and various enhancer trap backgrounds ( G04609-heart , G12424-serosa , KT650-serosa , HC079-amnion ) , including heterozygote crosses of HC079 with each of G04609 , G12424 , and nGFP .", "( Background autofluorescence was sufficient for scoring early development in the absence of specific GFP signal . ) 'Frequency by stage' refers to the beginning of rotation; all embryos beginning during ( 16% ) or after ( 41% ) the SW stage completed rotation before the GBE stage .", "Rarely , a moderate degree of rotation occurred during dorsal closure ( 4% ) .", "( B ) .", "Chi-square statistics for specified comparisons .", "For significant results , text color-coding indicates the nature of the interaction .", "Longitudinal rotation ranged from approximately 20 to 100 degrees , with two-thirds of embryos rotating approximately 90° .", "Embryos laying on their dorsal or ventral surface ( 'poles' ) were more likely ( 69% ) to rotate 90° , while embryos in lateral aspect ( 85% ) tended to only rotate 45° .", "While overall there was no bias in direction of rotation , embryos initially laying on their right sides tended to rotate anticlockwise ( 70% ) while embryos on their left sides rotated clockwise ( 78% ) .", "Direction of rotation was from the perspective of the embryo’s posterior ( i . e . , clockwise is right , anticlockwise is left ) .", "These directional biases are applicable across the full range from ventral-lateral through dorsal-lateral , such that rotation tended to result in more strictly lateral final orientations ( 64% ) .", "Meanwhile , most non-rotating embryos began in a lateral orientation ( 89% ) .", "( C ) Pie charts representing the eight 45°-sectors of the egg circumference ( D , dorsal; DL-L and -R , dorsal-lateral-left and -right; L-L and -R , lateral-left and -right; VL-L and -R , ventral-lateral-left and -right; V , ventral ) , color-coded for frequency of occurrence according to the heat map . DOI: http://dx . doi . org/10 . 7554/eLife . 13834 . 01510 . 7554/eLife . 13834 . 016Figure 6—figure supplement 2 . Separation during degeneration: the amnion and serosa resolve into two distinct dorsal organ structures . Images are dorsal ( A , A1 , B1 ) or lateral ( B , B2 , C ) , with anterior up ( A , B , B2 , C ) , or upper-left ( A1 , B1 ) , shown as maximum intensity projections ( A , A1 , B , B1 ) or sagittal optical sections ( B2 , C ) .", "Tissue-specific EGFP labels two distinct dorsal organ structures , each characterized by apical F-actin enrichment around a hollow center or cleft ( arrow: amniotic; double-headed arrow: serosal ) .", "Scale bars are 100 µm ( A , B ) and 25 µm ( A1 , B1 , B2 ) .", "After the stage shown here , the two dorsal organs will separate: the serosa remains medial , while the amniotic dorsal organ migrates anteriorly to a position behind the head before undergoing final degeneration ( Panfilio et al . , 2013 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 13834 . 016 Given that rupture invariantly occurs in the cap region in the amnion , variable longitudinal rotation of the embryo and amnion away from where they had detached from the early serosa therefore precludes an impetus for EE rupture from any eggshell or serosa-specific landmark associated with initial EE tissue separation ( contra Strobl and Stelzer , 2014 ) .", "Rather than the serosa causing regionalization in the amnion , it may well be the other way around .", "We previously observed a subtle morphological change in serosal cells over the yolk compared to those over the amnion , and this change only arises during germband retraction ( Koelzer et al . , 2014 ) , which correlates with the time of EE tissue apposition ( Figure 2A ) .", "Thus , the region of amniotic specialization for rupture may be autonomous ( e . g . , corresponding to where it ultimately closed at the serosal window stage ) or induced by signals from the underlying embryo , but is unlikely to be determined by the serosa .", "Once the EE tissues have ruptured anterior-ventrally , they pull back from the embryo as an everting sack that folds up to a dorsal-medial position , similar to a pillowcase being turned inside out as it is peeled off of a pillow ( Figure 6D ) .", "The serosa appears to be the driving force for withdrawal .", "We had previously observed that the serosa facilitates the final stages of withdrawal during dorsal closure , making the process more robust and efficient ( Panfilio et al . , 2013 ) .", "Here , we find that this is true throughout these morphogenetic movements , and furthermore that this is an inherent property of this tissue .", "Firstly , we again use the serosa-less situation after Tc-zen1RNAi to assess the serosa’s normal contribution .", "Wild type withdrawal is rapid , with visible squeezing of the embryo’s abdomen as the posterior EE tissue edge pulls back and clears the posterior pole .", "In tracking the edge of the EE tissues during withdrawal , the squeezing is manifest in the degree to which the tissue pulls away from the eggshell ( Figure 5A: bracketed track segment represents 1 min at 21°C ) .", "This is due to serosa-specific enrichment in apical F-actin as the cells undergo a drastic shape change to become pseudocolumnar ( Figure 5D , also Figure 3F′ ) .", "Indeed , wild type specimens occasionally exhibited a transient extra fold in the posterior EE tissue ( Figure 3C , C′: double-headed arrow ) .", "This likely reflects a degree of stochasticity in tissue relaxation after snapping over the abdomen , similar to kinetic descriptions in other species ( e . g . , Patten , 1884 ) .", "Both the histological and kinetic situations are altered in the absence of the serosa .", "In Tc-zen1RNAi embryos the amnion does withdraw dorsally , but over a much longer time scale and without sufficient force to squeeze the abdomen away from the eggshell ( Figure 5B: entire track represents 3 . 9 hr at 21°C ) .", "The slow progression is in part due to the tissue ruffling into folds rather than everting via apical constriction ( Figure 5C: red bracket corresponds to the red track segment in Figure 5B ) .", "Consistent with a lack of apical constriction , the amnion alone shows no significant F-actin enrichment or increase in cell height ( Figure 5E ) .", "We then used RNAi against Tc-zen2 , the functionally diverged paralogue of Tc-zen1 , as a second approach to functionally test the serosa’s role , as strong RNAi knockdown of Tc-zen2 completely blocks EE rupture ( van der Zee et al . , 2005 ) .", "In the absence of rupture , the embryo remains confined within the amniotic cavity at the time when dorsal closure is initiated by the epidermal flanks .", "With nowhere else to go , the flanks grow along the inner surface of the intact amnion until they meet at the ventral midline , resulting in a ventral closure of the body over the legs ( Panfilio , 2008; Sander , 1976; Truckenbrodt , 1979; van der Zee et al . , 2005 ) .", "This manipulation allowed us to examine the serosa’s inherent morphogenetic properties without rupture as a trigger event .", "We find that the Tc-zen2RNAiserosa still contracts strongly even in the absence of rupture .", "Although the amniotic cavity remains unopened and there are no free EE tissue edges , the serosa contracts until it tears ectopically , withdrawing ventrally over the amnion ( Figure 5F–G: shown mid contraction , when the serosa only occupies a small surface area over the egg ) .", "We infer that the serosa remains attached throughout the bilayer region and pulls on the underlying amnion , which would account for the displacement of the embryo’s head away from the anterior pole ( Figure 5G: note yolk anterior to the head ) .", "This phenotype and the autonomous nature of serosal contractility in Tribolium are conserved compared to Oncopeltus ( Panfilio , 2009 ) .", "As this contrasts with the differences between these species in amnion structure and behavior , discussed above , it appears that there is a degree of modularity or independence in how the two epithelia have evolved .", "This is an unexpected conclusion , given the need for tight coordination between these tissues in any one species .", "Inter-tissue coordination is further demonstrated in weaker Tc-zen2RNAi phenotypes in which rupture does occur but withdrawal morphogenesis is defective .", "Ectopic tearing of the Tc-zen2RNAi EE tissue produces a constrictive EE 'belt' around the embryo .", "Whether visualized for the serosa ( Figure 5H ) or amnion ( Figure 5I ) , the nature of embryonic constriction is consistent with the intact portions of the tissues maintaining adhesion throughout the region of apposition and therefore contracting together .", "Thus , we propose a model in which correct rupture of the amnion is required for directed EE tissue withdrawal , while the serosa autonomously provides the motive force to achieve this .", "Adhesion of the EE tissues throughout the region of apposition provides the means to couple these two functions .", "In many insects , the dorsal organ is a transient , hollow structure formed by the serosa as it sinks into the yolk and degenerates at the end of its life ( Panfilio , 2008 ) .", "Importantly , it has previously been described as comprising only serosal tissue .", "The amnion is restricted to attachment to the serosa at its lateral edges in many species ( Enslee and Riddiford , 1981; Panfilio and Roth , 2010; Tojo and Machida , 1997 ) .", "Alternatively , it was regarded as fully enveloping the serosa as a smooth outer layer ( Rempel and Church , 1971 ) , if a distinct amnion was recognized ( see first Results and discussion subsection ) .", "The persistent bilayered structure of the Tribolium EE tissues , however , results in both a serosal dorsal organ and a nested , amniotic dorsal organ ( Figure 6E , Figure 6—figure supplement 2 ) .", "In light of these observations , re-analysis of late serosa-less Tc-zen1RNAi embryos indicates that the previously observed region of anterior-medial amniotic F-actin enrichment ( Panfilio et al . , 2013 ) corresponds to the amniotic dorsal organ , demonstrating that this structure also forms in the absence of a serosa .", "Thus , at the end of the EE tissues’ lifetimes , they once again function as independent structures , involving separation of the bilayer as the tissues degenerate separately ( Figure 6—figure supplement 2 ) .", "Altogether , the tissue reorganizations for insect EE withdrawal have complex implications for inter-epithelial attachment , balancing a requirement for tissue continuity over the yolk and coordinated withdrawal with enabling the amnion and the serosa to follow their own morphogenetic programs .", "It was previously known that the Tribolium EE tissues arise from the same blastodermal cell sheet before enclosing the embryo and detaching from one another ( Figure 6A; Benton et al . , 2013; Handel et al . , 2000; Koelzer et al . , 2014 ) .", "After those early stages , the structure and arrangement of the amnion had been obscure .", "Here , we have characterized a new genetic resource in the form of the HC079 enhancer trap line that literally illuminates this enigmatic tissue for the first time .", "Following its early detachment from the serosa , the amnion often rotates relative to the serosa ( Figure 6B ) before reattaching to form the bilayer ( Figure 6C ) .", "Then , given that the EE tissues withdraw from the embryo as a single unit ( Figure 6D ) , the re-establishment of their independence during final degeneration at the dorsal organ stage is striking ( Figure 6E ) .", "The mechanical requirements of these morphogenetic events imply a precisely regulated and dynamic mode of epithelial attachment .", "The apposed surfaces ( Figure 6C–D ) present a basal-basal interface where they presumably interact via the basement membrane , which is known to influence both cell adhesion and shape changes at tissue folds during embryogenesis in many animal systems ( Daley and Yamada , 2013 ) .", "Furthermore , the bilayer structure and its rupture in Tribolium have intriguing parallels with vulval development in C . elegans , where two apposed epithelial sheets must also be opened .", "In the roundworm , the uterine and ventral epidermal layers are juxtaposed but separated by their respective basement membranes ( Morrissey et al . , 2014 ) .", "This barrier is breached when the specialized anchor cell of the inner uterine tissue initiates a sequence of extracellular remodeling events to connect the two layers ( Hagedorn and Sherwood , 2011 ) .", "It will be interesting to see whether similar approaches to local basement membrane removal and gap widening ( Ihara et al . , 2011 ) also occur during insect EE rupture , a rapid event that occurs nearly an order of magnitude more quickly than anchor cell invasion ( Figure 4F; Hagedorn and Sherwood , 2011 ) .", "Also , in contrast to a system of invasive cell behavior in which cell cycle regulation and the balance of mitosis among neighboring cells contribute to basement membrane breach and widening , respectively ( Matus et al . , 2014; 2015 ) , the insect EE tissues enter the endocycle and mature to a polyploid state before rupture ( Panfilio et al . , 2013 ) .", "The withdrawing EE tissues of Tribolium thus provide a new system for investigating restructuring of the basement membrane and the cellular-extracellular matrix in simple epithelia .", "Our working model for how the amnion and serosa cooperate to achieve withdrawal also generates several testable hypotheses regarding the nature of their morphogenetic interactions .", "For example , we observe a difference in rupture initiation between the amnion and serosa ( Figure 4F–G , Video", "3 ) that we interpret as a delay before the serosa ruptures and then catches up relative to the amnion .", "Curiously , serosal cells seem to flatten ( lose peripheral EGFP signal ) prior to rupture ( compare Figure 4E1-E2 ) , and partial dissociation of some serosal cells ( Figure 4E2: blue cells ) is observed during rupture , although epithelial integrity is maintained during withdrawal .", "Might this represent a more brittle approach to rupture in which the serosa effectively shatters , compared to the flexibility of opening amniotic cells ?", "In future work , it will be important to elucidate how cells in the amnion and serosa differ in their mechanical properties ( Koehl , 1990 ) .", "In Drosophila it was recently shown that non-muscle myosin II plays a nuanced role in tuning the mechanical properties of the peripodial epithelium so that it can rupture and withdraw from the wing imaginal disc columnar tissue ( Aldaz et al . , 2013 ) .", "The future development of comparable genetic tools in Tribolium will permit the direct testing of our model .", "Taking a step back from rupture itself , it will also be important to uncover the upstream genetic determinants for this event .", "For example , Tc-zen2 not only promotes rupture but is also expressed in the anterior amnion in early development ( van der Zee et al . , 2005 ) , which approximately prefigures the region in which we later detect the morphologically distinct amniotic cap cells .", "However , this transcription factor is also expressed throughout the serosa , and we find that its knockdown impairs both EE tissues without specifically affecting amnion anterior-ventral differentiation ( data not shown ) , indicating that other regulators control cap formation .", "Identifying the molecular cues for amniotic regionalization and the proximate triggers for rupture itself will further clarify the amnion’s role .", "Meanwhile , the bilayered EE arrangement in Tribolium is thus far unique among insects and resolves a long-standing ambiguity regarding amnion structure and function .", "While limited data are available for holometabolous insects with complete EE tissues , hemimetabolous insects have been more extensively studied and have a different arrangement .", "Hemimetabolous EE withdrawal , known as katatrepsis , involves the embryo being pulled out of the yolk by the EE tissues .", "The requirements for katatrepsis restrict amnion-serosa connection to a distinct border region with lateral-lateral cell contact ( Panfilio , 2009; Panfilio and Roth , 2010 ) , but katatrepsis was lost at the base of the holometabolous insect radiation ( Panfilio , 2008 ) .", "The longitudinal rotation of the young Tribolium germband embryo and amnion is akin to the more extensive movements of these tissues within the yolk in hemimetabolous insects ( Cobben , 1968; Rakshpal , 1962; Wheeler , 1889 ) .", "In contrast , the basal rather than lateral nature of reattachment in Tribolium represents a qualitatively different physical environment for the amnion during degeneration ( Figure 6E , Figure 6—figure supplement 2; Horn et al . , 2015 ) .", "Consistent with this topographical change , we observe that whereas the morphogenetic properties of the serosa are conserved between Tribolium and the hemimetabolous bug Oncopeltus , the structure and mode of preparation for rupture in the amnion in these species are decidedly different .", "Further taxonomic sampling will reveal whether Tribolium represents the norm or one of multiple possible EE configurations within the Holometabola .", "Loss of katatrepsis in this insect lineage may have relaxed constraints on extraembryonic developmental strategies and permitted a degree of independence in the evolution of the insect amnion and serosa ." ], [ "Analyses were performed in the San Bernardino wild type strain , EFA-nuclear-GFP ( nGFP ) line ( Sarrazin et al . , 2012 ) , and selected GEKU screen enhancer trap lines ( Trauner et al . , 2009 ) : G12424 ( 'serosa' ) and G04609 ( 'heart': cardioblasts and segmental domains ) , as described ( Koelzer et al . , 2014 ) ; and HC079 ( 'amnion' , characterized in this study , see also Figure 1—figure supplement 1 , Video 1 ) .", "The KT650 serosal line ( Koelzer et al . , 2014 ) was also used in assessing pre-rupture serosal cell morphology throughout the region of apposition with the amnion ( as in Figure 4A ) .", "Parental RNA interference ( RNAi ) for Tc-zen1 and Tc-zen2 was performed as previously described , with adult females injected with double-stranded RNA of ≥688 bp at 1 µg/µl concentration ( Panfilio et al . , 2013; van der Zee et al . , 2005 ) .", "Embryos were dechorionated and mounted on slides in halocarbon oil for conventional microscopy ( Panfilio et al . , 2013 ) .", "Additionally , a light sheet fluorescence microscope ( mDSLM model: Strobl and Stelzer , 2014 ) was used for high temporal and spatial resolution recordings .", "Here , embryos were dechorionated and embedded in low melt agarose and filmed in anterior-ventral aspect ( see Supplementary file 1 for acquisition details for data in Figure 4D , E , G , Figure 2—figure supplement 1D–E , and Video 3 ) .", "Temporal resolution was improved both by image acquisition speed of this microscope system , and , for certain applications , by slowing the rate of embryogenesis via temperature regulation .", "Whereas embryogenesis takes approximately three days at the customary stock maintenance temperature of 30°C ( Koelzer et al . , 2014 ) , selected embryos were filmed for up to 24 hr at 19 . 5 ± 1°C , slowing development roughly by a factor of four ( Sokoloff , 1974 ) .", "Subsequent hatching was confirmed in all cases to verify that recordings documented healthy development .", "With this criterion , we analyzed rupture with light sheet fluorescence microscopy in 14 embryos , across homozygous and heterozygous genetic backgrounds .", "In fact , of the 17 embryos observed , only one died prior to hatching , while in two cases we were unable to confirm hatching due to post-acquisition handling difficulties .", "Cell tracking was performed on maximum intensity projection ( MIP ) time-lapse movies with MTrackJ ( Meijering et al . , 2012 ) .", "Cell outlines were drawn manually in ImageJ with the segmented line and polygon selection tools , based on observed cell morphology .", "Embryos were recorded every 3 . 5 min at 27 . 5–28°C on a DeltaVision RT microscope ( Applied Precision/GE Healthcare Life Sciences , Issaquah , Washington , USA ) , with simultaneous recording of up to 11 embryos each of the serosa-heart and amnion-heart crosses in each of three experiments .", "Using the MIP movie output , rupture initiation was determined as the interval between the following events .", "The start of rupture was recorded as either the first frame in which two or more cells clearly diverged , followed by the appearance of a hole in the EE tissue at the same position in subsequent frames , or as the first frame after a sudden collapse of the anterior EE membrane , whichever came first .", "The end of rupture was recorded as the first frame in which the retracting membrane cleared the head dorsally .", "Embryos were analyzed from time-lapse data sets of at least 15 . 75 hr duration at 30°C .", "Orientation and degree of rotation were determined by eye from MIP movies , and categorized into eight sectors of 45° around the egg circumference .", "See Figure 6—figure supplement 1 for further details .", "To assess early amnion topography , fuchsin staining after standard fixation and methanol shock without complete devitellinization was performed based on standard protocols ( Wigand et al . , 1998 ) .", "For fluorescent imaging of endogenous EGFP , embryos were fixed , manually devitellinized , optionally stained with fluorescently conjugated phalloidin or wheat germ agglutinin ( WGA ) , and embedded in Vectashield mountant with DAPI as described previously ( Panfilio et al . , 2013 ) ." ] ]

[ "Unlike passive rupture of the human chorioamnion at birth , the insect extraembryonic ( EE ) tissues – the amnion and serosa – actively rupture and withdraw in late embryogenesis .", "Withdrawal is essential for development and has been a morphogenetic puzzle .", "Here , we use new fluorescent transgenic lines in the beetle Tribolium castaneum to show that the EE tissues dynamically form a basal-basal epithelial bilayer , contradicting the previous hypothesis of EE intercalation .", "We find that the EE tissues repeatedly detach and reattach throughout development and have distinct roles .", "Quantitative live imaging analyses show that the amnion initiates EE rupture in a specialized anterior-ventral cap .", "RNAi phenotypes demonstrate that the serosa contracts autonomously .", "Thus , apposition in a bilayer enables the amnion as 'initiator' to coordinate with the serosa as 'driver' to achieve withdrawal .", "This EE strategy may reflect evolutionary changes within the holometabolous insects and serves as a model to study interactions between developing epithelia ." ]

[ "Early in development a protective fluid-filled sac forms around an embryo .", "In humans , this sac bursts during birth , but the sac surrounding insect embryos ruptures long before these animals begin to emerge from their eggs .", "This early rupture is important for insects to develop normally: if an insect embryo’s sac remains intact too long , the animal’s back will not close properly .", "The sac that surrounds insect embryos has two layers: an inner layer called the amnion , and a tough outer layer called the serosa .", "However , it has been difficult to study what happens to the amnion as the insect embryo develops because it is hard to distinguish it from the serosa .", "Now , Hilbrant et al . have used genetically engineered red flour beetles in which the cells of the amnion produce a fluorescent protein that can be viewed under a microscope .", "This allowed the amnion to be observed in living specimens during beetle embryo development , and revealed that the amnion attaches and detaches from the serosa more than once as the embryo develops .", "Furthermore , the amnion and serosa remain as distinct tissues as they withdraw from the embryo .", "Hilbrant et al . also found that the cells in part of the amnion near the head of the beetle embryo have a special shape before the sac ruptures .", "This region of the amnion breaks apart first , and the serosa breaks open a few minutes afterwards .", "Once the two layers have broken , they pull back from the embryo like a pillowcase being turned inside out as it is removed from a pillow .", "In normal beetles , this process is quite rapid and squeezes the embryo’s abdomen .", "But in beetles genetically manipulated to lack a serosa the process is slower because the amnion is not strong enough by itself to squeeze the embryo .", "Overall , the experiments show that the amnion starts the rupture of the red flour beetle embryo’s protective sac and the serosa drives the process of the sac being peeled back .", "Further research will now investigate the mechanics behind the two tissues’ roles and whether the amnion and serosa display similar behaviors in other related insect species ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "developmental biology", "neuroscience" ]

[ [ "The Hedgehog ( Hh ) pathway is essential in the morphogenesis and patterning of many tissues ( Briscoe and Therond , 2013 ) .", "Hh signaling is transduced through a series of negative regulatory interactions .", "Binding of a Hh ligand such as Sonic hedgehog ( Shh ) to its receptor Patched ( Ptch ) releases Ptch inhibition of Smoothened ( Smo ) .", "Activated Smo then triggers a signaling cascade that eventually activates Gli transcription factors , which enable Hh target gene transcription ( Briscoe and Therond , 2013 ) .", "Abnormalities in Hh signaling are responsible for certain birth defects and contribute to a wide range of tumors , including medulloblastoma ( MB ) , the most common malignant pediatric brain tumor ( Taylor et al . , 2012 ) .", "For example , the loss of the negative regulator Ptch , causes Smo to be inappropriately active , and this leads to skin cancer and MB ( Johnson et al . , 1996; Stone et al . , 1996 ) .", "Current treatment for MB , surgical removal followed by chemotherapy and radiotherapy , cures about 2-thirds of patients , but often leaves survivors suffering from devastating neurocognitive consequences ( Fouladi et al . , 2005 ) .", "The Smo inhibitor , vismodegib , is the only FDA-approved drug that targets the Hh pathway .", "However , the effect of vismodegib tends to be transient , and irreversible drug resistance and tumor relapse often ensue ( Rudin et al . , 2009; Yauch et al . , 2009 ) .", "Genomic sequencing results showed that the resistance is due to mutations in Smo that reduce its binding affinity to vismodegib , or cause Smo to be constitutively active in tumor cells .", "Thus novel drug targets downstream of Smo are critically needed .", "We previously discovered that Neuropilins ( Nrp1&2 ) positively regulate Hh transduction downstream of Smo ( Hillman et al . , 2011 ) .", "Nrps are co-receptors for Semaphorin 3 ( Sema3 ) in axon guidance ( Gu et al . , 2002; Appleton et al . , 2007 ) , and for VEGF that controls cardiovascular development and angiogenesis ( Pellet-Many et al . , 2008 ) .", "Nrps are also widely involved in many types of cancers .", "In particular , they are implicated in the growth and spread of MB ( Hayden Gephart et al . , 2013; Snuderl et al . , 2013 ) .", "However , how Nrps regulate Hh transduction at the molecular level remains unknown , which limits our understanding of developmental signal integration and hampers the development of Nrp-related therapeutics for Hh-related tumors .", "MB results from the over-proliferation of granule neuron precursors ( GNPs ) in the developing cerebellum .", "GNP proliferation during normal development is stimulated by Hh signaling .", "Shh released by Purkinje neurons acts as a mitogen for GNPs ( Dahmane and Ruiz i Altaba , 1999; Wallace , 1999; Wechsler-Reya and Scott , 1999 ) .", "Activating mutations in the Hh pathway are responsible for about one-quarter of MB cases .", "Nrp1 and Nrp2 are highly expressed in developing cerebellum and in human MBs ( Snuderl et al . , 2013 ) .", "One way that Nrps can affect tumor growth is by facilitating VEGF-driven vascularization events that prevent tumor suffocation .", "However , several lines of evidence suggest that Nrps promote tumor growth through mechanisms in addition to VEGF-mediated angiogenesis .", "Abolishing Nrp function in tumor cells using function-blocking antibodies suffices to suppress MB growth and metastasis ( Snuderl et al . , 2013 ) .", "Selective inhibition of Nrp function by RNAi specifically in MB tumor cells , that is not in vasculature , blocks growth of MB allografts ( Hayden Gephart et al . , 2013 ) .", "However , despite extensive research on each pathway , it remains unclear whether and how Nrps interact with Hh pathways to control GNP proliferation and MB formation .", "Here we describe a molecular mechanism that integrates Sema3/Nrp signaling with Hh transduction .", "We found that Sema3 , a member of a secreted ligand family , enhances Hh signaling .", "This is achieved by activation of Phosphodiesterase 4D ( PDE4D ) , which reduces intracellular cAMP levels through hydrolysis .", "The subsequent inhibition of Protein Kinase A ( PKA ) activity promotes Hh transduction .", "We demonstrate that this molecular interplay operates in the developing cerebellum .", "Genetic removal of Sema3/Nrp signaling severely impairs GNP proliferation .", "Furthermore , inhibiting PDE4D suppresses the growth of Hh-related MB .", "These findings reveal a hitherto unknown transduction mechanism that links Sema3/Nrp signaling with Hh pathway , 2 major pathways in development and disease , and highlight PDE4D as a new therapeutic target for Hh-related tumors ." ], [ "Nrps are single transmembrane proteins with five extracellular domains and a short cytoplasmic tail .", "The binding of Sema3 and VEGF to Nrps is ascribed to the extracellular A1/A2 and B1/B2 domains , respectively ( Figure 1A ) .", "Little is known about the function of the Nrp cytoplasmic domain , though it is evolutionarily conserved .", "To identify which domain of Nrps is required to promote Hh transduction , we silenced endogenous Nrps in NIH3T3 cells with lentivirus-mediated RNAi and over-expressed truncated Nrp1 to rescue the Hh signaling .", "We found that both extracellular and cytoplasmic domains are required to promote Hh signaling , since Nrp mutants without either domain were expressed stably but failed to rescue Hh transduction ( Figure 1B ) .", "At the extracellular side , A1/A2 domains are required for Nrps to enhance Hh transduction , whereas B1/B2 domains are dispensable ( Figure 1B ) .", "We then explored what Hh transduction events are mediated by the requisite domains . 10 . 7554/eLife . 07068 . 003Figure 1 . Signaling downstream of Sema3-Nrp enhances Hh transduction .", "( A ) Schematic drawing of Nrps protein structure .", "( B ) In NIH3T3 cells in which Nrp1&2 were silenced by RNAi , Hh signaling could be rescued by full-length ( FL ) Nrp1 construct , but not by Nrp1 constructs that lack the entire extracellular domain ( ΔExt ) , cytoplasmic domain ( ΔCyt ) , A1 ( ΔA1 ) , or A2 ( ΔA2 ) domain .", "Western blot shows that truncated Nrps were expressed with expected molecular weights .", "( C , D )", "Hh signaling activity in NIH3T3 cells treated with increasing concentrations of recombinant Shh in conjunction with a constant concentration of Sema3A , Sema3F ( 3 μg/ml ) , or VEGF165 ( 100 ng/ml ) for 24 hr ( E , F ) Western blot shows that lentivirus-mediated expression of shRNA against Nrp1 and Nrp2 abolished the expression of endogenous Nrps ( E ) .", "On day 3 , Hh signaling activity was evaluated after cells were treated with Shh in conjunction with Sema3A or Sema3F for 24 hr ( F ) .", "In all experiments Gli1 transcript level was measured by qPCR to evaluate Hh signaling activity; a . u . , arbitrary unit .", "All error bars represent SEM .", "Statistics: Student's t-Test .", "*p < 0 . 05 . DOI: http://dx . doi . org/10 . 7554/eLife . 07068 . 00310 . 7554/eLife . 07068 . 004Figure 1—figure supplement 1 . Sema3 signals through Nrps to enhance Hh transduction .", "( A , B )", "Hh signaling activity in NIH3T3 cells treated with increasing concentrations of Shh ( A ) or SAG ( B ) in conjunction with a constant concentration of Sema3 recombinant proteins ( 3 μg/ml ) for 24 hr . ( C , D ) Western blot showing that VEGFR1 and Sema3F are expressed in 3T3 cells .", "( E ) Diagram of function-blocking Nrp antibodies that specifically block Nrp binding with the ligands Sema3 and VEGF .", "( F , G )", "Hh signaling activity in NIH3T3 cells incubated with function-blocking Nrp antibodies ( 50 μg/ml ) together with Shh or SAG for 4 hr . ( H ) Western blot shows that lentivirus-mediated expression of shRNA ( pair #2 ) against Nrp1 and Nrp2 abolished the expression of endogenous Nrps .", "( I ) On day 3 , Hh signaling activity was evaluated after cells were treated with Shh in conjunction with Sema3A or Sema3F for 24 hr .", "In all experiments Gli1 transcript level was measured by qPCR to evaluate Hh signaling activity; a . u . , arbitrary unit .", "All error bars represent SEM .", "Statistics: Student's t-Test .", "*p < 0 . 05 , **p < 0 . 01 , ***p < 0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 07068 . 004 The Nrp A1/A2 domains interact with Sema3 secreted protein family .", "We therefore tested the effect of Sema3 on Hh transduction .", "We treated NIH3T3 cells with Sema3A or 3F , two well-characterized ligands for Nrp1 and 2 , respectively .", "An increasing concentration of Shh was used to induce Hh target gene ( Gli1 ) expression .", "Both Sema3A and 3F dramatically enhanced Shh-induced transcription of Gli1 ( Figure 1C ) .", "The most prominent amplification of Shh response was seen when Sema3F was added to cells in the presence of a high concentration of Shh ( 2 μg/ml ) .", "Sema3A and 3F were not able to induce Gli1 expression in the absence of Shh .", "Three other Sema3 isoforms , 3B , 3C and 3E , had similar effects ( Figure 1—figure supplement 1A ) .", "Similar results were obtained when Gli1 was induced by SAG , a small molecule that binds directly to Smo and triggers target gene expression ( Figure 1—figure supplement 1A ) .", "Together , these results demonstrate that activities of proteins in the Sema3 family contribute to Hh transduction .", "Since a single isoform of the Sema3 family is able to promote Hh transduction , blocking all Sema3 isoforms may be necessary in order to fully compromise Hh signaling .", "We performed such experiments by silencing both Nrps and by employing antibodies that block the Sema3-Nrp interaction ( Figure 1F , Figure 1—figure supplement 1F , G ) .", "Future studies using genetic approaches ( e . g . , genetic knockdown or knockout ) to lower multiple Sema3 ligands will be required to further define the role of Sema3 proteins in enhancing Hh signaling .", "In our previous study , using NIH3T3 cells stably transfected with a Gli-responsive luciferase reporter ( Shh-Light2 cells ) , we did not find a noticeable effect of Sema3A on Hh transduction ( Hillman et al . , 2011 ) .", "In the current study , we used unmodified NIH3T3 cells and new batches of recombinant Sema3 , and employed a more sensitive method ( qPCR ) to detect Hh signal activity .", "With this approach we detected robust Sema3-mediated enhancement of Hh signaling .", "In contrast to the strong Sema effects , recombinant VEGF had no impact on Shh-induced Gli1 transcription ( Figure 1D ) .", "To make sure that the inactivity of VEGF was not due to absence of VEGF receptor , a co-receptor with Nrps in angiogenesis , we confirmed expression of VEGF receptor ( VEGFR1 ) in NIH3T3 cells ( Figure 1—figure supplement 1B ) .", "Taken together , we found a hitherto unknown synergy between Sema3 and Hh transduction .", "In NIH3T3 cells , we detected the expression of Sema3F by Western blot ( Figure 1—figure supplement 1C ) .", "We also detected the expression of other Sema3 isoforms by next generation RNA sequencing ( data not shown ) .", "It is likely that these endogenous Sema3 contribute to the baseline level of Hh transduction .", "To block the binding of the endogenous Sema3 with Nrps , we used function-blocking antibodies that specifically blocked ligand-binding sites in Nrps .", "The antibody NrppanA occupies Sema3's binding site in both Nrp1 and Nrp2; antibodies Nrp1B and Nrp2B occupy VEGF's binding sites in Nrp1 and Nrp2 , respectively ( Appleton et al . , 2007 ) .", "These antibodies were previously used to block tumor growth by interfering with tumor vascularization ( Liang et al . , 2007; Pan et al . , 2007 ) .", "We treated NIH3T3 cells with each antibody together with Shh or SAG .", "As expected , NrppanA significantly reduced Shh- and SAG-induced Gli1 transcription , whereas Nrp1B and Nrp2B had no significant effect ( Figure 1—figure supplement 1D–F ) .", "Thus blocking Sema3F from binding to Nrp prevented Sema3F from enhancing Hh transduction , while control antibodies did not affect Hh transduction .", "We then silenced expression of both Nrp genes with shRNA-expressing lentiviruses ( Figure 1F ) .", "As reported before ( Hillman et al . , 2011 ) , Nrp loss reduced Gli1 transcription to ∼30% of the control level .", "Furthermore , Sema3A and 3F failed to enhance Shh-induced target gene transduction in the absence of Nrps ( Figure 1E–F ) .", "To exclude possible off-target effects of RNAi , we used another pair of ShRNAs to silence Nrp expression , and got similar results ( Figure 1—figure supplement 1G , H ) .", "These data suggest that Sema3 acts through receptor Nrps to promote Hh signal transduction .", "The short cytoplasmic domains of Nrps have had no clear role in transducing any signal .", "However , recently this domain was found to be required for the survival and growth of MB cells ( Snuderl et al . , 2013 ) , and our data suggest that this domain is indispensable for robust Hh transduction ( Figure 1B ) .", "To discover how the cytoplasmic domains of Nrps may operate in the Hh pathway , we performed yeast two-hybrid screening using the Nrp1 cytoplasmic domain as the ‘bait’ .", "Phosphodiesterase 4D ( PDE4D ) emerged multiple times from the screen .", "PDE4D belongs to the superfamily of PDE4 , enzymes that degrade the second messenger cyclic AMP ( cAMP ) .", "Multiple isoforms of PDE4D with distinct molecular weights are generated through alternative RNA splicing ( Richter et al . , 2005 ) .", "We detected three main isoforms expressed in NIH3T3 cells: PDE4D2/6 ( 68KD ) , PDE4D1 ( 78KD ) and PDE4D4 ( 110KD ) ( Figure 2—figure supplement 1A ) .", "We then used co-immunoprecipitation to determine whether the interaction between Nrp1 and PDE4D observed in yeast is indicative of an interaction in NIH3T3 cells .", "We found that Nrp1 interacted with PDE4D , in particular the isoform PDE4D2/6 ( Figure 2A ) . 10 . 7554/eLife . 07068 . 005Figure 2 . Nrp1 interacts with PDE4D , and Sema3 promotes this interaction .", "( A ) Immunoblot showing that the endogenous PDE4D isoform 2/6 from NIH3T3 cells was immunoprecipated by Nrp1 antibody , but not by an unrelated GFP antibody .", "( B , C )", "Immunoblots showing the amount of PDE4D2/6 immunoprecipitated by Nrp1 antibody from cells treated with Sema3 ( a combination of Sema3A+3F ) for different periods of time .", "( B ) .", "Band intensity was normalized to the input at the corresponding time point , and then normalized to time 0 ( C ) .", "( D , E )", "Immunoblots showing the amount of Nrp1 and PDE4D2/6 in the membrane and cytosolic fractions from cells treated with Sema3A+3F for different periods of time .", "( D ) .", "Band intensity was normalized to time 0 ( E ) .", "Quantification in ( C ) and ( E ) were from three independent experiments .", "Error bars represent SEM .", "Statistics: Student's t-Test .", "*p < 0 . 05 , **p < 0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 07068 . 00510 . 7554/eLife . 07068 . 006Figure 2—figure supplement 1 . The cytoplasmic domain of Nrps mediates their interactions with PDE4D2 . ( A ) Major PDE4D isoforms expressed in NIH3T3 cells recognized by a PDE4D antibody against the consensus C-terminus of all PDE4D isoforms .", "( B , C )", "Flag- tagged PDE4D2 co-immunoprecipitates with full length EGFP-tagged Nrp1/2 after they are overexpressed in NIH3T3 cells , but not with cytoplasmic domain-truncated Nrps ( Nrp1/2-Δcyto ) .", "( D ) Immunoblots showing the amount of Nrp1/2 and PDE4D2/6 in the membrane fractions from cells treated with Shh , Sema3A+3F , or the combination of Shh and Sema3s for 30 min . ( E ) Band intensity was normalized to vehicle .", "( F ) After Nrp1+2 were silenced by RNAi , membrane fractions were isolated from the cell .", "Immunoblots showing the amount of Nrp1/2 and PDE4D2/6 in the membrane fractions from cells treated with Sema3 ( 3A+3F ) for 30 min . ( E ) Band intensity was normalized to vehicle .", "Quantification in ( E ) and ( G ) were from three independent experiments .", "Error bars represent SEM .", "Statistics: Student's t-Test .", "*p < 0 . 05 , **p < 0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 07068 . 00610 . 7554/eLife . 07068 . 007Figure 2—figure supplement 2 . Sema3-Nrp signaling reduces intracellular cAMP levels .", "( A ) cAMP standard curve generated by cAMP-Glo assay showing the linear relationship between cAMP concentration and the luminescence intensity change ( ΔLuminescence ) .", "( B ) ΔLuminescence was read out after cells were treated with control ( BSA ) , forskolin ( 1 μM ) or Sema3F ( 3ug/ml ) for 30 min ΔLuminescence was calculated as Luminescencetreated—Luminescenceuntreated .", "( C ) Cells were infected with lentivirus expressing shRNA against Nrp1&2 for 72 hr ΔLuminescence was read out after cells were treated with control ( BSA ) , or Sema3F ( 3ug/ml ) for 30 min .", "Statistics: Non-parametric Mann–Whitney test .", "All error bars represent SEM .", "*p < 0 . 05 , **p < 0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 07068 . 007 Since Nrp1 and Nrp2 play partially redundant roles in the regulation of Hh transduction ( Hillman et al . , 2011 ) , we tested whether both Nrps interact with PDE4D .", "We overexpressed the tagged protein Nrp1/2-EGFP and PDE4D2-Flag , and found that both Nrps immunoprecipitated PDE4D2 ( Figure 2—figure supplement 2A , B ) .", "To further determine the involvement of the cytoplasmic domain of Nrps in the interaction with PDE4D , we deleted this domain from both Nrps .", "The truncated Nrp proteins failed to immunoprecipitate PDE4D2 .", "Thus , the interactions of PDE4D2 with Nrp1 and Nrp2 depends upon their cytoplasmic domains .", "The interaction of the Nrp cytoplasmic domain with PDE4D could be related to the Sema3 signaling that affects Hh transduction .", "To test the connection , NIH3T3 cells were treated with Sema3F , and Nrp1 was immunoprecipitated .", "Sema3F treatment increased the amount of PDE4D2/6 that was immunoprecipitated by Nrp1 antibody in a time-dependent manner ( Figure 2B–C ) .", "Thus binding of Sema3F to Nrp1 facilitates the interaction between Nrp1 and PDE4D .", "Nrp1 is a transmembrane protein , primarily located in the plasma membrane .", "We hypothesized that through interacting with Nrp1 , PDE4D is recruited to the plasma membrane .", "Since Sema3F promotes the Nrp-PDE4D interaction , the net effect would be that Sema3 promotes plasma membrane localization of PDE4D .", "We tested this hypothesis by subcellular fractionation , examining the intensity of PDE4D2/6 in the cytosolic and membrane fractions .", "30 min after cells were treated with Sema3F , the amount of PDE4D2/6 in the membrane fraction increased significantly , while the amount in the cytosolic fraction decreased slightly ( Figure 2D–E ) .", "Note that the time scale is in agreement with the co-immunoprecipitation; recruitment of PDE4 to the membrane coincides with the increased co-immunoprecipitation ( Figure 2B–C ) .", "As a conclusion , Sema3 promotes recruitment of PDE4D to the cell membrane .", "To test the effect of Shh on Sema3-mediated PDE4D translocation to the cell membrane , we treated cells with Shh in the presence or absence of Sema3 prior to cell fractionation .", "Shh by itself did not recruit PDE4D to the membrane , nor did it increase Sema3's effect on PDE4D membrane translocation ( Figure 2—figure supplement 1D , E ) .", "We then determined whether Nrps are required for Sema3's effect on PDE4D membrane translocation .", "We silenced both Nrps with lentivirus-mediated RNAi , and found that Sema3 could not promote PDE4D membrane association without Nrps ( Figure 2—figure supplement 1F , G ) .", "We concluded that Sema3 signals through Nrps to promote PDE4D translocation to the cell membrane .", "PDE4D hydrolyzes cAMP , which is produced by adenylate cyclase ( AC ) at the plasma membrane ( Beavo and Brunton , 2002 ) .", "The recruitment of PDE4D by Sema3-Nrps to the plasma membrane may bring it close to the site of cAMP production , where it can efficiently degrade cAMP .", "To test this hypothesis , we measured the change of intracellular cAMP level using a cell-based assay ( Kumar et al . , 2007; Dunn et al . , 2013; Kokkinaki et al . , 2013 ) .", "Sema3F significantly reduced intracellular cAMP levels compared to a BSA control ( Figure 2—figure supplement 2B ) .", "When we silenced Nrps expression using lentivirus-mediated RNAi , the intracellular cAMP level significantly increased , and Sema3F failed to reduce it ( Figure 2—figure supplement 2C ) .", "In conclusion , Sema3 signals through Nrps to reduce intracellular cAMP levels .", "cAMP activates multiple protein effectors in the cell , such as PKA , Epac , and cyclic nucleotide-gated ion channels ( Bradley et al . , 2005; Borland et al . , 2009 ) .", "PKA is a well-known negative regulator of the Hh pathway .", "Genetic removal of PKA leads to full activation of the Hh pathway in the developing neural tube ( Epstein et al . , 1996; Tuson et al . , 2011 ) .", "The lowering of intracellular cAMP by Sema3F-Nrp signaling has the potential to inhibit PKA , so we examined PKA activity in NIH3T3 cells .", "Previous publications suggest that phosphorylation of PKA at T197 is essential for PKA activity ( Adams et al . , 1995; Steichen et al . , 2010 ) , and that the level of phosphorylation of PKA at T197 correlates with PKA activation ( Moore et al . , 2002; Barzi et al . , 2010 ) .", "Thus , we used an antibody that specifically recognizes the phosphorylation of PKA at T197 ( pPKA-T197 ) and performed immunofluorescence .", "In agreement with previous reports ( Barzi et al . , 2010; Tuson et al . , 2011 ) , phospho-PKA-T197 accumulates at the centrosome , and is distributed throughout the entire cytoplasm in a punctate pattern ( Figure 3A ) .", "Sema3F treatment for 30 min dramatically reduced the pPKA-T197 level in the cytoplasm ( Figure 3B ) .", "The pool of PKA at the cilium base , marked by Pericentrin antibody , also went down ( Figure 3C ) ; this pool of PKA has been hypothesized to directly participate in Hh regulation ( Barzi et al . , 2010; Tuson et al . , 2011 ) .", "To determine whether the reduction in pPKA-T197 level is due to decreased total PKA , we stained the cells with an antibody that detects total PKA .", "Sema3F did not induce any change in total PKA level in the cytoplasm or at the centrosome ( Figure 3—figure supplement 1A–C ) .", "Therefore , Sema3F reduced the detectable level of PKA phosphorylation at T197 .", "This effect was dependent on the presence of Nrps , since Sema3F did not reduce pPKA-T197 after Nrp expression was silenced by lentivirus-mediated RNAi ( Figure 3D–E ) , and the total PKA level remained unchanged ( Figure 3—figure supplement 1D , E ) . 10 . 7554/eLife . 07068 . 008Figure 3 . Sema3-Nrp signaling inhibits PKA activity .", "( A ) Immunofluorescence showing active PKA in NIH3T3 cells after Sema3F treatment .", "Cells were infected with lentiviruses expressing control shRNA or shRNA against Nrp1&2 for 72 hr .", "Active PKA is recognized by the antibody against phospho-PKA T197 ( green ) .", "Scale bar , 10 μm .", "( B–E )", "Quantification of phospho-PKA level at the cytoplasm and centrosome .", "Data are shown as mean ± SD .", "Statistics: Kruskal–Wallis non-parametric One-Way ANOVA .", "( F ) Western blot showing the phospho-PKA and total PKA levels in NIH3T3 cells treated with Sema3F or Forskolin for indicated time periods .", "Quantification of relative active PKA level ( mean ± SEM ) from three independent experiments was shown at the bottom of each blot .", "( G ) Western blot showing the levels of PKA substrate phosphorylation in NIH3T3 cells treated with Sema3F or Forskolin for indicated time periods .", "Quantification of the relative intensity of all bands in each lane ( mean ± SEM ) from three independent experiments was shown at the bottom . DOI: http://dx . doi . org/10 . 7554/eLife . 07068 . 00810 . 7554/eLife . 07068 . 009Figure 3—figure supplement 1 . Sema3-Nrp signaling does not change total PKA level .", "( A ) Immunofluorescence showing total PKA in NIH3T3 cells after Sema3F treatment .", "Cells were infected with lentiviruses expressing control shRNA or shRNA against Nrp1&2 for 72 hr .", "The antibody that recognizes total PKA stains for both phosphorylated and non-phosphorylated PKA ( green ) .", "Scale , 10 μm .", "( B–E )", "Quantification of total PKA level at the centrosome .", "Data are shown as mean ± SD .", "Statistics: Kruskal–Wallis non-parametric One-Way ANOVA . DOI: http://dx . doi . org/10 . 7554/eLife . 07068 . 00910 . 7554/eLife . 07068 . 010Figure 3—figure supplement 2 . Sema3-PKA signaling enhances Shh-induced Gli2 enrichment to the cilia tip , but does not affect Gli3 processing .", "( A , B )", "NIH3T3 cells were infected with lentivirus expressing control or Nrp shRNA for 72 hr .", "After Shh treatment , cells were stained with Gli2 and acetylated tubulin ( Red ) .", "Gli2 intensity at the cilia tips ( arrows ) was measured ( B ) .", "( C ) 72 hr after lentivirus-mediated RNAi , cells were treated with Shh for 24 hr .", "Cell lysate was then used in Western Blot to detect Gli1 induction and Gli3 processing .", "( D–F )", "Quantification of the intensity of Gli1 , Gli3 full length and Gli3 repressor from 3 independent experiments .", "Data are mean ± SD .", "Statistics: non-parametric Mann–Whitney test .", "**p < 0 . 01 , ****p < 0 . 0001 .", "Scale , 2 μm .", "Abbreviation: Gli3FL , Gli3 full length .", "Gli3R: Gli3 repressor . DOI: http://dx . doi . org/10 . 7554/eLife . 07068 . 010 To further investigate the effect of Sema3 on PKA activity , we detected the pPKA-T197 level using protein blots of NIH3T3 cell lysates .", "When cells were treated with forskolin , a drug that elevates intracellular cAMP levels , the detectable level of pPKA-T197 increased by 50% ( Figure 3F ) .", "This result suggests that the detectable level of pPKA-T197 is a valid measurement for inferring the PKA activity level .", "Sema3F treatment reduced the detectable level of pPKA-T197 by ∼20% ( Figure 3F ) .", "Next , we further evaluated PKA activity by examining PKA substrate phosphorylation using an antibody that recognizes the phosphorylated PKA consensus motif ( Rxx[S/T]P ) .", "Substrate phosphorylation level increased by ∼130% upon forskolin treatment , and decreased by ∼27% when treated with Sema3F ( Figure 3G ) .", "The results of the experiments using pPKA-T197 measurements suggest that Sema3F acts through Nrps to reduce PKA activity , thus stimulating Hh transduction .", "The transcription factors Gli2 and Gli3 are known PKA targets in Hh transduction .", "Both Gli2 and Gli3 can be phosphorylated by PKA , but the effects of PKA differ qualitatively and quantitatively with respect to the two Gli proteins .", "PKA-driven phosphorylation leads to proteolytic processing of Gli3 into Gli3R , a potent transcription repressor of Hh target genes ( Humke et al . , 2010; Tukachinsky et al . , 2010 ) .", "PKA has a distinct effect on Gli2 , affecting proteolysis only very slightly but more dramatically controlling its accumulation at cilia tips , a step that is required for Gli2 activation ( Pan et al . , 2006; Hui and Angers , 2011; Tuson et al . , 2011; Niewiadomski et al . , 2013 ) .", "We evaluated the effect of Sema3-Nrp signaling on Gli3 proteolytic processing using protein Blots .", "After silencing Nrps by lentivirus-mediated RNAi , we did not detect any obvious change in Gli3R production in response to Shh treatment ( Figure 3—figure supplement 2C–F ) .", "We examined the effect of Sema3-Nrp signaling on Gli2 enrichment at the cilia tips .", "Consistent with previous reports ( Tukachinsky et al . , 2010; Niewiadomski et al . , 2013 ) , Shh increased Gli2 levels at cilia tips within a few hours .", "However , after the expression of both Nrp genes was silenced by RNAi , this increase was significantly attenuated when compared with control RNAi treated cells ( Figure 3—figure supplement 2A , B ) .", "We conclude that Sema3/Nrp-mediated PKA inhibition promotes Hh transduciton by facilitating Gli2 activation , but leaves the level of Gli3 repressor unaffected .", "Our data suggest that signaling downstream of Sema3-Nrp promotes PDE4D activity to inhibit PKA , which eventually enhances Hh transduction ( Figure 4A ) .", "This model indicates that inhibiting PDE4D will suppress Hh transduction .", "To test this idea we treated cells with rolipram , which inhibits all PDE4 families; GEBR-7b , which specifically inhibits the PDE4D subfamily ( Bruno et al . , 2011 ) ; or roflumilast , which targets all PDE4 proteins and is approved by the FDA for treating inflammatory lung disease .", "Shh was added simultaneously with each individual drug to the cells , and Gli1 transcription levels were measured to assess Hh transduction .", "All three drugs inhibited Shh- or SAG-induced Hh target gene activation in a dose-dependent manner ( Figure 4B , Figure 4—figure supplement 1B ) .", "To exclude the possibility that PDE4D inhibitors are toxic to the cell , we assessed Wnt signaling by measuring transcripts of the Wnt target gene Axin2 .", "Roflumilast and GEBR-7b had no effect on Wnt3a-induced Axin2 transcription at all concentrations tested , nor did rolipram at concentration lower than 1uM ( Figure 4C ) . 10 . 7554/eLife . 07068 . 011Figure 4 . PDE4D inhibitors suppress Hh signal transduction .", "( A ) Schematic diagram showing that Sema3-Nrp signaling regulates Hh pathway through controlling the activity of PDE4D and PKA .", "( B ) The Hh signaling activity in NIH3T3 cells incubated with PDE4D inhibitors together with Shh conditioned medium for 4 hr Gli1 transcript levels were measured by qPCR to evaluate the Hh signal transduction .", "( C ) The Wnt signaling activity in NIH3T3 cells incubated with PDE4D inhibitors together with Wnt3a for 4 hr Axin2 transcript levels were measured by qPCR to evaluate the Wnt signal transduction .", "All data are mean ± SEM .", "Statistics: Student t-Test , in comparison to the condition where cells were treated with DMSO .", "**p < 0 . 01 , ***p < 0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 07068 . 01110 . 7554/eLife . 07068 . 012Figure 4—figure supplement 1 . PDE4D inhibitors suppress SAG-induced Hh signal transduction .", "( A ) Chemical structure of the PDE4D inhibitors used in the assays .", "( B ) The Hh signaling activity in NIH3T3 cells incubated with PDE4D inhibitors together with SAG for 4 hr Gli1 transcript levels were measured by qPCR to evaluate the Hh signal transduction .", "All data are mean ± SEM .", "Statistics: Student t-Test , in comparison to the condition where cells were treated with DMSO .", "**p < 0 . 01 , ***p < 0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 07068 . 01210 . 7554/eLife . 07068 . 013Figure 4—figure supplement 2 . PDE4D knockdown reduces Hh signaling activity .", "( A ) Western blot shows that lentivirus-mediated expression of shRNA against PDE4D abolished the expression of endogenous PDE4D2/6 .", "( B ) On day 3 , Hh signaling activity was evaluated after cells were treated with Shh .", "Gli1 transcript level was measured by qPCR to evaluate Hh signaling activity .", "Data are mean ± SEM .", "Statistics: Student's t-Test .", "**p < 0 . 01 , ***p < 0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 07068 . 013 We then used shRNA to silence the expression of PDE4D in NIH3T3 cells , and assessed Hh signal transduction .", "Without PDE4D , the Shh-induced Hh signal transduction was significantly reduced ( Figure 4—figure supplement 2 ) .", "These data are fully consistent with a view of PDE4D as a positive regulator of Hh transduction .", "The SAG result indicates that PDE4D operates in the Hh pathway at the level of Smo or downstream .", "MB derives from over-proliferation of GNPs in the developing cerebellum ( Barakat et al . , 2010 ) , and Shh is a strong mitogen that stimulates GNP proliferation ( Dahmane and Ruiz i Altaba , 1999; Wallace , 1999; Wechsler-Reya and Scott , 1999 ) .", "To test whether Sema3-Nrp-PDE4D signaling controls GNP proliferation , we cultured GNPs in dishes and evaluated their proliferation with BrdU incorporation assay ( Figure 5A ) .", "The BrdU incorporation rate was doubled by SAG , and further increased by Sema3F .", "The effect of Sema3F was blocked by the NrppanA antibody , which interferes with Sema3-Nrp interaction , but not by the Nrp2B antibody , which blocks VEGF .", "Activating PKA with forskolin or inhibiting PDE4D with roflumilast completely abolished the Sema3F effect and reduced GNP proliferation to the baseline level ( Figure 5B ) .", "Thus the molecular mechanisms linking Sema3-Nrp signaling with the Hh pathway control GNP proliferation . 10 . 7554/eLife . 07068 . 014Figure 5 . GNP proliferation is promoted by Sema3 and inhibited by hyperactive PKA .", "( A ) GNP proliferation was assayed by BrdU incorporation in GNPs cultured in vitro for 20 hr .", "GNPs were cultured in medium with indicated reagents and BrdU was added 1 hr before the cells were fixed .", "Scale: 20 μm .", "( B ) BrdU incorporation rate was calculated as the number of BrdU-positive cells divided by total cell number .", "Error bars represent SEM , Statistics: Kruskal–Wallis non-parametric One-Way ANOVA .", "*p < 0 . 05 , ***p < 0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 07068 . 014 After establishing the molecular mechanism that integrates Nrp and Hh signaling in vitro , we explored the interplay of the two signaling pathways in vivo .", "To this end , we genetically removed both Nrp1 and Nrp2 .", "To circumvent the early lethality of Nrp1 knockout mice ( Kawasaki et al . , 1999 ) , we introduced a Math1-Cre allele into Nrp1floxed/floxed , Nrp2−/− mice ( Giger et al . , 2000; Gu et al . , 2003 ) .", "The Math1 enhancer drives Cre production in GNPs early in development ( Matei et al . , 2005 ) .", "Immunofluorescent staining of P7 cerebellum showed that Nrp1 is expressed in the external granule cell ( EGL ) layer , which contains GNP cell bodies , in the molecular layer ( ML ) , which contains parallel fibers of differentiated granule neurons , and in blood vessels ( Figure 6A ) .", "In Nrp1floxed/floxed , Nrp2−/− , Math1-Cre mice ( DKO ) , Nrp1 fluorescent signals disappeared from EGL and ML , but remained in blood vessels ( Figure 6A ) , demonstrating the specific knock-out of Nrp1 in GNPs .", "A protein blot of P7 cerebellar lysate from these mice showed elimination of detectable Nrp2 and little to no Nrp1 ( Figure 6B ) . 10 . 7554/eLife . 07068 . 015Figure 6 . Loss of Nrps impairs GNP proliferation in the developing cerebellum .", "( A ) Immunostaining of Nrp1 ( green ) in the P7 cerebellum of wild type and Nrp1/2 DKO mouse show that Nrp1 was specifically knocked out from GNP cells in EGL and parallel fibers in ML , but remains in blood vessels .", "Scale , 20 μm .", "( B ) Immunoblots showing that Nrp1 and 2 are knocked out from DKO cerebellum , and that Gli1 expression was much lower in the cerebellum of Nrp1/2 DKO mice compared to wild type littermates .", "Nrp1-cko: Math1Cre , Nrp1floxed/floxed .", "Nrp2-null: Nrp2-/- .", "( C ) The Hh pathway activity in the cerebellum of Nrp1/2 DKO mice and littermates was evaluated by Gli1 transcript level .", "( D , E )", "GNP proliferation in the EGL ( circled by dotted lines ) of Nrp1/2 DKO mice and littermates was evaluated by BrdU incorporation assay .", "Scale: 50 μm .", "( F , G )", "Immunostaining and quantification of Phospho-H3 ( green ) in EGL of wild type and Nrp1/2 DKO cerebellum at P7 .", "Scale , 100 μm .", "All error bars represent SEM .", "Statistics: non-parametric Mann–Whitney test .", "*p < 0 . 05 , **p < 0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 07068 . 01510 . 7554/eLife . 07068 . 016Figure 6—figure supplement 1 . Loss of Nrps in the developing cerebellum leads to PKA hyperactivation .", "( A ) Immunoblots showing that phospho-PKA level was significantly higher in Nrp1/2 DKO mice at P7 as indicated by phospho-PKA antibody , but the total PKA levels remained unchanged .", "Quantification of relative active PKA level ( mean ± SEM ) from 3 animals of each phenotype was shown at the bottom of each blot .", "( B ) Immunoblot showing that PKA substrate phosphorylation increased in Nrp1/2 DKO cerebellum comparing to wild type littermates . DOI: http://dx . doi . org/10 . 7554/eLife . 07068 . 016 Hh transduction was severely impaired in GNPs in Nrp double knock-out ( DKO ) mice , as assessed by Gli1 transcript and protein levels ( Figure 6B , C ) .", "BrdU incorporation was used to assess GNP proliferation .", "BrdU density was much lower in the EGL of DKO mice , reflecting GNP proliferation defects ( Figure 6D , E ) .", "The density of mitotic cells indicated by mitotic marker PH3 also significantly decreased in DKO mice ( Figure 6F , G ) .", "Thus genetically eliminating Sema3-Nrp signaling compromises Hh transduction in vivo , and consequently impairs GNP proliferation .", "We examined PKA activity in cerebella of Nrp DKO mice .", "In agreement with the results from NIH3T3 cells , PKA activity , assessed with pPKA-T197 antibody , was increased by 1 . 4 fold in the DKO , whereas the total PKA level remained unchanged ( Figure 6F—figure supplement 1A ) .", "Blots with the antibody that recognizes the phosphorylated PKA consensus motif showed that phosphorylation of PKA substrates was greatly increased in DKO cerebellum ( Figure 6—figure supplement 1B ) .", "In conclusion , eliminating Sema3-Nrp signaling from the cerebellum increases PKA activity .", "Next , we tested the effect of blocking Sema3-Nrp-PDE4D signaling on MB growth .", "We targeted PDE4D because roflumilast , the FDA-approved PDE4D inhibitor , blocks Hh transduction ( Figure 4 ) and is readily available for patients .", "Since PDE4D regulates Hh transduction downstream of Smo , roflumilast would be an ideal drug to treat vismodegib-resistant tumors .", "We tested this hypothesis in mouse MB tumors containing the Smo mutation ( SmoD477G ) that blocks vismodegib binding with Smo ( Kim et al . , 2013 ) ( Figure 7A ) .", "In mouse MB allografts , roflumilast dramatically inhibited tumor growth starting from the first day after treatment .", "In contrast , vismodegib had no effect on tumor growth , as expected ( Figure 7B , C ) .", "Roflumilast treatment also significantly reduced cell proliferation in tumors as evaluated by BrdU incorporation ( Figure 7C ) .", "Hh signal transduction dramatically decreased , as assessed by Gli1 transcript level in tumors ( Figure 7E ) .", "Thus roflumilast inhibited Hh signal transduction and suppressed the growth of vismodegib-resistant tumors . 10 . 7554/eLife . 07068 . 017Figure 7 . PDE4 inhibitors suppress Hh signal transduction and Hh-related tumor growth .", "( A ) Schematic diagram of the MB tumor allograft experiment in mouse .", "Drug treatment started 6 day after injection when the size of tumors could be accurately measured .", "( B ) The relative tumor size is defined as the tumor volume on the indicated day divided by that on day 0 .", "For each drug 9–10 mice were used .", "( C , D )", "BrdU density in tumors after drug treatment .", "Scale: 50 μm .", "( E ) Hh signal transduction in tumors was assessed by Gli1 transcription level through qPCR .", "RFM: roflumilast .", "Error bars represent SEM .", "Statistics: Student's t-Test .", "**p < 0 . 01 , ***p < 0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 07068 . 01710 . 7554/eLife . 07068 . 018Figure 7—figure supplement 1 . Schematic view of the integration of Sema3-Nrp with the Hh signal pathway by controlling PKA and PDE4 . ( A ) Without Sema3 or Nrps , the majority of PDE4D resides in the cytoplasm rather than being associated with the cell membrane , where cAMP is produced by AC .", "As peri-membrane cAMP is not easily accessible to cytoplasmic PDE4D for degradation , its concentration remains high , which leads to high PKA activity and inhibition of Hh signaling .", "( B ) In the presence of Sema3 and Nrps , PDE4D is recruited to the cell membrane and thus brought into close proximity to its target cAMP .", "This results in reduced cAMP concentration in the entire cell and at the cilium base .", "Consequently PKA is inhibited , which promotes Hh signal transduction .", "In this process , inhibition of PKA by Sema3-Nrp signal per se is insufficient to turn on Hh signaling; instead it enhances Hh signaling once Shh turns on the pathway .", "Abbreviations: AC: Adenylyl Cyclase; PDE4D: Phosphodiesterase 4D; Nrp: Neuropilin . DOI: http://dx . doi . org/10 . 7554/eLife . 07068 . 018" ], [ "Hh and Sema3-Nrp signaling are simultaneously involved in many developmental processes and diseases .", "The interaction between the two pathways was revealed only recently by our previous study ( Hillman et al . , 2011 ) .", "We found that Nrps positively regulate Hh transduction , and Hh in turn induces Nrp1 expression .", "In this previous study , we excluded the possibility that Nrps regulate binding or processing of the Hh ligand , and pinpointed Nrp participation in the Hh pathway at steps between Smo and SuFu .", "Beyond these findings , how the two pathways interact at the molecular level has been unknown .", "In the present study we discovered that the Sema3-Nrp pathway integrates with the Hh pathway by controlling the subcellular localization of PDE4D and consequently cAMP levels and PKA activity .", "Sema3 enhances the recruitment of PDE4D to the cell membrane by promoting its binding to the Nrp cytoplasmic domain ( Figure 7—figure supplement 1 ) .", "As most cAMP is produced by AC at the cell membrane , we speculate that the recruitment of PDE4D brings the enzyme into close proximity to the site of cAMP production and may enable more efficient hydrolysis of synthesized cAMP before it diffuses throughout the entire cytoplasm , including the cilium base .", "Consequently PKA is inhibited , which in turn promotes Hh transduction .", "Sema3 by itself cannot activate Hh signal transduction ( Figure 1C ) , but does enhance Hh signaling that has been activated by Shh or SAG ( Figure 1C , Figure1—figure supplement 1A , B ) .", "Therefore , we believe that the Sema3-Nrp signaling is modulatory rather than permissive for the Hh pathway , but nevertheless is a powerful facilitator of Hh transduction .", "Sema3-Nrp signaling is likely to contribute to Hh transduction in many tissues in vivo .", "During the development of many tissues , Sema3 genes and Shh are co-expressed , and the Hh pathway functions together with Nrps .", "For example , in the developing neural tube Sema3 and Shh act together to control the midline crossing of commissural axons ( Parra and Zou , 2010 ) .", "In developing skin where Hh signaling is critical for hair follicle development , Nrps are co-expressed in hair follicle cells with Hh signaling components such as Smo ( Hillman et al . , 2011 ) .", "In developing cerebellum Sema3 genes are co-expressed with Shh in Purkinje neurons .", "The two kinds of ligands may be released together to stimulate proliferation of GNPs in the cerebellar external granule layer ( http://www . gensat . org/imagenavigator . jsp ? imageID=50656 ) .", "Indeed , we found that genetic knockout of both Nrp genes blocks Sema3 signaling and impairs GNP proliferation in vivo .", "Finally , RNA-seq results showed that in pediatric medulloblastoma , Sema3 and Nrp1 expression levels are significantly elevated ( Cho et al . , 2011 ) .", "This elevated expression may contribute to increased Hh signaling activity that promotes tumor growth .", "Nrps are known as multi-functional co-receptors that bind to different ligands in different cell types ( Gu et al . , 2003; Gitler et al . , 2004; Pellet-Many et al . , 2008 ) .", "Sema3 and VEGF family proteins , both ligands for Nrp receptor complexes , activate distinct transduction cascades .", "At least three pathways lie downstream of Nrps .", "First , Sema3 activates a small GTPase of the Rho family , and regulates the reorganization of actin cytoskeleton in neurons .", "This pathway is implicated in growth cone collapse ( Chen et al . , 1998; Polleux et al . , 1998; Zanata et al . , 2002 ) .", "Second , VEGF family proteins signal through phospholipase C ( PLC ) to activate the pro-survival/pro-growth factor ERK1/2 and PI3K/Akt , and contribute to endothelial stimulation and pathologic angiogenesis ( Soker et al . , 1998; Bielenberg et al . , 2006 ) .", "Third , Sema3 signaling reduces PKA activity in growing axons via unknown mechanisms ( Parra and Zou , 2010; Shelly et al . , 2011 ) .", "The signaling mechanism downstream of Nrps that contributes to MB growth also involves these signaling pathways .", "Snuderl et al . ( 2013 ) found that the placental growth factor ( PLGF ) , a member of the VEGF family , acts through Nrp1 to promote MB growth .", "Their study suggested that the PLGF-Nrp1 signaling is involved in all subgroups of MB .", "At the molecular level , PLGF does so by activating ERK1/2 and PI3K/Akt .", "In our study , we found that Sema3 signals through Nrps to promote PDE4D activity and inhibit PKA , which ultimately potentiates Hh target gene activation .", "The Sema3-Nrp-PDE4D signaling is involved in the Shh subgroup of MB .", "Although the two studies revealed different signaling mechanisms downstream of Nrps focusing on different subgroups of MBs , both studies highlight the involvement of Nrps in the development of MB , and highlight Nrp1 as a promising therapeutic target for this devastating brain tumor .", "It has been proposed that cAMP-PKA activity at the base of cilia is crucial in regulating Hh signal transduction .", "The concentration of cAMP is controlled by two enzymes: the AC that produces cAMP , and the phosphosdiesterase ( PDE ) that hydrolyzes it .", "A recent study ( Mukhopadhyay et al . , 2013 ) reported a mechanism through which GPR161 activates Gαs-AC to produce cAMP at the cilia base , thereby switching off Hh signal transduction .", "Shh triggers the internalization of GPR161 from cilia , which switches on Hh signaling .", "This landmark work provides mechanistic insights into the coupling of cAMP-PKA activity to the on and off switch of the Hh pathway .", "Our study revealed the mechanism of cAMP control in Hh pathway—PDE hydrolysis .", "Furthermore , we found that Sema3-Nrp signal-mediated cAMP degradation does not switch on Hh transduction , but rather potentiates already activated Hh transduction .", "We believe that the mechanism of cAMP control by the GPR161-Gαs-AC and by Sema3-Nrp-PDE4D signaling coexist in the same cell , and may act at different stages of Hh signaling: GPR161 as a switch , and Sema3-Nrp as an amplifier .", "The substrates of PKA on Hh pathway are Gli transcription factors .", "In the absence of Hh ligand , PKA phosphorylates Gli3 and subsequently Gli3 is proteolytically processed into Gli3 repressor ( Wang et al . , 2000 ) .", "Gli2 is the major Hh pathway transcriptional activator; its activation is controlled by translocation to the cilium ( Humke et al . , 2010; Tukachinsky et al . , 2010 ) .", "From our experiments we conclude that Sema3-Nrp signaling inhibits PKA and that inhibition of Nrp functions impairs Shh-induced Gli2 enrichment at cilia tips .", "These observations support the idea that overactive PKA following Nrp RNAi is the mechanism of reduced Gli2 enrichment at cilia tips .", "This result is consistent with two previous findings .", "First , activation of PKA by Forskolin inhibits cilium translocation of the SuFu–Gli2/3 complex ( Humke et al . , 2010; Tukachinsky et al . , 2010 ) .", "Second , Gli2 localization to cilia tips is elevated in PKA-null mice ( Tuson et al . , 2011 ) .", "These studies also suggest that , within cilia , the SuFu–Gli complex is dissociated by active Smo , and that only after the dissociation can free Gli2 be activated and translocated into the nucleus to turn on target gene transcription .", "Therefore , without activation by Hh ligand , Gli2 cannot be active even if it is transported to cilia .", "This may explain why Sema3-Nrp signaling cannot , on its own , turn on Hh target genes , but does amplify Hh-triggered transcription .", "Intriguingly , we found that Sema3-Nrp is not involved in the production of Gli3R ( Figure 3—figure supplement 2C–F ) .", "Our finding is compatible with the existence of a PKA-independent mechanism of Gli3 processing .", "As shown by Tuson et al . ( 2011 ) , a significant portion of Gli3 is processed into Gli3R in PKA-null mice .", "This mechanism of Gli3 processing may kick in when PKA activity is inhibited by a Sema3-Nrp signal .", "Alternatively , PKA may control Gli2 and Gli3 at distinct subcellular locations , and Sema3-Nrp only modulates PKA activity where it controls Gli2 activation .", "A similar divergence between Gli2 activation and Gli3R regulation has been reported in Arl13B-null mice ( Caspary et al . , 2007 ) .", "In these mice , Gli3R is produced normally , whereas Gli2 is hyperactive .", "Another example is Eya1 , a phosphatase that positively regulates Hh signaling .", "Eya1 mutants exhibited reduced Hh transduction , but Gli3 processing was normal ( Eisner et al . , 2015 ) .", "These findings suggest that Gli2 activation and Gli3 repressor formation are independently regulated during Hh signal transduction .", "Finally , a third possibility exists: Gli3 processing and Gli2 inhibition could be sensitive to different levels of PKA activity .", "In cells without Nrps , Shh could still be able to lower PKA activity at the cilium base by inhibiting GPR161 to block Gli3 processing , whereas residual PKA activity could be sufficient to suppress Gli2 activation .", "In this model , the Sema3-Nrp-PDE4D pathway we describe here might act in parallel to Shh-Gpr161 pathway to more robustly reduce PKA activity in cilium , leading to more robust Gli2 activation .", "Nrps typically form complexes with the co-receptors Plexin and VEGF receptor to transduce signals from Sema3 and VEGF .", "However , accumulating evidence suggest that Nrps support intracellular signaling independent of their known co-receptors ( Pellet-Many et al . , 2008 ) .", "The short cytoplasmic domain of Nrp1 has been reported to bind to a PDZ-domain containing protein GIPC1 ( Cai and Reed , 1999 ) .", "We could not detect an interaction between Nrp1 and GIPC1 in immunoprecipitation assays ( data not shown ) , perhaps due to the different cell type used in our experiment .", "We discovered another protein that interacts with the Nrp1 cytoplasmic domain: PDE4D .", "Sema3 enhances the Nrp-PDE4D interaction and promotes the recruitment of PDE4D to the plasma membrane .", "Subcellular localization of PDE4D is an important mechanism for the precise control of cAMP concentration in the cell ( Houslay , 2010 ) .", "In airway epithelium PDE4D localizes to the apical domains of cells , where it degrades cAMP and prevents cAMP diffusion into other cellular locations ( Barnes et al . , 2005 ) .", "In cardiac myocytes PDE4D is rapidly recruited to the cell membrane by activated β2-adrenergic receptor to precisely control the spatial and temporal cAMP concentration ( Perry et al . , 2002 ) .", "These previous findings are consistent with our proposed model in which PDE4D efficiently degrades cAMP at the site of cAMP production , thereby reducing cAMP concentration throughout the cytosol and at the cilium base .", "We showed that this reduced cAMP concentration inhibited PKA activity , thus promoting Hh transduction .", "Our discovery was confirmed by a recent study in zebrafish showing that a small molecule that inhibits PDE4 promotes Hh signal transduction ( Williams et al . , 2015 ) .", "In additional to PKA , cAMP activates EPAC proteins , guanine nucleotide exchange factors for Ras-like GTPases ( Borland et al . , 2009 ) , and cyclic nucleotide-gated ion channels in the cilia of the olfactory neurons ( Bradley et al . , 2005 ) .", "It will be intriguing to see whether these cAMP targets also contribute to Hh transduction or other Hh-triggered effects on cells .", "Recently genome wide sequencing studies implicated mutations in PDE4D and PKA in adult Hh-related MB ( Kool et al . , 2014 ) , independently suggesting PDE4D and PKA as potential therapeutic targets .", "Our studies elucidate a mechanism that may well underlie these genetic phenomena .", "Remarkably , we found that targeting PDE4D to inhibit the newly discovered Sema3-Nrp-PDE4D-PKA pathway powerfully blocks the growth of Hh-related MBs that are resistant to Smo inhibitors .", "One of the PDE4D inhibitors , roflumilast , is used in clinics to treat chronic obstructive pulmonary disease ( COPD ) ( Giembycz and Maurice , 2014 ) .", "In addition to roflumilast , many PDE4D inhibitors are under development to improve cognitive and psychological performances , some of which are in clinical trials ( Gavalda and Roberts , 2013 ) .", "Our findings highlight the priority of repurposing PDE4D inhibitors as solo or combination therapies for Hh-related MB ." ], [ "Human Nrp1 and Nrp2 cDNA were subcloned into pEGFP-N1 vector .", "Human PDE4D2 cDNA and a triplicated Flag ( 3×Flag ) sequence ( DYKDHDG-DYKDHDI-DYKDDDDK ) were cloned into the pPBbrs2 vector , using Gibson assembly to place the 3×Flag tag at the C-terminus of PDE4D2 .", "ShhN conditional medium was obtained using a HEK 293 cell line that stably secretes ShhN .", "Recombinant Shh , Wnt3a and all Sema3 proteins were obtained from R&D Systems ( MN , USA ) , and reconstituted according to the instructions of the manufacture .", "SAG , Forskolin , Rolipram , Roflumilast and GEBR-7b were obtained from Millipore ( NY , USA ) .", "Mouse strains of Nrp1 conditional knockout ( Nrp1f/f ) , Nrp2−/− , and Math1-Cre were described previously ( Gu et al . , 2003; Matei et al . , 2005 ) .", "GNPs were isolated from P7 cerebellum using a protocol modified from Wechsler-Reya and Scott ( 1999 ) .", "Briefly , cerebella from P7 mice were removed , cut into small pieces with razor blades , and incubated at 37°C for 30 min in digestion buffer consisting of 1x HBSS ( ThermoFisher , NY , United States , 14 , 185 ) with 10 U/ml papain ( Worthington , NJ , United States , LSOO3126 ) and 250 U/ml DNase ( Sigma , MO , United States , D4627 ) .", "At the end of the incubation , the digestion buffer was removed and replaced with 1x HBSS containing 8 mg/ml Ovomucoid ( Worthington , NJ , United States , LK003182 ) , 8 mg/ml bovine serum albumin ( Sigma ) , and 250 U/ml DNase .", "Tissues were then triturated using Pasteur pipettes to obtain a single-cell suspension .", "Cells were centrifuged at room temperature and resuspended in PBS containing 0 . 02% BSA ( PBS/BSA ) .", "The cell suspension was passed through a cell strainer ( VWR , 21 , 008-952 ) to remove debris .", "The cell suspension was then underlaid with a step gradient of 35% and 65% Percoll ( Sigma , P4937 ) and centrifuged at high speed for 12 min at room temperature .", "Granule neuron precursors were harvested from the 35/65% interface and washed in PBS/BSA .", "Cells were then resuspended in Neurobasal medium ( Life Technologies , CA , United States , 21 , 103-0449 ) supplemented with GlutaMax ( ThermoFisher , 35 , 050-061 ) and Penicillin/streptomycin , and plated on tissue culture dishes coated with Poly-D-Lysine ( Millipore , MA , United States , A-003-E ) and Laminin ( Sigma , L2020 ) .", "Drugs were added at the time when cells were plated .", "Sema3F ( R&D , MN ) was used at 3 μg/ml , VEGF165 ( R&D , MN ) was used at 100 ng/ml , Nrp antibodies ( provided by Genentech , CA , United States ) were used at 50 μg/ml .", "SAG: 100 μM; Forskolin: 5 μM; Rolipram: 1 μM; Roflumilast: 1 μg/ml .", "2 hr BrdU pulse label was done 24 hr after plating .", "Cells were fixed with 4% paraformaldehyde , and underwent BrdU immunofluorescence .", "Mouse anti-BrdU ( monoclonal , 1:500 , Dakocytomation , CA , United States ) was used .", "NIH3T3 cells were plated 24 hr before each experiment at 70% confluency .", "The cultures were treated with vehicle ( 0 . 1% carrier protein BSA in PBS ) or Sema3F ( 3 μg/ml ) for 30 min or 1 hr .", "Cells were fixed with 4% paraformaldehyde for 10 min at room temperature .", "For Pericentrin staining , cells were then incubated with pre-cooled methanol at −20 °C for 8 min .", "After that cells underwent immunofluorescence staining .", "Primary antibodies were phosphoPKA-T197 ( Abcam , MA ) , PKA Cα ( Cell signaling , MA ) , pericentrin ( BD Biosciences , CA ) , acetylated tubulin ( Sigma , MO ) .", "Images were taken with an inverted laser-scanning confocal microscope ( DMIRE2; Leica ) .", "Confocal imaging stacks of Z-section scanning were superimposed into one image .", "The cytoplasmic levels of PhosphoPKA and PKA were analyzed using the open-source software program CellProfiler ( Broad Institute , Cambridge , MA ) .", "The process had four main steps: identification of nuclei ( Primary objects ) , identification of nuclei plus cytoplasm ( Secondary objects ) , determination of cytoplasm ( Tertiary objects ) , and measurement of signals in tertiary objects .", "Once the nuclei were identified , the secondary objects were determined by dilating primary objects by 15 pixels .", "The cytoplasm was identified by subtracting the nuclei from the secondary objects .", "Finally the mean gray level in the cytoplasm was calculated and exported to a spreadsheet .", "The levels of phosphoPKA and PKA in centrosome were measured using a customized program written in Matlab ( MathWorks , Natick , MA ) .", "First the contour of pericentriolar area was delineated based on the signal intensity in the red channel ( pericentrin staining ) .", "The program then measured the mean gray value of the enclosed area in the green channel ( PhosphoPKA and PKA staining ) ( V1 ) .", "The contours of each pericentriolar area were then manually moved to a nearby extracellular region , and the mean gray level of the enclosed area in the green channel was measured as background ( V2 ) .", "The final values of PhosphoPKA and PKA were calculated as V = V1−V2 .", "For each condition , 80–100 cells were measured , and data from one of the three independent experiments were shown .", "Subcellular fractionation was modified from the protocol in ( Humke et al . , 2010 , Genes Dev ) .", "Briefly , cells were cross-linked with 0 . 1 mM DSP at room temperature for 15 min , washed with 100 mM Tris buffer pH 7 . 5 , and scraped off the plate .", "After centrifugation at 1000×g , 10 min , the cell pellet was re-suspended and incubated in hypotonic solution ( 10 mM HEPES , pH 7 . 9 ) for 10 min on ice .", "Cells were pelleted again at 1000×g , and homogenized in SEAT buffer ( 10 mM Triethanolmine pH 7 . 4 , 10 mM Acetic Acid , 1 mM EDTA , 250 mM Sucrose , Protease Inhibitors ) with a Dounce homogenizer .", "Nuclei were separated by centrifugation at 900×g for 5 min , twice , and the supernatant was then spun in an ultracentrifuge at 95 , 000×g for 20 min at 4°C .", "The supernatant was kept as the cytosolic fraction , and was concentrated 10-fold using an Amicon centrifugal filter ( 10KD ) .", "The pellet was designated as the membrane fraction , and was resuspended in RIPA buffer .", "For each fraction , the protein concentration was determined using a BCA kit ( ThermoFisher ) , and an equal amount of total protein was loaded into each lane of the gel .", "For immunoblot to detect endogenous protein , cells were lysed in RIPA buffer ( 25 mM Tris pH 7 . 4 , 150 mM NaCl , 1% NP-40 , 1% sodium deoxycholate , 0 . 1% SDS , 1 mM DTT , 1 mM PMSF , 10 mM NaF , 50 mM Na-pyrophosphate , 5 mM EDTA , Roche protease inhibitor cocktail , Roche PhosphoSTOP cocktail ) for 30 min at 4°C .", "The lysate was clarified by centrifugation at 14 , 000 rpm for 30 min .", "Protein concentrations of the supernatants were determined using the detergent-insensitive BCA kit ( Pierce ) .", "Equal amounts of total protein from the samples were supplemented with 6× SDS sample buffer , and boiled for 5 min .", "Proteins were resolved using SDS-PAGE , and processed for immunoblotting .", "Primary antibodies used for immunoblot: Gli1 , Nrp2 , GAPDH , phospho- PKA-T197 , and PKA substrate are from Cell Signaling ( MA ) ; Nrp1 and VEGFR1 were from AbCam ( MA ) .", "Sema3F is from R&D ( MN ) .", "The intracellular cAMP concentration was measured using the cAMP-Glo assay kit from Promega according to the manufacture's instruction .", "The cAMP standard curve was generated using purified cAMP , from which the relative intracellular level of cAMP was inferred .", "For each drug treatment , 3 biological repeats were used , and each experiment was repeated 2–3 times .", "All data are expressed as mean ± SEM unless otherwise indicated .", "For statistical analysis of active and total PKA levels , Prism statistical analysis software was used ( GraphPad Software , CA ) .", "Kruskal–Wallis non-parametric One-Way ANOVA was used to assess significance of multiple data points .", "Non-parametric Mann–Whitney test was used between data comparing only two groups .", "For qPCR , the data were analyzed in Excel with Student t-Test .", "We consider a p value less than 0 . 05 statistically significant ." ] ]

[ "Alterations in Hedgehog ( Hh ) signaling lead to birth defects and cancers including medulloblastoma , the most common pediatric brain tumor .", "Although inhibitors targeting the membrane protein Smoothened suppress Hh signaling , acquired drug resistance and tumor relapse call for additional therapeutic targets .", "Here we show that phosphodiesterase 4D ( PDE4D ) acts downstream of Neuropilins to control Hh transduction and medulloblastoma growth .", "PDE4D interacts directly with Neuropilins , positive regulators of Hh pathway .", "The Neuropilin ligand Semaphorin3 enhances this interaction , promoting PDE4D translocation to the plasma membrane and cAMP degradation .", "The consequent inhibition of protein kinase A ( PKA ) enhances Hh transduction .", "In the developing cerebellum , genetic removal of Neuropilins reduces Hh signaling activity and suppresses proliferation of granule neuron precursors .", "In mouse medulloblastoma allografts , PDE4D inhibitors suppress Hh transduction and inhibit tumor growth .", "Our findings reveal a new regulatory mechanism of Hh transduction , and highlight PDE4D as a promising target to treat Hh-related tumors ." ]

[ "A communication system in cells called the Hedgehog signaling pathway plays an essential role in the formation of tissues and organs in animal embryos .", "The activity of the pathway is carefully controlled during development and if Hedgehog signaling is disrupted it can lead to developmental defects and particular types of cancer .", "Some of these cancers can be treated with a drug called vismodegib , which targets a particular molecule in the Hedgehog signaling pathway .", "However , tumor cells can become resistant to this drug , so researchers are hoping to find new therapies that target other aspects of the signaling pathway .", "Hedgehog signaling promotes the division of brain cells called granule neuron precursor cells ( or GNP cells for short ) .", "If the signaling pathway is over-active it can trigger the GNP cells to divide more than they should .", "This can lead to medulloblastoma , which is the most common type of brain tumor that affects children .", "Proteins called Neuropilins—which bind to molecules known as Semaphorins—promote Hedgehog signaling and the formation of medulloblastoma , but it was not clear how this works .", "Here Ge et al . studied the role of Neuropilin in cultured cells and in the cerebellum of mice .", "The experiments show that Semaphorin 3 promotes the accumulation of an enzyme called PDE4D at the cell membrane .", "PDE4D interacts with Neuropilin and blocks the activity of another enzyme that normally inhibits Hedgehog signaling .", "In mice that lack Neuropilin and Semophorin 3 , the GNP cells are less able to divide , which leads to abnormal development of the cerebellum .", "Further experiments show that drugs that target PDE4D inhibit both the Hedgehog pathway and the growth of tumors that are resistant to vismodegib treatment .", "Ge et al . 's findings uncover a new way in which Hedgehog signaling is regulated and highlight a potential new strategy for treating medulloblastoma and other similar tumors .", "Current PDE4D inhibitors are associated with severe side effects , so the next challenge is to develop new drugs that have fewer side effects ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "plant biology" ]

[ [ "Plants are colonized by a wide range of microorganisms .", "While some microbes enter the plant and establish endophytic interactions with a broad range of outcomes from beneficial to pathogenic , plant surfaces harbor a large variety of microbial organisms .", "Recent research has focused largely on the importance of the rhizosphere microbiota in nutrient acquisition , protection from pathogens , and boosting overall plant growth and development ( Ritpitakphong et al . , 2016; Walker et al . , 2003; Bulgarelli et al . , 2013 ) .", "However , the above ground parts of the plant including the phyllosphere are colonized by diverse groups of microbes that also assist in plant protection and immunity ( Busby et al . , 2016; Mikiciński et al . , 2016 ) .", "The environment has a major impact on the microbial communities of the leaf surface , ultimately influencing their interactions with the host ( Stone et al . , 2018 ) .", "Scale-free network analysis was performed with the leaf microbial population of Arabidopsis thaliana ( Agler et al . , 2016 ) .", "The majority of the interactions between kingdoms , e . g . fungi and bacteria , were found to be negative , consistent with the fact that rather the antagonistic interactions stabilize a microbial community ( Coyte et al . , 2015 ) .", "Phyllosphere network analysis of A . thaliana identified a small number of microbes as ‘hub’ organisms , i . e . influential microbes that have severe effects on the community structure .", "The major hub microbe in the A . thaliana phyllosphere is the oomycete Albugo laibachii , which is a pathogenic symbiont biotrophic of Arabidopsis ( Agler et al . , 2016 ) .", "This pathogen has been shown to significantly reduce the bacterial diversity of epiphytic and endophytic leaf habitats .", "Since bacteria generally comprise a large proportion of the phyllosphere microbiome ( Vorholt , 2012 ) , phylogenetic profiling of A . thaliana was also directed toward identifying a small group of bacteria that frequently colonize A . thaliana leaves .", "The analysis helped to develop a synthetic community of bacteria for experiments in gnotobiotic plants .", "Besides bacteria and oomycetes , the microbiota of the A . thaliana leaf also comprises a broad range of fungi .", "Among those fungi , basidiomycete yeasts are frequently found and the most frequent ones are the epiphytic basidiomycete genus Dioszegia ( Agler et al . , 2016 ) , as well as an anamorphic yeast associated with A . laibachii infection belonging to the Ustilaginales .", "This order includes many pathogens of important crop plants , for example corn smut and loose smut of oats , barley , and wheat are caused by Ustilago maydis , U . avenae , U . nuda , and U . tritici , respectively .", "Generally , the pathogenic development of smut fungi is linked with sexual recombination and plant infection is only initiated upon mating , when two haploid sporidia form a dikaryotic filament ( Brefort et al . , 2009 ) .", "Ustilaginales Pseudozyma sp .", "yeasts , however , are found mostly in their anamorphic stage in nature .", "They tend to epiphytically colonize a wide range of habitats , where an infrequent sexual recombination might occur when they meet on a susceptible host ( Kruse et al . , 2017 ) .", "Phylogenetic reconstruction ( Kruse et al . , 2017; Wang et al . , 2015 ) showed that the smut pathogen of millet , Moesziomyces bullatus and four species of Pseudozyma , namely P . antarctica , P . aphidis , P . parantarctica , and P . rugulosa form a monophyletic group .", "The latter do represent anamorphic and culturable stages of M . bullatus and , hence , can be grouped to this genus .", "Moesziomyces strains have been reported in a number of cases to act as microbial antagonists .", "A strain formerly classified as Pseudozyma aphidis ( now M . bullatus ) inhibited Xanthomonas campestris pv .", "vesicatoria , X . campestris pv .", "campestris , Pseudomonas syringae pv .", "tomato , Clavibacter michiganensis , Erwinia amylovora , and Agrobacterium tumefaciens in vitro and also led to the activation of induced defense responses in tomato against the pathogen ( Barda et al . , 2015 ) .", "It was reported that P . aphidis can parasitize the hyphae and spores of Podosphaera xanthii ( Gafni et al . , 2015 ) .", "Pseudozyma churashimaensis was reported to induce systemic defense in pepper plants against X . axonopodis , Cucumber mosaic virus , Pepper mottle virus , Pepper mild mottle virus , and broad bean wilt virus ( Lee et al . , 2017 ) .", "In the present study , we explored the antagonistic potential of an anamorphic Ustilaginales yeast within the leaf microbial community of A . thaliana .", "We show that Moesziomyces bullatus ex Albugo on Arabidopsis ( which will be referred to as MbA from further on ) prevents infection by the oomycete pathogen A . laibachii and identified fungal candidate genes that were upregulated in the presence of A . laibachii , when both microbes were co-inoculated to the host plant .", "A knockout mutant of one of the candidates , which belongs to the glycoside hydrolase – family 25 ( GH25 ) – was found to lose its antagonistic activity toward A . laibachii , providing mechanistic insights into fungal-oomycete antagonism within the phyllosphere microbiota .", "Functional characterization of GH25 will be an important step toward establishing MbA as a suitable biocontrol agent ." ], [ "Genome sequence of MbA was analyzed by Single Molecule Real-Time sequencing ( Pacific Biosciences , Menlo Park , CA ) , which lead to 69 , 674 mapped reads with an accuracy of 87 . 3% and 8596 bp sub-read length .", "Sequence assembly using the HGAP-pipeline ( Pacific Biosciences ) resulted in 31 contigs with an N50 Contig Length of 705 kb .", "The total length of all contigs results in a predicted genome size of 18 . 3 Mb ( Table 1 ) .", "Gene prediction for the MbA genome with Augustus ( Stanke et al . , 2004 ) identified 6653 protein coding genes , of which 559 carry a secretion signal .", "Out of these 559 , 380 are predicted to be secreted extracellularly ( i . e . they do not carry membrane domains or cell-wall anchors ) ( Table 1 ) .", "The small genome size and high number of coding genes result in a highly compact genome structure with only small intergenic regions .", "These are features similarly found in several pathogenic smut fungi such as U . maydis and S . reilianum ( Table 1 ) .", "Remarkably , both MbA and Anthracocystis flocculosa , which is another anamorphic and apathogenic yeast , show a similarly high rate of introns , while the pathogenic smut fungi have a significantly lower intron frequency ( Table 1 ) .", "To gain better insights in the genome organization of MbA , we compared its structure with the U . maydis genome , which served as a manually annotated high-quality reference genome for smut fungi ( Kämper et al . , 2006 ) .", "Out of the 31 MbA contigs , 21 show telomeric structures and a high synteny to chromosomes of U . maydis , with three of them displaying major events of chromosomal recombination ( Figure 2A ) .", "Interestingly , the Moesziomyces contig 2 , on which also homologs to pathogenic loci like the U . maydis virulence cluster 2A ( Kämper et al . , 2006 ) can be found , contains parts of three different U . maydis chromosomes ( Chr . 2 , 5 , and 20 ) ( Figure 2—figure supplement 1 ) .", "The second recombination event on contig six affects the U . maydis leaf-specific effector protein See1 , which is required for tumor formation ( Redkar et al . , 2015 ) .", "This recombination event is also found in the genome of the maize head smut S . reilianum , wherein the U . maydis chromosomes 5 and 20 recombined in the promoter region of the see1 gene ( Figure 2B ) .", "In this respect it should be noted that S . reilianum , although infecting the same host , does not produce leaf tumors as U . maydis does ( Schirawski et al . , 2010 ) .", "Also the third major recombination event , affecting MbA contig 8 , changes the genomic context genes encoding essential virulence factors in U . maydis ( stp1 and pit1/2 ) , as well as the A mating type locus , which is important for pheromone perception and recognition of mating partners ( Bölker et al . , 1992 ) .", "Based on the strong antibiotic activities of MbA , we mined the genome of MbA for the presence of secondary metabolite gene clusters .", "Using AntiSMASH , we were able to predict 13 of such clusters , of which three can be assigned to terpene synthesis , three contain nonribosomal peptide synthetases , and one cluster has a polyketide synthase as backbone genes ( Figure 3—figure supplement 1 ) .", "Interestingly , the secondary metabolite cluster that is involved in the production of the antimicrobial metabolite ustilagic acid in other Ustilaginomycetes is absent in MbA ( Figure 3—figure supplement 1B ) .", "On the contrary , we could identify three MbA-specific metabolite clusters that could potentially be involved in the antibacterial activity of MbA ( Figure 3—figure supplement 1C ) .", "A previous genome comparison of the related Ustilaginales yeast A . flocculosa with U . maydis concluded that this anamorphic strain had lost most of its effector genes , reflecting the absence of a pathogenic stage in this organism ( Lefebvre et al . , 2013 ) .", "In contrast , MbA contains 1:1 homologs of several known effectors with a known virulence function in U . maydis ( Table 2 ) .", "We previously found that Moesziomyces sp .", "possess functional homologues of the pep1 gene , a core virulence effector of U . maydis ( Sharma et al . , 2019 ) , suggesting that such anamorphic yeasts have the potential to form infectious filamentous structures by means of sexual reproduction ( Kruse et al . , 2017 ) .", "To assess the potential virulence activity of MbA effector homologs , we expressed the homolog of the U . maydis core effector Pep1 in an U . maydis pep1 deletion strain ( SG200∆01987 ) .", "This resulted in complete restoration of U . maydis virulence , demonstrating that MbApep1 encodes a functional effector protein ( Figure 3—figure supplement 2 ) .", "A hallmark of the U . maydis genome structure is the presence of large clusters with effector genes , the expression of which is only induced during plant infection ( Kämper et al . , 2006 ) .", "To assess the presence of potential virulence clusters in MbA , we compared all U . maydis effector gene clusters to the MbA genome , based on homology .", "This revealed that the 12 major effector clusters of U . maydis are present in MbA .", "However , while many of the clustered effector genes are duplicated in pathogenic smut fungi , MbA carries only a single copy of each effector gene .", "This results in the presence of ‘short’ versions of the U . maydis gene effector clusters ( Figure 3—figure supplement 3 ) .", "This gets particularly obvious for the biggest and most intensively studied virulence cluster of smut fungi , the effector cluster 19A ( Schirawski et al . , 2010; Brefort et al . , 2014; Dutheil et al . , 2016 ) .", "In MbA , only 5 out of the 24 effector genes present in U . maydis are conserved in this cluster ( Figure 3 ) .", "Interestingly , some anamorphic yeasts like Kalmanozyma brasiliensis and A . flocculosa completely lost virulence clusters , while another non-pathogenic member of the Ustilaginales , Pseudozyma hubeiensis , shows an almost complete set of effectors when compared to U . maydis ( Figure 3 ) .", "To perform reverse genetics in MbA , we established a genetic transformation system based on protoplast preparation and polyethylene glycol ( PEG ) -mediated DNA transfer .", "In preliminary transformation assays , we expressed a cytosolic GFP reporter-gene under control of the constitutive o2tef-Promoter ( Figure 4A ) .", "For the generation of knockout strains , a split marker approach was used to avoid ectopic integrations ( Figure 4B ) .", "To allow generation of multiple knockouts , we used a selection marker-recycling system ( pFLPexpC ) that allows selection marker excision at each transformation round ( Khrunyk et al . , 2010 ) .", "We decided to apply the transformation system to study the MbA mating type loci in more detail .", "Although phylogenetically closely related to U . hordei , which has a bi-polar mating system , MbA owns a tetrapolar mating system whereby both mating type loci are physically not linked .", "This situation is similar to the mating type structure in the pathogenic smut U . maydis ( Figure 4A ) .", "The a-locus , which encodes a pheromone receptor system that is required for sensing and fusion of compatible cells , is located on contig 6 .", "The b-locus can be found on contig 1 .", "This multiallelic mating locus contains two genes ( b-East and b-West ) , which code for a pair of homeodomain transcription factors .", "Upon mating of compatible cells , pathogenic and sexual development are triggered by a heterodimeric bE/bW complex ( Brefort et al . , 2009 ) .", "Since the MbA genome is completely equipped with mating type genes , we first deployed a screen for potential mating partners .", "To this end , we screened wild M . bullatus isolates to find a suitable mating partner , but we could not observe any mating event ( Figure 5—figure supplement 1 ) .", "To test if MbA is able to undergo pathogenic differentiation in the absence of mating , we generated a self-compatible strain ( CB1 ) that carries compatible b-mating alleles: to construct the CB1 strain , we used compatible alleles of the b-East and b-West genes of the barley smut U . hordei , a pathogen that is the phylogenetically most closely related to MbA and amenable to reverse genetics .", "The native MbA locus was replaced by the compatible U . hordei b-East and b-West gene alleles via homologous recombination ( Figure 5B ) .", "Incubation of the MbA CB1 on charcoal plates led to the formation of aerial hyphae with the characteristic fluffy phenotype of filamentous strains like the self-compatible , solopathogenic U . maydis SG200 strain ( Figure 5C ) .", "A second established method to induce filament formation in smuts is on hydrophobic parafilm ( Mendoza-Mendoza et al . , 2009 ) .", "Quantification after 18 hr incubation of MbA CB1 on parafilm resulted in the formation of filaments comparable to those of the U . maydis SG200 strain ( Figure 5D ) .", "While about 17% of MbA wild-type cells showed filaments , the CB1 strain with compatible b-genes showed 38% filamentous growth .", "Formation of appressoria is a hallmark of pathogenic development in smut fungi ( Mendoza-Mendoza et al . , 2009 ) .", "While the switch from yeast-like growth to filamentous development is the first step in the pathogenic development of smut fungi , host penetration is accompanied by the formation of a terminal swelling of infectious hyphae , termed ‘appressoria’ .", "Induction of appressoria formation in vitro can be induced by adding 100 µM of the cutin monomer 16-hydroxyhexadecanoic acid ( HDD ) to the fungal cells prior to cell spraying onto a hydrophobic surface ( Mendoza-Mendoza et al . , 2009 ) .", "In the absence of HDD , only about 8% of the U . maydis SG200 cells and 14% of the MbA cells formed appressoria on parafilm 24 hr after spraying ( Figure 5E ) .", "Addition of 100 µM HDD resulted in a significant induction of appressoria in both U . maydis and MbA , demonstrating that MbA does hold the genetic repertoire to form infection structures in vitro .", "Together , the analysis of the recombinant CB1 strain indicates that MbA can sense pathogenesis-related surface cues and produce penetration structures to a similar level as that seen for the pathogenic model organism U . maydis .", "To study the transcriptomic response of MbA to different biotic interactions , RNA sequencing was performed .", "The MbA transcriptome was profiled in five different conditions ( Figure 6A; cells in axenic culture versus cells on-planta , on-planta + SynCom , on-planta + A . laibachii , on-planta + SynCom + A . laibachii ) .", "Inoculations of A . thaliana leaves were performed as described above for A . laibachii infection assays ( Figure 6A ) .", "For MbA RNA preparation , the epiphytic microbes were peeled from the plant tissue by using liquid latex ( see Materials and methods section for details ) .", "The libraries of the 15 samples ( five conditions in three biological replicates each ) were generated by using a poly-A enrichment and sequenced on an Illumina HiSeq4000 platform .", "The paired end reads were mapped to the MbA genome by using Tophat2 ( Kim et al . , 2013 ) .", "The analysis revealed that MbA cells on A . thaliana leaves ( on-planta ) downregulated 1300 and upregulated 1580 genes compared to cells in axenic culture ( Figure 6B , Supplementary file 2 ) .", "Differentially expressed genes were determined with the ‘limma’-package in R on ‘voom’ ( Figure 6—figure supplement 1 ) using a False discovery rate threshold of 0 . 05 and log2FC > 0 .", "A gene ontology ( GO ) terms analysis revealed that , among the downregulated genes , 50% were associated with primary metabolism ( Figure 6—figure supplement 2 ) .", "In the two conditions in which A . laibachii was present , we observed upregulation of 801 genes .", "Among these genes , 411 genes were specific to co-incubation of MbA with A . laibachii and SynCom while 174 were specific to incubation with A . laibachii only .", "A set of 216 genes was shared in both conditions ( Figure 6B ) .", "In the presence of A . laibachii , mainly metabolism- and translation-dependent genes were upregulated , which might indicate that MbA can access a new nutrient source in the presence of A . laibachii ( Figure 6—figure supplement 2 ) .", "Among all A . laibachii-induced MbA genes , 18 genes encode proteins carrying a secretion signal peptide and having no predicted transmembrane domain ( Figure 6C ) .", "After excluding proteins being predicted to be located in intracellular organelles , nine candidate genes remained as potential microbe–microbe-dependent effectors , i . e . MbA genes that are induced by A . laibachii , show no or low expression in axenic culture , and encode for putative secreted proteins ( Figure 6C ) .", "Interestingly , four of these genes encode putative glycoside hydrolases .", "Furthermore , two genes encode putative peptidases , one gene likely encodes an alkaline phosphatase and two encode uncharacterized proteins ( Figure 6C ) .", "To directly test the eventual antagonistic function of those genes toward A . laibachii , we selected the two predicted glycoside hydrolases-encoding genes g5 and g2490 ( GH43 and GH25 ) and the gene encoding the uncharacterized protein g5755 for gene deletion in MbA .", "The respective mutant strains were tested in stress assays to assess , whether the gene deletions resulted in general growth defects .", "Wild-type and mutant MbA strains were exposed to different stress conditions including osmotic stress ( sorbitol , NaCl ) , cell wall stress ( calcofluor , Congo-red ) , and oxidative stress ( H2O2 ) .", "Overall , in none of the tested conditions we observed a growth defect of the deletion mutants in comparison to wild-type MbA ( Figure 7—figure supplement 1 ) .", "To test an eventual impact of the deleted genes in the antagonism of the two microbes , the MbA deletion strains were each pre-inoculated on A . thaliana leaves prior to A . laibachii infection .", "Deletion of g5 resulted in a significant but yet marginal increase of A . laibachii disease symptoms , while deletion of g5755 had no effect on A . laibachii .", "We therefore considered these two genes being not important for the antagonism of MbA toward A . laibachii .", "Strikingly , the MbA Δg2490 strain almost completely lost its biocontrol activity toward A . laibachii .", "This phenotype was reproduced by two independent g2490 deletion strains ( Figure 7A ) .", "To check if this dramatic loss of microbial antagonism is specific to the deletion of g2490 , in-locus genetic complementation of strain Δg2490_1 was performed via homologous recombination .", "The resulting strain MbA Δg2490/compl regained the ability to suppress A . laibachii infection , confirming that the observed phenotype specifically resulted from the deletion of the g2490 gene ( Figure 7B ) .", "Together , these results demonstrate that the biocontrol of the pathogenic oomycete A . laibachii by the basidiomycete yeast MbA is determined by the secretion of a previously uncharacterized GH25 enzyme , which is transcriptionally activated specifically when both microbes are co-colonizing the A . thaliana leaf surface .", "To characterize the protein function of the GH25 encoded by MbA g2490 , we were using Pichia pastoris for heterologous expression .", "The recombinant protein was tagged with polyhistidine tag for Ni-NTA affinity purification .", "The purified protein was detected at an expected size of 27 kDa ( Figure 7—figure supplement 2 ) .", "In addition , via site directed mutagenesis a mutated version of the protein was generated , carrying a single amino exchange at the predicted active site ( GH25_D124E ) .", "Both active and mutated versions of the GH25 hydrolase were subjected to a quantitative lysozyme activity assay using the fluorogenic substrate Micrococcus lysodeikticus with commercial Hen egg-white lysozyme ( HEWL ) as a control .", "We noticed a concentration-dependent increase in relative fluorescence unit ( RFU ) /min for the active GH25 in molar concentrations from 2 µM to 10 µM .", "Whereas , for similar concentrations , mutated GH25 ( GH25mut ) showed no significant increase in RFU/min compared to the active version .", "Commercial HEWL showed a steady increase in RFU/min from 1 µM to 5 . 5 µM concentrations ( Figure 7C; Figure 7—figure supplement 2 ) .", "Thus , the recombinant protein represents a functional GH25 hydrolase with a lysozyme activity .", "To test for a direct function of the GH25 lysozyme , we treated A . laibachii-infected Arabidopsis plants with the recombinant protein .", "To quantify the impact of GH25 treatment on A . laibachii infection , we performed quantitative PCR to determine the relative A . laibachii biomass on Arabidopsis in response to GH25 .", "Strikingly , we observed a significant reduction of A . laibachii colonization in leaves treated with the active GH25 lysozyme , while the mutated enzyme GH25_D124E did not significantly influence infection ( p-value of <0 . 0001 and an R2 value of 98 . 88% ) ( Figure 7D ) .", "Overall , treatment with the GH25 lysozyme reduced the amount of A . laibachii to about 50% ." ], [ "Healthy plants in natural habitats are extensively colonized by microbes , therefore it has been hypothesized that the immune system and the microbiota may instruct each other beyond the simple co-evolutionary arms race between plants and pathogens ( Vannier et al . , 2019 ) .", "Community members as individuals or in a community context have been reported to confer extended immune functions to their plant host .", "Root endophytic bacteria for example were found to protect A . thaliana and stabilize the microbial community by competing with filamentous eukaryotes ( Durán et al . , 2018 ) .", "A large inhibitory interaction network was found in the leaf microbiome of A . thaliana and genome mining was used to identify over 1000 predicted natural product biosynthetic gene clusters ( BGCs ) ( Helfrich et al . , 2018 ) .", "In addition , the bacterium Brevibacillus sp .", "leaf 182 isolate was found to inhibit half of the 200 strains isolated from A . thaliana phyllosphere .", "Further analysis revealed that Brevibacillus sp .", "leaf 182 produces a trans-acyltransferase polyketide synthase-derived antibiotic , macrobrevin along with other putative polyketide synthases ( Helfrich et al . , 2018 ) .", "In this study , we describe the role of the basidiomycete yeast MbA , which we previously co-isolated with the oomycete pathogen A . laibachii and now characterized as an antagonistic driver in the A . thaliana phyllosphere .", "A . laibachii inhibits in vitro growth of seven members of a bacterial leaf SynCom and , most strikingly , strongly suppresses disease progression and reproduction of the pathogenic oomycete A . laibachii on A . thaliana .", "MbA is a member of the Ustilaginales , which had previously been classified into the group of pathogenic smut fungi of the Moesziomyces bullatus species ( Kruse et al . , 2017 ) .", "Our genome analysis identified the anamorphic yeasts M . rugulosus , M . aphidis , and M . antarcticus , which had previously been classified as ‘Pseudozyma sp . ’ , as the closest relatives of MbA .", "Anamorphic Ustilaginales yeasts are long known and have been used for biotechnological applications and also biocontrol ( Boekhout , 2011 ) .", "Mannosylerythritol lipids produced by M . antarcticus are known to act as biosurfactants and are of great interest for pharmaceutical applications ( Kitamoto et al . , 1990; Morita et al . , 2007 ) .", "Glycolipids like flocculosin produced by A . flocculosa or ustilagic acid characterized in the smut fungus U . maydis have antifungal activity .", "Those compounds destabilize the membrane of different fungi and thus serve as biocontrol agents against powdery mildews or gray mold ( Cheng et al . , 2003; Mimee et al . , 2005; Teichmann et al . , 2007 ) .", "We identified 13 potential secondary metabolite gene clusters in MbA , including non-ribosomal peptide synthases and polyketide synthetase .", "Interaction among microbes within the same habitat is believed to have given rise to a variety of secondary metabolites ( Schroeckh et al . , 2009; Rutledge and Challis , 2015 ) .", "The presence of Streptomyces rapamycinicus was shown to activate an otherwise silent polyketide synthase gene cluster , fgnA , in Aspergillus fumigatus .", "The resultant compound proved to be a potent fungal metabolite that inhibited the germination of S . rapamycinicus spores ( Stroe et al . , 2020 ) .", "Therefore , secondary metabolite gene clusters and their corresponding products may confer a competitive advantage to fungi over the bacteria that reside in the same environment .", "What is still under investigation is the relation of anamorphic yeasts with the related pathogenic smuts .", "Many smut fungi including the model species U . maydis are dimorphic organisms .", "In their saprophytic phase they grow as haploid non-pathogenic yeast cells .", "Only on appropriate host surfaces , haploid cells switch to filamentous growth and expression of pathogenicity-related genes is only activated upon mating in the filamentous dikaryon .", "A prime prerequisite for pathogenic development is therefore the ability of mating ( Bölker , 2001; Nadal and Gold , 2010 ) .", "Our genome analysis identified a tetrapolar mating system with a complete set of mating genes in MbA .", "Looking more closely on the phylogeny of different mating genes it appears that all sequenced Moesziomyces strains have the same pheromone receptor type ( Figure 7—figure supplement 3 ) .", "Together with our unsuccessful mating assays , this suggests that all sequenced strains of this species have the same mating type and , therefore , are unable to mate .", "Mating type bias after spore germination was reported for Ustilago bromivora , which leads to a haplo-lethal allele linked to the MAT-2 locus ( Rabe et al . , 2016 ) .", "In this case , an intratetrad mating event rescues pathogenicity in nature as the second mating partner is not viable after spore germination .", "Together with the observation that anamorphic Moesziomyces yeasts are ubiquitous in nature , one could hypothesize that these fungi are highly competitive in their haploid form and antagonism might have led to the selection of one viable mating type .", "This eventually adapted to the epiphytic life style .", "At the same time , one should not dismiss the possibility that in the presence of a viable mating partner and suitable host surface , the yeasts can undergo potential sexual reproduction and thereby revitalizing the gene pool from time to time .", "Transcriptome analysis showed that epiphytic growth of MbA on A . thaliana leads to massive transcriptional changes particularly in primary metabolism , which might reflect adaptation to the nutritional situation on the plant surface .", "Moreover , MbA showed specific transcriptional responses to a bacterial community , as well as to A . laibachii when being co-inoculated on plant leaves .", "The presence of A . laibachii resulted in the induction of primary metabolism and biosynthesis pathways , which might reflect enhanced growth of MbA in the presence of A . laibachii .", "A set of MbA genes encoding secreted hydrolases was induced by A . laibachii and one of these genes which encodes a putative GH25 hydrolase with similarity to Chalaropsis type lysozymes appeared to be essential for the biocontrol of A . laibachii .", "Initially discovered in the fungi Chalaropsis sp .", ", this group of proteins is largely present in bacteria as well as phages , for example the germination-specific muramidase from Clostridium perfringens S40 ( Chen et al . , 1997 ) .", "The bacterial muramidase , cellosyl from Streptomyces coelicolor ( Rau et al . , 2001 ) , also belongs to the Chalaropsis type of lysozyme .", "These proteins are proposed to cleave the β-1 , 4-glycosidic bond between N-acetylmuramic acid ( NAM ) and N-acetylglucosamine ( NAG ) in the bacterial peptidoglycan .", "Specifically , the β-1 , 4 N , 6-O-diacetylmuramidase activity allows the Chalaropsis type lysozyme to degrade the cell wall of Staphylococcus aureus , in contrast to the commercially available HEWL ( Rau et al . , 2001 ) .", "Despite differences in structure and molecular weight from HEWL , the GH25 of MbA has lysozyme activity against the gram positive bacterium Micrococcus lysodeikticus in a fluorogenic assay .", "This highlights the overall biochemical functionality of the recombinant glycoside hydrolase .", "The glycoside Hydrolase 25 family is predicted to have an active site motif DXE that is highly conserved across the fungal kingdom ( Figure 7—figure supplement 4 ) .", "The structure of glycoside hydrolase family 25 from Aspergillus fumigates was characterized and the presence of N-terminal signal peptide was considered to indicate an extracellular secretion of the protein with possible antimicrobial properties ( Korczynska et al . , 2010 ) .", "The role of the secreted hydrolase in the fungal kingdom is not completely explored yet .", "The presence of such hydrolases has in many cases been hypothesized to be associated with hyperparasitism of fungi parasitizing fungi ( Hyde et al . , 2019 ) or oomycetes parasitizing oomycetes ( Horner et al . , 2012 ) .", "Our results might therefore indicate a cross kingdom hyperparasitism event between a fungus and an oomycete .", "Previous work on microbial communities has indicated that negative interactions stabilize microbial communities .", "Hyperparasitism is such a negative interaction with a strong eco-evolutionary effect on pathogen–host interactions and therefore on community stability ( Parratt and Laine , 2016 ) .", "MbA might therefore regulate A . laibachii infection and reduce disease severity .", "The qPCR evaluation of oomycete biomass strongly points toward the idea that A . laibachii is a direct target of antagonism for MBA .", "Since we observed reduced formation of A . laibachii in the presence of MbA , we also tested if the GH25 lysozyme would suppress zoospore germination .", "However , we could not detect a significant reduction of A . laibachii zoosporangia germination upon treatment with active GH25 lysozyme ( Figure 7—figure supplement 5 ) , suggesting that the GH25 lysozyme interferes with A . laibachii at a later stage of infection .", "As A . laibachii has been shown to reduce microbial diversity ( Agler et al . , 2016 ) , MbA might increase diversity through hyperparasitism of A . laibachii .", "At the same time this increased diversity might have caused the need for more secondary metabolites to evolve in the MbA genome to defend against niche competitors .", "Through its close association with A . laibachii , MbA could be a key regulator of the A . thaliana microbial diversity and therefore relevant for plant health beyond the regulation of A . laibachii infection .", "In conclusion , the secreted hydrolase we identified as a main factor of A . laibachii inhibition has great potential to act as antimicrobial agent .", "The isolated compound is not only valuable per se in an ecological context .", "It can further lay the grounds for exploring other microbial bioactive compounds that mediate inter-species and inter-kingdom crosstalk .", "A main goal of our future studies will be to understand on the mechanistic level , how the GH-25 suppresses A . laibachii , and at which developmental step the oomycete infection is blocked .", "It will be particularly interesting to elucidate if and how the GH25 enzyme activity directly interferes with the A . laibachii cell wall .", "While the canonical lysozyme substrate is found in bacterial cell walls , a detailed biochemical characterization of substrate specificity will be required to pinpoint potential target sites in the oomycete cell wall .", "Also , to the best of our knowledge there is no detailed information on the A . laibachii cell wall structure and composition .", "One could speculate if GH25 activity directly affects cell wall integrity of A . laibachii , or if a modification of the cell wall structure interferes with pathogenic development , e . g . by interfering with cellular differentiation , blocking signal perception , or by triggering a host defense response .", "Since the GH-25 enzyme is well conserved among Ustilaginales including pathogenic species , it will also be tempting to elucidate whether the species-specific antagonism identified here is broadly conserved among Ustilaginales fungi and oomycetes .", "We further will investigate potential responses by the host plant and how this impacts A . laibachii growth upon MbA colonization .", "Functional investigation of these interactions can provide meaningful insights as to why certain yeasts prefer to colonize specific environments .", "At the same time , it will be worth exploring how the basidiomycete yeasts influence the bacterial major colonizers of the phyllosphere ." ], [ "MbA wild-type strain was isolated from A . laibachii infected A . thaliana leaves [7] .", "Wild-type MbA ( at 22° ) and U . maydis ( at 28° ) strains were grown in liquid YEPS light medium and maintained on potato dextrose agar plates .", "King’s B medium was used for culturing Syn Com bacterial members at 22° .", "All the strains were grown in a rotary shaker at 200 rpm .", "All the recipes for medium and solutions can be found in Supplementary file", "3 . Stress assays for fungi: wild-type and mutant strains of MbA grown to an optical density ( 600 nm ) of 0 . 6–0 . 8 were centrifuged at 3500 rpm for 10 min and suspended in sterile water to reach an OD of 1 . 0 .", "Next , a dilution series from 100 to 10–4 was prepared in sterile H2O .", "In the end , 5 μl of each dilution was spotted on CM plates supplemented with the indicated stress agents .", "The plates were incubated for 2 days at 22°C .", "Confrontation assays: at first , MbA and SynCom bacterial strains were grown to an O . D of 0 . 8–1 .", "MbA cultures ( 10 μl ) were dropped in four quadrants of a potato dextrose agar plate , previously spread with a bacterial culture .", "Plates were incubated for 2–4 days at 22°C .", "Fungal strains were grown in YEPSL at 22°C in a rotary shaker at 200 rpm until an O . D . of 0 . 6 was reached and centrifuged for 15 min at 3500 rpm .", "The cells were washed in 20 ml of SCS ( Supplementary file 3 ) and further centrifuged for 10 min at 3000 rpm , before being treated with 3 ml SCS solution with 20 mg/ml of Glucanex ( Lysing Enzyme from Trichoderma harzianum , # L1412 , Sigma ) .", "After 20 min of incubation at room temperature , as cell wall lysis occurred , cold SCS was added to the mixture and protoplasts spun down for 10 min at 2400 rpm .", "They were then washed twice with SCS and resuspended with 10 ml STC ( Supplementary file 3 ) to be centrifuged at 2000 rpm for 10 min .", "Finally , the pellet was dissolved in 500 µl STC and stored in aliquots of 50 µl at −80°C .", "Five micrograms of plasmid DNA along with 15 µg heparin was added to 50 µl protoplasts .", "After incubation on ice for 10 min , STC/40% PEG ( 500 µl ) was added to it and mixed gently by pipetting up and down; this step was followed by another 15 min on ice .", "The transformation mix was added to 10 ml of molten regeneration ( reg ) agar and poured over a layer of already solidified reg agar containing appropriate antibiotic solution .", "For the bottom layer , we used 400 µg/ml hygromycin/8 µg/ml carboxin/300 µg/ml nourseothricin ( NAT ) .", "Plasmids were cloned using Escherichia coli DH5α cells ( Invitrogen , Karlsruhe , Germany ) .", "Construction of deletion mutants was performed by homologous recombination; the 5’ and 3’ flanking regions of the target genes were amplified and ligated to an antibiotic resistance cassette ( Kämper , 2004 ) .", "The ligated fragment was subsequently transformed into MbA .", "Homologous integration of the target gene was verified via PCR on the antibiotic resistant colonies .", "Oligonucleotide pairs for knockout generation and verification can be found in Supplementary file", "4 . PCR amplification was done using Phusion DNA polymerase ( Thermo Scientific , Bonn , Germany ) , following the manufacturer’s instructions , with 100 ng of genomic DNA or cDNA as template .", "Nucleic acids were purified from 1% TAE agarose gels using Macherey-Nagel NucleoSpin Gel and PCR Clean-up Kit .", "Haploid strains of MbA were grown in liquid cultures , mixed , and drops arranged on PD plates with charcoal to induce filament formation .", "Plate with the haploid U . maydis strains FB1 and FB2 and the solopathogenic strain SG200 served as internal control .", "The complete b-locus of the solopathogenic U . hordei strain DS200 was amplified ( Figure 1—figure supplement 2 ) and inserted into the MbA b-locus by homologous recombination .", "The strain obtained , known as compatible b1 ( CB1 ) , was tested positive by amplification of the right border and left border areas with primers specific for the genomic locus and for the plasmid region .", "Additionally , two primers specific for the MbA bE and bW genes were chosen to amplify parts of the native locus .", "To induce filament and appressoria formation in vitro we used a Moesziomyces YEPSL culture at OD600 0 . 6–0 . 8 .", "The cells were diluted to an OD600 of 0 . 2 in 2% YEPSL ( for appressoria formation 100 µM 16-hydroxyhexadecanoic acid [Sigma-Aldrich] or 1% ethanol was added ) and sprayed the yeast like cells on parafilm which mimics the hydrophobic plant surface .", "After 18 hr of incubation at 100% humidity the number of cells grown as filaments ( or generating appressoria ) was determined relative to the total number of total cells by using a light microscope .", "Sterilized Arabidopsis thaliana seeds were subjected to cold treatment for 7 days and sown on 1/2 strength Murashige Skoog ( MS ) medium ( Supplementary file 3 ) .", "The MS plates are directly transferred to growth chambers having 22°C on a short-day period ( 8 hr light ) with ( 33–40% ) humidity and grown for 4 weeks before inoculation .", "Overnight liquid cultures of MbA and SynCom bacterial strains were grown to an OD600 of 0 . 6 .", "The cultures were spun down at 3500 rpm for 10 min and the pellets dissolved in MgCl2 .", "Five hundred microliters of each culture was evenly sprayed on 3-week old A . thaliana seedlings using airbrush guns .", "Two days later , a spore solution of A . laibachii was then sprayed on the seedlings following the protocol of Ruhe et al . , 2016 .", "Two weeks later , the disease symptoms on the leaves were scored as a percentage between infected and non-infected leaves .", "Four weeks old A . thaliana seedlings on MS plates were sprayed with A . laibachii as a control and GH25+ A . laibachii and Mut_GH25+A .", "laibachii as treatments .", "After 10 dpi , the seedlings were harvested , frozen in liquid nitrogen , and kept at −80°C .", "For DNA extraction , the frozen plant material was ground into a fine powder with mortar and pestle and treated with extraction buffer ( 50 mM Tris pH 8 . 0 , 200 mM NaCl , 0 . 2 mM ethylenediaminetetraacetic acid [EDTA] , 0 . 5% SDS , 0 . 1 mg/ml proteinase K [Sigma–Aldrich] ) .", "This was followed by centrifugation after the addition of one volume phenol/chloroform/isoamylalkohol , 25:24:1 ( Roth ) .", "The top aqueous layer was removed and added to one volume of isopropanol to precipitate the nucleic acids .", "DNA pellet obtained after centrifugation was washed with 70% EtOH and finally dissolved in 50 µl nuclease-free water .", "For qPCR measurements , 10 µl of GoTaq qPCR 2× Master Mix ( Promega , Waltham , Madison , USA ) , 5 µl of DNA ( ~50 ng ) , and 1 µl of forward and reverse primer ( 10 µM ) up to a total volume 20 µl were used .", "Samples were measured in triplicates in a CFX Connect real-time PCR detection system ( Bio-Rad ) following the protocol of Ruhe et al . , 2016 .", "Amount of A . laibachii DNA was quantified using the following oligonucleotide sequences: A . thaliana EF1-α: 5′-AAGGAGGCTGCTGAGATGAA-3′ , 5′-TGGTGGTCTCGAACTTCCAG-3′; Oomycete internal transcribed spacer ( ITS ) 5 . 8 s: 5′-ACTTTCAGCAGTGGATGTCTA-3′ , 5′-GATGACTCACTGAATTCTGCA-3′ .", "Cq values obtained in case of the oomycete DNA amplification was normalized to A . thaliana DNA amplicon and then the difference between control ( only Albugo ) and treatment ( Albugo+ GH25/Mut_GH25 ) was calculated by ddCq .", "The relative biomass of Albugo was analyzed by the formula ( 2−ddCq ) .", "Each data point in the graph represents three independent biological replicates .", "RNA-Extraction of Latex-peeled samples: Four weeks old A . thaliana plants were fixed between two fingers and liquid latex was applied to the leaf surface by using a small brush .", "The latex was dried using the cold air option of a hair dryer , carefully peeled off with a thin tweezer , and immediately frozen in liquid nitrogen .", "Afterwards , the frozen latex pieces were grinded with liquid nitrogen and the RNA was isolated by using Trizol Reagent ( Invitrogen , Karlsruhe , Germany ) according to the manufacturer’s instructions .", "Turbo DNA-Free Kit ( Ambion , Life Technologies , Carlsbad , California , USA ) was used to remove any DNA contamination in the extracted RNA .", "Synthesis of cDNA was performed using First Strand cDNA Synthesis Kit ( Thermo Fischer scientific , Waltham , Massachusetts , USA ) according to recommended instruction starting with a concentration of 10 µg RNA .", "QIAprep Mini Plasmid Prep Kit ( QIAGEN , Venlo , The Netherlands ) was used for isolation of plasmid DNA from bacteria after the principle of alkaline lysis .", "Genomic DNA was isolated using phenol–chloroform extraction protocol ( Kämper et al . , 2006 ) .", "RT-qPCR oligonucleotide pairs were designed with Primer3 Plus .", "The oligonucleotide pairs were at first tested for efficiency using a dilution series of genomic DNA .", "The reaction was performed in a Bio-Rad iCycler system using the following conditions: 2 min at 95°C , followed by 45 cycles of 30 s at 95°C , 30 s at 61°C , and generation of melting curve between 65°C and 95°C .", "Sequence assembly of MbA strains was performed using the HGAP pipeline ( Pacific Biosciences ) .", "MbA genome was annotated with the Augustus software tool .", "Secretome was investigated using SignalP4 . 0 .", "Analysis of functional domains in the secreted proteins was done by Inter-Pro Scan .", "AntiSmash was used to predict potential secondary metabolite clusters .", "RNA sequencing was done at the Cologne Center for Genomics ( CCG ) by using a poly-A enrichment on an Illumina HiSeq4000 platform .", "The achieved paired end reads were mapped to the MbA and A . thaliana TAIR10 genome by using Tophat2 ( Kim et al . , 2013 ) .", "RNA-Seq reads of MbA axenic cultures were used to generate exon and intron hints and to start a second annotation with Augustus .", "Heat maps were performed using the heatmap . 2 function of the package gplots ( version 3 . 0 . 1 ) in R-studio ( R version 3 . 5 . 1 ) .", "An analysis of variance ( ANOVA ) model was used for pairwise comparison of the conditions , with Tukey's HSD test to determine the significant differences among them ( p-values <0 . 05 ) .", "The Pichia pastoris KM71H-OCH gene expression system was used to produce MBA_GH25 domain tagged with an N-terminal Polyhistidine tag ( 6xHis ) and a C-terminal peptide containing the c-myc epitope and a 6xHis tag .", "The His-MspGH25 cloned into pGAPZαA vector ( Invitrogen , Carlsbad , CA , USA ) under the control of a constitutive promotor with an α-factor signal peptide for secretion .", "Expression and purification of recombinant proteins were performed according to manufacturer’s instructions ( Invitrogen Corporation , Catalog no . K1710-01 ) : YPD medium supplemented with 100 μg ml−1 zeocin was used for initial growth of P . pastoris strains at 28°C and 200 rpm ( for liquid cultures ) .", "Production of the recombinant protein was performed in 1 L buffered ( 100 mM potassium phosphate buffer , pH 6 . 0 ) YPD medium with 2% sucrose at 28°C for 24 hr with 200 rpm shaking .", "Next the protein was subjected to affinity purification with a Ni-NTA-matrix according to manufacturer’s instructions ( Ni-Sepharose 6 Fast-Flow , GE-Healthcare; Freiburg , Germany ) .", "After purification , the His-MspGH25 protein was dialyzed in an exchange buffer ( 0 . 1 M NaPi , 0 . 1 M Nacl , pH = 7 . 5 ) .", "The purified protein was kept in 100 µl aliquots at 4°C .", "Site-directed mutagenesis was performed on pGAPZα-His- MspGH25 vector according to the instructions of the QuikChange Multi Site-Directed Mutagenesis Kit ( Agilent Technologies , Santa Clara , United States ) with primers targeting nucleotides of the active site of GH25 .", "Purified glycoside hydrolase of MBA from P . pastoris was quantified according to a sensitive fluorescence-based method using Molecular Probes EnzChek Lysozym-Assay-Kit ( ThermoFisher Scientific , Katalognummer: E22013 ) .", "DQ lysozyme substrate ( Micrococcus lysodeikticus ) stock suspension ( 1 . 0 mg/ml ) and 1000 units/ml HEWL stock solution were prepared according to the manufacturer's instruction .", "Molar concentration of the HEWL stock solution was calculated using the following website ( https://www . bioline . com/media/calculator/01_04 . html ) and was found to be 11 µM .", "Protein concentration of MspGH25 both active and mutated version was measured in the Nanodrop 2000c spectrophotometer ( Thermo Fischer scientific , Waltham , Massachusetts , USA ) according to the manufacturer’s instructions using 100 µl of sample after using 100 µl of the appropriate buffer as a blank control in glass cuvette .", "The molar concentrations of recombinant proteins were also calculated as above .", "At the start of the reaction 50 μl of the DQ lysozyme substrate working suspension was added to each microplate well containing reaction buffer with either HEWL ( in molar concentrations ranging from 0 . 1 to 5 . 5 µM ) or MspGH25 ( in molar concentration from 0 . 5 to 17 . 5 µM ) .", "Fluorescence intensity of each reaction was measured every 5 min to follow the kinetic of the reaction at 37°C for 60 min , using fluorescence microplate reader with fluorescein filter Tecan Infinite 200 Pro plate reader ( Tecan Group Ltd . , Männendorf , Switzerland ) .", "Digestion products from the DQ lysozyme substrate have an absorption maximum at ~494 nm and a fluorescence emission maximum at ~518 nm ." ] ]

[ "Plants are not only challenged by pathogenic organisms but also colonized by commensal microbes .", "The network of interactions these microbes establish with their host and among each other is suggested to contribute to the immune responses of plants against pathogens .", "In wild Arabidopsis thaliana populations , the oomycete pathogen Albugo laibachii plays an influential role in structuring the leaf phyllosphere .", "We show that the epiphytic yeast Moesziomyces bullatus ex Albugo on Arabidopsis , a close relative of pathogenic smut fungi , is an antagonistic member of the A . thaliana phyllosphere , which reduces infection of A . thaliana by A . laibachii .", "Combination of transcriptomics , reverse genetics , and protein characterization identified a GH25 hydrolase with lysozyme activity as a major effector of this microbial antagonism .", "Our findings broaden the understanding of microbial interactions within the phyllosphere , provide insights into the evolution of epiphytic basidiomycete yeasts , and pave the way for novel biocontrol strategies ." ]

[ "Much like the ‘good bacteria’ that live in our guts , many microscopic organisms can co-exist with and even benefit the plants they live on .", "For instance , the yeast Moesziomyces bullatus ex Albugo ( MbA for short ) can shield the leaves of its plant host against white rust , a disease caused by the organism Albugo laibachii .", "Studies have started to unveil how the various microbes at the surface of leaves interact and regulate their own community , yet the genetic mechanisms at play are less well-known .", "To investigate these processes , Eitzen et al . examined the genes that were switched on when MbA cells were in contact with A . laibachii on a leaf .", "This experiment revealed a few gene candidates that were then deleted , one by one , in MbA cells .", "As a result , a gene emerged as being key to protect the plant from white rust .", "It produces an enzyme known as the GH25 hydrolase , which , when purified , could reduce A . laibachii infections on plant leaves .", "Bacteria , fungi and other related microorganisms cause many diseases which , like white rust , can severely affect crops .", "Chemical methods exist to prevent these infections but they can have many biological and ecological side effects .", "A solution inspired by natural interactions may be safer and more effective at managing plant diseases that affect valuable crops .", "Harnessing the interactions between microbes living on plants , and the GH25 enzyme , may offer better disease control ." ]

[ "Introduction", "Results", "Discussion", "Material and methods" ]

[ "short report", "neuroscience" ]

[ [ "The sense of body ownership ( i . e . the belief that a specific body part belongs to one’s own body ) ( Gallagher , 2000 ) is a fundamental aspect of self-consciousness .", "Apparently , in normal conditions , the feeling of body ownership does not need any particular explanation; it is immediate and even obvious .", "However , both pathological cases after brain damage ( somatoparaphrenia [Romano et al . , 2014] and pathological embodiment [Pia et al . , 2016; Fossataro et al . , 2016; Garbarini et al . , 2013 , 2014 , 2015; Pia et al . , 2013; Garbarini and Pia , 2013] ) and experimental manipulations in healthy subjects ( e . g . the rubber hand illusion – RHI [Botvinick and Cohen , 1998] ) suggest that body ownership , as we normally experience it , is the product of many different and complex operations .", "It has been suggested that the feeling that our body belongs to us presumably depends on multisensory integration processes arising within a fronto-parietal network , where sensory inputs coming from different modalities are realigned in a unique reference frame ( Blanke et al . , 2015 ) .", "Within this network , the ventral premotor cortex seems to play a crucial role , thus establishing , both in monkeys ( Graziano , 1999 ) and in humans ( Makin et al . , 2008; Ehrsson et al . , 2004 ) , an anatomical link between the sense of body ownership and the motor system .", "Furthermore , it has been proposed that voluntary motor activity of body parts contributes critically to the subjective experience of body ownership ( Tsakiris et al . , 2010 ) .", "Within this context , we asked whether the subjective and sometimes illusory sense of body ownership influences objective measures of the sensory-motor system .", "We took advantage of the RHI paradigm in order to provide a physiological counterpart of the interaction between body awareness and motor control , investigating the relationship between body ownership alterations , such as those occurring during the RHI , and modulation of primary motor cortex excitability .", "During the RHI , the subject’s real hand is out of view , while a realistic rubber hand ( RH ) is positioned in its place .", "When the experimenter synchronously strokes the index finger of both the real and the fake hand , most subjects , after a few seconds of viewing the fake hand’s finger being touched , attribute their tactile sensation to the RH hand , which they start to perceive as their own .", "During the illusion , the subject’s hand-centered reference frame shifts towards the RH , and so it has been proposed that , as a consequence , the real hand is subject to a sort of disembodiment ( Ehrsson et al . , 2004 ) .", "Accordingly , a feeling of disownership of one’s own hand has been reported as an important behavioural component of the RHI ( Longo et al . , 2008 ) , while a decrease in the temperature of the real ( disembodied ) hand has been observed as a neurophysiological correlate of the RHI ( Moseley et al . , 2008 ) .", "Moreover , it has been demonstrated that cooling the subject’s hand increases the strength of the RHI , whereas warming the hand decreases it ( Kammers et al . , 2011 ) .", "However , another study found that hand-cooling can be present in both the experimental ( synchronous ) and the control ( asynchronous ) condition , thus suggesting that it is not a reliable correlate of the subjective feeling of hand disownership in the RHI ( Rohde et al . , 2013 ) .", "A further study proposed that somatosensory changes observed in the participants' hand while experiencing the RHI can be explained by cross-modal mismatch between the seen and felt position of the hand , and are not necessarily a signature of disownership ( Folegatti et al . , 2009 ) .", "In the present study , in the main behavioral experiment , we employed a classical RHI procedure to investigate the presence of the illusory experience in our subject sample .", "In addition , in the control behavioral experiment , the complementary presence of both embodiment of the RH and disembodiment of the real hand was explicitly investigated .", "Moreover , during the main physiological experiment , we studied the excitability modulation of motor circuits to the real ( stimulated ) hand during RHI .", "While subjects received visual-tactile stimulations , either synchronous ( to induce the illusion ) or asynchronous ( control condition ) , motor evoked potentials ( MEPs ) were elicited by a single-pulse of transcranial magnetic stimulation ( TMS ) over the left primary motor cortex ( M1 ) and recorded from the right first dorsal interosseous muscle ( FDI ) .", "See details in Material and methods and in Figure 1A . 10 . 7554/eLife . 14972 . 003Figure 1 . Experimental setup . The white square indicates the opening in the experimental wooden box through which the rubber hand is visible to the subject .", "Subjects could see only the rubber hand being stroked by the experimenter’s right hand .", "In A , main experiment , MEPs were acquired from the stimulated ( right ) hand’s FDI muscle; in B , control experiment , MEPs were acquired from non-stimulated ( left ) hand’s FDI muscle .", "In C , timeline of the study and experimental conditions are plotted . DOI: http://dx . doi . org/10 . 7554/eLife . 14972 . 003 We hypothesized that , in the motor domain , a disembodiment effect during the RHI might be measurable as a lower excitability of motor pathways to the real hand , i . e . a situation in which a stronger voluntary command is needed to bring enough motor neurons to threshold and thus to elicit movement .", "Thus , a decrease of FDI MEP amplitude compared to the baseline , specific to the real ( disembodied ) hand , was expected in the synchronous condition ( where the subjects experienced the RHI ) and not in the asynchronous ( control ) condition .", "Furthermore , according to behavioral studies reporting an increased illusory experience over time ( Lewis and Lloyd , 2010; Valenzuela Moguillansky et al . , 2013 ) , this inhibitory motor response was expected to increase during exposure to the illusion .", "Finally , we expected the MEP amplitude decrease to be specific for the stimulated ( right ) hand , i . e . no amplitude modulation in the non-stimulated ( left ) hand ( see Figure 1B; control physiological experiment ) ." ], [ "The main behavioral results showed that both proprioceptive drift towards the RH and embodiment questionnaire rating ( emb-q-rating ) were significantly higher in the synchronous than in the asynchronous condition ( drift=mean ± sd: 4 . 51 ± 4 . 2 cm vs . 2 . 08 ± 2 . 75 cm; t ( 23 ) =2 . 783 , p=0 . 0105 , dz=0 . 58; emb-q-rating=mean ± sd: 2 . 4 ± 0 . 64 vs . −2 ± 0 . 9 , Z=4 . 2857 , p=0 . 000018 , dz=3 . 88; Figure 2A1 and A2; see also Figure 2—source data 1 ) .", "No significant correlation was found between emb-q-rating and proprioceptive drift . 10 . 7554/eLife . 14972 . 004Figure 2 . Behavioral results following asynchronous and synchronous condition . The average values for proprioceptive drift and emb-q-rating are plotted in A1 and A2 , respectively , for the main experiment , and in B1 and B2 , respectively , for the control experiments .", "In B3 are reported average values for disemb-q-rating .", "Error bars indicate 95% CI .", "Significant levels: *p<0 . 05; ***p<0 . 0001 .", "Linear regressions between emb-q rating and disemb-q-rating in both syncronous and asyncronous conditions and in the delta syncronous minus asyncronous are plotted in C1 , C2 , C3 , respectively .", "All subjects behavioural data are available in the additional source data file ( see Figure 2—source data 1 and 2 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 14972 . 00410 . 7554/eLife . 14972 . 005Figure 2—source data 1 . Main experiment behavioral results following asynchronous and synchronous condition .", "( A ) MAIN EXPERIMENT .", "For each subject , the proprioceptive drift ( estimation of right index finger felt position ) mean values , calculated as the difference between pre and post stimulation in synchronous ( mean ± sd = 4 . 51 ± 4 . 2 ) and asynchronous ( mean ± sd = 2 . 08 ± 2 . 75 ) , are reported .", "( B ) MAIN EXPERIMENT .", "For each subject , the mean rating value of the three ownership statements in synchronous ( mean ± sd = 2 . 4 ± 0 . 64 ) and asynchronous ( mean ± sd = -2 . 04 ± 0 . 9 ) are reported . DOI: http://dx . doi . org/10 . 7554/eLife . 14972 . 00510 . 7554/eLife . 14972 . 006Figure 2—source data 2 . Control experiment behavioral results following asynchronous and synchronous condition .", "( A ) CONTROL EXPERIMENT .", "For each subject , the proprioceptive drift ( estimation of right index finger felt position ) mean values , calculated as the difference between pre and post stimulation in synchronous ( mean ± sd = 2 . 47 ± 2 . 707 ) and asynchronous ( mean ± sd = 0 . 075 ± 2 . 461 ) , are reported .", "( B ) CONTROL EXPERIMENT .", "For each subject , the mean rating value of the three ownership statements in synchronous ( mean ± sd = 2 ± 0 . 763 ) and asynchronous ( mean ± sd = -0 . 97 ± 1 . 387 ) are reported .", "( C ) CONTROL EXPERIMENT .", "For each subject , the mean rating value of the three disownership statements in synchronous ( mean ± sd = 0 . 153 ± 1 . 427 ) and asynchronous ( mean ± sd = -1 . 15 ± 1 . 371 ) are reported . DOI: http://dx . doi . org/10 . 7554/eLife . 14972 . 006 In the control behavioral experiment , similar results were found for both proprioceptive drift and emb-q-rating ( drift=mean ± sd: 2 . 47 ± 2 . 707 cm vs . 0 . 075 ± 2 . 461 cm; t ( 19 ) =5 . 275 , p=0 . 000043 , dz=1 . 18; emb-q-rating=mean ± sd: 2 ± 0 . 763 vs . −0 . 97 ± 1 . 387; t ( 19 ) =9 . 357 , p=0 . 0000001 , dz=-2 . 1; Figure 2 , B1 and B2; see also Figure 2—source data 2 ) .", "Furthermore , the disembodiment questionnaire rating ( disemb-q-rating ) was significantly higher in the synchronous than in the asynchronous condition ( disemb-q-rating=mean ± sd: 0 . 153 ± 1 . 427 vs . −1 . 15 ± 1 . 371; t ( 19 ) =3 . 835 , p=0 . 00116 , dz==0 . 86; Figure 2 , B3; see also Figure 2—source data 2 ) .", "Finally , significant correlations were found between emb-q-rating and disemb-q-rating in both synchronous and asynchronous conditions and in the delta synchronous minus asynchronous ( respectively: r-=-0 . 4911 , p=0 . 0279; r-=-0 . 7128 , p=0 . 0004; r-=-0 . 5537 , p=0 . 0113; Figure 2 , C1 , C2 , C3 ) .", "No significant correlations was found between proprioceptive drift and either emb-q-rating or disemb-q-rating .", "For the physiological data , the Friedman non-parametric ANOVA showed a significant effect of condition ( χ2 [2 , n=24]=9 , 000 , 000; p= 0 . 01111 ) , suggesting a difference between baseline , synchronous and asynchronous conditions , when MEPs were recorded from the stimulated ( right ) hand .", "Wilcoxon matched pairs tests , after Bonferroni correction , revealed a significant MEP decrease in the synchronous condition with respect to both the asynchronous ( mean ± sd: −0 . 367 ± 0 . 362 vs . 0 . 205 ± 0 . 395; Z=3 . 3143 , p=0 . 000919; dz=0 . 85 ) and the baseline ( mean ± sd: −0 . 367 ± 0 . 362 vs . 0 . 277 ± 0 . 691; Z=3 . 1428 , p=0 . 001673; dz=0 . 74 ) conditions ( Figure 3A1; see also Figure 3—source data 1 ) .", "No significant difference was found between asynchronous and baseline conditions ( 0 . 205 ± 0 . 395 vs . 0 . 277 ± 0 . 691; Z=0 . 1714 , p=0 . 863887; dz=0 . 08 ) .", "Examples of MEPs recorded from the FDI muscle of a representative subject are shown in Figure 3 .", "Interestingly , the MEP time-course analysis showed that the inhibitory motor response increases over time , with lower values measured at each time-point .", "Note that Wilcoxon matched pairs tests , after Bonferroni correction , showed a significant difference between TIME 1 ( first five trials ) and TIME 4 ( last five trials ) ( mean ± sd: −0 . 23963 ± 0 . 425821 vs . −0 . 5481 ± 0 . 394248; Z=2 . 942857; p=0 . 003252; dz=0 . 72 ) , Figure 3A2; see also Figure 3—source data 1 . 10 . 7554/eLife . 14972 . 007Figure 3 . Physiological results for the baseline , asynchronous and synchronous conditions . Average MEP amplitude variation in the FDI muscle recorded across all subjects are plotted in A1 , for the main experiment , and in B1 for the control experiments .", "Histograms represent the peak-to-peak MEP mean amplitude ( normalized ) ± 95% CI in the baseline , asynchronous and synchronous conditions , respectively .", "Significant levels: **p<0 . 005; ***p<0 . 0001 .", "Average MEP amplitude profile recorded across all subjects in the synchronous condition areplotted in A2 for the main experiment and in B2 for the control experiment; points represent the peak-to-peak MEP mean amplitude ( normalized ) , ± 95% CI , at four time-points after induction of the illusion ( 90 s , 180 s , 270 s 360 s ) ; significance level: **p<0 . 005 .", "Examples of average raw MEPs recorded from two representative subjects ( for the main and control experiments ) in the baseline ( main: 609 µVolt; control: 619 µVolt ) , asynchronous ( main: 771 µVolt; control: 601 µVolt ) and synchronous ( main:150 µVolt; control:583 µVolt ) conditions .", "All subjects' physiological data are available in an additional source data file ( see Figure 3—source data 1 and 2 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 14972 . 00710 . 7554/eLife . 14972 . 008Figure 3—source data 1 . Main experiment physiological results during baseline , asynchronous and synchronous condition .", "( A ) MAIN EXPERIMENT .", "For each subject , the mean MEPs amplitude ( row values in µV ) , recorded during baseline ( mean ± sd = 945 . 204 ± 535 . 076 ) , asynchronous condition ( mean ± sd = 918 . 075 ± 635 . 777 ) and synchronous condition ( mean ± sd = 527 . 328 ± 325 . 998 ) , are reported .", "( B ) MAIN EXPERIMENT .", "For each subject , the mean MEPs amplitude ( normalized z-scores ) , recorded during baseline ( mean ± sd = 0 . 277 ± 0 . 691 ) , asynchronous condition ( mean ± sd = 0 . 205 ± 0 . 395 ) and synchronous condition ( mean ± sd = -0 . 367 ± 0 . 362 ) , are reported .", "In the z-scores computation , the mean and the sd of the three conditions were used to normalized row data according to the formula x-mean/sd .", "( C ) MAIN EXPERIMENT .", "For each subject , mean amplitude of 5 MEPs ( normalized z-scores ) at four time-point recorded during synchronous condition are reported ( respectively mean ± sd of TIME 1 , 2 , 3 , 4: -0 . 239 ± 0 . 425 , -0 . 284 ± 0 . 620 , -0 . 389 ± 0 . 385 , -0 . 548 ± 0 . 394 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 14972 . 00810 . 7554/eLife . 14972 . 009Figure 3—source data 2 . Control experiment physiological results during baseline , asynchronous and synchronous condition .", "( A ) CONTROL EXPERIMENT .", "For each subject , the mean MEPs amplitude ( row values in µV ) , recorded during baseline ( mean ± sd = 725 . 665 ± 365 . 104 ) , asynchronous condition ( mean ± sd = 734 . 398 ± 416 . 262 ) and synchronous condition ( mean ± sd = 685 . 368 ± 469 . 286 ) , are reported .", "( B ) CONTROL EXPERIMENT .", "For each subject , the mean MEPs amplitude ( normalized z-scores ) , recorded during baseline ( mean ± sd = 0 . 057 ± 0 . 368 ) , asynchronous condition ( mean ± sd = 0 . 045 ± 0 . 262 ) and synchronous condition ( mean ± sd = -0 . 104 ± 0 . 399 ) , are reported .", "In the z-scores computation , the mean and the sd of the three conditions were used to normalized row data according to the formula x-mean/sd .", "( C ) CONTROL EXPERIMENT .", "For each subject , mean amplitude of 5 MEPs ( normalized z-scores ) at four time-point recorded during synchronous condition are reported ( respectively mean ± sd of TIME 1 , 2 , 3 , 4: −0 . 092 ± 0 . 483 , −0 . 224 ± 0 . 435 , −0 . 093 ± 0 . 656 , −0 . 009 ± 0 . 587 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 14972 . 009 By contrast , in the control physiological experiment , in the three-level one-way ANOVA , no significant effect of condition was detected ( F ( 2 , 38 ) =0 , 894943; p=0 . 417068 ) , suggesting that for the non-stimulated ( left ) hand there is no difference between the synchronous condition and either the asynchronous ( mean ± sd: −0 . 104 ± 0 . 399 vs . 0 . 045 ± 0 . 262; p=0 . 820737; dz=0 . 26 ) or baseline ( mean ± sd: −0 . 104 ± 0 . 399 vs . 0 . 0572 ± 0 . 368; p=0 . 711483; dz=0 . 22 ) conditions ( Figure 3B1; see also Figure 3—source data 2 ) .", "Moreover , in the four-level one-way ANOVA adopted for analysis of MEP time-course , no significant effect of TIME was found ( F ( 3 , 57 ) =0 , 842673; p=0 . 476191 ) , suggesting that in the non-stimulated ( left ) hand , MEP amplitude is not modulated over time ( Figure 3B2; see also Figure 3—source data 2 ) .", "Additionally , the Mann-Whitney U-test revealed a significant difference between the delta synchronous minus asynchronous obtained in each experiment , showing a strong physiological effect of the RHI in the main compared to the control experiment ( mean ± sd: −390 . 746 ± 591 . 662 vs . − 49 . 0296 ± 238 . 332; Z=2 . 074180; p=0 . 038063 ) ." ], [ "In the present study , in order to investigate the link between body-ownership and motor system , we took advantage of the well-established RHI paradigm ( Botvinick and Cohen , 1998; Ehrsson et al . , 2004; Tsakiris et al . , 2010; Longo et al . , 2008; Moseley et al . , 2008; Kammers et al . , 2011; Rohde et al . , 2013; Folegatti et al . , 2009; Lewis and Lloyd , 2010; Valenzuela Moguillansky et al . , 2013; Tsakiris and Haggard , 2005 ) , a useful tool to manipulate the sense of body ownership in normal subjects .", "In the main behavioral experiment , our data show a very strong embodiment effect in the synchronous condition ( with a mean rating of 2 . 4 , on a scale of −3 to +3 ) , while none of the subjects reported the illusory experience in the asynchronous condition ( with a mean rating of −2 , on a scale of −3 to +3 ) .", "A complementary disembodiment effect ( Longo et al . , 2008 ) was investigated and measured in the control behavioral experiment , showing a significant correlation between the reported ownership of the fake hand and disownership of the subject’s own hand .", "Most importantly , the physiological results provide the first evidence that , during the RHI , the motor excitability of corticospinal hand circuits for the real stimulated hand is greatly reduced .", "This effect is absent for the real non-stimulated hand .", "In addition , consistent with behavioral studies reporting an increased illusory experience over time ( Lewis and Lloyd , 2010; Valenzuela Moguillansky et al . , 2013 ) , the time-course analysis revealed that motor cortex excitability decreases as time of exposure to the illusion increases .", "The link between body ownership and motor system activation has been investigated with different behavioral paradigms within the RHI framework .", "Recently , using a moving version of the RHI , it has been shown that voluntary ( but not passive ) movement of the real hand decreases the perceptual shift towards the rubber hand , suggesting that the subjective sense of agency strongly contributes to a coherent sense of body ownership ( Tsakiris et al . , 2010 ) .", "On the other hand , patients with schizophrenia , who show a specific deficit in predicting the consequences of their voluntary actions ( Voss et al . , 2010 ) , as well as an altered sense of agency ( Garbarini et al . , 2016; Daprati et al . , 1997; Maeda et al . , 2012 ) , are more susceptible to the RHI ( Peled et al . , 2003; Asai et al . , 2011; Thakkar et al . , 2011 ) .", "Other studies on clinical populations with movement disorders suggest that patients with focal hand dystonia ( Fiorio et al . , 2011 ) or partial or complete paralysis because of spinal cord injury ( Scandola et al . , 2014; Tidoni et al . , 2014 ) seem to have some impairment of body ownership , according to their susceptibility to the RHI paradigm .", "A recent study ( Burin et al . , 2015 ) , investigating the RHI in movement disorder after brain-damage , shows that hemiplegic patients display a weaker/more flexible sense of body ownership for the affected ( paralyzed ) hand ( where the strength of the RHI is increased ) when compared to controls , but an enhanced/more rigid sense of body ownership for the healthy hand ( where the strength of the RHI is decreased ) .", "In other words , the prolonged absence of movement makes the paralyzed limb more readily disowned , while the healthy limb seems to be more strongly owned .", "Furthermore , other studies ( Kilteni et al . , 2016; Schütz-Bosbach et al . , 2006 ) have investigated the modulation of corticospinal excitability during different experimental manipulations related to the sense of body ownership , though none with the specific hypothesis of linking a decrease in subjective ownership with a decrease in motor excitability .", "Kilteni and colleagues ( 2016 ) found that healthy people can experience a pseudo-amputation illusion during a virtual reality procedure , suggesting that this experimental manipulation causes corticospinal excitability changes in muscles associated with the virtually amputated body-part .", "Schütz-Bosbach and colleagues ( 2006 ) studied how an action observation task can induce different changes in corticospinal excitability , depending on the level of ownership experienced with respect to the observed moving hand .", "Ownership was previously manipulated using the RHI procedure: after asynchronous stimulation , observing others’ actions facilitated the motor system , whereas after synchronous stimulation , identical observed actions , now illusorily attributed to the subject's own body , evoked smaller MEPs .", "In light of our findings , the absence of facilitatory effect during observation following synchronous stimulation can be interpreted as resulting from the decreased corticospinal excitability induced by the illusion .", "Taken together , these results suggest that body ownership and the motor system are mutually interactive and both contribute to the dynamic construction of bodily self-awareness in healthy and pathological brains .", "In the present study , we found that the excitability of motor pathways in response to stimulation of the real ( disembodied ) hand is significantly decreased ( i . e . , MEP amplitude was significantly reduced ) when subjects experience the artificial hand as their own .", "This suggests that an experimental manipulation of the sense of body ownership is accompanied by a coherent modulation of the motor system .", "However , as the experimental design was not suited to investigate correlations ( see details in Material and methods ) , the present data do not allow us to address the question of whether subjects who experience a larger subjective illusion also show a larger decrease in MEPs .", "The presence or absence of linear correlations between MEP amplitude and behavioral measures , including both embodiment of the rubber hand and disembodiment of the real hand , will be investigated in future studies , in which physiological parameters of responder and non-responder subjects can be also compared .", "It has been suggested that active movements integrate distinct body parts into a unitary body representation ( Tsakiris et al . , 2010 ) .", "When this unified representation is altered , as during the visual-tactile conflict induced by the illusion , the excitability of the primary motor cortex is also altered , suggesting that the motor readiness of the real ( disembodied ) hand could also be reduced .", "We ascribe the excitability decrease in the primary motor cortex ( M1 ) to cortical inhibitory processes tied to the central processing of hand ownership , possibly reaching M1 via inhibitory input from the premotor cortex ( which is known to play a crucial role in the multisensory integration processes that give rise to the sense of body ownership ) ( Ehrsson et al . , 2004 ) .", "The present findings , which shed new light on our understanding of the different aspects that contribute to the formation of a coherent self-awareness , suggest that bodily self-consciousness strictly depends on the possibility of movement .", "The bodily self is primarily and originally construed in terms of motor potentiality for actions ( Gallese and Sinigaglia , 2010 ) .", "If I believe that the hand is mine , then I must be ready to use it; if not , then the activity of the motor system is accordingly down-regulated ." ], [ "Twenty-six ( ten male ) volunteers took part in the behavioral main experiment ( mean age ± SD=24 ± 5 years ) ( the sample size estimation was performed according to an a priori power analysis , see details in Supplementary file 1 ) .", "Additionally , a different sample of 26 subjects ( ten male ) ( mean age ± SD=24 ± 4 years ) were recruited for the behavioural control experiment .", "Subjects participated in the physiological experiments only if , in the embodiment questionnaire , they gave a rating higher than zero in the synchronous condition .", "According to this criterion , 24 out of 26 subjects participated in the physiological main experiment; 20 out of 26 subjects participated in the physiological control experiment .", "All participants were right-handed , as assessed with the Edinburgh Handedness Inventory , and were screened to exclude a family history of psychiatric , neurological or medical disease .", "The experimental protocol was approved by the Ethics Committee of the University of Milano and written informed consent was obtained from each subject in compliance with the rules of the 1964 Declaration of Helsinki .", "In both main ( Figure 1A ) and control ( Figure 1B ) experiments , the RHI was evoked by the synchronous stroking of the rubber hand and of the participant’s own hidden hand ( the location of stroking on the two hands was carefully matched ) using the traditional visual-tactile stimulation ( Botvinick and Cohen , 1998 ) .", "Asynchronous stroking of a participant own hand and the rubber hand was utilized as a control condition , in which strokes were delivered spatially and temporally out of phase between the two hands .", "Participants sat with their forearms resting on a table , with their right hand inserted , palm down , in one of two identical compartments of a wooden box ( 59 cm × 33 cm × 15 cm ) ; the rubber hand was placed in the left compartment , in egocentric position and aligned with the participant’s shoulder .", "The upper lid of the box could be lifted or lowered to either reveal or occlude the participant’s view of the rubber hand in the left compartment , while his/her right hand was always out of view .", "The participant was able to see only the rubber hand being stroked by the experimenter’s right hand , while the subject's right hand and the experimenter’s left hand were always out of subject's view .", "The distance between the index finger of the rubber hand and the participant’s own right index finger was 20 cm .", "A cloth was placed so as to hide both the participant’s shoulder and the proximal end of the rubber hand .", "The behavioral RHI effect was measured in two ways: ( 1 ) by asking participants to localize the position of their unseen hand along the horizontal plan in front of them , and thus obtaining a measure of the proprioceptive drift towards the rubber hand , ( 2 ) with a questionnaire investigating their feeling of ownership of the rubber hand as a consequence of the experimental manipulation .", "Moreover , in the control experiment , we also measured the disembodiment experience of the stimulated hand with a questionnaire investigating the feeling of loss of own hand ( Longo et al . , 2008 ) .", "The physiological effect was measured by recording motor-evoked potentials ( MEPs ) , utilized to evaluate the excitability modulation of cortical and spinal motor neurons during the RHI .", "In the main experiment , MEPs were recorded from the real stimulated ( right ) hand , while in the control experiment , MEPs were recorded from the contralateral non-stimulated ( left ) hand .", "Behavioral and physiological experiments were done in separate sessions: recording of MEPs in the three experimental conditions required about 40 min , and inserting the hand-ownership evaluation questions would have prolonged the experiment beyond a reasonable and feasible time , increasing the probability that subjects move their head with respect to the coil or lose their concentration on the task .", "In the main experiment , MEPs were elicited by single-pulse transcranial magnetic stimulation ( TMS ) of the hand area in the left M1 and recorded by self-adhesive bipolar surface electrodes that were placed over the belly of the right first dorsal interosseous muscle ( FDI ) .", "Electromyographic ( EMG ) signals were amplified , filtered ( 10 Hz to 1 kHz ) , digitally converted ( sampling rate 5 kHz ) and stored in a computer for offline analysis .", "The head of each subject was restrained by a comfortable pillow wrapping around the neck and supported by a fixed head rest .", "A mechanical arm held a figure-of-eight-shaped coil connected to a magnetic stimulator ( Magstim 200; Magstim Co . Ltd , Whitland , UK; maximal power 2 . 2 T ) .", "The coil was positioned and fixed on the left primary motor cortex with the handle pointing backwards at 45% from the midline so as to activate the selected muscle , and the stimulator output was set at about 110% of each subject’s motor threshold ( defined as the intensity giving three MEP responses out of six stimuli ) ( Rossini et al . , 1994; Borroni et al . , 2008 ) .", "The absence of voluntary contraction before each TMS pulse was verified by continuous monitoring of the EMG signal .", "We replicated the same procedure in the control experiment , but in this case , MEPs were elicited by single-pulse TMS of the hand area in the right M1 and recorded with self-adhesive bipolar surface electrodes over the left FDI belly .", "In both main and control experiments , the mean value of the three ownership statements used in the subjective rating questionnaire , in the synchronous and asynchronous conditions , was obtained and used as a dependent variable ( emb-q-rating ) ; in the control group , we also obtained and used as a dependent variable the mean value of the three disownership statements , in the synchronous and asynchronous conditions ( emb-q-rating and disemb-q-rating ) .", "For the proprioceptive drift , the difference between the indicated location of the participant’s right index finger before and after the visual-tactile stimulation ( in both synchronous and asynchronous conditions ) was taken as a measure of perceptual relocation , which was averaged and used as a dependent variable .", "All data were assessed for normal distribution using the Shapiro-Wilk test ( p>0 . 05 ) .", "In the main experiment , for the emb-q-rating in both synchronous and asynchronous conditions , the residuals were not normally distributed ( p=0 . 00146 and p=0 . 0092 ) , so the Wilcoxon signed-rank test was used for pairwise comparisons .", "In the control experiment , in both synchronous and asynchronous conditions for the emb-q-rating ( p=0 . 08448 and p=0 . 60427 ) and the disemb-q-rating ( p=0 . 88009 and p=0 . 34168 ) , the residuals were normally distributed , so comparisons between synchronous and asynchronous stimulations were computed by means of a paired T-test ( two tailed ) .", "In both experiments , residuals for the proprioceptive drift were normally distributed ( main: p=0 . 91975 and p=0 . 71247; control: p=0 . 90201 and p=0 . 37482 ) , so comparisons between synchronous and asynchronous stimulation were computed by means of a paired T-test ( two tailed ) .", "For each test performed , we reported mean , standard deviation , p ( significance ) value and Cohen’s d value ( calculated as within-subjects effect sizes using G Power matched pairs statistical tests ) .", "All subjects' behavioural data are available in an additional source data file ( see Figure 2—source data 1 and 2 ) .", "MEP amplitude of the FDI muscle was measured as the peak-to-peak distance ( in µV ) , and MEPs of amplitude lower than 50 µV were discarded from analysis ( Rossini et al . , 1994; Borroni et al . , 2008 ) .", "For each subject , 20 measurements of MEP baseline values were acquired at the very beginning of the experiment in order to provide a reference value that could be used", "a ) to verify that , during the on-line data acquisition , the cortical excitability was unchanged in the second experimental block of visual-tactile stimulation compared to the first one and", "b ) to compare , during data analysis , the obtained MEP values in the experimental blocks ( in order to discriminate between facilitation or inhibition effects ) .", "Normal distribution of the residuals was checked using the Shapiro-Wilk test ( p>0 . 05 ) , and the appropriate non-parametric tests were applied when one or more of the corresponding data sets failed to meet the criteria for normal distribution .", "In both experiments , for each subject , we used MEPs recorded in each experimental condition ( baseline , asynchronous and synchronous trials , a total of 60 trials ) , to obtain a grand-mean and a grand-standard-deviation .", "Then , each single trial was transformed in z-scores , according to the following formula: ( x – grand-mean ) / ( grand-standard-deviation ) , where x indicates a single trial value .", "The obtained values were averaged for each subject and entered into two separate ( for the main and control experiments ) three-level ( baseline , asynchronous , synchronous ) one-way ANOVAs .", "In this analysis , for the main experiment , the distribution of residuals in the synchronous condition was not normal ( respectively: p=0 . 70962 , p=0 . 08347 , p=0 . 00604 ) .", "Thus , we performed Friedman non-parametric ANOVA in order to detect significant differences across the three conditions ( baseline , asynchronous , synchronous ) ; therefore , Wilcoxon signed-rank tests were used for pairwise comparisons .", "Finally , for each pairwise comparison ( N = 3 ) , a Bonferroni correction was applied ( a value/n of comparisons: 0 . 05/3 = 0 . 017 ) .", "In the control experiment , residuals of the three conditions were normally distributed ( respectively: p=0 . 60988 , p=0 . 44773 , p=0 . 66546 ) and the ANOVA normality assumption was satisfied .", "Furthermore , we investigated the time course of MEP change in the synchronous condition of both experiments in order to describe , for each subject , a MEP amplitude time-profile during the illusion .", "MEP z-scores for all participants were divided into four blocks of five MEPs each .", "The obtained values were averaged starting from 0 to 5 ( TIME 1 ) , from 6 to 10 ( TIME 2 ) , from 11 to 15 ( TIME 3 ) and from 16 to 20 ( TIME 4 ) , and entered in a four-level one way ANOVA ( TIME: one , two , three , four ) .", "Residuals for the main experiment were not normally distributed , so Wilcoxon signed-rank tests were used for within comparisons of the four-level time variable; for each pairwise comparison ( N = 4 ) , Bonferroni correction was applied ( a value / n of comparisons: 0 . 05/4 = 0 . 0125 ) .", "Finally , in order to compare MEP modulation in the two experiments , we avoided classical ANOVA because residuals in the main experiment were not normally distributed .", "So , we calculated a delta on raw MEP amplitude between synchronous minus asynchronous for all subjects in each experiment; then the obtained values were analyzed with Mann-Whitney U-test .", "For each statistical test , we reported mean , standard deviation , p value and Cohen’s d ( calculated as within-subjects effect sizes using G Power matched pairs statistical tests ) .", "All subjects' physiological data are available in additional source data file ( see Figure 3—source data 1 and 2 ) .", "In the main experiment , linear regressions were performed between emb-q rating and proprioceptive drift in both synchronous and asynchronous conditions and in the delta synchronous minus asynchronous .", "In these correlations , the distribution of residuals , checked with Shapiro-Wilk test , was not normal , so we adopted the Spearman rank-order correlation .", "In the control experiments , linear correlations were performed between emb-q rating and disemb-q-rating in both synchronous and asynchronous conditions and in the delta synchronous minus asynchronous; moreover , linear regressions between proprioceptive drift and emb-q rating /disemb-q-rating in both synchronous and asynchronous conditions and in the delta synchronous minus asynchronous were performed .", "In these correlations , residuals , when checked with the Shapiro-Wilk test , were normally distributed .", "We acknowledge that the present experimental design was not ideally suited to investigate correlations , due to the fact that behavioural and physiological data were acquired in two separate sessions .", "Indeed , we could not obtain the behavioural responses during the registration of each MEP recording ( due to time constraints during MEP acquisition ) and , therefore , we could not have a point-by-point matching between those data .", "Furthermore , only responder subjects were admitted to the physiological experiment , i . e . we use only subjects who gave ratings higher than zero in the synchronous condition in the embodiment questionnaire administered during the behavioural experiment .", "Thus , in the present sample , which only includes responder subjects , correlations between physiological parameters and the presence/absence of the illusion cannot be investigated .", "However , to investigate whether responder subjects who experience a larger subjective illusion also show a larger decrease in MEP amplitude , we computed correlations , using either ratings of the questionnaire or proprioceptive drift values .", "In both cases , we used two different approaches:", "a ) we normalized MEP values , ratings and drift values by using z-scores to compute independent correlations for synchronous and asynchronous conditions: significant correlations with MEPs were not found , neither for questionnaire ratings nor for proprioceptive drift;", "b ) for MEP values , ratings and drift values , we computed a delta ( synchronous minus asynchronous ) value and performed correlations on these values: again , no significant correlations with MEPs were found for questionnaire ratings or for proprioceptive drift ." ] ]

[ "During the rubber hand illusion ( RHI ) , subjects experience an artificial hand as part of their own body , while the real hand is subject to a sort of 'disembodiment' .", "Can this altered belief about the body also affect physiological mechanisms involved in body-ownership , such as motor control ?", "Here we ask whether the excitability of the motor pathways to the real ( disembodied ) hand are affected by the illusion .", "Our results show that the amplitude of the motor-evoked potentials recorded from the real hand is significantly reduced , with respect to baseline , when subjects in the synchronous ( but not in the asynchronous ) condition experience the fake hand as their own .", "This finding contributes to the theoretical understanding of the relationship between body-ownership and motor system , and provides the first physiological evidence that a significant drop in motor excitability in M1 hand circuits accompanies the disembodiment of the real hand during the RHI experience ." ]

[ "The feeling of body ownership — that the various parts of your body are all part of you — is something that we typically take for granted .", "However , brain damage can disrupt this sensation and leave individuals convinced that an arm or a leg is no longer their own .", "Even in healthy people , the ‘rubber hand illusion’ can temporarily produce a similar phenomenon .", "Individuals watch a lifelike rubber hand being touched while their own hand – which is hidden from view – is touched at the same time .", "This creates the feeling that the artificial hand has become part of their body , while their real hand is left in a ‘disembodied’ state .", "How does the brain generate this illusory sense of ownership and accompanying sense of disembodiment ?", "A person’s ability to move their body is thought to contribute to their feeling of body ownership .", "Therefore , della Gatta , Garbarini et al . asked whether the brain’s ability to move the real hand changes during the rubber hand illusion .", "In the experiments , the region of the brain that controls hand movement was artificially stimulated in a number of volunteers .", "When an individual had been primed by the rubber hand illusion to perceive a fake hand as part of their own body , their brain was temporarily less able to activate the muscles of their real hand .", "This is as if the brain no longer considered the real hand as part of the body .", "Thus , the altered sense of body ownership experienced during the rubber hand illusion is not a bizarre fantasy , but corresponds to a physiological reaction that accompanies changes in brain activity .", "The next step is to further define and quantify the relationship between the sense of body ownership and the control of body movement .", "Specifically , how does activity in the brain areas that control movement contribute to the sense of body ownership ?", "And how do these brain regions communicate with one another to generate a sense of self ?" ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "cell biology", "immunology and inflammation" ]

[ [ "Immune cells eliminate pathogens and diseased cells while limiting damage to healthy cells .", "Macrophages , professional phagocytes and key effectors of the innate immune system , play an important role in this process by engulfing opsonized targets bearing ‘eat me’ signals .", "One of the most common ‘eat me’ signals is the immunoglobulin G ( IgG ) antibody , which can bind foreign proteins on infected cells or pathogens .", "IgG is recognized by Fcγ receptors ( FcγR ) in macrophages that drive antibody-dependent cellular phagocytosis ( ADCP ) ( DiLillo et al . , 2014; Erwig and Gow , 2016; Nimmerjahn and Ravetch , 2008 ) .", "ADCP is a key mechanism of action for several cancer immunotherapies including rituximab , trastuzumab , and cetuximab ( Chao et al . , 2010; Uchida et al . , 2004; Watanabe et al . , 1999; Weiskopf et al . , 2013; Weiskopf and Weissman , 2015 ) .", "Exploring the design parameters of effective antibodies could provide valuable insight into the molecular mechanisms driving ADCP .", "Activation of multiple FcγRs is required for a macrophage to engulf a three-dimensional target .", "FcγR-IgG must be present across the entire target to drive progressive closure of the phagocytic cup that surrounds the target ( Griffin et al . , 1975 ) .", "In addition , a critical antibody threshold across an entire target dictates an all-or-none engulfment response by the macrophage ( Zhang et al . , 2010 ) .", "Although the mechanism of this thresholded response remains unclear , receptor clustering plays a role in regulating digital responses in other immune cells ( Berger et al . , 2020; Davis and van der Merwe , 2006; Holowka and Baird , 1996; Kato et al . , 2020; Ma et al . , 2020; Veneziano et al . , 2020 ) .", "FcγR clustering may also regulate phagocytosis ( Goodridge et al . , 2012 ) .", "High-resolution imaging of macrophages has demonstrated that IgG-bound FcγRs form clusters ( resolution of >100 nm ) within the plasma membrane ( Lin et al . , 2016; Lopes et al . , 2017; Sobota et al . , 2005 ) .", "These small clusters , which recruit downstream effector proteins such as Syk-kinase and phosphoinositide 3-kinase , eventually coalesce into larger micron-scale patches as they migrate towards the center of the cell-target synapse ( Jaumouillé et al . , 2014; Lin et al . , 2016; Lopes et al . , 2017; Sobota et al . , 2005 ) .", "Prior observational studies could not decouple ligand clustering from other parameters , such as ligand number or receptor mobility .", "As a result , we do not have a clear picture of how ligand number or molecular spacing regulates signal activation .", "To directly assess such questions , we have developed a reconstituted system that utilizes DNA origami to manipulate ligand patterns on a single-molecule level with nanometer resolution .", "We found that tightly spaced ligands strongly enhanced phagocytosis compared to the same number of more dispersed ligands .", "Through manipulating the number and spacing of ligands on individual origami pegboards , we found that eight or more ligands per cluster maximized FcγR-driven engulfment , and that macrophages preferentially engulfed targets that had receptor-ligand clusters spaced ≤7 nm apart .", "We demonstrated that tight ligand clustering enhanced receptor phosphorylation , and the generation of PIP3 and actin filaments – critical downstream signaling molecules – at the phagocytic synapse .", "Together , our results suggest that the nanoscale clustering of receptors may allow macrophages to discriminate between lower density background stimuli and the higher density of ligands on opsonized targets .", "These results have implications for the design of immunotherapies that involve manipulating FcγR-driven engulfment ." ], [ "To study how isolated biochemical and biophysical ligand parameters affect engulfment , we sought to develop a well-defined and tunable engulfment system .", "Our lab previously developed a synthetic T cell signaling system , in which we replaced the receptor-ligand interaction ( TCR-pMHC ) with complimentary DNA oligos ( Taylor et al . , 2017 ) .", "We applied a similar DNA-based synthetic chimeric antigen receptor to study engulfment signaling in macrophages .", "In our DNA-CARγ receptor , we replaced the native extracellular ligand-binding domain of the FcγR with an extracellular SNAP-tag that covalently binds a benzyl-guanine-labeled single-stranded DNA ( ssDNA ) ( receptor DNA; Figure 1a; Morrissey et al . , 2018 ) .", "The SNAP-tag was then joined to the CD86 transmembrane domain followed by the intracellular signaling domain of the FcRγ chain ( Nimmerjahn and Ravetch , 2008 ) .", "We expressed the DNA-CARγ in the macrophage-like cell line RAW264 . 7 and the monocyte-like cell line THP-1 .", "As an engulfment target , we used silica beads coated with a supported lipid bilayer to mimic the surface of a target cell .", "The beads were functionalized with biotinylated ssDNA ( ligand DNA ) containing a sequence complementary to the receptor DNA via biotin-neutravidin interactions ( Figure 1A ) .", "We used a ligand DNA strand that has 13 complementary base pairs to the receptor DNA , which we chose because the receptor-ligand dwell time ( ~24 s; Taylor et al . , 2017 ) was comparable to the dwell time of IgG-FcγR interactions ( ~30–150 s; Li et al . , 2007 ) .", "To test whether this synthetic system can drive specific engulfment of ligand-functionalized silica beads , we used confocal microscopy to measure the number of beads that were engulfed by each cell ( Figure 1B , C ) .", "The DNA-CARγ drove specific engulfment of DNA-bound beads in both RAW264 . 7 and THP-1 cells ( Figure 1C , Figure 1—figure supplement 1 ) .", "The extent of engulfment was similar to IgG-coated beads , and the ligand density required for robust phagocytosis was also comparable to IgG [Figure 1D , Figure 1—figure supplement 1; Bakalar et al . , 2018; Morrissey et al . , 2020] .", "As a control , we tested a variant of the DNA-CAR that lacked the intracellular domain of the FcRγ chain ( DNA-CARadhesion ) .", "Cells expressing the DNA-CARadhesion failed to induce engulfment of DNA-functionalized beads ( Figure 1C ) , demonstrating that this process depends upon the signaling domain of the FcγR .", "Together , these data show that the DNA-CARγ can drive engulfment of targets in a ligand- and FcγR-specific manner .", "DNA origami technology provides the ability to easily build three-dimensional objects that present ssDNA oligonucleotides with defined nanometer-level spatial organization ( Hong et al . , 2017; Rothemund , 2006; Seeman , 2010; Shaw et al . , 2019; Veneziano et al . , 2020 ) .", "We used DNA origami to manipulate the spatial distribution of DNA-CARγ ligands in order to determine how nanoscale ligand spacing affects engulfment .", "We used a recently developed two-tiered DNA origami pegboard that encompasses a total of 72 ssDNA positions spaced 7 nm and 3 . 5 nm apart in the x and y dimensions , respectively ( Dong et al . , 2021; Figure 2A , Figure 2—figure supplement 1 ) .", "Each of the 72 ligand positions can be manipulated independently , allowing for full control over the ligand at each position ( Figure 2—figure supplement 1 ) .", "The DNA origami pegboard also contains fluorophores at each of its four corners to allow for visualization , and 12 biotin-modified oligos on the bottom half of the pegboard to attach it to a neutravidin-containing supported lipid bilayer or glass coverslip ( Figure 2A , B , Figure 2—figure supplement 1 ) .", "To determine if the DNA origami pegboards could successfully activate signaling , we first tested whether receptors were recruited to the origami pegboard in a ligand-dependent manner .", "Using TIRF microscopy , we quantified the fluorescence intensity of the recruited GFP-tagged DNA-CARγ receptor to origami pegboards presenting 0 , 2 , 4 , 16 , 36 , or 72 ligands ( Figure 2B–E ) .", "Using signal from the 72 ligand ( 72L ) origami pegboard as an internal intensity standard of brightness , and thus correcting for differences in illumination between wells , we found that the average fluorescence intensity correlated with the number of ligands presented by individual origami pegboards ( Figure 2D , E ) .", "In addition , we measured Syk recruitment to individual DNA origami pegboards and found that Syk intensity also increased as a function of the number of ligands present on each origami pegboard ( Figure 2C , Figure 2—figure supplement 2 ) .", "These results confirmed that our DNA origami system provides a platform that allows quantitative receptor recruitment and the analysis of downstream signaling pathways .", "FcγR cluster upon ligand binding , but the functional importance of such clustering for phagocytosis has not been directly addressed , and whether a critical density of receptor-ligand pairs is necessary to initiate FcγR signaling is unclear ( Duchemin et al . , 1994; Jaumouillé et al . , 2014; Lin et al . , 2016; Lopes et al . , 2017; Sobota et al . , 2005 ) .", "To address these questions , we varied the size of ligand clusters by designing DNA origami pegboards presenting 2–36 ligands .", "To ensure a constant total number of ligands and origami pegboards on each bead , we mixed the signaling origami pegboards with 0-ligand ‘blank’ origami pegboards in appropriate ratios ( Figure 3A ) .", "We confirmed that the surface concentration of origami pegboards on the beads was comparable using fluorescence microscopy ( Figure 3—figure supplement 1 ) .", "We found that increasing the number of ligands per cluster increased engulfment , but that engulfment plateaued at a cluster size of eight ligands ( Figure 3b ) .", "We confirmed that the observed engulfment phenotype was both ligand , receptor , and FcγR signaling dependent ( Figure 3C , D ) .", "Together , these data reveal that FcγR clustering strongly enhances engulfment up to a cluster size of eight ligands .", "Next , we examined whether distance between individual receptor-ligand molecules within a signaling cluster impacts engulfment .", "For this experiment , we varied the spacing of four ligands on the origami pegboard .", "The 4-ligand tight origami ( 4T ) contains four ligands clustered at the center of the pegboard ( 7 nm by 3 . 5 nm square ) , the medium origami ( 4M ) has ligands spaced 21 nm by 17 . 5 nm apart , and the spread origami ( 4S ) has four ligands positioned at the four corners of the pegboard ( 35 nm by 38 . 5 nm square ) ( Figure 4A ) .", "We found that the efficiency of macrophage engulfment was approximately twofold higher for the 4T-functionalized beads when compared to the 4M or 4S beads ( Figure 4A ) .", "We confirmed via fluorescence microscopy that the concentration of origami pegboards on the surface was similar , and therefore ligand numbers on the beads were similar ( Figure 4—figure supplement 1 ) .", "Human THP-1 cells expressing the DNA-CARγ showed the same ligand spacing dependence ( Figure 4—figure supplement 1 ) .", "In addition , we generated DNA-CAR constructs containing the FcRγ and α chain transmembrane domains that would be present in the endogenous receptor complex ( Figure 4—figure supplement 1 ) .", "To minimize dimerization between FcRγ transmembrane domains , we either made a C25Aγ chain mutation , as this cysteine forms a disulfide bridge between y chains , or truncated the transmembrane domain before this residue .", "We found that the efficiency of macrophage engulfment was dependent on ligand spacing for all constructs tested ( Figure 4—figure supplement 1 ) .", "Expression of the various DNA-CARs at the cell cortex was comparable , and engulfment of beads functionalized with both the 4T and the 4S origami platforms was dependent on the FcγR signaling domain ( Figure 4—figure supplement 1 ) .", "Together , these results demonstrate that macrophages preferentially engulf targets with tighter ligand clusters .", "Tightly spaced ligands could potentially increase phagocytosis by enhancing the avidity of receptor-ligand interactions within each cluster .", "Such a hypothesis would predict that tightly spaced ligands increase DNA-CARγ-BFP occupancy at the phagocytic cup .", "However , when we measured the total fluorescence intensity of receptors at the phagocytic cup , we did not detect a difference in DNA-CARγ-BFP recruitment to 4T and 4S beads ( Figure 6A , B ) .", "However , to eliminate any potential contribution of avidity , we created 4T and 4S origami pegboards with very high-affinity 16mer DNA ligands that are predicted to dissociate on a time scale of >7 hr ( Taylor et al . , 2017; Figure 4B ) .", "Using these 16mer high-affinity ligands , we found that 4T origami beads were still preferentially engulfed over 4M or 4S origami beads ( Figure 4B , Figure 4—figure supplement 1 ) .", "These results suggest that an avidity effect is not the cause of the preferential engulfment of targets having tightly spaced ligands .", "We next determined how ligand spacing affects the kinetics of engulfment .", "Using data from live-cell imaging , we subdivided the engulfment process into three steps: bead binding , engulfment initiation , and engulfment completion ( Figure 5A , Video 1 ) .", "To compare engulfment dynamics mediated by 4T and 4S origami pegboards in the same experiment , we labeled each pegboard type with a different colored fluorophore , functionalized a set of beads with each type of pegboard , and added both bead types to macrophages at the same time ( Figure 5B , Video 2 ) .", "Macrophages interacted with beads functionalized with the 4T and 4S pegboards with comparable frequency ( 46 ± 7% total bead-cell contacts vs . 54 ± 7% total bead-cell contacts , respectively ) .", "However , the probability of engulfment initiation was significantly higher for the 4T ( 95 ± 5% of bead contacts ) versus 4S ( 61 ± 9% of bead contacts ) beads , and the probability that initiation events resulted in successful completion of engulfment was higher for 4T ( 69 ± 9% of initiation events ) versus 4S ( 39 ± 11% of initiation events ) beads ( Figure 5A ) .", "Initiation events that failed to induce successful engulfment either stalled after progressing partially over the bead or retracted the extended membrane back to the base of the bead .", "In addition , for beads that were engulfed , the time from contact to engulfment initiation was ~300 s longer for beads functionalized with 4S origami pegboards than beads containing 4T origami pegboards ( Figure 5C ) .", "However , once initiated , the time from initiation to completion of engulfment did not differ significantly for beads coated with 4T or 4S origami ( Figure 5D ) .", "Overall , 66 ± 8% of 4T bead contacts resulted in successful engulfment compared to 24 ± 8% for 4S beads ( Figure 5E ) .", "The DNA-CARadhesion macrophages rarely met the initiation criteria , suggesting that active signaling from the FcγR is required ( Figure 5—figure supplement 1 ) .", "Together , these data reveal that tighter spacing between ligands within a cluster enhances the probability and kinetics of initiating engulfment , as well as the overall success frequency of completing engulfment , but does not affect the rate of phagosome closure once initiated .", "We next determined how the 4T or 4S origami pegboards affect signaling downstream of FcγR binding by measuring fold enrichment at the phagocytic cup compared to the rest of the cortex of ( 1 ) a marker for receptor phosphorylation ( the tandem SH2 domains of Syk; Bakalar et al . , 2018; Morrissey et al . , 2018 ) , ( 2 ) PIP3 ( via recruitment of the PIP3 binding protein Akt-PH-GFP ) , and ( 3 ) filamentous actin ( measured by rhodamine-phalloidin binding , Figure 6A , B ) .", "We found that 4T phagocytic cups recruited more tSH2-Syk than the 4S beads , indicating an increase in receptor phosphorylation by nanoclustered ligands .", "Generation of PIP3 and actin filaments at the phagocytic cup also increased at 4T relative to 4S synapses ( Figure 6B ) .", "This differential recruitment of downstream signaling molecules to 4T versus 4S origami beads was most apparent in early and mid-stage phagocytic cups; late-stage cups showed only a slightly significant difference in tSH2-Syk recruitment and no significant differences in generation of PIP3 or actin filaments ( Figure 6—figure supplement 1 ) .", "Together , these data demonstrate that nanoscale ligand spacing affects early downstream signaling events involved in phagocytic cup formation .", "We next sought to understand why distributing ligands into tight clusters enhanced receptor phosphorylation and engulfment .", "One possibility is that the clustering of four complete receptors is needed to drive segregation of the inhibitory phosphatase CD45 and allow sustained phosphorylation of the FcγR immune receptor tyrosine-based activation motif ( ITAM ) ( Bakalar et al . , 2018; Freeman et al . , 2016; Goodridge et al . , 2012; Schmid et al . , 2016 ) .", "Alternatively , the 4-ligand cluster may be needed to obtain a critical intracellular concentration of FcγR ITAM signaling domains .", "To test for the latter possibility , we designed a synthetic receptor ( DNA-CAR-4xγ ) that contains four repeats of the intracellular domain of the DNA-CARγ connected by a GGSG linker between each repeat ( Figure 6C ) .", "We confirmed that this DNA-CAR-4xγ receptor was more potent in activating engulfment than an equivalent receptor ( DNA-CAR-1xγ−3xΔITAM ) in which the three C-terminal ITAM domains were mutated to phenylalanines ( Figure 6C , D ) .", "Keeping the number of intracellular ITAMs constant , we compared the engulfment efficiency mediated by two different receptors: ( 1 ) the DNA-CAR-4xγ that interacted with beads functionalized with 1-ligand origami and ( 2 ) the DNA-CAR-1xγ−3xΔITAM that interacted with beads coated with equivalent amounts of 4T origami ( Figure 6C ) .", "While the DNA-CAR-1xγ−3xΔITAM-expressing macrophages engulfed 4T origami beads , the DNA-CAR-4xγ macrophages failed to engulf the high-affinity 1-ligand origami beads ( Figure 6D , Figure 6—figure supplement 1 ) .", "To ensure that all four ITAM domains on the DNA-CAR-4xγ were signaling competent , we designed two additional DNA-CARs that placed the functional ITAM at the second and fourth position ( Figure 6—figure supplement 1 ) .", "These receptors were able to induce phagocytosis of 4T origami beads , indicating that the DNA-CAR-4xγ likely contains four functional ITAMs .", "Collectively , these results indicate that the tight clustering of multiple receptors is necessary for engulfment and increasing the number of intracellular signaling modules on a single receptor is not sufficient to surpass the threshold for activation of engulfment ." ], [ "Macrophages integrate information from many FcγR-antibody interactions to discriminate between highly opsonized targets and background signal from soluble antibody or sparsely opsonized targets .", "How the macrophage integrates signals from multiple FcγR binding events to make an all-or-none engulfment response is not clear .", "Here , we use DNA origami nanostructures to manipulate and assess how the nanoscale spatial organization of receptor-ligand interactions modulates FcγR signaling and the engulfment process .", "We found that tight ligand clustering increases the probability of initiating phagocytosis by enhancing FcγR phosphorylation .", "Phagocytosis requires IgG across the entire target surface to initiate local receptor activation and to ‘zipper’ close the phagocytic cup ( Freeman et al . , 2016; Griffin et al . , 1975 ) .", "Consistent with this zipper model , incomplete opsonization of a target surface , or micron-scale spaces between IgG patches , decreases engulfment ( Freeman et al . , 2016; Griffin et al . , 1975 ) .", "Initially suggested as an alternative to the zipper model , the trigger model proposed that engulfment occurs once a threshold number of receptors interact with IgG ( Ben M'Barek et al . , 2015; Griffin et al . , 1975; Swanson and Baer , 1995 ) .", "While this model has largely fallen out of favor , more recent studies have found that a critical IgG threshold is needed to activate the final stages of phagocytosis ( Zhang et al . , 2010 ) .", "Our data suggest that there may also be a nanoscale density-dependent trigger for receptor phosphorylation and downstream signaling .", "Taken together , these results suggest that both tight nanoscale IgG-FcγR clustering and a uniform distribution of IgG across the target are needed to direct signaling to ‘zipper’ close the phagocytic cup .", "Why might macrophages use this local density-dependent trigger to dictate engulfment responses ?", "Macrophages constantly encounter background ‘eat me’ signals ( Gonzalez-Quintela et al . , 2008 ) .", "This hyper-local density measurement may buffer macrophages against background stimuli and weakly opsonized targets that are unlikely to have adjacent bound antibodies , while still robustly detecting and efficiently engulfing highly opsonized targets .", "Our findings are consistent with previous results demonstrating that FcγR crosslinking correlates with increased ITAM phosphorylation ( Huang et al . , 1992; Kwiatkowska and Sobota , 2001; Lin et al . , 2016; Sobota et al . , 2005 ) .", "While our data pinpoints a role for ligand spacing in regulating receptor phosphorylation , it is possible that later steps in the phagocytic signaling pathway are also directly affected by ligand spacing .", "The mechanism by which dense-ligand clustering promotes receptor phosphorylation remains an open question , although our data rule out a couple of models .", "Specifically , we demonstrate that nanoscale ligand clustering does not noticeably affect the amount of ligand-bound receptor at the phagocytic cup , and that ligand spacing continues to affect engulfment when avidity effects are diminished through the use of high-affinity receptor-ligands .", "Collectively , these data reveal that changes in receptor binding or recruitment caused by increased avidity are unlikely to account for the increased potency of clustered ligands .", "Our data also exclude the possibility that receptor clustering simply increases the local intracellular concentration of FcγR signaling domains as arranging FcγR ITAMs in tandem did not have the same effect as clustering multiple receptor-ligand interactions .", "However , it remains possible that the geometry of the intracellular signaling domains could be important for activating or localizing downstream signaling , and that tandem ITAMs on the same polypeptide cannot produce the same engulfment signals as ITAMs on separate parallel polypeptides .", "One possible model to explain the observed ligand-density dependence of signaling involves the ordering of lipids around the FcγR .", "Segregated liquid-ordered and liquid-disordered membrane domains around immune receptor clusters have been reported to promote receptor phosphorylation ( Bag et al . , 2020; Dinic et al . , 2015; Eggeling et al . , 2009; Kabouridis , 2006; Simons and Ikonen , 1997; Sohn et al . , 2006; Stone et al . , 2017 ) .", "FcγR clusters are associated with liquid-ordered domains ( Beekman et al . , 2008; Katsumata et al . , 2001; Kwiatkowska and Sobota , 2001 ) .", "Liquid-ordered domains recruit Src family kinases , which phosphorylate FcγRs , while liquid-disordered domains are enriched in the transmembrane phosphatase CD45 , which dephosphorylates FcγRs ( Bag et al . , 2020; Sohn et al . , 2006; Stone et al . , 2017 ) .", "Thus , lipid ordering could provide a mechanism that leads to receptor activation if denser receptor-ligand clusters are more efficient in nucleating or associating with ordered lipid domains .", "As an alternative model , a denser cluster of ligated receptors may enhance the steric exclusion of the bulky transmembrane proteins like the phosphatases CD45 and CD148 ( Bakalar et al . , 2018; Goodridge et al . , 2012; Zhu et al . , 2008 ) .", "CD45 is heavily glycosylated , making the extracellular domain 25–40 nm tall ( Davis and van der Merwe , 2006; McCall et al . , 1992; Woollett et al . , 1985 ) .", "Because of its size , CD45 is excluded from close cell-cell contacts , such as those mediated by IgG-FcγR , which have a dimension of 11 . 5 nm ( Bakalar et al . , 2018; Burroughs et al . , 2011; Carbone et al . , 2017; Chung et al . , 2013; Lu et al . , 2011; Schmid et al . , 2016 ) .", "IgG bound to antigens ≤ 10 . 5 nm from the target surface induces CD45 exclusion and engulfment ( estimated total intermembrane distance of ≤22 nm Bakalar et al . , 2018 ) .", "Our DNA origami structure is estimated to generate similar intermembrane spacing , consisting of hybridized receptor-ligand DNA ( ~9 . 4 nm ) , the origami pegboard ( 6 nm ) , and neutravidin ( 4 nm ) ( Rosano et al . , 1999 ) .", "A higher receptor-ligand density constrains membrane shape fluctuations ( Krobath et al . , 2009; Krobath et al . , 2011; Różycki et al . , 2010 ) , and this constraint may increase CD45 exclusion ( Schmid et al . , 2016 ) .", "Both the lipid ordering and the steric exclusion models predict at least a partial exclusion of the CD45 from the zone of the receptor cluster .", "However , the dimension of the tight cluster in particular is very small ( 7 by 3 . 5 nm ) and measurement of protein concentration at this level is currently not easily achieved , even with super-resolution techniques .", "Overall , our results establish the molecular and spatial parameters necessary for FcγR activation and demonstrate that the spatial organization of IgG-FcγR interactions alone can affect engulfment decisions .", "How does our synthetic DNA-CARγ receptor compare to endogenous FcγRs ?", "Our DNA-CARs are single-chain receptors that recruit one intracellular signaling domain per ligand , similar to the single-chain human FcγRIIA receptor ( Nimmerjahn and Ravetch , 2006 ) .", "FcγRIIA is ubiquitously expressed on human myeloid cells , and high-affinity FcRIIA alleles correlate with an increase in effectiveness of the ADCP-inducing drug rituximab and lupus susceptibility ( Bruhns and Jönsson , 2015; Nimmerjahn and Ravetch , 2006 ) .", "The majority of FcγR family members , including all activating mouse FcγRs and the human FcγRI and FcγRIIIA , are multimeric complexes composed of a ligand-binding α chain and a dimerized signaling γ chain .", "This results in two signaling γ chains recruited to each IgG ligand .", "The different stoichiometry between ligand-binding and intracellular signaling domains may affect some parameters like optimal cluster size .", "A second difference between the DNA-CARγ and the endogenous system is the presence of the CD86 transmembrane domain .", "We found that ligand spacing had a similar effect on phagocytosis when we replaced the CD86 transmembrane domain with the Fcα or Fcγ transmembrane domain .", "However , the Fcγ transmembrane domain construct triggered more bead internalization across all conditions .", "We hypothesize this could be because the transmembrane domain retains some ability to dimerize , recruiting more signaling domains to each ligand , or because it is better able to associate with lipid-ordered domains .", "Future studies that pattern either endogenous Fc receptor complex or IgG ligand could clarify these questions .", "How does the spacing requirements for FcγR nanoclusters compare to other signaling systems ?", "Engineered multivalent Fc oligomers revealed that IgE ligand geometry alters Fcε receptor signaling in mast cells ( Sil et al . , 2007 ) .", "DNA origami nanoparticles and planar nanolithography arrays have previously examined optimal inter-ligand distance for the T cell receptor , B cell receptor , NK cell receptor CD16 , death receptor Fas , and integrins ( Arnold et al . , 2004; Berger et al . , 2020; Cai et al . , 2018; Deeg et al . , 2013; Delcassian et al . , 2013; Dong et al . , 2021; Veneziano et al . , 2020 ) .", "Some systems , like integrin-mediated cell adhesion , appear to have very discrete threshold requirements for ligand spacing while others , like T cell activation , appear to continuously improve with reduced intermolecular spacing ( Arnold et al . , 2004; Cai et al . , 2018 ) .", "Our system may be more similar to the continuous improvement observed in T cell activation as our most spaced ligands ( 36 . 5 nm ) are capable of activating some phagocytosis , albeit not as potently as the 4T .", "Interestingly , as the intermembrane distance between T cell and target increases , the requirement for tight ligand spacing becomes more stringent ( Cai et al . , 2018 ) .", "This suggests that IgG bound to tall antigens may be more dependent on tight nanocluster spacing than short antigens .", "Planar arrays have also been used to vary inter-cluster spacing , in addition to inter-ligand spacing ( Cai et al . , 2018; Freeman et al . , 2016 ) .", "Examining the optimal inter-cluster spacing during phagosome closure may be an interesting direction for future studies .", "Our study on the spatial requirements of FcγR activation could have implications for the design of therapeutic antibodies or chimeric antigen receptors .", "Antibody therapies that rely on FcγR engagement are used to treat cancer , autoimmune , and neurodegenerative diseases ( Chao et al . , 2010; Nimmerjahn and Ravetch , 2005; Uchida et al . , 2004; Watanabe et al . , 1999; Weiskopf et al . , 2013; Weiskopf and Weissman , 2015 ) .", "Multimerizing Fc domains or targeting multiple antibodies to the same antigen may increase antibody potency ( Zhang et al . , 2016 ) .", "Interestingly , rituximab , a successful anti-CD20 therapy that potently induces ADCP , has two binding sites on its target antigen ( Zhao et al . , 2020 ) .", "Selecting clustered antigens or pharmacologically inducing antigen clustering may also increase antibody potency ( Chew et al . , 2020 ) .", "These results suggest that oligomerization may lead to more effective therapy; however , a systematic study of the spatial parameters that affect FcγR activation has not been undertaken ( Bakalar et al . , 2018 ) .", "Our data suggest that antibody engineering strategies that optimize spacing of multiple antibodies through leucine zippers , cysteine bonds , DNA hybridization ( Delcassian et al . , 2013; Seifert et al . , 2014; Sil et al . , 2007 ) , or multimeric scaffolds ( Divine et al . , 2020; Fallas et al . , 2017; Huang et al . , 2021; Ueda et al . , 2020 ) could lead to stronger FcγR activation and potentially more effective therapies ." ], [ "RAW264 . 7 macrophages were purchased from the ATCC and cultured in DMEM ( Gibco , Cat# 11965-092 ) supplemented with 1× penicillin-streptomycin-L-glutamine ( Corning , Cat# 30-009 Cl ) , 1 mM sodium pyruvate ( Gibco , Cat# 11360-070 ) , and 10% heat-inactivated fetal bovine serum ( Atlanta Biologicals , Cat# S11150H ) .", "THP1 cells were also purchased from the ATCC and cultured in RPMI 1640 Medium ( Gibco , Cat# 11875-093 ) supplemented with 1× Pen-Strep-Glutamine and 10% heat-inactivated fetal bovine serum .", "All cells were certified mycoplasma-free and discarded after 20 passages to minimize variation .", "All relevant information can be found in the Key resources table , including detailed descriptions of the amino acid sequences for all constructs .", "Lentiviral infection was used to express constructs described in the Key resources table in either RAW264 . 7 or THP1 cells .", "Lentivirus was produced by HEK293T cells or Lenti-X 293 T cells ( Takara Biosciences , Cat# 632180 ) transfected with pMD2 . G ( a gift from Didier Tronon , Addgene plasmid # 12259 containing the VSV-G envelope protein ) , pCMV-dR8 . 91 ( since replaced by second generation compatible pCMV-dR8 . 2 , Addgene plasmid #8455 ) , and a lentiviral backbone vector containing the construct of interest ( derived from pHRSIN-CSGW , see Key resources table ) using lipofectamine LTX ( Invitrogen , Cat# 15338-100 ) .", "The HEK293T media was harvested 60–72 hr post-transfection , filtered through a 0 . 45 µm filter , and concentrated using Lenti-X ( Takara Biosciences , Cat# 631232 ) via the standard protocol .", "Concentrated virus was added directly to the cells , and the plate was centrifuged at 2200×g for 45 min at 37°C .", "Cells were analyzed a minimum of 60 hr later .", "Cells infected with more than one viral construct were FACs sorted ( Sony SH800 ) before use to enrich for double infected cells .", "The DNA origami pegboard utilized for all experiments was generated as described in Figure 2—figure supplement 1 .", "The p8064 DNA scaffold was purchased from IDT ( Cat# 1081314 ) .", "All unmodified oligonucleotides utilized for the origami were purchased from IDT in 96-well plates with standard desalting purification and resuspension at 100 µM in water .", "Fluorophore and biotin-conjugated oligonucleotides were also purchased from IDT ( HPLC purification ) .", "All oligonucleotide sequences are listed in Supplementary file 1 , the assembly is schematized in Figure 2—figure supplement 1 , and the Cadnano strand diagram for the pegboard with 72 medium-affinity ligands is included in Figure 2—figure supplement 1 .", "Core staple oligonucleotides ( 200 nM ) ( plates 1 and 2 ) , ligand oligonucleotides ( 200 nM ) ( plates 3L , 3MA , and 3 HA ) , biotinylated oligonucleotides ( 200 nM ) , DNA scaffold ( 20 nM final concentration ) , and fluorophore-labeled oligonucleotides ( 200 nM final concentration ) were mixed in 1× folding buffer ( 5 mM Tris pH 8 . 0 , 1 mM EDTA , 5 mM NaCl , 20 mM MgCl2 ) .", "Origami folding reaction was performed in a PCR thermocycler ( Bio-Rad MJ Research PTC-240 Tetrad ) , with initial denaturation at 65°C for 15 min followed by cooling from 60°C to 40°C with a decrease of 1°C/hr .", "To purify excess oligonucleotides from fully folded DNA origami , the DNA folding reaction was mixed with an equal volume of PEG precipitation buffer ( 15% ( w/v ) PEG-8000 , 5 mM Tris-Base pH 8 . 0 , 1 mM EDTA , 500 mM NaCl , 20 mM MgCl2 ) and centrifuged at 16 , 000× rcf for 25 min at room temperature ( RT ) .", "The supernatant was removed , and the pellet was resuspended in 1× folding buffer .", "PEG purification was repeated a second time , and the final pellet was resuspended at the desired concentration in 1× folding buffer and stored at 4°C .", "5′-amine modified ( 5AmMC6 ) DNA oligonucleotides were ordered from IDT and diluted in 0 . 15 M HEPES pH 8 . 5 to a final concentration of 2 mM .", "N-hydroxysuccinimide ester ( BG-GLA-NHS ) -functionalized benzylguanine was purchased from NEB ( Cat# S9151S ) and freshly reconstituted in DMSO to a final concentration of 83 mM .", "To functionalize the oligonucleotides with benzylguanine , the two solutions were mixed so that the molar ratio of oligonucleotide-amine:benzylguanine-NHS is 1:50 and the final concentration of HEPES is between 50 mM and 100 mM .", "The reaction was left on a rotator overnight at RT .", "To remove excess benzylguanine-NHS ester , the reaction product was purified the next day with illustra NAP-5 Columns ( Cytiva , Cat# 17085301 ) , using H2O for elution .", "The molar concentration of the benzylguanine-conjugated oligonucleotides was determined by measuring the absorbance of the purified reaction at 260 nm with a Nanodrop .", "This reaction was further condensed with the Savant SpeedVac DNA 130 Integrated Vacuum Concentrator System , resuspended in water to a final concentration of 100 µM , aliquoted , and stored at −20°C until use .", "96-well glass-bottom MatriPlates were purchased from Brooks ( Cat# MGB096-1-2-LG-L ) .", "Before use , plates were incubated in 5% ( v/v ) Hellmanex III solution ( Z805939-1EA; Sigma ) overnight , washed extensively with Milli-Q water , dried under the flow of nitrogen gas , and covered with sealing tape ( ThermoFisher , Cat# 15036 ) .", "Wells used for experiment were unsealed , incubated with 200 µL of Biotin-BSA ( ThermoFisher , Cat# 29130 ) at 0 . 5 mg/mL in PBS pH 7 . 4 at RT for 2 hr overnight .", "Wells were washed 6× with PBS pH 7 . 4 to remove excess BSA and incubated for 30 min at RT with 100 μL neutravidin at 250 μg/mL in PBS pH 7 . 4 for origami quantification and 50 μg/mL for cellular experiments .", "Wells were again washed 6× with PBS pH 7 . 4 supplemented with 20 mM MgCl2 and incubated for 1–2 hr with the desired amount of DNA origami diluted in PBS pH 7 . 4 with 20 mM MgCl2 and 0 . 1% BSA .", "Five wells of a 96-well glass-bottom MatriPlate per origami reaction were prepared as described in ‘Functionalization of glass surface with DNA origami’ .", "The purified DNA origami reaction was serially diluted into PBS pH 7 . 4 with 20 mM MgCl2 and 0 . 1% BSA , and five different concentrations were plated and incubated for 1 . 5 hr before washing 5× with PBS pH 7 . 4 with 20 mM MgCl2 and 0 . 1% BSA .", "Fluorescent TIRF images were acquired in the channel with which the origami was labeled .", "100 sites per well were imaged using the High Content Screening ( HCS ) Site Generator plug-in in µManager ( Stuurman et al . , 2010 ) .", "The number of individual DNA origami per µm2 in each well was quantified using the Spot Counter plug-in in Fiji .", "This was repeated for all concentrations of origami plated .", "The final concentration of the origami reaction was measured as number of origami/µm2 and was calculated from a linear fit including all concentrations in which individual origami could be identified by the plug-in .", "96-well glass-bottom MatriPlates were functionalized with DNA origami as described and then washed into engulfment imaging media ( 20 mM HEPES pH 7 . 4 , 135 mM NaCl , 4 mM KCl , 1 mM CaCl2 , 10 mM glucose ) containing 20 mM MgCl2 .", "Approximately 100 , 000 dual-infected mNeonGreen-DNA-CARγ and BFP-Syk THP1 cells per well were pelleted via centrifugation , washed into engulfment imaging media , re-pelleted , and resuspended into 50 µL of engulfment imaging media .", "1 µL of 100 μM benzylguanine-labeled-receptor DNA stock was added per ~50 , 000 cells pelleted , and the cell-DNA mixture was incubated at RT for 15 min .", "Cells were subsequently washed twice via centrifugation with 10 mL of imaging buffer to remove excess benzylguanine-labeled DNA and resuspended in 200 μL per 100 , 000 cells of imaging buffer containing 20 mM MgCl2 .", "Cells were then immediately added to each well and imaged .", "Data was only collected from a central region of interest ( ROI ) in the TIRF field .", "The origami fluorescent intensities along the x and y axes were plotted to ensure there was no drop off in signal and thus no uniformity of illumination .", "Cells that expressed both the mNeonGreen-tagged DNA-CARγ receptor and the BFP-tagged Syk and had interactions with the 72-ligand origami were chosen for analysis in Fiji .", "An ROI was drawn around the perimeter of the cell-glass surface interaction , which was determined by the presence of receptor fluorescence .", "The ‘Spot Intensity in All Channel’ plug-in in Fiji ( https://github . com/nicost/spotIntensityAnalysis/; Stuurman , 2020 ) was used to identify individual origami pegboards , measure fluorescence intensity of the DNA-CARγ receptor and Syk at each origami pegboard , and subtract local background fluorescence .", "The intensity at each origami pegboard was normalized to the average intensity measured at 72-ligand origami pegboards in each well .", "Chloroform-suspended lipids were mixed in the following molar ratios: 96 . 8% POPC ( Avanti , Cat# 850457 ) , 2 . 5% biotinyl cap PE ( Avanti , Cat# 870273 ) , 0 . 5% PEG5000-PE ( Avanti , Cat# 880230 ) , and 0 . 2% atto390-DOPE ( ATTO-TEC GmbH , Cat# AD 390-161 ) for labeled lipid bilayers , or 97% POPC , 2 . 5% biotinyl cap PE , and 0 . 5% PEG5000-PE for unlabeled lipid bilayers .", "The lipid mixes were dried under argon gas and desiccated overnight to remove chloroform .", "The dried lipids were resuspended in 1 mL PBS , pH 7 . 2 ( Gibco , Cat# 20012050 ) and stored under argon gas .", "Lipids were formed into small unilamellar vesicles via ≥30 rounds of freeze-thaws and cleared via ultracentrifugation ( TLA120 . 1 rotor , 35 , 000 rpm/53 , 227×g , 35 min , 4°C ) .", "Lipids were stored at 4°C under argon gas in an Eppendorf tube for up to 2 weeks .", "To form bilayers on beads , 8 . 6 × 108 silica beads with a 4 . 89 µm diameter ( 10 µL of 10% solids , Bangs Labs , Cat# SS05N ) were washed 2× with water followed by 2× with PBS by spinning at 300 rcf and decanting .", "Beads were then mixed with 1 mM SUVs in PBS , vortexed for 10 s at medium speed , covered in foil , and incubated in an end-over-end rotator at RT for 0 . 5–2 hr to allow bilayers to form over the beads .", "The beads were then washed 3× in PBS to remove excess SUVs and resuspended in 100 μL of 0 . 2% casein ( Sigma , Cat# C5890 ) in PBS for 15 min at RT to block nonspecific binding .", "Neutravidin ( Thermo , Cat# 31000 ) was added to the beads at a final concentration of 1 μg/mL for 20–30 min , and the beads were subsequently washed 3× in PBS with 0 . 2% casein and 20 mM MgCl2 to remove unbound neutravidin .", "The indicated amounts of biotinylated ssDNA or saturating amounts of DNA origami pegboards were added to the beads and incubated for 1 hr at RT with end-over-end mixing to allow for coupling .", "Beads were washed two times and resuspended in 100 μL PBS with 0 . 2% casein and 20 mM MgCl2 to remove uncoupled origami pegboards or ssDNA .", "When functionalizing SUV-coated beads with anti-biotin Alexa Fluor 647-IgG ( Jackson ImmunoResearch Laboratories Cat# 200-602-211 , Lot# 137445 ) , the IgG was added to the beads at 1 μM immediately following the casein blocking step , and beads were incubated for 1 hr at RT with end-over-end mixing .", "To estimate the amount of ssDNA bound to each bead , we compared the fluorescence of Atto647-labeled DNA on the bead surface to calibrated fluorescent beads ( Quantum Alexa Fluor 647 , Bangs Lab ) using confocal microscopy ( Figure 1—figure supplement 1 ) .", "To determine saturating conditions of IgG and origami pegboards , we titrated the amount of IgG or origami in the coupling reaction and used confocal microscopy to determine the concentration at which maximum coupling was achieved .", "A comparable amount of origami pegboard coupling was also confirmed with confocal microscopy for beads used in the same experiment .", "30 , 000 RAW264 . 7 macrophages were plated in one well of a 96-well glass bottom MatriPlate ( Brooks , Cat# MGB096-1-2-LG-L ) between 12 and 24 hr prior to the experiment .", "Immediately before adding beads , 100 μL of a 1 μM solution of benzylguanine-conjugated receptor DNA in engulfment imaging media was added , incubated for 10 min at RT , and washed out four times with engulfment imaging media containing 20 mM MgCl2 , making sure to leave ~100 μL of media covering the cells between washes , and finally leaving the cells in ~300 μL of media .", "Approximately 8 × 105 beads were added to the well and engulfment was allowed to proceed for 45 min in the cell incubator .", "Cells were fixed with 4% PFA for 10 min and washed into PBS .", "For Figures 4C and 6D , 10 nM Alexa Fluor 647 anti-biotin IgG ( Jackson Immuno Labs , Cat# 200-602-211 ) diluted into PBS containing 3% BSA was added to each well for 10 min to label non-internalized beads .", "Wells were subsequently washed three times with PBS .", "Images were acquired using the HCS Site Generator plug-in in µManager and at least 100 cells were scored for each condition .", "When quantifying bead engulfment , cells were selected for analysis based on a threshold of GFP fluorescence , which was held constant throughout analysis for each individual experiment .", "For Figures 3 , 4 , and 6 , and Figure 4—figure supplement 1 , the analyzer was blinded during engulfment scoring using the position randomizer plug-in in µManager .", "For the THP1 cells , ~100 , 000 cells per condition were spun down , washed into engulfment imaging media , and coupled to benzylguanine-labeled receptor DNA as described under TIRF imaging .", "Cells were resuspended into 300 μL engulfment imaging media containing 20 mM MgCl2 in an Eppendorf tube , ~8 × 105 beads were added to the tube , and the tube was inverted 8× before plating the solution into a round-bottomed 96-well plate ( Corning , Cat# 38018 ) .", "Engulfment was allowed to proceed for 45 min in the cell incubator before the plate was briefly spun and the cells were fixed in 4% PFA for 10 min .", "Cells were subsequently washed 3× with PBS by briefly centrifuging the plate and removing the media , and finally moved into a 96-well glass-bottom MatriPlate for imaging .", "RAW264 . 7 macrophages were plated and prepared in wells of a 96-well glass bottom MatriPlate as described in ‘Quantification of engulfment’ .", "Using Multi-Dimensional Acquisition in µManager , four positions in the well were marked for imaging at 20 s intervals through at least seven z-planes .", "Approximately 4 × 105 Atto647N-labeled 4S origami functionalized beads and ~4 × 105 Atto550N-labeled 4T origami functionalized beads were mixed in an Eppendorf tube , added to the well , and immediately imaged .", "Bead contacts were identified by counting the number of beads that came into contact with the cells throughout the imaging time .", "Initiation events were identified by active membrane extension events around the bead .", "Engulfment completion was identified by complete internalization of the bead by the macrophage .", "The initiation time was quantified as the amount of time between bead contact ( the first frame in which the bead contacted the macrophage ) and engulfment initiation ( the first frame in which membrane extension around the bead was visualized ) and was only measured for beads that were completely internalized by the end of the imaging time .", "The engulfment time was quantified as the amount of time between engulfment initiation and engulfment completion ( the first frame in which the bead has been fully internalized by the cell ) .", "Phagocytic cups were selected for analysis based on clear initiation of membrane extension around the bead visualized by GFP fluorescence from the DNA-CARγ receptor .", "The phagocytic cup and the cell cortex ( areas indicated in schematic in Figure 6B ) were traced with a line ( six pixels wide for DNA-CARγ receptor and the tSH2 Syk reporter , and eight pixels wide for the Akt-PH reporter and phalloidin ) at the Z-slice with the clearest cross section of the cup .", "Images were acquired on a spinning disc confocal microscope ( Nikon Ti-Eclipse inverted microscope with a Yokogawa CSU-X spinning disk unit and an Andor iXon EM-CCD camera ) equipped with a 40 × 0 . 95 NA air and a 100 × 1 . 49 NA oil immersion objective .", "The microscope was controlled using µManager .", "For TIRF imaging , images were acquired on the same microscope with a motorized TIRF arm using a Hamamatsu Flash 4 . 0 camera and the 100 × 1 . 49 NA oil immersion objective .", "Statistical analysis was performed in Prism 8 ( GraphPad , Inc ) .", "The statistical test used is indicated in each relevant figure legend ." ] ]

[ "Macrophages destroy pathogens and diseased cells through Fcγ receptor ( FcγR ) -driven phagocytosis of antibody-opsonized targets .", "Phagocytosis requires activation of multiple FcγRs , but the mechanism controlling the threshold for response is unclear .", "We developed a DNA origami-based engulfment system that allows precise nanoscale control of the number and spacing of ligands .", "When the number of ligands remains constant , reducing ligand spacing from 17 . 5 nm to 7 nm potently enhances engulfment , primarily by increasing efficiency of the engulfment-initiation process .", "Tighter ligand clustering increases receptor phosphorylation , as well as proximal downstream signals .", "Increasing the number of signaling domains recruited to a single ligand-receptor complex was not sufficient to recapitulate this effect , indicating that clustering of multiple receptors is required .", "Our results suggest that macrophages use information about local ligand densities to make critical engulfment decisions , which has implications for the mechanism of antibody-mediated phagocytosis and the design of immunotherapies ." ]

[ "The word ‘phagocytosis’ means cellular eating .", "It is the process by which cells extend their membranes around foreign particles and engulf them .", "Macrophages , a type of immune cell found in every tissue of the body , perform phagocytosis to eat pathogens and diseased cells .", "To avoid eating healthy cells , macrophages focus on targets marked by proteins called antibodies .", "They look for cells coated with high levels of a type of antibody called immunoglobulin G , or IgG for short , but only eat cells coated with enough IgG , raising the question , can macrophages count ?", "Macrophages recognize IgG antibodies using cell surface receptors called Fc-gamma Receptors .", "When these receptors bind to IgG , they cluster together .", "Researchers do not yet know how the number of IgG antibodies per cluster , or the spacing between them , affects phagocytosis .", "To find this out , researchers need to be able to manipulate the clustering experimentally .", "One way to do this is using a technique called DNA origami .", "This technique creates nanoscale patterns of DNA strands on a target surface .", "If the part of a receptor that interacts with its target is then replaced with a complementary DNA strand to the strands on the target surface , the receptor will bind the surface following the nanoscale pattern .", "This allows researchers to generate synthetic targets with specific patterns of receptor-target interaction .", "Kern et al . replaced the part of the macrophage Fc-gamma Receptor that interacts with IgG with a strand of DNA .", "They then used DNA origami to arrange complementary DNA strands on pegboards and attached these pegboards to silica beads .", "The different arrangements of DNA on these pegboards mimicked the types of antibody clusters macrophages might encounter on the surfaces of the cells and particles they have to engulf in the body .", "Kern et al . found that tight clusters of the DNA targets on the pegboards made the macrophages most likely to begin phagocytosis , particularly clusters of eight or more DNA strands spaced less than seven nanometers apart .", "Macrophages encountering these tight clusters showed an increase in Fc-gamma receptor activation , which is crucial for macrophage attack .", "Whether or not macrophages can count , they can at least sense the level of clustering of IgG antibodies to determine if a target should be engulfed .", "Doctors use antibody therapies that rely on Fc-gamma receptor engagement to treat cancer , autoimmune and neurodegenerative diseases .", "Understanding how clustering affects phagocytosis could aid in the design of new antibody treatments .", "It could also help improve the design of synthetic receptors to create designer immune cells that can attack specific targets .", "The next step will be to recreate the results from the synthetic system used by Kern et al . with natural receptors and antibodies ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "cell biology", "structural biology and molecular biophysics" ]

[ [ "Kinesin superfamily proteins ( KIFs ) are microtubule-based molecular motors that hydrolyze ATP to provide energy to support various cellular functions , such as intracellular transport and the regulation of microtubule ( MT ) dynamics ( Hirokawa et al . , 2009; Miki et al . , 2005 ) .", "Most KIFs , including kinesin-1–kinesin-12 and kinesin-14 subfamily members , actively move along the MT toward either the plus or minus end to transport cellular cargoes , such as protein complexes , vesicles and mRNAs ( Hirokawa et al . , 2009 ) .", "In contrast , kinesin-13 proteins , such as KIF2A and KIF2C , are not actively motile along MTs but depolymerize MTs from both ends to control MT dynamics ( Desai et al . , 1999; Homma et al . , 2003; Ogawa et al . , 2004 ) .", "Kinesin-13 proteins reach the ends of MTs by a passive one-dimensional random diffusion ( Helenius et al . , 2006 ) or with the assistance of a MT plus end-tracking protein ( Honnappa et al . , 2009 ) .", "KIF19A , a kinesin-8 sub-family member , is unique in that it possesses both functions , MT-based active motility toward the plus-end and MT depolymerizing activity .", "Most kinesin-8 motor proteins play critical roles during the cell division process , for example in spindle length regulation ( Weaver et al . , 2011; Su et al . , 2013 ) or in the control of mitotic chromosome alignment ( Kline-Smith and Walczak , 2004; Stumpff et al . , 2008 ) .", "The most extensively studied kinesin-8 , Kip3p ( budding yeast kinesin-8; a homologue of mammalian KIF18 ) , requires its C-terminal tail for MT-binding to prevent its detachment from the MT lattice ( Mayr et al . , 2011; Stumpff et al . , 2011; Su et al . , 2011; Weaver et al . , 2011 ) .", "Kip3p is highly processive ( >5 μm ) and multiple Kip3ps act cooperatively to mediate length-dependent MT depolymerization .", "One possible model for a length-dependent action is proposed to involve the incoming Kip3p bumping off the paused motor at the MT plus-end ( Gupta et al . , 2006; Varga et al . , 2006 , 2009 ) .", "Structural studies of the motor domain of the human kinesin-8 , KIF18A , showed a bent conformation of the α4 relay helix and important loops , indicating the structural basis of the multi-tasking kinesin-8 motor ( Peters et al . , 2010 ) .", "However , the molecular mechanisms of the dual-functions of kinesin-8 proteins remain to be determined .", "We recently reported that KIF19A functions as a MT-depolymerizing kinesin in the control of cilium length ( Niwa et al . , 2012 ) .", "Kif19a-/- mice displayed hydrocephalus and female infertility phenotypes due to abnormally elongated cilia that cannot generate proper fluid flow .", "We also reported that , unlike KIF18A , a KIF19A dimer without the tail domain depolymerizes MTs mainly from the plus-end .", "Therefore , KIF19A possesses the key structural elements for the dual functions of the catalytic motor domain .", "Thus , to elucidate the molecular mechanism of the dual KIF19A functions , we performed crystal structure analysis of the mouse KIF19A motor domain as well as cryo-electron microscopy ( cryo-EM ) reconstruction of the KIF19A motor domain complexed with a MT . In combination with a structure-based mutagenesis analysis , the functional anatomy of KIF19A is reported .", "In the catalytic core of KIF19A , the KIF19A-specific feature of adopting two different interfaces for MTs and tubulins is utilized to achieve the dual functions ." ], [ "We previously reported that dimeric KIF19A-379 has dual activities: MT-based motility toward the plus-end and MT-depolymerizing activity mainly from the plus-end ( Niwa et al . , 2012 ) .", "To clarify which region is responsible for these dual functions , we made the monomeric construct KIF19A-353 ( 353WT ) and assessed its motility and MT-depolymerizing activities .", "353WT includes the motor domain followed by the neck-linker , but does not include the neck coiled coil , which is required for the dimerization of KIF19A ( Figure 1A ) .", "We first performed the in vitro MT gliding assay , in which tetramethylrhodamine ( TMR ) -labeled and polarity-marked MTs were used to show the tracking direction .", "The strongly-labeled MT minus-ends lead the MT gliding , suggesting that the monomeric 353WT moves toward the plus-end ( Video 1 ) .", "MT gliding velocity was 5 . 3 ± 1 . 2 nm/s ( n = 105 MTs from three independent preparations , mean ± SD , Figure 1B and C ) , which was slower than that of dimeric KIF19A-379 ( 21 ± 3 nm/s ) ( Niwa et al . , 2012 ) .", "An in vitro MT depolymerizing assay was also performed for KIF19A-353 ( Desai et al . , 1999 ) .", "GMPCPP-MTs were dose-dependently depolymerized by 353WT ( Figure 1D ) .", "The half-maximal effective concentration for MT depolymerization ( EC50 ) was 142 ± 2 nM , which was approximately half that of KIF19A-379 ( 253 nM ) ( Figure 1E ) .", "Considering that one of two motor domains will reach the plus-end of the MT , EC50 values of one catalytic unit for depolymerizing MTs might be similar between monomeric 353WT and dimeric KIF19A-379 .", "Either way , these in vitro experiments collectively indicate that the KIF19A monomer construct , 353WT , is a dual function motor that moves along and depolymerizes MTs . 10 . 7554/eLife . 18101 . 003Figure 1 . Characteristics of the dual function mouse KIF19A motor domain .", "( A ) Schematic of mouse KIF19A motor domain constructs .", "The KIF19A monomer KIF19A-353 ( referred to as 353WT ) was used in this study .", "NL , neck-linker .", "( B ) MT gliding assays of 353WT on taxol-stabilized MTs .", "Data are presented as the mean ± SEM , n = 105 MTs .", "( C ) Kymograph showing movement of 353WT along MTs as imaged by TIRF microscopy .", "Scale bars , 2 μM ( horizontal ) and 3 min ( vertical ) .", "The average MT length is 8 . 39 ± 2 . 71 μM .", "( D ) 1 . 5 μM GMPCPP-stabilized MTs were incubated with 250 nM 353WT in the presence of 5 mM ATP or AMP-PNP ( Top ) for 15 min .", "AMP-PNP was used as a control .", "1 . 5 μM GMPCPP-stabilized MTs were incubated with different concentrations of 353WT in the presence of 5 mM ATP for 15 min ( Bottom ) .", "The microtubules were pelleted by centrifugation to separate the free tubulin ( S ) and MT pellets ( P ) .", "SDS-PAGE and Coomassie brilliant blue staining were used to confirm the ratio of free tubulin and MTs .", "Representative data from three independent sample preparations are shown .", "( E ) Dose-response MT depolymerization curve for different concentrations of 353WT .", "Data are presented as the mean ± SD .", "The mean EC50 values of 353WT was 142 ± 2 nM .", "The data from three independent experiments were analysed . DOI: http://dx . doi . org/10 . 7554/eLife . 18101 . 00310 . 7554/eLife . 18101 . 004Figure 1—source data 1 . The data and analysis for 353WT . Sheet F1B: Motility Velocity of 353WT .", "Sheet F1E: The data and analysis of dose-response MT depolymerization curve for different concentrations of 353WT . DOI: http://dx . doi . org/10 . 7554/eLife . 18101 . 00410 . 7554/eLife . 18101 . 005Video 1 . Polarity-marked microtubules sliding on 353WT . 353WT was fixed on the coverslip .", "The strongly-labeled MT minus-ends lead the MT gliding , indicating that KIF19A motor proteins move toward the plus end .", "10 seconds intervals , total tracking time 15 minutes . DOI: http://dx . doi . org/10 . 7554/eLife . 18101 . 005 We investigated the molecular mechanism of the dual functions of the KIF19A 353WT monomeric motor by solving its crystal structure at 2 . 7 Å resolution ( Supplementary file 1 ) .", "At the initial stage of the 353WT structure determination process , the residues of the switch II helix α4 could not be determined because there was no corresponding density at the site where α4 is usually located at the center of the MT-binding interface ( Figure 2—figure supplement 1A ) .", "Instead , we found an unmodeled helical-like density , which was more distant from the KIF19A catalytic core ( Figure 2—figure supplement 1B ) .", "Long wavelength X-ray diffraction experiment was thus performed to investigate the property of this density ( Hendrickson and Teeter , 1981; Dauter et al . , 1999 ) , because in the switch II helix α4 , one cysteine residue exists .", "It successfully depicted the anomalous diffractions of sulfur or phosphorus atoms ( Figure 2—figure supplement 1C–E ) .", "Among them , one strong anomalous signal was detected close to the center of the corresponding helical density ( Figure 2—figure supplement 1E ) .", "When the Cys283 residue was assigned to this anomalous signal , all the residues in α4 and the following loop L12 were reasonably determined ( Figure 2—figure supplement 1F ) .", "In this way , the complete tertiary structure of 353WT was finally determined with a reasonable decrease of the R-factor to 22 . 2% ( Supplementary file 1 ) .", "The overall structure of the KIF19A 353WT motor domain shared a similar triangle-shape with other kinesins , which consists of a central β-sheet of eight strands , sandwiched between six α-helices , three on either side ( Figure 2A and B ) ( Sack et al . , 1999 ) .", "In the 353WT atomic structure , Mg2+-ADP was found embedded in the nucleotide-binding pocket .", "In comparison with previously solved motor domains of various KIFs , the remarkable features of the KIF19A motor domain are concentrated on its MT-binding side and include the long and wide L2 loop , the flexible L8 loop , the disordered α4 helix , and the short α6 helix ( Figure 2A and B ) .", "Loop L2 is located at the minus-end side ( rear side ) of the MT-binding surface and is atypically long .", "Loop L8 , which is the major MT-binding region at the plus-end side , is more retracted toward the catalytic core compared with that of kinesin-13 ( Figure 2C ) .", "The switch II helix α4 , which is considered the main contributor to MT-based kinesin motility , is more distant from the motor domain than those of other kinesins including kinesin-8 KIF18A ( Figure 2C and D ) .", "In comparison to the other kinesins , the first portion of helix α5 is more destabilized in KIF19A ( Figure 2F ) ; therefore , the loop region between α4 and α5 ( L12 ) is longer .", "This might allow the invasion of a neighboring molecule into the space between α4 and the motor domain in the crystal packing environment , resulting in the distant position of α4 ( Figure 2—figure supplement 1G ) .", "Helix α6 serves as a base for the neck-linker element , which is important for both the regulation of ATPase activity and MT-based kinesin motility ( Case et al . , 2000 ) .", "Its reduced length by more than one turn might be caused by the atypical glycine mutation in helix α6 , which is not seen in other typical plus end motors ( G341 , Figure 2E and Figure 2—figure supplement 2A ) .", "Glycine has high conformational flexibility and often tends to disrupt the α-helix , which might affect the conformation of the following neck-linker by terminating α6 prematurely . 10 . 7554/eLife . 18101 . 006Figure 2 . Crystal structure of the dual function mouse KIF19A motor domain .", "( A , B )", "Crystal structure of the KIF19A motor domain in the ADP-state seen from the MT binding side ( A ) and 90 degree rotation around the ordinate axis ( B ) .", "L2 ( yellow ) , L8-α3-L9 ( green ) , L11-α4-L12-α5 ( red ) and α6 ( blue ) are depicted .", "The Mg2+-ADP is shown as a ball and stick model .", "( C ) Superposition of the KIF19A motor domain and KIF2C-ADP ( PDB: 1V8J ) .", "The clusters with obviously different structures are colored red ( KIF19A ) and gold ( KIF2C ) .", "L2 is shown in the square .", "( D ) Superposition of KIF19A-ADP and KIF18A-ADP ( PDB: 3LRE ) .", "The clusters with obviously different structures are colored red ( KIF19A ) and light blue ( KIF18A ) .", "α4-L12-α5 is shown in the square .", "( E ) Sequence alignment of α6 between KIF19A and other typical moving kinesin motors .", "For other kinesin motors see Figure 2—figure supplement 2A .", "( F ) Zoom-in view of α4-L12-α5 of KIF19A and KIF18A motor domain in Figure 2D .", "N297 of KIF19A and P306 of KIF18A are shown as balls . DOI: http://dx . doi . org/10 . 7554/eLife . 18101 . 00610 . 7554/eLife . 18101 . 007Figure 2—figure supplement 1 . The unmodeled helix determination by the long wavelength X-ray diffraction .", "( A ) The 2Fo-Fc electron density map contoured at 1 . 0 σ ( blue ) is shown .", "The dashed line rectangle shows the common α4 position for all reported kinesin structures where no corresponding density was found in the KIF19A map .", "( B ) The Fo-Fc difference map contoured at 2 . 5 σ ( red ) shows the positive helical density in the dashed line rectangle .", "( C–E )", "The anomalous difference Fourier maps contoured at 3 . 8 σ ( red ) of ( C ) Met248 , ( D ) ADP and ( E ) Cys283 clearly show the signals from the sulfur or the phosphorus atoms .", "( F ) The 2Fo-Fc electron density map around α4 contoured at 1 . 0 s ( blue ) is shown with the final atomic model of KIF19A-ADP .", "( G ) Crystal packing environment of the KIF19A-ADP crystal . DOI: http://dx . doi . org/10 . 7554/eLife . 18101 . 00710 . 7554/eLife . 18101 . 008Figure 2—figure supplement 2 . Sequence alignment between representative kinesin members .", "( A ) Sequence Alignment around β8-L14-α6 between representative kinesin members . DOI: http://dx . doi . org/10 . 7554/eLife . 18101 . 008 To identify structural elements contributing to the dual functions of KIF19A , we first investigated the loop L2 .", "L2 of kinesin-13 is a key element contributing to MT depolymerization ( Ogawa et al . , 2004; Shipley et al . , 2004 ) .", "According to sequence alignments , KIF2C and KIF19A have L2 regions ( β1b-L2-β1c ) of comparable length in which the hydrophobic residue ( s ) around the tip is/are suggested to be surrounded by basic and acidic residue ( s ) ( Figure 3A ) .", "KIF2C has a slender L2 with a KVD finger at its tip that is vital to MT depolymerization ( Figure 3B ) ( Ogawa et al . , 2004; Shipley et al . , 2004 ) .", "In contrast , KIF19A has a long , fan-shaped L2 that has a hydrophobic tip ( I54-L55 ) sandwiched between acidic and basic clusters ( Figure 3B ) .", "Despite the acidic-hydrophobic-basic order being conserved between KIF2C and KIF19A , their shapes are distinct from each other .", "To test the contribution of these residues to MT-depolymerizing activity , we introduced a series of mutations: an alanine mutant of the basic cluster ( PC2A: R56A-H58A-R59A-R61A ) and hydrophobic mutants ( I54A , L55A , and IL2A: I54A-L55A ) .", "It should be noted that the alanine mutant of the acidic cluster was so unstable that we were unable to acquire reliable in vitro data .", "The acidic cluster is , however , expected to contribute to the depolymerization function . 10 . 7554/eLife . 18101 . 009Figure 3 . Basic and hydrophobic residues in L2 contribute to MT-depolymerizing activity .", "( A ) Top , sequence alignment of L2 between KIF19A and KIF2C .", "Bottom , the L2 swap mutation and other point mutation strategies are shown .", "( B ) β1b-L2-β1c structure diagram of KIF2C ( gold ) and KIF19A ( red ) .", "The acidic , hydrophobic and basic residues are colored red , black and blue , respectively .", "( C ) 1 . 5 μM GMPCPP-stabilized MTs were co-sedimented with 250 nM 353WT or its L2 mutants in the presence of 5 mM ATP or AMP-PNP .", "5 mM AMP-PNP was used as a control .", "( D ) Dose-response MT depolymerization curve for different concentrations of 353WT and its L2 mutants in the presence of 5 mM ATP .", "Data are presented as the mean ± SD .", "The mean EC50 values of 353WT , PC2A and L55A were 142 ± 2 nM , 4936 ± 15 nM and 409 ± 4 nM , respectively .", "The data from three independent experiments were analysed .", "( E ) Kymographs from MT depolymerization .", "Segment-marked microtubules incubated with 125 nM 353WT or its L2 mutants .", "Scale bars , 2 μM ( horizontal ) and time ( vertical ) .", "( F ) The depolymerization speeds of 353WT , L55A and PC2A were 10 . 9 ± 2 . 0 nm/s , 2 . 5 ± 0 . 5 nm/s , and 0 . 7 ± 0 . 2 nm/s , respectively .", "Each column represents the mean ± SEM ( n = 45 , 67 , 58 and 51 MTs for buffer , 353WT , L55A and PC2A , respectively . Data were analysed using a two-tailed t-test , ***p<0 . 001 , compared with 353WT ) .", "( G ) 1 . 5 μM GMPCPP-stabilized MTs were co-sedimented with 250 nM 353WT , KIF18A , KIF5C , and the swap mutants , whose L2 loops were replaced by the counterpart of KIF19A .", "5 mM AMP-PNP was used as a control .", "Representative data from three independent sample preparations are shown . DOI: http://dx . doi . org/10 . 7554/eLife . 18101 . 00910 . 7554/eLife . 18101 . 010Figure 3—source data 1 . The data and analysis for 353WT and L2 mutants . Sheet F3D: The data and analysis of dose-response MT depolymerization curve for different concentrations of 353WT and L2 mutants . DOI: http://dx . doi . org/10 . 7554/eLife . 18101 . 010 In vitro MT depolymerization assays of L2 mutants were performed using a saturated concentration ( 250 nM ) of 353WT ( Figure 3C ) .", "PC2A , L55A and IL2A markedly impaired depolymerization , while I54A had little effect ( Figure 3C ) .", "Different concentrations of 353WT and the L2 mutants that showed an effect ( PC2A and L55A ) were then incubated with GMPCPP-stabilized MTs to obtain EC50 values for MT depolymerization .", "The mean EC50 values of 353WT , PC2A and L55A in three independent experiments were 142 ± 2 nM , 4936 ± 15 nM and 409 ± 4 nM , respectively ( Figure 3D ) .", "The dose-reaction curve of L55A was shifted to the right of 353WT ( Figure 3D ) .", "For PC2A , even at the highest enzyme concentration used ( 5000 nM ) , approximately 50% of the wild-type depolymerization activity was achieved .", "We also observed MT depolymerization in the presence of 5 mM Mg-ATP by TIRF microscopy ( Figure 3E ) .", "The depolymerization was mainly observed at the MT plus-ends and the speeds were 10 . 9 ± 2 . 0 nm/s for 353WT , 2 . 5 ± 0 . 5 nm/s for L55A , and 0 . 7 ± 0 . 2 nm/s for PC2A .", "The depolymerization speed of PC2A was the lowest among them , and only a little higher than that achieved by the negative control ( 0 . 2 ± 0 . 02 nm/s ) ( Figure 3F ) .", "We then tested the effect of introducing KIF19A L2 into KIF18A ( another kinesin-8 member ) and KIF5C ( a typical plus-end directed motor ) ( Figure 3G ) .", "Consistent with a previous report , the KIF18A monomer had little MT depolymerization activity ( Weaver et al . , 2011 ) .", "The substitution of KIF19A L2 mildly increased the depolymerization activity of KIF18A .", "Note that a fraction of KIF18A moved to the soluble fraction via interaction with depolymerized tubulin-dimers .", "In contrast , the L2 of KIF19A did not make KIF5C a MT depolymerase .", "These data collectively suggested that the basic cluster of L2 , as well as the Leu 55 at the tip of L2 , play crucial roles in depolymerization from MT ends , although the simple swapping of L2 is not sufficient for transport motors such as KIF5C to attain MT depolymerization function .", "As described above , KIF19A not only depolymerizes MTs mainly from their plus-ends , but can also move slowly along MTs ( Niwa et al . , 2012 ) .", "Therefore , it is expected that KIF19A ATPase will be activated by both MTs and tubulins .", "To confirm this idea , we first examined the steady-state ATPase activity of the wild-type construct in the absence/presence of MTs or tubulins .", "The basal ATPase activities of 353WT , L55A , and PC2A in the absence of MTs or tubulins were all comparable to that of another kinesin-8 motor , Kip3p ( Figures 4A and Figure 4—figure supplement 1 ) ( Gupta et al . , 2006 ) .", "Activation of 353WT by MTs caused the ATPase to be activated ~300 times to reach a maximum rate of 1 . 43 ± 0 . 07 s−1 , while free tubulin-dimers caused the ATPase to be activated ~200 times to reach a maximum rate of 0 . 80 ± 0 . 06 s−1 ( Figures 4A and Figure 4—figure supplement 1 ) .", "Thus , the KIF19A ATPase is similarly activated by both MTs and tubulins .", "This characteristic is conserved among kinesin-8 proteins , including KIF18A ( Peters et al . , 2010 ) .", "However , the ratio of KIF19A ATPase activation by tubulins/MTs is much higher than that of KIF18A , suggesting that KIF19A is more adapted to tubulins to increase MT depolymerization activity . 10 . 7554/eLife . 18101 . 011Figure 4 . L2 also affects KIF19A kinetics and motility .", "( A ) Steady state ATPase kinetics of 100 nM 353WT and its L2 mutants .", "Data are presented as the mean ± SD ( n = 3 ) .", "( B ) Graph of the bound fraction of 353WT and L2 mutants plotted against the concentration of MTs .", "Fitting the data to a one site binding model gave a dissociation constant ( Kd ) .", "Data are presented as the mean ± SD ( n = 3 ) .", "( C ) MT sliding speed on 353WT and the L55A mutant .", "Data are presented as the mean ± SEM ( n = 69 MTs for L55A ) .", "( D , F , H )", "Kymographs show MTs gliding on L55A and other constructs as imaged by TIRF microscopy .", "Scale bars , 2 μM ( horizontal ) and time ( vertical ) .", "( E , G )", "Introduction of KIF19A L2 into KIF18A ( E ) and KIF5C ( G ) to generate two swap mutants .", "MT sliding speed of the wild type and swap mutants .", "Data are presented as the mean ± SEM ( n = 80 and 58 MTs for KIF18A WT and KIF18A swap , respectively , n = 60 and 48 MTs for KIF5C WT and KIF5C swap , respectively ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 18101 . 01110 . 7554/eLife . 18101 . 012Figure 4—source data 1 . The data and analysis for 353WT , L2 and swap mutants . Sheet F4A_F4FS1: The data and analysis of ATPase activity of 353WT and L2 mutants .", "Sheet F4B: The data and analysis of MT binding assay of 353WT and L2 mutants .", "Sheet F4C: Motility Velocity of 353WT and L55A mutant .", "Sheet F4E: Motility Velocity of KIF8A WT and KIF18A swap .", "Sheet F4G Motility Velocity of KIF5C WT and KIF5C swap . DOI: http://dx . doi . org/10 . 7554/eLife . 18101 . 01210 . 7554/eLife . 18101 . 013Figure 4—figure supplement 1 . ATPase kinetics of 353WT and Its L2 mutants .", "( A ) The basal ATPase kinetics of 353WT and its L2 mutants .", "( B ) The max basal ATPase rate of 353WT and L2 mutants .", "( C ) Microtubule-stimulated ATPase activity of 353WT and its L2 mutants .", "( D ) The max turnover rate ( Vmax ) of 353WT and its L2 mutants with MTs .", "( E ) The Michaelis-Menten constant ( KM ) of 353WT and its L2 mutants with MTs .", "( F ) Tubulin-stimulated ATPase activity of 353WT and its L2 mutants .", "( G ) The max turnover rate ( Vmax ) of 353WT and its L2 mutants with tubulins .", "( H ) The Michaelis-Menten constant ( KM ) of 353WT and its L2 mutants with tubulins .", "Data are presented as the mean ± SD ( n = 3 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 18101 . 01310 . 7554/eLife . 18101 . 014Figure 4—figure supplement 2 . Binding affinity difference between 353WT and PC2A .", "( A ) Incubate 10 ng/μl MTs with 353WT and different concentrations of PC2A .", "( B ) Incubate 4 ng/μl MTs with 353WT and different concentrations of PC2A .", "( C ) Incubate 2 ng/μl MTs with 353WT and different concentrations of PC2A .", "Representative data from three independent sample preparations are shown . DOI: http://dx . doi . org/10 . 7554/eLife . 18101 . 014 Compared to KIF19A 353WT , L55A exhibited relatively small effects on ATPase turnover in the absence or presence of MTs ( Figures 4A and Figure 4—figure supplement 1C ) , or in the presence of tubulins ( Figures 4A and Figure 4—figure supplement 1F ) .", "The affinities for MTs and tubulins were slightly affected by the L55A mutation ( Figure 4A and 4B ) .", "This indicates that the role of L55 in MT depolymerization is independent from that of the KIF19A catalytic cycle stimulated by MTs or tubulins .", "This L55 property seems similar to that of the valine residue of the KVD finger in kinesin-13 ( Ogawa et al . , 2004 ) .", "In contrast , PC2A significantly decreased the MT/tubulin stimulation of the KIF19A ATPase ( Figure 4A and Figure 4—figure supplement 1 ) .", "The PC2A mutation did not significantly affect the KM , tubulin , but markedly increased the KM , MT to approximately 30 times that of 353WT ( Figures 4A and Figure 4—figure supplement 1 ) , suggesting that the basic residues in L2 might strengthen the MT-lattice binding at the weak-binding state , similar to the K-loop of KIF1A ( Okada and Hirokawa , 2000 ) .", "The PC2A mutation also decreased the ATPase stimulation by tubulin without affecting the KM , tubulin .", "Hence , the KIF19A-specific basic residues of L2 might be involved in the tubulin-activated ATPase pathway , as described later .", "The alanine mutation in the basic L2 cluster in KIF19A weakens the affinity for MTs .", "This was confirmed by a MT co-sedimentation assay using KIF19A and a non-hydrolysable ATP-analogue , adenylyl-imidodiphosphate ( AMP-PNP ) .", "We found that L55A slightly decreased MT affinity ( 353WT , Kd , MT 0 . 6 ± 0 . 1 μM; L55A , Kd , MT 1 . 6 ± 0 . 3 μM ) , whereas PC2A severely decreased MT affinity ( PC2A , Kd , MT 5 . 8 ± 2 . 1 μM ) , consistent with the kinetics data above ( Figure 4B ) .", "We also observed the MT gliding motility of KIF19A .", "The speed of the L55A mutant was a little higher than that of 353WT described above ( L55A , 7 . 5 ± 0 . 6 nm/s , Figure 4C and D ) .", "However , bound MTs were rarely detected when the PC2A mutant , at the same concentration as L55A , was fixed on the coverslip .", "We thus checked the number of bound MTs to PC2A in the presence of AMP-PNP .", "Different concentrations of MTs were incubated with 353WT or PC2A in the presence of 5 mM AMP-PNP .", "Although PC2A bound fewer MTs than 353WT , increasing the concentration of PC2A ( 1 mg/ml ) enabled sufficient microtubules to fix to the glass surface ( Figure 4—figure supplement 2 ) .", "Even in these conditions , however , bound MTs glided distances that were too short to reliably calculate the gliding speed on the PC2A-coated coverslip .", "Thus , the PC2A mutation severely increased the dissociation constant for MTs so that few MTs were observed on PC2A attached to the coverslip .", "The KIF18A and KIF5C L2 substitution mutations were also introduced into KIF19A to check MT gliding motilities .", "Again , bound MTs were not detectable with KIF5C L2 and very few MTs were detected with KIF18A L2 , indicating that the L2 of KIF19A , especially its basic cluster , is necessary for 353WT to stay attached to MTs .", "We further investigated the role of loop L2 for MT-based motility by the reverse swapping of KIF19A L2 into other kinesin motors .", "The L2 of KIF19A was swapped into the kinesin-8 member , KIF18A , and the kinesin-1 member , KIF5C , to clarify its effect on motility .", "As a consequence , MTs glided much more slowly with the swap mutants compared with the wild-type motors ( KIF18A WT , 34 . 4 ± 0 . 3 nm/s; KIF18A swap , 10 . 2 ± 0 . 1 nm/s; KIF5C WT , 179 . 9 ± 2 . 5 nm/s; KIF5C swap , 68 . 9 ± 0 . 2 nm/s ) ( Figure 4E–H ) .", "The intensive ionic interactions between the L2 of KIF19A and MTs might reduce motility speed .", "KIF19A L2 is thus likely to interact electrostatically with the MT to tether the KIF19A motor domain on the MT , which in turn reduces motility speed on the MT . The most remarkable structural difference between KIF19A and other kinesins was found in the switch II region , α4-L12-α5 , which is destabilized and distant from the motor domain ( Figures 2A–B and 5B ) .", "Considering that switch II serves as a major interface for MTs and tubulins , this conformational difference is expected to contribute to the dual functions of KIF19A .", "To address this question , we investigated the specific roles of the KIF19A switch II region in motility and depolymerization activity . 10 . 7554/eLife . 18101 . 015Figure 5 . Basic residues in L12 assist KIF19A to effectively depolymerize microtubules .", "( A ) Top , sequence alignment of KIF19A , KIF5C and KIF18A .", "Bottom , the L12 swap mutation and other point mutation strategies are shown .", "( B ) L11-α4-L12-α5 structure of KIF18A ( light blue ) and KIF19A ( red ) .", "KIF18A P306 and KIF19A N297 are shown as balls .", "( C , D ) 1 . 5 μM GMPCPP-stabilized MTs were co-sedimented with 250 nM 353WT or its L12 swap mutants ( C ) and charged mutants ( D ) in the presence of 5 mM ATP or AMP-PNP .", "AMP-PNP ( 5 mM ) was used as a control .", "( E ) Dose-response MT depolymerization curve for different concentrations of 353WT and its L12 mutants in the presence of 5 mM ATP .", "Data are presented as the mean ± SD .", "The mean EC50 values of 353WT , GS2R and K2A were 142 ± 2 nM , 91 ± 2 nM and 678 ± 5 nM , respectively .", "The data from three independent experiments were analysed .", "( F ) Kymographs from MTs depolymerization .", "Segment-marked MTs incubated with 125 nM 353WT or its L12 mutants .", "Scale bars , 2 μM ( horizontal ) and time ( vertical ) .", "( G ) The depolymerization speeds of 353WT , GS2R and K2A were 10 . 9 ± 2 . 0 nm/s , 30 . 0 ± 4 . 9 nm/s , and 2 . 0 ± 0 . 5 nm/s , respectively .", "Each column represents the mean ± SEM ( n = 45 , 67 , 52 and 47 MTs for buffer , 353WT , GS2R , and K2A , respectively ) .", "Data were analyzed using the two-tailed t-test , ***p<0 . 001 , compared with 353WT ) .", "( H ) Steady state ATPase kinetics of 100 nM 353WT and its L12 mutants .", "Data are presented as the mean ± SD ( n = 3 ) .", "( I ) Graph of the bound fraction of 353WT and L12 mutants plotted against MT concentration .", "Data are presented as the mean ± SD ( n = 3 ) .", "( J ) MTs polymerized by tubulin and tubulin S were co-sedimented with different concentrations of KIF19A in the presence of 1 mM AMP-PNP .", "Representative data from three independent sample preparations are shown . DOI: http://dx . doi . org/10 . 7554/eLife . 18101 . 01510 . 7554/eLife . 18101 . 016Figure 5—source data 1 . The data and analysis for 353WT and L12 mutants . Sheet F5E: The data and analysis of dose-response MT depolymerization curve for different concentrations of 353WT and L12 mutants .", "Sheet F5H_F5FS1: The data and analysis of ATPase activity of 353WT and L12 mutants .", "Sheet F5I: The data and analysis of MT binding assay of 353WT and L12 mutants . DOI: http://dx . doi . org/10 . 7554/eLife . 18101 . 01610 . 7554/eLife . 18101 . 017Figure 5—figure supplement 1 . Steady state ATPase kinetics of 353WT and Its L12 mutants .", "( A ) The basal ATPase kinetics of 353WT and its L12 mutants .", "( B ) The max basal ATPase rate of 353WT and L12 mutants .", "( C ) Microtubule-stimulated ATPase activity of 353WT and its L12 mutants .", "( D ) The max turnover rate ( Vmax ) of 353WT and its L12 mutants with MTs .", "( E ) The Michaelis-Menten constant ( KM ) of 353WT and its L12 mutants with MTs .", "( F ) Tubulin-stimulated ATPase activity of 353WT and its L12 mutants .", "( G ) The max turnover rate ( Vmax ) of 353WT and its L12 mutants with tubulins .", "( H ) The Michaelis-Menten constant ( KM ) of 353WT and its L12 mutants with tubulins .", "Data are presented as the mean ± SD ( n = 3 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 18101 . 017 A sequence alignment of the switch II cluster in KIF19A , KIF5C and KIF18A shows marked differences that are concentrated in loop L12 .", "Thus we first constructed two KIF19A swap mutants , in which L12 was replaced with the counterpart of KIF18A or KIF5C ( Figure 5A ) .", "In comparison to 353WT , soluble tubulin was decreased in the presence of KIF5C L12 , but was slightly increased in the presence of KIF18A L12 ( Figure 5C ) .", "Within the KIF18A L12 sequence , three continuous basic residues ( K299 , R300 and R301 ) are present .", "Only one basic residue , K274 , was found in the L12 of KIF5C , whereas two basic residues , K290 and K294 , were found in the L12 of KIF19A .", "Therefore , to test the relationship between the number of basic residues in L12 and MT depolymerization ability , we made two more constructs: GS2R , in which both G291 and S292 were mutated to arginine , such that L12 had four basic residues , and K2A in which both K290 and K294 were replaced with alanine , such that L12 had no basic residues .", "As expected , GS2R exhibited a slight increase in MT depolymerization activity , similar to the KIF18A L2 swap mutant , whereas K2A without basic residues in L12 showed lower MT depolymerization ability , similar to the KIF5C L2 swap mutant ( Figure 5D–G ) .", "We also examined the ATPase of L12 mutants , GS2R and K2A .", "The basal ATPase activities of L12 mutants in the absence of MTs or tubulins were all comparable to the 353WT ( Figure 5—figure supplement 1 ) .", "The GS2R mutant showed only a small increase in MT- and tubulin-activated turnover rate , though it exhibited a four-times lower Michaelis-Menten constant for both MTs and tubulins ( Figure 5H ) .", "In contrast , K2A showed a lower MT- and tubulin-activated turnover rate and a higher Michaelis-Menten constant for both MTs and tubulins ( Figure 5H ) .", "Therefore , the change in the number of basic residues in L12 might alter the electrostatic affinities to the MT lattice or tubulins .", "To further confirm this idea , a MT co-sedimentation assay with Taxol-stabilized MTs was performed .", "The GS2R mutation did not significantly alter the affinity for MTs ( 353WT , Kd , MT 0 . 6 ± 0 . 1 μM; GS2R , Kd , MT 0 . 6 ± 0 . 06 μM ) .", "The K2A mutation , however , significantly weakened the affinity for MTs ( Kd 3 . 0 ± 0 . 6 μM ) ( Figure 5I ) .", "Thus , two basic residues of L12 contribute to the depolymerization ability of KIF19A through electrostatic interaction .", "Further addition of basic residues did not effectively increase MT affinity .", "This contribution of basic residues in loop L12 resembles that of the KIF1A K-loop ( Okada and Hirokawa , 2000 ) .", "Thus , the binding partner of KIF19A L12 was expected to be the E-hook , the C-terminal tail of tubulin that has an acidic cluster .", "To address whether the E-hook is responsible for the electrostatic interaction with the KIF19A motor domain , we incubated KIF19A with two kinds of MTs , normal tubulin polymers and tubulin-S polymers , in the presence of AMP-PNP .", "Tubulin-S , which was obtained by limited subtilisin proteolysis of tubulin dimer , lacks the cluster of negatively charged residues found in the E-hook of α- and β-tubulins .", "Whether the binding ratio was 1:1 or 1:2 . 5 , a greatly reduced quantity of KIF19A was seen in the tubulin S assembled MT pellet ( Figure 5J ) .", "Thus , the E-hook of tubulin is confirmed to be responsible for the KIF19A-MT interaction .", "We also investigated the asparagine in KIF19A at the junction of loop L12 and helix α5 ( Figure 6A ) .", "At this position , a proline residue is highly conserved among kinesin superfamily proteins except for KIF19A ( Figure 6—figure supplement 1A ) .", "It has been proposed that this proline serves as a 'helix starter' of α5 because of its restricted dihedral angles .", "Thus , a lack of proline at the starting position of α5 might destabilize helix α5 , further destabilizing the preceding L12 and α4 , as is observed in the crystal structure of 353WT in the absence of a MT ( Figure 2A and B ) .", "To test this hypothesis , Asn297 was mutated to proline to clarify its effect on depolymerization and motility along MTs ( Figure 6C–G ) .", "The resulting EC50 value of N297P for MT depolymerization ( EC50 201 ± 2 μM , Figure 6C ) was slightly increased compared to the wild type , with the slight decrease in the depolymerization speed ( 7 . 7 ± 1 . 3 nm/s , Figure 6D and E ) .", "In contrast , the MT-gliding velocity ( 6 . 5 ± 0 . 2 nm/s ) was slightly increased compared to the wild type in three independent experiments ( Figure 6F and G ) .", "Therefore , Asn297 in α5 has a slight advantage for the MT depolymerization but has a slight disadvantage for the motility over Pro297 . 10 . 7554/eLife . 18101 . 018Figure 6 . Contribution of KIF19A-Specific Asn297 . ( A ) Sequence alignment of KIF19A and other typical kinesins for the α4-L12-α5 region .", "For more kinesin member sequence alignments see Figure 6—figure supplement 1A .", "( B ) 1 . 5 μM GMPCPP-stabilized MTs were co-sedimented with 250 nM 353WT or N297P mutant in the presence of 5 mM ATP or AMP-PNP .", "Representative data from three independent sample preparations are shown .", "AMP-PNP was used as a control .", "( C ) Dose-response MT depolymerization curve for different concentrations of 353WT and N297P in the presence of 5 mM ATP .", "Data are presented as the mean ± SD .", "The mean EC50 values of 353WT , N297 were 142 ± 2 nM and 201 ± 2 nM , respectively .", "The data from three independent experiments were analyzed .", "( D ) The depolymerization speeds of 353WT and N297 were 10 . 9 ± 2 . 0 nm/s and 7 . 7 ± 1 . 3 nm/s , respectively .", "Each column represents the mean ± SEM ( n = 67 and 29 MTs for 353WT and N297P , respectively ) .", "Data were analyzed using the two-tailed t-test , **p<0 . 01 , compared with 353WT ) .", "( E ) Kymographs of MT depolymerization .", "Segment-marked MTs incubated with 125 nM 353WT or N297P mutant .", "Scale bars , 2 μM ( horizontal ) and 1 min ( vertical ) .", "( F ) MT gliding assays of KIF19A-353WT and N297P on taxol-stabilized MTs .", "Data are presented as the mean ± SEM , ( n = 113 MTs for N297P ) .", "( G ) Kymograph showing movement of N297P long MTs as imaged by TIRF microscopy .", "Scale bars , 2 μM ( horizontal ) and 3 min ( vertical ) .", "( H ) Steady state ATPase kinetics of 100 nM 353WT and N297P .", "Data are presented as the mean ± SD , ( n = 3 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 18101 . 01810 . 7554/eLife . 18101 . 019Figure 6—source data 1 . The data and analysis for 353WT and N297P mutant . Sheet F6C The data and analysis of dose-response MT depolymerization curve for different concentrations of 353WT and N297P mutant .", "Sheet F6F: Motility Velocity of 353WT and N297P mutant .", "Sheet F6H_F6FS2: The data and analysis of ATPase activity of 353WT and N297P mutant . DOI: http://dx . doi . org/10 . 7554/eLife . 18101 . 01910 . 7554/eLife . 18101 . 020Figure 6—figure supplement 1 . Sequence alignment between representative kinesin members .", "( A ) Sequence Alignment around L12-α5 between representative kinesin members . DOI: http://dx . doi . org/10 . 7554/eLife . 18101 . 02010 . 7554/eLife . 18101 . 021Figure 6—figure supplement 2 . Steady state ATPase kinetics of 353WT and N297P mutant .", "( A ) The Basal ATPase kinetics of 353WT and N297P mutant .", "( B ) The Basal ATPase rate of 353WT and N297P mutant .", "( C ) Microtubule-stimulated ATPase activity of 353WT and N297P mutant .", "( D ) The max turnover rate ( Vmax ) of 353WT and N297P mutant with MTs .", "( E ) The Michaelis-Menten constant ( KM ) of 353WT and N297P mutant with MTs .", "( F ) Tubulin-stimulated ATPase activity of 353WT and N297P mutant .", "( G ) The max turnover rate ( Vmax ) of 353WT and N297P mutant with tubulins .", "( H ) The Michaelis-Menten constant ( KM ) of 353WT and N297P mutant with tubulins .", "Data are presented as the mean ± SD ( n = 3 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 18101 . 021 We then checked the basal ATPase activity as well as stimulation by MTs or tubulins .", "Even though no significant difference was shown , the N297P mutation tended to increase the basal ATPase activity ( Figures 6H and Figure 6—figure supplement 2 ) .", "The destabilized conformation of switch II might be less favorable for the basal ATPase activity .", "The MT-stimulated ATPase rate did not alter significantly , although the Michaelis-Menten constant was increased ( Figures 6H and Figure 6—figure supplement 2 ) .", "In contrast , the tubulin-stimulated ATPase rate of N297P was markedly reduced to approximately half that of the wild type ( Figures 6H and Figure 6—figure supplement 2 ) .", "Hence , the KIF19A-specific asparagine residue , Asn297 , contributes to the increase of tubulin-stimulated ATPase activity but has minimal effect on MT-stimulated ATPase activity .", "Thus , Asn297 of KIF19A might be advantageous to adopting both the straight tubulin-dimer in the MT-lattice and the curved tubulin-dimer at the MT-ends .", "Asn297-induced destabilization of switch II helices might contribute to this adaptation to achieve the dual functions of KIF19A .", "Finally , we performed cryo-EM analysis of KIF19A in the nucleotide-free state complexed with a GDP-taxol MT . Cryo-EM reconstruction at a 7 . 0 Å resolution clarified the interactions between KIF19A specific structures and a straight MT ( Figure 7—figure supplement 1A ) .", "It should be noted that , presumably because of the weak or unstable interaction between KIF19A and a straight MT , stable and full decoration of KIF19A on the GDP-taxol MT was uncommon; the occupancy of KIF19A is thus lower than that of a tubulin-dimer in our reconstruction ( Figure 7—figure supplement 1B and C ) .", "However , almost all helices , β-sheets and important loops that contact a MT are reliably visualized in our reconstruction ( Figure 7—figure supplement 1D ) .", "To clarify the conformation of the nucleotide-free KIF19A on a MT , two crystal structures , the KIF19A-ADP structure solved here ( Figure 7A and B ) and the nucleotide-free KIF5 structure ( PDB: 4LNU ) ( Figure 7C and D ) ( Cao et al . , 2014 ) , were rigidly fitted into our reconstruction .", "The KIF5-free structure represented better fitting than the KIF19A-ADP structure , as indicated by the higher cross correlation values ( KIF19A-ADP , 0 . 83; KIF5-free , 0 . 89 ) .", "Most of the regions in the catalytic core of both KIF19A-ADP and KIF5-free are nicely fitted into the cryo-EM map of KIF19A-free on a MT ( gray in Figure 7A–D ) .", "However , marked differences were observed between the two structures at the MT-binding interfaces , loop L8 at the plus-end side , switch II α4-L12-α5 , and loop L2 at the minus-end side ( green or red in Figure 7A–D ) .", "The first two of these regions of the cryo-EM map take very similar conformations to those of KIF5-free on a MT ( red arrows in Figure 7C ) , whereas the L2 of KIF19A-ADP was nicely fitted into the extra-density observed at the minus-end side ( black arrow in Figure 7A ) .", "Thus , we finally created an atomic model of KIF19A-free on a MT that has the similar L8 and switch II conformations as KIF5-free and the similar L2 conformation as KIF19A-ADP ( Figure 7E ) .", "This model exhibits the highest cross correlation value of 0 . 90 with our cryo-EM structure . 10 . 7554/eLife . 18101 . 022Figure 7 . Cryo-EM reconstruction of KIF19A on a straight MT .", "( A–D )", "Cryo-EM reconstruction of KIF19A-nucleotide-free complexed with GDP-taxol-MT with three different contour levels ( grey , blue , cyan ) , and atomic models of KIF19A-ADP solved in this study ( green ) ( A and B ) and KIF5-nucleotide-free ( red , PDB: 4LNU ) ( C and D ) , seen from the right side ( A and C ) and the plus-end side ( B and D ) .", "Black arrow and dashed circles indicate the elongated L2 .", "Red arrows show the α4-L12 and L8 fitted in the corresponding densities .", "( E ) Atomic model of KIF19A-nucleotide-free on GDP-taxol-MT ( orange ) was created and rigidly fitted onto the cryo-EM map .", "The density of the helix H12 and the following E-hook of β-tubulin can be observed ( red dashed line ) , while the E-hook of α-tubulin is not clear .", "( F ) Conformation of the switch II ( α4–L12 ) and the E-hook of β-tubulin ( red dashed line ) .", "Atomic model of α4 fits perfectly in the density .", "( G and H )", "Comparison of the KIF19A-ADP ( green ) and KIF19A-nucleotide-free models on MT ( orange ) , viewed as in A and B . ( I ) L2 density was clearly observed at the inter-tubulin dimer interface .", "( J ) Zoom-in view around L2 .", "The acidic residues in L2 potentially interact with the basic residues in β-tubulin H11’ ( red region ) .", "The basic residues in L2 interact with the acidic residues in α-tubulin H12 ( blue region ) .", "Between these electrostatic clusters , the hydrophobic residues of L2 in KIF19A and H12 and H8-S7 loops in α-tubulin constitute the hydrophobic core ( yellow region ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 18101 . 02210 . 7554/eLife . 18101 . 023Figure 7—figure supplement 1 . Cryo-EM reconstruction of KIF19A-MT complex .", "( A ) FSC curves for standard FSC ( black ) , FSC noise ( high resolution noise substitution cutoff 10 Å , gray , Chen et al . , 2013 ) and FSC true ( cyan ) .", "The resolutions of FSC true according to the 0 . 143 and 0 . 5 criteria were estimated as 7 . 0 Å and 8 . 7 Å .", "To achieve the resolution , 471 , 380 particles were calculated in total .", "( B ) Example micrograph of nucleotide-free KIF19A complexed with GDP-taxol MT . ( C ) Cryo-EM reconstruction of a KIF19A-decorated GDP-taxol-MT .", "The occupancy of KIF19A was lower than that of the MT so that only the density for the MT can be visualized at the threshold-level 6 . 5 .", "( D ) Cryo-EM map at the threshold-level 2 . 7 .", "The secondary structural elements of KIF19A as well as the loops interacting between MT and KIF19A are visualized . DOI: http://dx . doi . org/10 . 7554/eLife . 18101 . 023 The L8 of KIF19A-free on a MT moves to interact with H12 residues of β-tubulin during binding with the MT-lattice and the release of ADP from the catalytic core ( Figure 7G ) .", "This movement is accompanied by approximately 7 degree counter-clockwise rotation of the following helix α3 , as also reported in kinesin-13 ( Figure 7H ) ( Ogawa et al . , 2004 ) .", "Thus , the interaction with the straight lattice of the MT and the MT-induced ADP release from KIF19A triggers the counter-clockwise rotation of the L8-α3 complex ( Figure 7H ) .", "The KIF19A conformation on the MT-lattice is similar to typical plus-end directed kinesins , such as KIF5 , and might close the nucleotide-binding pocket even on a straight MT , allowing access for ATP into the pocket .", "The switch II α4-L12-α5 of KIF19A-free is retracted to take a similar conformation to that of KIF5-free during MT-lattice binding and ADP release ( Figure 7G ) .", "The length , the location , and the angle of α4 in KIF5-free fit perfectly to the corresponding cryo-EM density of KIF19A-free ( Figure 7F ) .", "This means that KIF19A is able to adjust the switch II conformation to fit the straight tubulin interface on the MT-lattice .", "It is also notable that a clear density was found at the right side of α4 , corresponding to the E-hook of β-tubulin ( Figure 7E and F ) .", "As expected by the biochemical assays described above , the acidic residues in the E-hook ionically interact with the basic cluster of L12 in KIF19A , thus stabilizing the E-hook .", "In comparison with the E-hook of α-tubulin , that of β-tubulin is remarkably stabilized by interaction with L12 of KIF19A ( Figure 7E ) .", "Loop L2 extends toward the minus-end of the MT . No marked difference was observed between KIF19A-ADP and KIF19A-free on a MT ( Figure 7G ) .", "The corresponding density of L2 almost reaches the inter-tubulin-dimer groove close to the H11’-helix of β-tubulin at the minus-end side ( Figure 7I ) .", "Because L2 has the characteristic sequence in which the hydrophobic residues at the tip of L2 are sandwiched by an acidic cluster on the left side and a basic cluster on the right side , the possible interactions of these residues were estimated from the fitted atomic models of KIF19A and tubulin-dimers ( Figure 7J ) .", "The acidic cluster faces the H11’ helix of the neighboring β-tubulin , where basic H406 and K402 residues are located ( red region in Figure 7J ) .", "However , the Cα distance between L2 and H11’ is around 11 Å , which is slightly longer than a stable interacting distance .", "Hydrophobic residues presumably interact with the hydrophobic residues of the H8-S7 loop and the H12 helix in α-tubulin ( yellow region in Figure 7J ) .", "Consistent with the mutational studies , the L55 of L2 serves as the main interface of the hydrophobic contacts .", "The basic cluster then faces acidic clusters on the H12 helix of α-tubulin ( blue region in Figure 7J ) .", "Its Cα distances are within 9 Å , thus salt-bridges might be formed between them even on the straight MT lattice .", "When KIF19A is on the MT-lattice , therefore , loop L2 interacts with α-tubulin through the hydrophobic tip and basic cluster , though the acidic cluster will not contribute to the interaction with the MT ." ], [ "To achieve the dual functions , KIF19A should meet the following two requirements: ( 1 ) the ability to adopt two different interfaces for straight MTs and curved tubulins to enable KIF19A ATPase activation , and ( 2 ) the ability to stabilize the curved conformation of MT-ends to destabilize the MT protofilaments , as observed for kinesin-13 ( Figure 8A ) ( Ogawa et al . , 2004; Shipley et al . , 2004 ) .", "To accomplish these requirements , KIF19A-specific characteristics are concentrated on the MT-binding surface .", "As detailed in the following sections , for the 1st requirement , the location of the destabilized , retractable loop L8 and switch II ( α4-L12-α5 ) enable adjustment to both the straight tubulin interface on the MT-lattice [Figure 8C", "( i ) ] and the curved tubulin interface at the MT-ends [Figure 8C", "( iv ) ] .", "For the second requirement , the long and fan-shaped L2 is located at the minus-end side .", "It plays a central role in depolymerizing MTs from the ends through stabilization of the curved conformation of MT-ends .", "Helix α6 is atypically shorter than those of the other previously solved kinesins .", "Its functional meaning is still uncertain mainly because structural information for the neck-linker that follows α6 is still lacking .", "The short conformation of α6 is expected to affect the neck-linker conformation and further structural study of KIF19A in the ATP state is needed to address the role of the short helix α6 . 10 . 7554/eLife . 18101 . 024Figure 8 . Model of KIF19A motor domain function .", "( A ) Schematic diagram of the dual functions of KIF19A .", "( B ) Simulated 7 Å maps of straight and curved MTs created from the atomic models of 1JFF and 3RYC , respectively .", "The two structures were aligned at the position of the marked α-tubulins .", "( C ) Simulated 7 Å maps and atomic models of KIF19A on straight and curved MTs showing the adaptation of KIF19A-MT interfaces .", "Collisions between KIF19A and β-tubulin in", "( ii ) and", "( iii ) are highlighted in red .", "( D ) Zoom-in view around the KIF19A L2 and curved tubulin-dimer interface ( green ) .", "Atomic model of a straight MT ( grey ) is superimposed to compare the distance from L2 to β-tubulin H11’ .", "( E ) Functional anatomy for the dual function of KIF19A . DOI: http://dx . doi . org/10 . 7554/eLife . 18101 . 024 Tubulin-dimers have distinct conformations when located in the MT-lattice or in the MT-curved end ( Figure 8B ) .", "Most kinesins choose between the two , although kinesin-8 members are able to bind both .", "To investigate which structural alterations are needed to achieve this , in silico docking of KIF19A with a curved tubulin-dimer ( PDB: 3RYC ) was performed ( Nawrotek et al . , 2011 ) .", "The α-tubulin of the curved tubulin-dimer was aligned with that of the straight MT-lattice .", "On the MT-lattice , the interface of the KIF19A-free model is nicely fitted to the tubulin-dimer interface [Figure 8C", "( i ) ] .", "L8 , switch II , and L2 serve as main interfaces for the straight tubulin-dimer .", "The KIF19A-free model on the MT-curved end comes into collision with the β-tubulin surface at two regions , L8 and switch II L12-α5 [Figure 8C", "( ii ) ] .", "L8 in the KIF19A-ADP model , however , made better contact with the curved tubulin-dimer at the MT ends [Figure 8C", "( iii ) ] .", "Hence , the retractable L8 by the rotational movement of the L8-α3-L9 cluster is the first key feature to enable fitting to both the straight and curved interfaces of MTs .", "However , switch II still collides with curved β-tubulin at the junction of L12 and the first turn of helix α5 [Figure 8C", "( iii ) ] .", "At that point , the highly conserved proline residue is replaced in KIF19A with a unique asparagine residue ( Asn297 ) .", "A proline residue is often found at the start or end points of a helix to stabilize it .", "Thus , replacement of proline for an asparagine residue might destabilize α5 allowing it to melt into the flexible long L12 loop .", "In fact , the first turn of helix α5 melted in the crystal structure ( Figure 2F ) .", "Therefore , we expected that the flexible feature of the L12 and initial α5 might be caused by the KIF19A-specific Asn297 .", "This idea was supported by our biochemical experiment .", "The N297P mutant had a more than 40% decrease in tubulin-stimulated ATPase activity compared with the wild type , indicating that this mutation limits adjustment of the KIF19A interface to the curved tubulin-dimer ( Figure 6H ) .", "Thus , the destabilized conformation of switch II is the second key feature to avoid collision when binding to the curved MT end [Figure 8C", "( iv ) ] .", "To confirm this idea , a high resolution structure of KIF19A complexed with tubulin is required .", "The arrangements of the KIF19A MT-binding interface described above enable it to achieve dual functions .", "On the flip side , however , they result in decreased affinity of KIF19A for the MT-lattice or the MT-curved end .", "KIF19A has overcome this difficulty by introducing basic clusters into the L2 and L12 loops .", "The basic cluster at the right side of L2 tethers MTs by ionic interactions between L2 and the MT . The alanine mutant , PC2A , severely decreases the MT-binding affinity of KIF19A so that MTs were rarely found on the PC2A-bound coverslip .", "It should be noted here that the PC2A mutation only decreases the affinity for MTs but not for soluble tubulin-dimers ( Figure 4A ) .", "As described below , the additional salt bridges through the acidic cluster on the left side of L2 are only formed when interacting with a tubulin-dimer .", "This could possibly minimize the effect of the PC2A mutation on the affinity for tubulins , but not on the affinity for MTs .", "In contrast , when the basic cluster was introduced into the L2 of KIF5C or KIF18A , the motility speed decreased to nearly one third of wild type .", "The additional strong interaction between the basic cluster and MTs might slow down the detachment of KIF19A from the MT . Hence introducing the basic cluster of L2 to increase affinity for MTs has the consequence of reducing the speed of KIF19A motility .", "Similar to the basic cluster of L2 , the basic residues of L12 contribute to tethering KIF19A to the E-hook of a MT , in a similar fashion to the K-loop of KIF1A , ( Okada and Hirokawa , 2000 ) .", "Our data showed that more than two of the basic residues in L12 were required to show wild-type KIF19A MT-binding ability and MT-depolymerization activity .", "In kinesin-13 , alanine substitutions of charged residues in the switch II cluster showed lower depolymerization activity due to weak MT binding ability ( Shipley et al . , 2004 ) .", "We presume that the positively charged residues support both straight and curved MT binding , providing minor assistance to L2 .", "The shapes of the fan-like L2 of KIF19A and the slender L2 of kinesin-13 were different , while the length of loop L2 is comparable between KIF19A and kinesin-13 and the locations of the hydrophobic residues at the tip are conserved ( Figure 3A ) .", "Alanine mutation of Leu 55 , which caused a MT depolymerization deficiency with a small effect on MT-based motility , suggested its central role for depolymerizing MTs ( Figure 3 ) .", "The comparison of KIF19A binding to the MT-lattice and the MT-curved end indicates a further role for L2 [Figure 8C", "( i ) and", "( iv ) ] .", "The interactions between L2 of KIF19A and α-tubulin through the hydrophobic tip of L2 and the basic cluster of L2 are similarly observed for both straight and curved tubulins .", "For curved tubulins , however , interactions between L2 and the neighboring β-tubulin at the minus-end side would also be formed ( Figure 8D ) .", "The H11’ helix of β-tubulin gets closer to L2 by ~2Å compared with that in the straight conformation ( Cα distance is around 9 Å ) so that the acidic cluster could make salt bridges with the basic residues in the H11’ helix of β-tubulin .", "Therefore , the combination of acidic-hydrophobic-basic residues of L2 stabilizes the curved conformation of inter-tubulin-dimer interface .", "This could be the structural basis for MT depolymerization by KIF19A .", "Based on the structural properties of the KIF19A motor domain and substantial mutant analysis , we present a functional anatomy for the dual function of KIF19A ( Figure 8E ) .", "The destabilized switch II helices contribute to the adaptation to the two distinct interfaces of straight and curved MTs with a concomitant sacrifice of slightly decreased motility speed along the MT . The rotatable L8-α3-L9 cluster also supports adaptation to the two interfaces .", "The basic cluster of L2 , as well as the basic residues of L12 , enable KIF19A to tether to both straight and curved MTs via flexible ionic interactions with the acidic residues of H12 or the E-hook of tubulins .", "This assures its motility along the MT , although motility speed may be decreased .", "The hydrophobic tip of L2 , as well as the surrounding basic and acidic clusters , play critical roles in MT depolymerization .", "These residues stabilize the inter-tubulin-dimer interface of the curved MT protofilament .", "Thus , the curved conformation of MT ends is stabilized by L2 , resulting in the depolymerization of the MTs .", "In this way , KIF19A has acquired dual functions by introducing multiple strategies .", "The resulting KIF19A is a slow plus-end directed motor combined with mild MT-depolymerizing activity .", "The KIF19A motility speed and the MT depolymerization activity might be different on ciliary doublet MTs .", "Further studies are required to reveal how KIF19A is involved in length control of ciliary MTs in vivo ." ], [ "The coding sequence of the mouse KIF19A motor domain ( 1–353 ) and its related mutants were cloned into pET21b ( + ) ( Novagen , Germany ) with a 7×His-tag at the C terminal of the motor domain .", "All constructs were transformed into E . coli strain BL21 ( DE3 ) ( Novagen ) .", "Protein expression was induced by the addition of 0 . 4 mM IPTG to cultures followed by incubation at 24°C for 16 hr with vigorously shaking .", "Immobilized-metal affinity chromatography ( His-select Nickel Affinity Gel , Sigma-Aldrich , Saint Louis , MO ) and ion exchange chromatography ( Resource S , GE Healthcare , Japan ) were sequentially used for protein purification .", "The protein samples were aliquoted in small volumes for immediate use or flash frozen in liquid nitrogen for later use .", "The hanging drop vapor diffusion method was used .", "One microliter of KIF19A353WT protein sample at 10 mg/ml containing 0 . 1 mM ADP was mixed with 1 μl reservoir buffer and incubated at 20°C .", "Single Crystals are grown in 10% ethylene glycol , 2% PEG8000 , 50 mM Tris-Bicine ( pH 8 . 5 ) , 9 mM MgCl2 and 9 mM CaCl2 .", "X-ray diffraction data at 2 . 72 Å resolution were collected using a BL41XU beam-line ( SPring-8 , Japan ) , at a wavelength λ = 1 . 0 Å .", "The anomalous diffraction data were collected using a BL1A beamline ( Photon Factory , Japan ) , at the wavelength λ = 2 . 7 Å .", "The HKL2000 program package ( Otwinowski and Minor , 1997 ) was used to index , integrate and scale the data .", "The structure of 353WT was solved by molecular replacement using the Crystallography & NMR System ( CNS ) ( Brunger , 2007; Brünger et al . , 1998 ) and the atomic coordinates of KIF18AMD ( PDB: 3LRE ) as a search model .", "Several rounds of iterative model building and refinement were performed using COOT ( Emsley and Cowtan , 2004 ) ( RRID: SCR_014222 ) and Refmac5 ( Murshudov et al . , 2011 ) ( RRID: SCR_014225 ) .", "The final crystallographic model of 353WT at 2 . 7 Å resolution has a Rwork/Rfree of 0 . 222/0 . 302 .", "The data collection and refinement statistics are shown in Supplementary file 1 .", "UCSF Chimera ( RRID: SCR_004097 ) was used for structure alignment and visualization ( Pettersen et al . , 2004 ) .", "Porcine brain tubulin was purified and tetramethylrhodamine ( TMR ) labeled .", "The microtubule gliding assay was performed as previously described with some modifications ( Niwa et al . , 2012 ) .", "KIF19A 353WT and its mutants were immobilized on a coverslip using PentaHis antibody ( Qiagen , Netherlands , RRID: AB_2619735 ) .", "The surface of the coverslip was further coated with casein ( Wako Chemical , Japan ) to prevent nonspecific binding .", "After washing , 20 μl KIF19A protein solution ( 0 . 1 mg/ml ) was injected into the flow chamber and washed out after 3 min incubation .", "Then , TMR-labeled MTs in PEM buffer ( 100 mM PIPES-KOH pH 6 . 8 , 1 mM EGTA , 1 mM MgCl2 , 10 μM taxol ) were injected and allowed to bind for 5 min .", "Taxol-stabilized MTs were used to minimize the depolymerization of MTs by KIF19A .", "The coverslip was then turned face down for 3 min to reduce background fluorescence and to prevent extra MTs binding during data collection .", "Finally , the motility buffer was supplemented with oxygen-scavenger just before observation to minimize photo bleaching .", "Time-lapse observation was performed at 37°C , using the ELYRA P . 1 system ( Carl Zeiss , Germany ) in the TIRF mode .", "The data were collected every 10 seconds and tracking time was 15 min for 353WT and its mutants .", "The data were collected every 5 seconds and the tracking time was 7 . 5 min for KIF18A WT and KIF18A swap .", "The data were collected every 1 second tracking time was 7 . 5 min for KIF5C WT and KIF5C swap .", "GMCCPP-stabilized MTs were polymerized as previously described ( Yajima et al . , 2012 ) .", "Depolymerization assays were performed using GMPCPP MTs ( Hertzer et al . , 2006; Niwa et al . , 2012; Noda et al . , 2012 ) .", "250 nM wild-type or mutant KIF19A was incubated in BRB80 buffer with 5 mM ATP or AMPPNP and 1 . 5 μM GMPCPP MTs .", "To determine the EC50 , 353WT was titrated at different concentrations ( 0–5000 nM ) with 1 . 0 μM GMPCPP MTs .", "All reactions were incubated at 25°C for 15 min , and subsequently centrifuged in a TLA55A rotor ( Beckman Coulter , Brea , CA ) at 38 , 000 ×g for 10 min at 25°C .", "The supernatant was removed from the pellet , and the pellet was resuspended in the same volume of BRB80 buffer as the supernatant .", "Equal volumes of supernatant and pellet were electrophoresed on a 10% SDS-PAGE .", "The gel was stained with Coomassie Brilliant Blue and scanned .", "The fraction of free tubulin was quantified and analyzed using ImageJ ( NIH ) .", "The data were plotted and fitted to the four-parameter logistic equation and the EC50 was calculated using KaleidaGraph 4 . 0 software ( Synergy software ) .", "Time-lapse observation of microtubule depolymerization was performed as previously described ( Niwa et al . , 2012 ) .", "The steady state ATPase kinetics of KIF19A 353WT and mutants ( 100 nM ) were measured using an EnzCheck phosphate assay kit ( Molecular Probes , Eugene , OR ) ( Nitta et al . , 2008 ) .", "In the presence of inorganic phosphate , Pi , whose production is catalyzed by Kinesin , the substrate 2-amino-6-mercapto-7-methylpurine riboside ( MESG ) is enzymatically converted by purine nucleoside phosphorylase ( PNP ) to ribose 1-phosphate and 2-amino-6-mercapto-7-methylpurine .", "This process results in a spectrophotometric shift in maximum absorbance to 360 nm for the product .", "The absorbance at 360 nm was recorded at 25°C every 5 s for 300 s using a V-630 Bio spectrophotometer ( JASCO , Japan ) .", "The ATPase hydrolysis rate was calculated from the slope of the absorbance plot , and measured at different concentrations of MT or tubulin .", "Kinetic data were plotted and fitted the Michaelis-Menten model .", "Two micromoles of purified KIF19A protein were incubated with increasing amounts of MTs ( 0–40 μM ) in PEM buffer at 25°C for 15 min .", "To minimize the MT depolymerization by KIF19A , taxol-stabilized MTs were used .", "Samples were centrifuged at 38 , 000 ×g for 10 min at 25°C , in a TLA55 rotor ( Beckman Coulter ) .", "The separated supernatant and pellet fractions were loaded onto SDS-PAGE gels and resulting band intensities were analyzed using Image J ( NIH ) .", "The data were fitted using nonlinear regression and a one site-specific binding model .", "Dissociation constants ( Kd ) were calculated using KaleidaGraph 4 . 0 software ( Synergy software ) .", "Tubulin was diluted to 5 mg/ml in distilled water and GTP added to 1 mM final concentration .", "After pre-incubating in a centrifuge tube at 25°C for 5 min , subtilisin was added in a weight ratio of 1/100 , with further incubation at 25°C for 45 min .", "The reaction was stopped with PMSF to a final concentration of 0 . 05% and incubated on ice for 40 min .", "The samples were then centrifuged at 110 , 000 ×g at 4°C for 20 min ( Knipling et al . , 1999 ) .", "The supernatant was collected and the protein concentration determined by the BCA method ( ThermoFisher , Waltham , MA ) .", "Samples were used fresh or flash frozen in liquid nitrogen and stored at −80°C .", "Tubulin ( 20 µM ) was polymerized in polymerization buffer ( PEM-GTP and 7% DMSO ) at 37°C for 30 min .", "Taxol was added in a stepwise fashion to a final concentration of 20 μM .", "KIF19A 353WT was diluted to 100 μM in a dilution buffer ( 10 mM Hepes-NaOH pH 7 . 5 , 50 mM NaCl , 1 mM MgCl2 ) .", "A 5 µl drop of the polymerized microtubules ( 4 μM ) was placed onto a glow-discharging holey carbon grid ( R2/2 , Quantifoil ) .", "After 30 s , the solution was wicked away with a piece of Whatman no . 1 filter paper and a 5 µl drop of 353WT ( 28 μM ) was quickly applied .", "After another 60 s , the grid was reacted with PEM containing 10 U/ml apyrase for 90 s and plunge-frozen into liquid ethane using a semi-automated vitrification device ( Vitrobot Mark IV , FEI , Hillsboro , OR ) with 5 s in 100% humidity at 27°C .", "Data acquisition was performed using a 200 kV field emission cryo-electron microscope ( Tecnai Arctica , FEI ) at 78 , 000-fold nominal magnification with an FEI Falcon II direct detection camera under low-dose conditions using the data acquisition software , Serial EM ( Mastronarde , 2005 ) .", "All data were collected as a movie with seven subframes with a total electron dose of 50 e−1/Å2 at a pixel size of 1 . 28 Å/pixel .", "The defocus range of the data set was set to −1 . 5 to −2 . 5 μm .", "All the movie data were processed for motion correction using the software Unblur ( Grant and Grigorieff , 2015 ) .", "Motion corrected and summed images were analyzed for defocus and astigmatism using CTFFIND3 ( Mindell and Grigorieff , 2003 ) and images without significant drift and astigmatism were used for further analysis .", "To generate an initial low-resolution model ( ~20 Å ) , images of a 14-protofilament motor-microtubule complex were selected and semi-automatically straightened using the 'unbend' program of Ruby-Helix ( Metlagel et al . , 2007 ) .", "Three-dimensional structures were generated using asymmetric helical reconstruction ( Kikkawa , 2004 ) .", "This initial 3D structure was then used for single particle analysis as described previously ( Shang et al . , 2014 ) .", "Segments were extracted at a spacing of 80 Å using a box size of 768 × 768 pixels and initial XY shifts and Euler angles were determined by template matching to the 3D model .", "The position of the microtubule seam was determined using the set of segment images extracted from one microtubule as described ( Sindelar and Downing , 2007 ) .", "Subsequently , the parameters were refined and the 3D structures were reconstructed using the FREALIGN package ( Grigorieff , 2007 ) without imposing symmetry .", "The helical parameters were determined from the non-symmetrized map and the parameters were applied to the map .", "Twelve rounds of refinement with increasing resolution were performed .", "To assess the effective resolutions and possible over-fitting , the phases of high-resolution components ( >10 Å ) of individual images were randomized ( Chen et al . , 2013 ) and the 3D structures were reconstructed and analyzed by Fourier Shell Correlation ( FSC; Van Heel , 1987 ) ( Figures 7 and Figure 7—figure supplement 1 ) ." ] ]

[ "The kinesin-8 motor , KIF19A , accumulates at cilia tips and controls cilium length .", "Defective KIF19A leads to hydrocephalus and female infertility because of abnormally elongated cilia .", "Uniquely among kinesins , KIF19A possesses the dual functions of motility along ciliary microtubules and depolymerization of microtubules .", "To elucidate the molecular mechanisms of these functions we solved the crystal structure of its motor domain and determined its cryo-electron microscopy structure complexed with a microtubule .", "The features of KIF19A that enable its dual function are clustered on its microtubule-binding side .", "Unexpectedly , a destabilized switch II coordinates with a destabilized L8 to enable KIF19A to adjust to both straight and curved microtubule protofilaments .", "The basic clusters of L2 and L12 tether the microtubule .", "The long L2 with a characteristic acidic-hydrophobic-basic sequence effectively stabilizes the curved conformation of microtubule ends .", "Hence , KIF19A utilizes multiple strategies to accomplish the dual functions of motility and microtubule depolymerization by ATP hydrolysis ." ]

[ "The cells that line the airways and other passages in the body have hair-like structures called cilia on their surface .", "Maintaining the cilia at an appropriate length is key to allowing fluid to flow efficiently in these passages .", "A protein called tubulin forms scaffolds known as microtubules that give each cilium its shape and allow it to change length .", "Motor proteins are also found in cilia , and travel along the microtubules to transport substances .", "One of these microtubule-based motors , referred to as KIF19A , accumulates at the tip of cilia and controls their length .", "It does so by combining two actions: it moves along the microtubule to the tip of the cilium , and then removes tubulin molecules from the end .", "Microtubules are straight along their length and curved at the end , and it is thought that kinesin recognizes both of these shapes in order to carry out these roles .", "A single region of the KIF19A protein appears to be able to accomplish both roles , but the molecular changes that the protein undergoes to do so are not known .", "Wang et al . have now investigated these changes by determining the structure of the motor domain of KIF19A from mice using two experimental approaches: X-ray crystallography and cryo-electron microscopy .", "These structures showed that the specific structural features responsible for the protein's dual roles are indeed clustered on the side of the protein that binds to the microtubule .", "Wang et al . also identified the regions that make KIF19A flexible enough to fit this interface with both straight and curved microtubules .", "Next , Wang et al . found that other regions of KIF19A stop it detaching from the microtubule and allow it to stabilize the curved shape of microtubule ends; this stimulates the microtubule to disassemble , or “depolymerize” .", "The findings show that KIF19A uses multiple strategies to enable it to carry out its roles .", "To understand better how KIF19A depolymerizes the microtubule , a more detailed structure of KIF19A together with tubulin will be needed .", "Structural studies of KIF19A in cilia will also be useful to understand how the protein controls the length of microtubules ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "ecology" ]

[ [ "Sea urchin larvae with their elaborate calcareous endoskeleton have been studied by embryologists for over a century to promote our understanding of calcification in biological systems ( Boveri , 1901; Decker and Lennarz , 1988; Wilt , 2002 ) .", "Similar to mammalian osteoblasts that arise from mesenchymal stem cells ( MSC ) , the sea urchin larval skeleton is also produced by a specific cell line – the primary mesenchyme cells ( PMCs ) ( Wilt , 2002 ) .", "During the blastula stage , PMCs ingress into the blastocoel and migrate in a stereotypical pattern , forming a posterior ring around the blastopore ( Ettensohn , 1990; Gustafson and Kinnander , 1956 ) .", "Amorphous calcium carbonate ( ACC ) is precipitated within intracellular vesicles that exocytose their content into the lumen of a syncytial cable formed by the PMCs .", "This process involves at least 40 distinct skeletal matrix proteins supporting the formation of the mature calcite spicules within this extracellular space ( Beniash et al . , 1997; Benson et al . , 1986 ) .", "Some of these matrix proteins are also present in intracellular compartments where they may play a role in the stabilization of ACC ( Urry et al . , 2000; Wilt , 2002 ) .", "Furthermore , recent findings suggest that Ca2+ also enters calcification vesicles by endocytosis of seawater into a vesicular network within the PMCs ( Vidavsky et al . , 2016 ) .", "During de novo formation of the larval skeleton in the mid-gastrula stage , calcium uptake increases ten-fold , compared to the blastula stage .", "70% of this calcium is incorporated into the newly formed spicules ( Nakano et al . , 1963 ) , while the remaining 30% reflects the uptake of calcium into cell organelles , including mitochondria and the smooth endoplasmatic reticulum ( Decker et al . , 1987 ) .", "Besides the availability of Ca2+ , dissolved inorganic carbon ( DIC , i . e . CO2 , HCO3- and CO32- ) is required at equimolar levels for the precipitation of CaCO3 .", "Radioisotopic measurements demonstrate that up to 63% of carbon for spicule formation derives from respiratory CO2 , while the remaining DIC that is incorporated into the spicules derives from seawater ( Sikes et al . , 1981 ) .", "Based on the assumption that HCO3- rather than CO2 or CO32- supplies the remaining 37% of DIC for calcification , 1 . 6 protons are generated per molecule CO32- precipitated .", "An even stronger local acidification at the site of ACC formation can be expected considering CO2 as the major source of DIC .", "Based on these calculations , it has been suggested that PMCs are capable of efficiently handling this proton load using pH regulatory mechanisms ( Sikes et al . , 1981 ) .", "However , such potential acid-base regulatory mechanisms in PMCs remain poorly understood despite their importance in the formation of the larval skeleton .", "A previous study demonstrated that pHi regulation of PMCs is dependent on external Na+ as well as HCO3- to compensate for an intracellular acidosis indicating Na+-dependent HCO3- buffering mechanisms ( Stumpp et al . , 2012 ) .", "The importance of HCO3- transport in the calcifying PMCs is underlined by the observation that DIDS ( 4 , 4´-diisothiocyano-2 , 2´´-disulfonic acid stilbene ) , an inhibitor for most SLC4 family transporters ( Romero et al . , 2004 ) inhibits uptake and deposition of 45Ca into spicules ( Yasumasu et al . , 1985 ) .", "Accordingly , it has been concluded that these compounds block the supply of HCO3- for ACC formation rather than the ability of PMC cells to form their syncytium as shown for other blockers ( Basse et al . , 2015; Mitsunaga et al . , 1986 ) .", "Furthermore , H+-export pathways involving Na+/H+ exchange were suggested to remove protons from the cytosol of PMCs although amiloride , an inhibitor for Na+-dependent H+ exchange had no effect on spicule formation and pHi regulation of PMCs ( Mitsunaga et al . , 1987; Stumpp et al . , 2012 ) .", "Here , we hypothesize that HCO3- transport pathways , via a so far unidentified HCO3- transport mechanism affect the calcification process in different ways:", "( i ) HCO3- transport supplies the cell with substrate for the precipitation of CaCO3 and", "( ii ) the regulation of intracellular HCO3- homeostasis is critical to buffer excess protons generated by the intravesicular precipitation of CaCO3 .", "Apart from an endogenous generation of protons through biomineralization , pH regulatory mechanisms of sea urchin larvae have received considerable attention in the context of CO2 driven sea water acidification ( Byrne et al . , 2013; Stumpp et al . , 2012 ) .", "The sea urchin larva has been extensively studied with respect to their potential for physiological acclimation and evolutionary adaptation to predicted near-future ocean acidification .", "These studies suggested that energy allocations and ion regulatory efforts are key processes that determine the resilience to reductions in seawater pH ( Pan et al . , 2015; Pespeni et al . , 2013; Stumpp et al . , 2012 ) .", "These studies also indicated that despite moderate impacts at the organismal level , the compensatory reactions at the cellular level are substantial .", "Thus , a better mechanistic understanding for cellular processes affected by changes in seawater pH is essential to explain energy allocations in the sea urchin larva exposed to experimental ocean acidification .", "Four Slc4 transporters were identified in the genome of the purple sea urchin , Strongylocentrotus purpuratus ( Tu et al . , 2012 ) .", "To date , little is known regarding the function and tissue-specific localization of Slc4 transporters in the sea urchin larva .", "During the period of early skeleton formation in the sea urchin embryo ( 30–48 hr post fertilization ) , highest transcript abundance was detected for the SpSlc4a10 gene compared to all other Slc4 transporters ( Tu et al . , 2014 ) .", "This gene shares highest sequence identity with the mammalian Slc4a10 ( NBCn2 ) gene that encodes an electroneutral sodium-dependnet Cl-/HCO3- exchanger with Cl- self-exchange activity ( Parker et al . , 2008 ) .", "This prompted us to hypothesize that SpSlc4a10 may be critically involved in HCO3- transport during formation of the larval skeleton .", "Here , we test the role of SpSlc4a10 in the maintenance of intracellular pH homeostasis and as a DIC concentration mechanism in the calcifying PMCs of the sea urchin embryo .", "Since pHi regulation is intrinsically linked to precipitation of ACC , these mechanisms will fill an important knowledge gap regarding the fundamental principles of biomineralization in the sea urchin larva ." ], [ "The SpSlc4a10 has widespread expression in blastula embryos at 1 day post-fertilization ( dpf ) , including the PMCs surrounding the blastopore ( Figure 1A ) .", "In the late gastrula ( prism stage , 2 dpf ) , SpSlc4a10 is highly expressed in the PMCs during the formation of the syncycial cables .", "Besides a strong staining within the main cell bodies , SpSlc4a10 is also expressed in the syncycial cables and filopodia of PMCs ( Figure 1—figure supplement 1 ) .", "In the pluteus larva ( 3 dpf ) expression of SpSlc4a10 was exclusively detected in PMCs located at the tips of the primary and secondary rods .", "Negative control using sense probes validate the specificity of the staining ( Figure 1A lower panel ) .", "Among the four SLC4 transporters identified in the sea urchin genome ( SpSlc4a3 , SpSlc4a2 , SpSlc4a11 and SpSlc4a10 ) the PMC specific SpSlc4a10 clusters within the clade of the Slc4a7-10 , electroneutral Na+-coupled HCO3- transporters found in vertebrates ( Figure 1—figure supplement 2 ) .", "The amino acid sequence of the sea urchin SpSlc4a10 shares 44% similarity to the mammalian Slc4a10 ( NBCn2 ) orthologue ( Figure 1—figure supplement 3 ) .", "During the first 5 days of development , SpSlc4a10 expression peaks at 2 dpf , accompanied by the onset of larval Ca2+ accumulation ( Figure 1C ) .", "After this , expression levels decrease , while high Ca2+ uptake rates are maintained up to 5 dpf .", "Light microscopic analyses using polarized light ( birefringence ) demonstrated a disturbed formation of the larval skeleton in morphants compared to KCl injected control larvae ( Figure 2A ) or scramble MO control larvae ( 150 µM , Figure 2—figure supplement 1 ) .", "The length of the primary rod is decreased in a dose-dependent manner with increasing SpSlc4a10 morpholino ( MO ) concentrations ( Figure 2B ) , while the tri-partite digestive tract develops normally .", "This reduction in primary rod length is accompanied by a decreased total Ca2+ content in SpSlc4a10 morphants ( Figure 2B , inset ) .", "Besides the predominant expression of SpSlc4a10 in PMCs , a Na+/K+-ATPase ( SpAtp1a3 ) and a Na+/H+-exchanger ( SpSlc9a2 ) isoform that are mainly expressed in stomach epithelial cells are also expressed in PMCs of late gastrula and pluteus larvae ( Figure 2C ) .", "A four-fold up-regulation of SpSlc4a10 expression levels in the morphants compared to control larvae at 3dpf demonstrates the specificity of the morpholino applied .", "In addition , SpSlc9a2 was significanly downregulated in the morphants ( Figure 2D ) .", "No differences were detected for the Na+/K+-ATPase coding gene SpAtp1a3 between control larvae and the SpSlc4a10 morphants ( Figure 2D ) .", "In situ hybridization using the SpSlc4a10 anti-sense probe demonstrates an irregular pattern of SpSlc4a10 positive PMCs in the morphants compared to control larvae ( Figure 2E ) .", "Immunohistological analyses using a costom made antibody designed against the sea urchin SpSlc4a10 protein demonstrate strong immunoreactivity with primary mesenchyme cells and their syncytial cables ( Figure 3A ) .", "High-magnification confocal microscopy indicates a localiazation of the protein in the plasma membrane as well as subcellular structures within the cytosol ( Figure 3B ) .", "Negative controls by blocking the primary antibody with the immunizing peptide demonstrated no unspecific binding of the secondary antibody ( Figure 3C ) .", "Westernblot analyses demonstrated positive immunoreactivity of our SpSlc4a10 antibody with a 135 KDa protein which is in the predicted size range of this protein ( 1183 amino acids; ≈130 KDa ) .", "Western-blot analyses demonstrate decreased SpSlc4a10 protein levels in MO-injected larvae when compared to control animals validating the knock-down by our SpSlc4a10 morpholino .", "Under control conditions ( pH 8 . 1 ) , sea urchin embryos increase total Ca2+ content at high rates of 1 . 75 nmol larva−1 day−1 during the formation of the larval skeleton ( 2–4 dpf ) ( Figure 4A ) .", "CO2-induced reductions in seawater pH by 0 . 4 and 0 . 6 pH units resulted in a reduction in developmental rates ( Figure 4—figure supplement 1 ) associated with a reduction in Ca2+ accumulation ( Figure 4A ) .", "Normalization of larval Ca2+ content to bodylength ( correction for developmental delay [Stumpp et al . , 2011] ) demonstrated no effect on the ability to accumulate Ca2+ under acidified conditions ( Figure 4B ) .", "Maintained calcification of larvae under acidified conditions is accompanied by an increased expression of SpSlc4a10 at the onset of skeleton formation at 2 dpf ( Figure 4C ) .", "This up-regulation of SpSlc4a10 at the onset of skeleton formation is still evident when expression levels were normalized for body length correcting for the developmental delay ( Figure 4D ) .", "Intracellular pH ( pHi ) regulatory abilities of PMCs were assessed by real-time ratiometric fluometry using the pH-sensitive dye BCECF-AM ( Figure 5A ) .", "Exposure of PMCs from control animals to seawater containing 20 mM NH4Cl evoked an intracellular alkalosis by increasing pHi from 6 . 8 to 7 . 6 .", "A slight compensation of the ammonia ( NH3/NH4+ ) induced alkalosis was observed during a period of 10 min .", "Removal of ammonia resulted in an intracellular acidosis ( pHi = 6 . 1 ) that was efficiently compensated within 5 min ( Figure 5A , B ) .", "SpSlc4a10 morphants had a significantly decreased pHi ( 6 . 66 ± 0 . 17 ) compared to control larvae ( 6 . 87 ± 0 . 13 ) ( Figure 5B; Supplementary file 1-Table S2 ) , and a significantly reduced ability to recover from the ammonia-induced acidosis during washout of NH3/NH4+ .", "While control PMCs recover at a rate of 0 . 14 ± 0 . 05 pH units min−1 that of the morphants was sigificantly decreased to 0 . 05 ± 0 . 02 pH units min−1 ( Figure 5B , C ) .", "Ratios were translated into pH values using the nigericin calibration method in combination with the depolarization of the membrane potential by high ( 150 mM ) external [K+] ( Figure 5D ) .", "Pharmacological experiments demonstrated that the recovery rate from an intracellular acidosis is sensitive to the anion exchange inhibitor 4 , 4'-Diisothiocyano-2 , 2'-stilbenedisulfonic acid ( DIDS ) in a dose-dependent manner ( Figure 5E ) .", "Despite full inhibition of the DIDS-sensitive component of pHi regulation , PMCs remained capable of partly restoring pHi at lower rates in a similar fashion to that seen in the SpSlc4a10 morphants ( Figure 5B , F ) .", "We tested for Ca2+ incorporation abilities in SpSlc4a10 morphants ( Figure 6A , B ) and in larvae treated with the anion exchange inhibitor DIDS ( Figure 6C , D ) in comparison to control larvae .", "Late gastrula stage larvae were incubated in calcein for 3 hr and fluorescence intensities monitored using confocal microscopy .", "In addition to skeletal deformations , calcein incorporation was decreased in the SpSlc4a10 morphants at the three different locations including posterior rod: PR ( −43% ) , junction: JC ( −31% ) and anterior rod: AR ( −35% ) .", "In the presence of 10 µM and 100 µM DIDS , fluorescence intensities in the three spicule sections , were also significantly decreased .", "Average values of different spicule sections ( inset Figure 6B ) demonstrated 40% and 50% reductions in spicule fluorescence intensity in the presence of 10 µM and 100 µM DIDS , respectively ." ], [ "Here , we show that bicarbonate transport by a Slc4 family transporter is required for pHi regulation in sea urchin PMCs critical for the normal development of the larval skeleton .", "Interestingly , besides their importance in bone formation of mammals and vertebrates , the occurrence of specific bicarbonate transporters has been suggested to be a key step for the evolution of biomineralization in basal metazoans ( Zoccola et al . , 2015 ) .", "A putative electroneutral Na+-independent Cl-/HCO3- cotransporter SLC4γ has been exclusively associated with calcifying tissues of scleratinian corals that is lacking in other non-calcifying cnidarians .", "Based on the findings of the present work , future studies will use the sea urchin larva as a model to identify other PMC-specific acid-base transporters ( e . g . Na+/H+-exchangers ) that may be critically involved in the calcification process .", "Here , special attention will also be dedicated to the ability of PMCs to re-absorb the larval skeleton close to metamorphosis , which is also likely associated with pH modulations in the PMC syncycium .", "Since pH regulation is ultimately linked to the biological precipitation of CaCO3 , the present work provides a first step toward characterizing ion-regulatory mechanisms in PMCs critical for biomineralization the sea urchin embryo .", "This knowledge can then serve as a basis to identify conserved mechanisms of biomineralization in marine species and their potential for physiological buffering in times of rapid environmental change ." ], [ "Adult purple sea urchins ( Stongylocentrotus purpuratus ) were collected from the California coast ( Kerckhoff marine Laboratory , California Institute of Technology ) , transferred to the Helmholtz Centre for Ocean Research Kiel ( GEOMAR ) , and maintained in a re-cirulating natural seawater system at 11°C .", "Animals were fed with Laminaria sp .", "Spawning of males and females was induced by shaking and larval cultures were maintained as previously described ( Stumpp et al . , 2013; Stumpp et al . , 2015 ) .", "From 3 days post fertilization ( dpf ) larvae were fed daily with Rhodomonas spp .", "at a concentration of 10 , 000 cells mL−1 .", "pH perturbation experiments were performed as previously described ( Stumpp et al . , 2012 ) .", "Briefly , larval cultures with three replicates per pH treatment were continuously aerated with CO2-enriched air providing constant pCO2 levels of 400 µatm ( pH 8 . 1 ) , 1250 µatm ( pH 7 . 7 ) and 4000 µatm ( pH 7 . 5 ) .", "Seawater physicochemical parameters including salinity , pH and temperature were monitored on a daily basis and samples for the determination of total dissolved inorganic carbon ( CT ) were collected at two time points .", "The seawater carbonate system was calculated from pHNBS and CT using CO2sys as previously described ( Stumpp et al . , 2013 ) ( see Supplementary file 1-Table S3 ) .", "Larval densities were determined and paraformaldehyde ( 4% in filtere seawater ) fixed samples were collected every day to monitor mortality and morphometry of larvae along the experimental period of 5 days .", "The amino acid sequences for phylogenetic analyses were collected from the Ensembl ( www . ensembl . org ) and EchinoBase ( www . echinobase . org ) databases .", "Collected sequences were submitted to the online tool MAFFT ( www . ebi . ac . uk/Tools/msa/mafft/ ) for alignment and phylogenetic analysis .", "The results of the phylogenetic analysis were plotted using the phylogenetic tree generation tool provided on the iTOL website ( itol . embl . de ) .", "For molecular cloning , the transcript sequences of sea urchin SpSlc4a10 , SpSlc9a2 and SpATP1a3 were PCR amplified using primers provided in Supplementary file 1-Table S4 .", "The amplicon sequence was then cloned into pGEM-Teasy ( Promega ) cloning vector and used to synthesize the RNA probes for in situ hybridization .", "Micro injections were performed according to the protocol provided in Warner and McClay , 2014 .", "The gene-specific morpholino-substituted antisense oligonucleotides ( MO ) 5`-GTTCAAGTTGTTTCTCAGTTCTCGT-3´ complementary to the start codon region of the sea urchin Slc4a10 gene as well as the srambled morpholino were obtained from Gene Tools ( Oregon ) .", "The MO was dissolved in 0 . 5 M KCl solution and was injected into the fertilized egg ( one-cell stage ) using a microinjection system ( Picospritzer III , Parker ) mounted on an inverse microscope ( Zeiss ObserverD1 ) equipped with a cooling stage .", "RNA from control and MO injected larvae was isolated by using the Direct-zol RNA MicroPrep kit ( Zymo Research ) .", "RNA samples were reverse transcribed by Super Scipt IV cDNA synthesis kit ( Invitrogen , Waltham , USA ) for quantitative PCR ( qPCR ) .", "Expression levels of the target genes were measured by qPCR using the 7500 Fast Real-Time PCR system ( Applied Biosystems ) and normalized to the housekeeping gene SpZ12 .", "qPCR primers used in this study are listed in Supplementary file 1-Table S5 .", "Whole mount in situ hybridization was performed as previously described ( Stumpp et al . , 2015 ) .", "IF and WB analysis was performed as previously described ( Stumpp et al . , 2013; Stumpp et al . , 2015 ) .", "Briefly , for IF larvae ware fixed in 4% paraformaldehyde dissolved in filtered seawater for 15 min and postfixed in ice-cold methanol for 1 hr .", "The polyclonal primary antibody was generated against a synthetic peptide corresponding to an internal region ( LTRHRHHKQKKKKEPENKAYNKGRRKS ) of the sea urchin SpSlc4a10 protein .", "The affinity chromatography purified antibody was diluted 1:250 and samples were incubated over night at 4°C .", "After washing , samples were incubated in the secondary antibody for 1 hr , and pictures were taken on a confocal microscope ( Axiovert 200M , Zeiss , Germany ) .", "For WB , 250 larvae were collected , weighted and extracted by gentle pipetting in 1:10 wt/vol of Lämmli loading buffer .", "Proteins were fractionated by SDS PAGE on 6 . 5% polyacrylamide gels , and transferred to nitrocellulose membranes ( Bio-Rad ) , using a tank blotting system ( Bio-Rad ) .", "Blots were preincubated for 1 hr at room temperature in TBS-Tween buffer containing 5% ( wt/vol ) bovine serum albumin .", "Blots were incubated at 4°C overnight in a 1:7500 dilution of the primary antibody ( see previous section ) .", "After washing with TBS-T , the blots were incubated for 1 hr with horseradish peroxidase-conjugated goat anti-rabbit IgG antibody ( Santa Cruz Biotechnology , Santa Cruz , CA ) diluted 1:14 , 000 in TBS-T .", "Protein signals were visualized with the ECL Western Blotting Detection Reagents ( GE Healthcare , Munich , Germany ) and photographed with a Gel Doc 2000 system equipped with a CCD camera ( BioRad ) .", "For the peptide compensation assays used in whole mount IF and WB analysis , the primary antibody was pre-absorbed with the immunization peptide at a concentration of 0 . 1 mg/ml for 12 hr at 4°C .", "Intracellular pH ( pHi ) measurements and ammonia pulse technique pHi determinations and ammonia pulse experiments were conducted as previously described ( Stumpp et al . , 2012 ) .", "Control and morphant larvae were measured in an alternate mode and from each larva 4–5 cells were simultanously recorded and treated as one replicate ( n = 1 ) .", "Nigericin in combination with high external [K+] ( 150 mM ) was used to calibrate pHi with our detected emission ratio of BCECF in living PMCs as previously described ( Stumpp et al . , 2012 ) .", "For the ammonia pulse experiments , all larvae were exposed to filtered sea water ( NSW ) followed by the 20 mM NH3/NH4+ prepulse ( NSW +20 mM NH4Cl ) .", "Acidosis was induced by washout using NSW or by using NSW containing different concentrations of 4 , 4´-Diisothiocyanatostilbene-2 , 2´-disulfonic acid disodium salt ( DIDS ) or 0 . 1% DMSO as vehicle control , respectively .", "To investigate the calcium content of larvae subjected to different pH levels , 50 ml of water from the larval cultures with known larval density was collected and larvae were concentrated by centrifugation .", "For the determination of Ca2+ content in control and SpSlc4a10 morphants 250 larvae were picked using a pipette and centrifuged to remove the sea water .", "Larvae were quickly washed three times in distilled water and the supernatant was removed after the last centrifugation .", "Samples were dried for 24 hr at 37°C .", "The samples were dissolved in 10 M HCl for 15 min and samples were analyzed using a flame photometer ( EFOX 5053 , Eppendorf ) .", "Calcium concentrations were normalized per larva and expressed as nmol larva−1 .", "Late gastrula stage larvae ( 2dpf ) were used for calcein labeling experiments .", "Control and MO injected larvae were bathed in filtered seawater ( NSW ) containing 160 µM calcein ( Sigma ) for 3 hr .", "For inhibitor experiments , larvae were additionally exposed to different DIDS concentrations ( 0 , 10 and 100 µM ) in the calcein solution .", "Vehicle controls contained 0 . 1% of DMSO .", "After 3 hr of incubation , larvae were washed four times by centrifugation and replacement of the calcein containing NSW by fresh NSW with the respective DIDS or DMSO concentration .", "Larvae were further incubated for 4 hr to remove calcein caught in the extracellular matrix of the larvae .", "Life larvae were mounted to microscope slides using synthetic cotton filaments ( 30–50 µm diameter ) as spacers and covered with a coverslip .", "5 Z-stacks images with a section thickness of 10 µm were aquired for every larva using a confocal microscope ( Zeiss LSM 510 ) and fluorescence intensities were determined using the associated Zeiss imaging software ( ZEN ) .", "Data were tested for normality and homogeneity and significance levels were analyzed using paired t-tests .", "For the analyses of gene expression in CO2-treated larvae as well as comparisons of calcein fluorescence intensities in morphants and DIDS-treated larvae one-way ANOVA followed by Holm-Sidak post-hoc test was used .", "Regression analyses were performed on log transformed data using linear models .", "ANCOVA followed by the Holm-Sidak method was performed to test for sigificant diffrences in Ca2+ content under different CO2 treatments .", "Statistical analyses were conducted using Sigma Stat 13 ( Systat Software ) ." ] ]

[ "Efficient pH regulation is a fundamental requisite of all calcifying systems in animals and plants but with the underlying pH regulatory mechanisms remaining largely unknown .", "Using the sea urchin larva , this work identified the SLC4 HCO3- transporter family member SpSlc4a10 to be critically involved in the formation of an elaborate calcitic endoskeleton .", "SpSlc4a10 is specifically expressed by calcifying primary mesenchyme cells with peak expression during de novo formation of the skeleton .", "Knock-down of SpSlc4a10 led to pH regulatory defects accompanied by decreased calcification rates and skeleton deformations .", "Reductions in seawater pH , resembling ocean acidification scenarios , led to an increase in SpSlc4a10 expression suggesting a compensatory mechanism in place to maintain calcification rates .", "We propose a first pH regulatory and HCO3- concentrating mechanism that is fundamentally linked to the biological precipitation of CaCO3 .", "This knowledge will help understanding biomineralization strategies in animals and their interaction with a changing environment ." ]

[ "Many marine organisms such as mussels , sea urchins or corals , have skeletons and shells , which – due to their beautiful colors and shapes – are often desirable collector pieces .", "These structures are made from calcium and carbonate ions that react to form calcium carbonate crystals in a process known as biomineralization .", "In sea urchin larvae , for example , the skeleton is built by so-called primary mesenchyme cells , which work similar to the bone forming cells in mammals .", "These mesenchyme cells use calcium from the sea water , which travels to the site where the shell starts to form .", "About half of the carbonate comes from carbon dioxide that the animals make as they breathe , but it is not known how the other half gets to the site of biomineralization .", "Producing a skeleton generates acid , and marine animals need to be able to regulate their pH levels , as too acidic environments can dissolve the calcium carbonate and threatening to destroy the developing shell .", "How cells accumulate enough carbonate to make their shells , and how they cope with acidity is still poorly understood .", "Here , Hu et al . address this problem by studying purple sea urchin larvae , revealing that they use ion transporters to gather bicarbonate from seawater .", "These structures are part of a group of bicarbonate transporters known as the ‘SLC4 transporter family’ , which sit across the membrane of the mesenchyme cells and move the bicarbonate ions along .", "As the sea urchin larvae develop , the levels of the transporter protein start to rise in mesenchyme cells , peaking around the time they are producing the skeleton .", "Stopping the production of the transporter hindered the larvae from building normal skeletons and also made their cells more acidic .", "It turns out that bicarbonate is more than a skeleton ingredient – it also helps to buffer the acid made in the process .", "Bicarbonate ions can combine with acidic molecules to form water and carbon dioxide .", "Bicarbonate pumped in from the sea neutralises the acidic molecules made during calcium carbonate formation , which helps to stabilize pH levels .", "When the acidity of the water was experimentally increased , it prompted the sea urchins to produce more of the SLC4 transporters , revealing that they may have another role to play .", "Their acid-neutralizing capability helped the animals to cope with changes in their environment .", "Taking on more bicarbonate could therefore help to compensate for rising acidity , allowing skeleton production to carry on as normal .", "This last finding is important in the context of ocean acidification .", "As the amount of carbon dioxide in the atmosphere increases , more of the gas dissolves in the sea .", "The chemical reactions that follow make the water more acidic and decreases the pH levels of the sea .", "Understanding how animals make their skeletons and shells , and manage acid , could reveal how they will cope as the environment changes in the future ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "cell biology", "cancer biology" ]

[ [ "Checkpoints function throughout the cell cycle to ensure accurate and timely coordination of cell cycle events ( Hartwell and Weinert , 1989 ) .", "The abscission ( NoCut ) checkpoint is active during cytokinesis when cells are connected by a narrow intercellular bridge containing a microtubule-rich structure termed the midbody .", "Abscission checkpoint signaling ceases if no errors are detected , reflecting checkpoint satisfaction .", "However , if residual mitotic errors are detected , then the checkpoint remains unsatisfied and the final cut of cytokinesis is delayed ( Norden et al . , 2006; Steigemann et al . , 2009 ) .", "To date , this checkpoint has been shown to be responsive to four error conditions: chromatin bridges within the midbody , reduced levels of particular nuclear pore proteins , tension at the intercellular bridge , and previous DNA replication stress ( Lafaurie-Janvore et al . , 2013; Mackay et al . , 2010; Mackay and Ullman , 2015; Petsalaki and Zachos , 2019; Steigemann et al . , 2009 ) .", "Left unchecked , these conditions have the potential to disrupt new daughter cell functions .", "This is most clear in the case of chromatin bridges , which can break under tension forces in the absence of the abscission checkpoint ( Petsalaki and Zachos , 2021 ) and even break and re-fuse in the absence of p53 , ultimately leading to chromothripsis , a mutational signature commonly found in cancer genomes ( Maciejowski et al . , 2015; Umbreit et al . , 2020 ) .", "Loss of the abscission checkpoint accelerates the time to abscission and induces genomic instability in cultured cells ( Sadler et al . , 2018 ) .", "Thus , the abscission checkpoint appears to protect cells from accumulating damage arising from mitotic errors , which otherwise have the potential to promote tumorigenesis .", "Once the abscission checkpoint is satisfied , cytokinetic abscission is mediated by the Endosomal Sorting Complexes Required for Transport ( ESCRT ) pathway , at least in transformed , cultured mammalian cells ( Carlton and Martin-Serrano , 2007; Gatta and Carlton , 2019; Morita et al . , 2007; Vietri et al . , 2020 ) .", "The ESCRT adaptor protein CEP55 provides an ESCRT recruiting platform ( Carlton and Martin-Serrano , 2007; Morita et al . , 2007; Bastos and Barr , 2010 ) at a protein-rich structure called the Flemming body ( Capalbo et al . , 2019; Skop et al . , 2004 ) centrally located within the midbody .", "CEP55 forms rings on either side of the Flemming body and recruits the early-acting ESCRT factors , TSG101 ( a component of the ESCRT-I complex ) and ALIX , through a shared binding site ( Lee et al . , 2008 ) .", "SEPT9 has also recently been implicated as a second , TSG101/ESCRT-I-specific midbody adaptor ( Karasmanis et al . , 2019 ) .", "ALIX and TSG101/ESCRT-I , in turn , ultimately recruit late-acting ESCRT-III factors , including CHMP4B and IST1 through two parallel pathways ( Christ et al . , 2016 ) .", "ESCRT-III subunits polymerize into membrane-constricting filaments and interact with the AAA-ATPase VPS4 to sever membranes in abscission zones on either side of the Flemming body ( Carlton and Martin-Serrano , 2007; Christ et al . , 2016; Morita et al . , 2007 ) .", "The abscission checkpoint must , therefore , inhibit ESCRT recruitment , polymerization and/or constriction to delay abscission .", "Aurora B kinase ( AurB ) functions as the master regulator of the abscission checkpoint .", "AurB phosphorylates many different targets at the midbody , and thereby enforces abscission delay and stabilizes the midbody ( Hegemann et al . , 2014; Kettenbach et al . , 2011; Steigemann et al . , 2009 ) .", "AurB is activated by phosphorylation ( pAurB ) , both by the collective action of CLK1 , 2 , and 4 kinases and by autophosphorylation ( Petsalaki and Zachos , 2016; Yasui et al . , 2004 ) .", "When the abscission checkpoint is satisfied , AurB is dephosphorylated and abscission proceeds ( Bhowmick et al . , 2019; Capalbo et al . , 2019 ) .", "Many pAurB targets likely remain to be identified , but one significant target is the regulatory ESCRT-III subunit , CHMP4C , a factor required for abscission checkpoint activity ( Capalbo et al . , 2012; Carlton et al . , 2012 ) .", "pAurB phosphorylates three sites within a region unique to CHMP4C that is required for the abscission checkpoint ( Figure 1A; Capalbo et al . , 2012; Carlton et al . , 2012 ) , and the ESCRT-binding kinase ULK3 phosphorylates other site ( s ) outside this insert region ( Caballe et al . , 2015 ) .", "A naturally occurring variant allele of CHMP4C that reduces ALIX binding abrogates the abscission checkpoint , induces DNA damage accumulation , and correlates with increased susceptibility to several different cancers ( Pharoah et al . , 2013; Sadler et al . , 2018 ) .", "Thus , CHMP4C phosphorylation and its ALIX binding activity are necessary for abscission checkpoint function .", "Checkpoint-induced abscission delay appears to be a multistep process , and previous studies have implicated: ( 1 ) phosphoregulation of several ESCRT-III activities ( Caballe et al . , 2015; Capalbo et al . , 2012; Carlton et al . , 2012 ) , ( 2 ) actin patch formation to stabilize the intercellular bridge and prevent chromatin bridge breakage ( Bai et al . , 2020; Dandoulaki et al . , 2018; Steigemann et al . , 2009 ) , and ( 3 ) sequestration of VPS4 in a single ring within the Flemming body , away from the abscission zones ( Thoresen et al . , 2014 ) .", "In the latter case , phosphorylated CHMP4C acts together with the adaptor protein ANCHR to sequester VPS4 ( Thoresen et al . , 2014 ) .", "Yet , the finding that CHMP4C must be able to bind ALIX for the checkpoint to function ( Sadler et al . , 2018 ) suggests additional crucial roles for CHMP4C .", "It remains unknown , however , where such interactions take place and whether cytoplasmic checkpoint regulatory mechanisms complement those at the midbody ." ], [ "The abscission checkpoint is normally active in only a small fraction of cultured cells because at any given time , few cells are in the cell cycle phase of cytokinesis and , further , because checkpoint activity is normally transient .", "We have overcome these experimental hurdles by combining treatments that ( 1 ) prevent checkpoint satisfaction ( Mackay et al . , 2010 ) , using siRNA-mediated depletion of the nuclear pore basket proteins Nup153 and Nup50 ( referred to throughout as siNups ) and ( 2 ) synchronize the cell cycle , using thymidine addition to arrest cells in G1/S phase , followed by release and synchronous progression through the remaining cell cycle ( Figure 1B , C ) .", "These conditions elicited an active checkpoint in up to ~80% of HeLa cells in culture ( Figure 1D , E ) .", "We did not observe increased chromatin bridges in these checkpoint-active samples ( Figure 1—figure supplement 1A , B ) , indicating that lagging chromatin did not significantly contribute to sustained checkpoint activity .", "Importantly , cells enriched with an active abscission checkpoint by this method remained competent for abscission , as they all completed division within 60 min following deactivation of the checkpoint with an AurB inhibitor ( AurBi , ZM 447439 ) ( Figure 1D , E ) or after >5 hr without treatment ( not shown ) .", "Using this new checkpoint enrichment protocol , we tested whether ESCRT factor recruitment to midbodies was altered by the abscission checkpoint .", "The ESCRT adaptor CEP55 and one of the two early-acting ESCRT factors , TSG101/ESCRT-I , localized normally to midbodies when the checkpoint was active ( Figure 2A , B , Figure 2—figure supplement 1A , Figure 2—figure supplement 2A , B ) .", "In contrast , checkpoint activity significantly delayed midbody recruitment of the other early-acting ESCRT factor , ALIX .", "This delay was particularly pronounced in early-stage midbodies ( Figure 2C , D ) and was still striking when all midbodies were tracked over time ( Figure 2—figure supplement 1B ) , despite a similar decrease in abundance of early-stage midbodies in both siCon and siNups samples ( e . g . , 15 hr post-thymidine , Figure 2—figure supplement 1C ) .", "To control for potential cell-to-cell variation , we also quantified CEP55 and ALIX intensity within the same cells to determine their relative ratios at individual midbodies .", "This experiment confirmed that an active checkpoint delays recruitment of ALIX to the midbody relative to CEP55 , with an approximately threefold decrease in the relative ratio 11 hr post-thymidine release ( Figure 2E , F , Figure 2—figure supplement 1D ) , and an approximately threefold decrease relative to TSG101/ESCRT-I in an analogous experiment ( Figure 2—figure supplement 2C , D ) .", "By 16 hr after thymidine release , ALIX levels at the midbody had largely , but not completely recovered ( Figure 2D–F , Figure 2—figure supplements 1B , D and 2D ) .", "This is consistent with the duration of abscission delay under these conditions , which starts to lift after 16 hr ( not shown ) .", "AurBi treatment rapidly rescued ALIX recruitment at 11 hr ( Figure 2G ) , confirming that delays in ALIX recruitment were checkpoint dependent .", "We also found that checkpoint activity delayed recruitment of IST1 , an ESCRT-III subunit that functions downstream of ALIX and is required for abscission ( Figure 2—figure supplement 3A , B; Agromayor et al . , 2009; Bajorek et al . , 2009 ) .", "IST1 recruitment was likewise restored by brief AurBi treatment ( Figure 2—figure supplement 3C ) .", "Taken together , these observations reveal a new dimension of regulation that helps explain why abscission is delayed when this checkpoint is not satisfied .", "Both ALIX and IST1 localize to midbodies and are required for cytokinesis in HeLa cells ( Agromayor et al . , 2009; Bajorek et al . , 2009; Carlton and Martin-Serrano , 2007; Morita et al . , 2007 ) , and abscission timing is therefore expected to be delayed when their recruitment is delayed .", "We next investigated what other cellular changes occur concomitantly with delayed ALIX recruitment .", "We have previously shown that cells expressing CHMP4C with mutations that inhibit ALIX binding bypass the abscission checkpoint ( Sadler et al . , 2018 ) , indicating that interaction of CHMP4C and ALIX is key to checkpoint activity .", "We therefore first tested whether CHMP4C localization was also altered .", "Stably expressed HA-CHMP4C localized to midbodies regardless of checkpoint status , whereas ALIX recruitment was again delayed when the checkpoint was active ( Figure 3—figure supplement 1A–D ) .", "Thus , consistent with previous reports ( Sadler et al . , 2018 ) , bulk CHMP4C protein localization does not change with checkpoint activity .", "We next examined the localization of specific AurB-activated phospho-isoforms of endogenous CHMP4C using phospho-specific antibodies ( Capalbo et al . , 2016 ) .", "As reported previously ( Capalbo et al . , 2016 ) , singly phosphorylated CHMP4C ( pCHMP4C ) formed a single ring at the Flemming body and was recruited with the same timing whether or not the checkpoint was active ( Figure 3—figure supplement 1E–G ) .", "Tri-phosphorylated CHMP4C ( pppCHMP4C ) also formed single Flemming body rings and , additionally , formed double rings , one on either side of the Flemming body ( Capalbo et al . , 2016 ) , particularly when the checkpoint was active ( Figure 3—figure supplement 1G ) .", "However , checkpoint-dependent changes in pppCHMP4C distribution were much more dramatic at cytoplasmic sites ( Figure 3A , B ) .", "In control midbody-stage cells , pppCHMP4C exhibited a granular nuclear localization , whereas cells with sustained checkpoint activity accumulated pppCHMP4C in 0 . 5–2 µm diameter cytoplasmic foci that we have named Abscission Checkpoint Bodies ( ACBs ) .", "Pan-CHMP4C antibodies decorated ACBs , whereas antibodies specific to singly phosphorylated pCHMP4C did not , consistent with the tri-phosphorylated subpopulation of CHMP4C preferentially localizing to this site ( Figure 3—figure supplement 1E , Figure 3—figure supplement 2A ) .", "Note , however , that detection of endogenous CHMP4C in ACBs with the pan-antibody required removing soluble cytoplasmic proteins by pre-fixation treatment with buffer ( PHEM ) to preserve general cell morphology followed by detergent to pre-permeabilize cell membranes .", "This treatment leaves larger cellular structures , including ACBs , intact while removing cytoplasmic background to increase ACB staining clarity .", "ACBs were observed in the majority of checkpoint-active cells throughout the entire post-thymidine time course ( Figure 3C ) .", "ACBs were abundant ( 26 ± 14 per midbody-stage cell ) , and the number of ACBs per midbody-stage cell remained constant throughout the time course ( Figure 3—figure supplement 2B ) .", "In summary , previously uncharacterized bodies that contain the abscission checkpoint factor pppCHMP4C form in the cytoplasm of cells in which the abscission checkpoint is active due to Nup153/50 depletion .", "Initial screens for additional ACB components revealed that a second CHMP4 isoform , CHMP4B , was also present at this site .", "These two CHMP4 isoforms have similar sequences , yet perform quite different roles in cell division: CHMP4C regulates abscission timing but is not required for abscission , whereas CHMP4B is required for abscission but does not have a regulatory role ( Capalbo et al . , 2012; Carlton et al . , 2012; Carlton et al . , 2008 ) .", "CHMP4B is an abundant cytosolic ESCRT-III protein , and like bulk CHMP4C , its ACB localization was also best visualized after pre-permeabilizing cells to remove soluble cytoplasmic proteins ( Figure 3D ) .", "An antibody specific for the T232 phosphorylated , active form of AurB ( pAurB ) ( Yasui et al . , 2004 ) also stained ACBs ( Figure 3D ) , explaining our previous observation that abscission checkpoint activity caused pAurB to localize to cytoplasmic foci of unknown composition ( Mackay et al . , 2010 ) .", "In the absence of checkpoint activation , CHMP4B exhibited a punctate cytoplasmic distribution in pre-permeabilized cells , but these puncta were significantly smaller than ACBs and did not colocalize with either pppCHMP4C or pAurB .", "Hence , in addition to pppCHMP4C , pAurB and CHMP4B are ACB components , and in each case , their ACB localization was strongly checkpoint dependent ( Figure 3E ) .", "We further characterized ACBs by testing whether they corresponded to a variety of known cellular assemblies and organelles ( Figure 4—figure supplement 1A–C ) .", "ACBs did not colocalize with a host of organelles , including P-bodies , which appeared the most similar in character and appearance yet were clearly distinct ( Figure 4—figure supplement 1D ) .", "Unexpectedly , our survey revealed that ACBs are specifically detected with an antibody that recognizes SR domain-containing splicing factors as well as an antibody ( mAb SC35 ) reported to primarily recognize the splicing factor SRRM2 ( Ilik et al . , 2020; Figure 4A , B Figure 4—figure supplement 2A , B ) .", "These antibodies are known to detect cytoplasmic bodies termed mitotic interchromatin granules ( MIGs ) ( Fu and Maniatis , 1990; Li and Bingham , 1991; Reuter et al . , 1985 ) .", "During interphase , MIG components reside in a nuclear compartment called nuclear speckles where these components are hypothesized to be concentrated to increase splicing efficiency ( Beck , 1961; Chen and Belmont , 2019 ) .", "Both MIGs and nuclear speckles are compartments with liquid–liquid phase separation characteristics ( Rai et al . , 2018; Strom and Brangwynne , 2019 ) , and they primarily contain factors that function in mRNA biogenesis , particularly splicing ( Mintz et al . , 1999; Saitoh et al . , 2004; Uversky , 2017 ) .", "After being released from nuclei upon mitotic nuclear envelope disassembly , these factors associate into small cytoplasmic MIG foci during the metaphase to anaphase transition .", "MIGs then normally disappear in late telophase , concomitant with stepwise reassembly of nuclear speckles within newly formed nuclei ( Prasanth et al . , 2003; Reuter et al . , 1985; Spector and Lamond , 2011; Spector and Smith , 1986; Tripathi and Parnaik , 2008 ) .", "In contrast , extended abscission checkpoint activity promoted the appearance of ACBs , which are larger than MIGs and become apparent in late midbody-stage cells ( Figure 4B ) .", "Nuclear speckle assembly in the nucleus was delayed concurrently with ACB appearance ( Figure 4A , B ) .", "ACBs were not an artifact of fixation as we observed them in live cells expressing SRRM2-mCherry ( Figure 4C ) .", "We therefore propose that cytokinetic ACBs are derived from MIGs and that abscission checkpoint signaling plays a pivotal role in inducing the transition .", "When abscission checkpoint satisfaction occurred promptly , pAurB , pppCHMP4C , and CHMP4B , newly discovered here to be MIG constituents , relocalized individually to different sites in control cells at telophase , rather than transitioning to ACBs .", "Interestingly , the checkpoint maintenance factors pAurB and pppCHMP4C relocalized to nuclei where they partially colocalized with nuclear speckles ( Figure 4A , Figure 4—figure supplement 2A , B ) .", "Nuclear speckle localization was not observed for CHMP4B , which remained cytoplasmic and also localized to midbodies ( Figure 3D , Figure 4—figure supplement 1B; Capalbo et al . , 2012; Carlton et al . , 2012 ) .", "Abscission checkpoint activity delayed timely nuclear relocalization of pAurB and pppCHMP4C , as well as splicing factors recognized by the canonical MIG marker mAb SC35 , until abscission or just prior ( Figure 4A , Figure 4—figure supplement 2A , B ) .", "These observations indicate that MIGs/ACBs play a role in constraining the migration of their constituents to various different cellular sites .", "To probe for direct ACB functions in abscission delay , we tested whether abscission timing was altered when we artificially stimulated their formation/maintenance without otherwise preventing abscission checkpoint satisfaction .", "This was accomplished by inhibiting DYRK3 , a kinase that prevents premature MIG formation in metaphase ( Rai et al . , 2018 ) , or by inhibiting CLK1 , a kinase that promotes component release from nuclear speckles and thus may similarly regulate MIG stability , allowing them to persist and at least partially mimic their maturation into ACBs ( Araki et al . , 2015; Colwill et al . , 1996 ) .", "CLK inhibition was previously reported to accelerate abscission ( Petsalaki and Zachos , 2016 ) , but our studies employed a CLK1/2-specific inhibitor ( CLK1i ) rather than a pan-CLK inhibitor , and our treatment windows were much shorter than those employed previously ( 30 min vs . 5 hr ) .", "For our studies , we treated with CLK1 inhibitor or DYRK3 inhibitor for 30 min to limit effects on preceding cell-cycle stages .", "Under these conditions , inhibition of either CLK1 or DYRK3 kinase increased the percentage of midbody-stage cells with SC35-positive cytoplasmic foci from 13% ( control ) to 23% ( DYRK3i ) or 21% ( CLK1i ) ( Figure 4D–F ) and increased median abscission times in the bulk population by 11 min ( DYRK3i , 19% delay ) or 6 min ( CLK1i , 10% delay ) , as measured in live cell imaging experiments ( Figure 4G ) .", "Similarly , the time course of midbody resolution in a synchronized cell population was delayed upon treatment with either inhibitor ( Figure 4—figure supplement 3 ) .", "Conversely , when we overexpressed CLK1 in a background of Nup153/50 depletion , midbody-stage cells decreased ~25% ( Figure 4H ) .", "CLK1 overexpression did not completely dissolve ACBs , but did decrease the number of ACBs per midbody pair ( Figure 4I , Figure 4—figure supplement 4A ) , and concomitantly increased the proportion of SRRM2 nuclear staining ( Figure 4—figure supplement 4B , C ) , indicating that CLK1 overexpression induced SRRM2 relocalization from ACBs into nuclei .", "Thus , treatments that promote MIG/ACB formation and maintenance delay abscission in cells independently of perturbations that prevent the abscission checkpoint from being satisfied , and treatments that promote ACB dissolution accelerate abscission , even when the abscission checkpoint is active .", "These effects are modest , but the experiments target just one aspect of abscission regulation in isolation and therefore support the model that ACBs have a functional role in abscission delay .", "To confirm that ACBs are a broadly relevant component of the abscission checkpoint , we investigated their formation under the three other conditions known to prevent abscission checkpoint satisfaction: replication stress , intercellular tension , and lagging chromatin bridges within the midbody ( Lafaurie-Janvore et al . , 2013; Mackay and Ullman , 2015; Steigemann et al . , 2009 ) .", "We observed that ACB levels significantly increased in midbody-stage cells under conditions of replication stress ( Figure 5—figure supplement", "1 ) or high intercellular tension ( Figure 5—figure supplement 2 ) , but not in the presence of chromatin bridges ( Figure 5—figure supplement 3 ) .", "Delayed ALIX midbody recruitment followed this same trend ( Figure 5—figure supplements 1G , 2D , and 3D ) .", "Thus , ACB formation and delayed recruitment of ALIX to the midbody were correlated and both were seen under three of four different conditions known to prevent satisfaction of the abscission checkpoint .", "To test whether this newly identified checkpoint mechanism is deployed in other cellular contexts , we probed the non-transformed epithelial cell-line RPE1 for key checkpoint hallmarks .", "RPE1 cells exhibited abscission checkpoint activity in response to Nup depletion , as measured by an increase in midbody-stage cells and a delay in ALIX recruitment to the midbody ( Figure 5—figure supplement 4A–C ) .", "This response was less robust than observed in HeLa cells , suggesting that corrective or compensatory pathways may mitigate errors that keep the abscission checkpoint from being satisfied or that RPE1 cells may be less dependent on the ESCRT pathway for abscission .", "Nonetheless , ACBs were once again detected in nearly all midbody-stage RPE1 cells subjected to the checkpoint enrichment protocol and contained pppCHMP4C , pAurB , CHMP4B , and splicing factors recognized by mAb SC35 ( Figure 5A–C , Figure 5—figure supplement 4D ) .", "These observations demonstrate that checkpoint-dependent delay of ALIX midbody recruitment and ACB formation occur in distinct cell types and are broadly relevant as abscission checkpoint mechanisms .", "To probe the relationship between the abscission checkpoint and ACBs further , we depleted the key abscission checkpoint regulator CHMP4C and assayed ACBs .", "As expected , co-depletion of CHMP4C in Nup-depleted cells abrogated the checkpoint and reduced midbody-stage cells ( Figure 5D , full bars ) .", "This treatment also significantly reduced the percentage of midbody-stage cells with ACBs ( Figure 5D , shaded regions ) .", "The dependence of ACB formation on CHMP4C reinforces the connection between abscission checkpoint signaling and ACB appearance .", "The suppression of ACB formation in the absence of CHMP4C , despite Nup depletion , also demonstrates that ACBs do not form due to altered function of the nuclear pore per se ( Figure 5D , Figure 5—figure supplement 5 ) .", "To investigate the connection between the presence of ACBs and the delay of ALIX recruitment to the midbody , we tested whether ALIX itself might target to ACBs .", "To avoid the strong signal for ALIX present in the cytoplasm ( Figure 2C ) , we again used pre-permeabilization treatment to remove soluble cytoplasmic content .", "Using this method , we detected ALIX in ACBs in both HeLa and RPE1 cells ( Figure 6A , B ) .", "To determine whether this localization was specific , we performed a co-depletion with siALIX and siNups , which was informative at several levels .", "First , as previously established , ALIX depletion alone resulted in cytokinesis failure ( Carlton and Martin-Serrano , 2007; Morita et al . , 2007; Figure 6—figure supplement 1A , B ) , and when abscission checkpoint satisfaction was prevented by concurrent treatment with siNups , enrichment of midbody-stage cells still took place ( Figure 6C , full bars ) .", "This checkpoint activity corresponded to a significant reduction in cytokinesis failure ( Figure 6—figure supplement 1A–B ) .", "These observations are consistent with the conclusion that reduced ALIX activity prevents abscission during checkpoint regulation .", "Second , depletion of ALIX confirmed its localization to ACBs as its detection at ACBs diminished concordantly ( Figure 6—figure supplement 2A , B ) .", "Third , in the absence of ALIX , although ACB-like structures still persist in late midbody-stage cells in response to checkpoint activation ( Figure 6C , shaded regions ) , the average cross-sectional area of these structures decreased by ~35% ( corresponding to ~50% reduction in volume ) , as measured using two different ACB markers ( Figure 6D , Figure 6—figure supplement 1C ) .", "Concomitantly , the number of the focal ACB-like structures increased in checkpoint-active midbody-stage cells depleted of ALIX ( Figure 6—figure supplement 1D ) .", "Thus , ALIX is a component of ACBs and is required to create full-sized ACBs or to maintain their integrity .", "Interestingly , although pAurB and pppCHMP4C clearly localize to MIGs ( Figure 6—figure supplement 2E ) , we could not detect ALIX in MIGs ( Figure 6—figure supplement 2C–D ) .", "These observations indicate that ALIX is present in ACBs and , although we cannot rule out that our detection method is limiting for MIGs , it appears that ALIX recruitment contributes to ACB maturation from MIGs .", "The presence of ALIX in ACBs suggested that the checkpoint-dependent delay in ALIX recruitment to the midbody may occur because a modified subpopulation of ALIX that would normally be targeted to the midbody is instead retained in ACBs .", "To determine whether ACB formation and ALIX midbody recruitment delay are connected , cells were scored for both the presence of ACBs and the intensity of ALIX at the midbody , under conditions that enrich for abscission checkpoint-active cells .", "Consistent with a direct ALIX sequestration model , we found that the presence of ACBs strongly correlated with decreased ALIX levels at the midbody in individual cells ( Figure 6E , Figure 6—figure supplement 2F ) .", "This correlation was striking even late in the post-thymidine time course , when overall ALIX midbody recruitment has begun to recover ." ], [ "We have developed a new experimental strategy that is ideally suited for elucidating the regulation of abscission timing .", "Employing this assay , we found that abscission checkpoint activity can delay ALIX recruitment to the midbody and identified ACBs as a previously uncharacterized cytoplasmic body that contributes a new facet of abscission regulation .", "Specifically , we find that the abscission checkpoint restrains recruitment of ALIX and the downstream factor IST1 to the midbody , helping to explain how abscission is stalled in response to mitotic errors .", "Furthermore , we identified ACBs as a checkpoint-dependent compartment that concentrates splicing , checkpoint , and abscission factors , including mAb SC35-reactive splicing factors , pAurB , pppCHMP4C , CHMP4B , and ALIX .", "ACBs appear to be derived from MIGs , a compartment whose components and cell cycle behavior had previously been partially characterized , but whose functional role ( s ) is unknown at a mechanistic level ( Ferreira et al . , 1994; Prasanth et al . , 2003; Rai et al . , 2018; Reuter et al . , 1985; Spector and Lamond , 2011; Tripathi and Parnaik , 2008; Turner and Franchi , 1987 ) .", "ACBs also share similarities with cytoplasmic granules that have been observed in mouse testes ( Saitoh et al . , 2012 ) .", "In this study , the presence of splicing factor-enriched cytoplasmic granules was found to correlate with decreased levels of particular nucleocytoplasmic transport factors .", "In light of our findings here , it will be of interest to resolve whether the cytoplasmic granules seen in spermatids are ACBs and , further , whether similar alteration of nucleocytoplasmic transport factors prevents abscission checkpoint satisfaction .", "Although much remains to be learned about MIG/ACB architecture and function , we found here that ACB formation/maintenance requires the abscission checkpoint factor CHMP4C and that the cytokinesis factor ALIX plays a distinct role in their maturation .", "CHMP4C and pAurB colocalize within ACBs , which may favor formation and maintenance of the pppCHMP4C isoform because CHMP4C is an AurB substrate ( Capalbo et al . , 2012; Carlton et al . , 2012 ) .", "At least two ACB components , CHMP4C and CHMP4B , can bind ALIX ( McCullough et al . , 2008 ) , and CHMP4C mutations that cripple or eliminate ALIX binding impair the checkpoint ( Sadler et al . , 2018 ) , highlighting the functional importance of the CHMP4C–ALIX interaction in checkpoint maintenance , and raising the possibility that this interaction could target either the CHMP4 proteins or ALIX to ACBs .", "In functional terms , targeting of ALIX to ACBs may sequester a particular subpopulation of this protein away from the midbody , contributing to the checkpoint-dependent abscission delay .", "Sequestration may serve to prevent both ALIX-mediated ESCRT-III recruitment to the midbody and stimulation of VPS4 activity , a role for ALIX that is suggested by recent results focused on its orthologue in yeast , Bro1 ( Tseng et al . , 2021 ) .", "Previous studies have demonstrated that in order to bind CHMP4 proteins and function in abscission , ALIX must be activated by phosphorylation of two serine residues in its autoinhibitory C-terminal tail ( Sun et al . , 2016; Zhai et al . , 2011 ) .", "Phospho-activated ALIX is therefore an attractive candidate for the subpopulation of ALIX that is sequestered within ACBs .", "Interestingly , the distal end of this autoinhibitory tail also houses an intrinsically disordered region capable of higher order interactions that can form amyloids and viscous gels in vitro ( Elias et al . , 2020 ) .", "This property may contribute to ALIX targeting to ACBs and/or its role in ACB growth .", "The key role of ALIX in the abscission of cultured , transformed cells is now well established ( Carlton and Martin-Serrano , 2007; Christ et al . , 2016; Morita et al . , 2007 ) and is reinforced by recent studies elucidating its stepwise recruitment to the abscission zone , where it works in a complex with syndecan4 and syntenin to recruit other ESCRT factors required for the scission event ( Addi et al . , 2020 ) .", "However , mouse knockout models reveal a more complex picture in vivo .", "In these mouse models , ALIX and CEP55 were found to be required for normal brain and kidney development and cell division , whereas other organs appear largely unaffected ( Campos et al . , 2016; Tedeschi et al . , 2020 ) .", "Similarly , humans without functional ALIX display microcephaly and kidney defects but are otherwise healthy and can live into their 20's ( Khan et al . , 2020 ) .", "We reconcile these observations with our model of the abscission checkpoint in the following ways: ( 1 ) Redundant pathways may ensure that abscission ( and an abscission checkpoint ) takes place in vivo; indeed , a recent report argues that ESCRT proteins , including ALIX , are still recruited to the midbody in the CEP55 knockout mouse ( Little et al . , 2021 ) , and in cases where ALIX is absent , TSG101 may dominate in driving ESCRT activity ( Christ et al . , 2016; Karasmanis et al . , 2019 ) .", "( 2 ) It is also possible that tumor-derived cells are more dependent on the ESCRT pathway for abscission than non-cancerous cells , albeit with the exception of the developing brain and kidney .", "In line with this , ESCRT factors are often overexpressed in cancer ( Jeffery et al . , 2016; Lin et al . , 2020 ) , and our results show a more robust abscission checkpoint response in HeLa cells compared to non-transformed RPE1 cells .", "This important issue requires further study , but could potentially be used to advantage in therapeutic approaches .", "ALIX targeting to ACBs is the first known cytoplasmic mechanism for abscission checkpoint regulation , but several lines of evidence indicate that this is just one of a repertoire of mechanisms that can be integrated to accomplish abscission delay .", "For example , the ACB mechanism does not function measurably when the checkpoint is induced by lagging chromatin bridges ( Figure 5—figure supplement 3 ) .", "Consistent with that observation , ALIX does not appear delayed in its recruitment to the midbody under these circumstances .", "Thus , the chromosomal bridge structure appears to connect to AurB-dependent abscission delay via distinct mechanisms .", "Indeed , a histone acetyltransferase complex plays an integral role in response to chromatin in the cell cleavage plane in the analogous NoCut checkpoint in yeast ( Mendoza et al . , 2009 ) , and condensin has been proposed to contribute to abscission delay in the presence of chromatin bridges in C . elegans ( Bembenek et al . , 2013 ) .", "There is also other precedent for modularity in abscission checkpoint mechanisms elicited by different cues .", "For example , although phosphorylation of IST1 by ULK3 is required for an abscission delay in response to chromatin bridges or Nup depletion , it does not appear to be required for tension-mediated abscission regulation ( Caballe et al . , 2015 ) .", "Furthermore , other abscission delay mechanisms , such as ANCHR-dependent sequestration of the ATPase VPS4 away from the abscission zone ( Thoresen et al . , 2014 ) , may be deployed in particular combinations depending on the error present .", "Finally , our demonstration that late midbody-stage ACBs are derived from telophase MIGs begs the question of why these bodies colocalize factors that function in both abscission and mRNA biogenesis .", "Interphase nuclear speckles are hypothesized to be transcription hubs that mediate efficient splicing especially of active genes , and molecular detail of their function is emerging ( Smith et al . , 2020 ) .", "The functional roles of MIGs are currently less clear .", "Abnormal MIG assembly triggers metaphase arrest ( Rai et al . , 2018; Sharma et al . , 2010 ) , implying a role in mitotic progression , but the mechanism is not yet understood .", "We have shown that artificially promoting ACB assembly triggers an abscission delay ( Figure 4D–G , Figure 4—figure supplement", "3 ) and that reducing ACB formation accelerates abscission ( Figure 4H , I , Figure 4—figure supplement 4 ) , indicating that ACBs have a functional role in cytokinetic progression .", "Intriguingly , our observations suggest that ACBs are normally remodeled and nuclear speckle reformation initiated before abscission takes place .", "It will therefore be of interest to determine whether sequestration and coordinated release of the splicing factors or other regulatory factors present within ACBs are required for abscission regulation and cytokinetic progression ." ], [ "Details regarding antibodies used in this study can be found in Supplementary file", "1 . Details regarding plasmids used in this study can be found in Supplementary file", "2 . DNA was amplified using PCR and ligated into the pLVX-inducible vector using Gibson Assembly according to the manufacturer’s instructions ( NEB , Rowley , MA ) .", "HeLa cells were cultured and maintained at 37°C and 5% CO2 in DMEM supplemented with 10% FBS .", "The Tet-On HeLa dox-inducible and HA-CHMP4C ( Carlton et al . , 2012 ) cell lines were supplemented with 100 µg/ml G418 to maintain Tet-On or HA-CHMP4C expression respectively ( Invitrogen , Carlsbad , CA ) .", "RPE1 cells were supplemented with 10 µg/ml hygromycin ( Invitrogen ) to maintain hTERT expression .", "The SRRM2-mCherry cell line was maintained in 100 µg/ml G418 to maintain Tet-On expression and 10 µg/ml puromycin ( InvivoGen , San Diego , CA ) for SRRM2-mCherry expression .", "At the outset of these studies , HeLa , HeLa Tet-On , and RPE1 cells were tested and found negative for mycoplasma using a PCR mycoplasma detection kit ( ABM , Bellingham , WA ) .", "Cell types were authenticated by sequencing 24 loci ( University of Utah Sequencing Core ) .", "Unless otherwise labeled in individual panels or figure legends , all experiments used HeLa cells .", "To generate a stable cell line , Day 1: Hela Tet-On cells were plated in a 24-well dish .", "Day 2: cells were transfected with 500 ng/well of the SRRM2-mCherry plasmid using Lipofectamine LTX with Plus Reagent according to the manufacturer’s instructions ( Thermo Fisher Scientific , Waltham , MA ) .", "Day 3: cells were split into a 10 cm dish with 500 μg/ml G418 and 10 μg/ml Puromycin for selection .", "After drug selection for 14 days , colonies were harvested into individual wells of 12-well plates .", "Clones were validated for inducible SRRM2-mCherry expression using immunofluorescence imaging and an antibody specific to mCherry ( Abcam , Cambridge , UK , ab167453 ) .", "Cells were singly transfected with siRNA for 42–72 hr , as indicated in figure legends , using Lipofectamine RNAiMax ( ThermoFisher ) according to the manufacturer’s instructions .", "Media was exchanged 24 hr after transfection , and cells were incubated between 18 and 48 hr before harvesting .", "For co-transfections , cells were seeded with combinations of ( 1 ) siALIX , siCon , or siNup153/50 , or ( 2 ) siCHMP4C , siCon , or siNup153/50 .", "Media was exchanged 24 hr after transfection .", "Cells were fixed 72 hr post-transfection to allow adequate time for CHMP4C or ALIX knockdown .", "Details regarding siRNAs used in this study are provided in Supplementary file", "2 . Cells were seeded on acid-washed , 10 µg/ml fibronectin-treated , glass coverslips and then treated according to the individual experimental protocol .", "To fix , cells were removed from the incubator , washed once in PBS pH 7 . 4 , and then fixed by one of the three methods as notated in Supplementary file 1: After fixing , cells were rinsed twice with 2 ml PBS each time and then blocked ( 3% FBS and 0 . 1% Triton X-100 in PBS ) for 30 min on the bench top .", "Primary antibodies were applied at the dilution noted in Supplementary file 1 for at least 1 hr , 23°C in blocking solution .", "After 1 wash with 2 ml PBS , secondary antibodies ( Thermofisher ) were applied for 45 min to 1 hr , and cells were washed in 2 ml PBS .", "For Figure 2E , Figure 2—figure supplement 2C , DNA was stained with Hoechst for 10 min at 23°C .", "For all other images , coverslips were mounted with ProLong Gold Antifade Reagent with DAPI ( Thermofisher ) on a microscope slide .", "Images were acquired using four different microscopes: The SRRM2-mCherry-inducible cell line was plated in four-well LabTek chamber slides .", "Day 1: cells were treated with 2 μg/ml Doxycycline to induce SRRM2-mCherry expression .", "Day 2: cells were treated with siCon or siNups as above .", "Day 4: cells were treated with one drop NucBlue reagent ( ThermoFisher ) per 2 ml of imaging media and 1 μM Tubulin Tracker Green ( Thermofisher ) 45 min prior to imaging in HEPES buffered , Phenol Red-free DMEM/F-12 ( ThermoFisher ) containing 10% FBS .", "Cells were imaged on a Leica SP8 White Light Nikon Ti-E widefield inverted microscope at 60× objective .", "Midbody-stage cells were identified by α-tubulin staining and were always counted as one cell .", "Cell populations were counted and sorted into interphase , midbody-stage , mitotic , recently abscised pair ( counted as one cell ) , multinucleate , and failed bridge ( counted as one cell ) .", "Cells were counted unblinded while on the microscope .", "Midbodies were designated as ‘early’ or ‘not early’ using α-tubulin staining before scoring for the examined phenotype .", "Midbodies were classified as early based on midbody width , level of midbody-pinching , nuclear area , and flatness of cells , see Figure 4B for illustration .", "Midbodies were scored as having ACBs if they had at least one ACB .", "However , most midbody-stage cells with ACBs had 10–40 ACBs .", "For most figures , cells were counted as having or not having ACBs , unblinded while on the microscope .", "In Figure 3—figure supplement 2B and Figure 4I , ACBs were counted from images using ‘Find Maxima’ with Fiji software ( NIH ) .", "To quantify ALIX signal in ACBs , deconvolved z-slices were stacked and then uniformly adjusted for brightness and contrast .", "The SC35 or pAurB channels were uniformly thresholded , and then cytoplasmic objects between 0 . 025 and 5 µm2 were marked as regions of interest ( ROIs ) .", "ROIs were used to measure individual ACB intensity and area in the non-thresholded ALIX channel .", "Values were background corrected with large ROIs in the cytoplasm that excluded ACBs .", "Quantification of fluorescence staining intensity at the midbody was also done with Fiji software ( NIH ) .", "The freehand selection tool was used to outline the region of interest at the midbody , and staining intensity within this area was measured .", "Signals were background corrected using measurements from adjacent regions .", "In cases where the protein was not visible at the midbody , these methods sometimes generated a small negative intensity value after background correction because the Flemming body is a natural dark zone .", "In these cases , intensity was valued at zero .", "To quantify SRRM2 subcellular distribution , the DAPI channel was used to threshold the nuclei and create ROIs using Fiji software .", "These ROIs were then used to determine the mean SRRM2 nuclear intensity .", "For each midbody-stage cell , an N:C ratio was determined by dividing the average mean nuclear intensity by the baseline cytoplasmic signal ( the mean cytoplasmic intensity measured in an area devoid of ACBs ) .", "Cells were lysed in NP40 lysis buffer ( 50 mM Tris pH 7 . 4 , 250 mM NaCl , 5 mM EDTA , 50 mM NaF , 1 mM Na2VO4 , 1% Nonidet P40 , 0 . 02% NaN3 , with freshly added PMSF [1 mM] , aprotinin , leupeptin ) for 30 min , 4°C , vortexing every 10 min .", "Lysates were clarified by spinning at 13 , 200 g for 10 min at 4°C , and protein contents in clarified lysates were quantified by Bradford assay before gel loading .", "Gel and western conditions are listed in Supplementary file", "1 . Twenty to 35 µg lysate per sample was prepared with SDS loading buffer , resolved by SDS–PAGE , and following wet transfer for 2 hr at 40 V , membranes were blocked with 5% milk in Tris-buffered saline ( TBS ) for at least 30 min , 23°C , and were incubated with primary antibodies in 5% milk in TBS-T ( 0 . 1% Tween 20/TBS ) overnight at 4°C .", "Membranes were washed in TBS-T and incubated with the corresponding secondary antibodies conjugated with HRP ( Thermofisher ) or near-infrared fluorescent dyes ( Abcam , Cambridge , UK ) in TBS-T for 1 hr , 23°C , and washed again .", "Blots were detected with Western Lightning PLUS ECL ( PerkinElmer , Waltham , MA ) on Hyblot CL film ( Thomas Scientific , Swedesboro , NJ ) .", "Proteins with infrared fluorescent dyes were detected using a Li-Cor Odyssey Infrared scanner and Image Studio version 5 . 2 software .", "The abscission checkpoint was kept active using one of four different methods .", "For fixed-imaging time courses , HeLa cells were seeded on glass coverslips in 24-well dishes , and 2 mM thymidine was added 8 hr post-seeding .", "After 24 hr , thymidine was washed out with PBS .", "Thirteen hours post-thymidine release , after most cells had completed metaphase , 1 µM DYRK3 inhibitor ( GSK 626616 , Tocris , Minneapolis , MN ) , 1 µM CLK1/2 inhibitor ( 534350 , Millipore , Burlington , MA ) , or DMSO were each added individually to six separate wells .", "Coverslips from wells corresponding to each treatment were fixed directly after inhibitor/vehicle was added for the first timepoint at 13 hr post-thymidine release and then at hourly intervals until 18 hr post-thymidine release .", "Live-imaging experiments to determine abscission timing used HeLa cells expressing H2B-mCherry and GFP-α-tubulin .", "Cells were seeded in a Lab-Tek II 8-chambered #1 . 5 German Coverglass System and incubated for 48 hr .", "Stage positions were first set on the microscope , and then 1 µM DYRK3i , 1 µM CLK1/2i , or DMSO were added just prior to initiation of imaging .", "Imaging was carried out for 3 hr on a Nikon Ti-E widefield inverted microscope ( Nikon 20x N . A . dry objective lens ) equipped with Perfect Focus system and housed in a 37°C chamber ( OKOLAB , Ambridge , PA ) with 5% CO2 .", "Multiple fields of view were selected at various x and y coordinates , and images were acquired using a high-sensitivity Andor Zyla CMOS camera ( Andor , Manchester , CT ) controlled by NIS-Elements software .", "Images were acquired every 5 min , and abscission time was measured as the time from midbody formation to disappearance .", "In parallel , cells were seeded on glass coverslips , incubated for 48 hr , and either DYRK3i , CLK1i , or DMSO were added for 60 min prior to fixing and detection of foci labeled with mAb SC35 or antibodies against pAurB .", "On Day 1 , cells were seeded into 24-well dishes both with and without glass coverslips and transfected with either siCon or siNups siRNA as described above .", "On Day 2 , cells were transfected with 800 ng/well of empty vector DNA or Myc-CLK1-WT DNA using Lipofectamine LTX according to the manufacturer’s instructions ( Thermofisher ) .", "At the time of transfection , 2 µg/ml doxycycline was added to the medium to induce expression .", "On Day 3 , media was changed and fresh media added with 2 µg/ml doxycycline .", "On Day 4 , cells were harvested for immunofluorescence and immunoblot as described above .", "Five statistical tests were used to evaluate the significance of data .", "When comparing two samples , p-values were calculated using an unpaired t-test or Mann–Whitney test for non-normally distributed datasets ( Figure 4I , Figure 1—figure supplement 1A , Figure 4—figure supplement 4B , Figure 5—figure supplement 2A , B , Figure 5—figure supplement 4A ) .", "When comparing two samples with multiple cells in each replicate , p-values were calculated using Stratified Analysis with Nonparametric Covariable Adjustment to adjust for replicate-level variation ( Figure 4G , Figure 6D , E , Figure 5—figure supplement 1C , G , Figure 5—figure supplement 2D , Figure 5—figure supplement 3D , Figure 6—figure supplement 1D , Figure 6—figure supplement 2B , D ) .", "When comparing two complete time curves , p-values were calculated using Welch’s t-test ( Figure 3C ) .", "When comparing datasets with three or more samples , p-values were calculated using one-way ANOVA with Sidak’s multiple comparisons test ( Figure 2F , G , Figure 3E , Figure 4F , H Figure 5C , D , Figure 6C , Figure 2—figure supplement 1D , Figure 2—figure supplement 2D , Figure 2—figure supplement 3C , Figure 3—figure supplement 2B , Figure 4—figure supplement 3C , D , Figure 5—figure supplement 3B , Figure 6—figure supplement 1B ) .", "When comparing two or more datasets over a time course or multiple categories within a sample , p-values were calculated using two-way ANOVA with Sidak’s multiple comparisons test ( Figure 1—figure supplement 1B , Figure 3—figure supplement 1G , Figure 4—figure supplement 3B , Figure 5—figure supplement 1B , E Figure 5—figure supplement 4C ) ." ] ]

[ "The abscission checkpoint regulates the ESCRT membrane fission machinery and thereby delays cytokinetic abscission to protect genomic integrity in response to residual mitotic errors .", "The checkpoint is maintained by Aurora B kinase , which phosphorylates multiple targets , including CHMP4C , a regulatory ESCRT-III subunit necessary for this checkpoint .", "We now report the discovery that cytoplasmic abscission checkpoint bodies ( ACBs ) containing phospho-Aurora B and tri-phospho-CHMP4C develop during an active checkpoint .", "ACBs are derived from mitotic interchromatin granules , transient mitotic structures whose components are housed in splicing-related nuclear speckles during interphase .", "ACB formation requires CHMP4C , and the ESCRT factor ALIX also contributes .", "ACB formation is conserved across cell types and under multiple circumstances that activate the checkpoint .", "Finally , ACBs retain a population of ALIX , and their presence correlates with delayed abscission and delayed recruitment of ALIX to the midbody where it would normally promote abscission .", "Thus , a cytoplasmic mechanism helps regulate midbody machinery to delay abscission ." ]

[ "When a cell divides , it must first carefully duplicate its genetic information and package these copies into compartments housed in the two new cells .", "Errors in this process lead to genetic mistakes that trigger cancer or other harmful biological events .", "Quality control checks exist to catch errors before it is too late .", "This includes a final ‘abscission’ checkpoint right before the end of division , when the two new cells are still connected by a thin membrane bridge .", "If cells fail to pass this ‘no cut’ checkpoint , they delay severing their connection until the mistake is fixed .", "A group of proteins called ESCRTs is responsible for splitting the two cells apart if nothing is amiss .", "The abscission checkpoint blocks this process by altering certain proteins in the ESCRT complex , but exactly how this works is not yet clear .", "To find out more , Strohacker et al . imaged ESCRT factors in a new experimental system in which the abscission checkpoint is active in many cells .", "This showed that , in this context , certain ESCRT components were rerouted from the thread of membrane between the daughter cells to previously unknown structures , which Strohacker et al . named abscission checkpoint bodies .", "These entities also sequestered other factors that participate in the abscission checkpoint and factors that contribute to gene expression .", "These results are key to better understand how cells regulate their division; in particular , they provide a new framework to explore when this process goes wrong and contributes to cancer ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "developmental biology", "neuroscience" ]

[ [ "The evolutionary expansion and elaboration of a gyrated neocortex underlie the complex cognition and development of intellect that characterize primates , especially humans .", "Emerging evidence suggests that differences in the type , abundance , and mode of division of neural stem and progenitor cells contribute to the diversity in the size and shape of the mammalian neocortex across species ( Lui et al . , 2011; Paridaen and Huttner , 2014; Sun and Hevner , 2014; Dehay et al . , 2015 ) .", "At the onset of neurogenesis , neuroepithelial cells ( neural stem cells ) become radial glia .", "These more committed neural progenitor cells reside in the ventricular zone ( VZ ) and are referred to as apical radial glia ( aRG ) .", "Their apical processes are integrated into the ventricular surface while their basal fibers extend radially to the pial basement membrane ( Götz and Huttner , 2005 ) .", "aRG generate neurons directly or indirectly via becoming intermediate progenitor cells ( IPC ) that occupy the subventricular zone ( SVZ ) .", "In contrast to their limited proliferation potential in lissencephalic rodents ( Fietz and Huttner , 2011 ) , IPC undergo multiple rounds of self-renewing division in species with enlarged and folded neocortices ( Fietz et al . , 2010; Hansen et al . , 2010; Betizeau et al . , 2013; Gertz et al . , 2014 ) .", "The increased proliferation of IPC expands the SVZ , which is subdivided into inner and outer compartments in primates ( Smart et al . , 2002; Zecevic et al . , 2005 ) .", "The outer SVZ may be responsible for the increased number of upper-layer neurons and thus the tangential expansion of neocortical surface area ( Lui et al . , 2011; Reillo et al . , 2011; Reillo and Borrell , 2012 ) .", "Recent advances in cell labeling and imaging have identified other basal progenitors in addition to IPC .", "At mid-neurogenesis , some aRG selectively lose their apical processes and move their soma to the outer SVZ ( Fietz et al . , 2010; Hansen et al . , 2010; Reillo et al . , 2011; Martínez-Martínez et al . , 2016 ) .", "These newly generated basal radial glia ( bRG , also called outer radial glia ) are abundantly present in gyrencephalic cortex ( Fietz et al . , 2010; Hansen et al . , 2010; Reillo et al . , 2011 ) but are relatively rare in the lissencephalic mouse cortex ( Shitamukai et al . , 2011; Wang et al . , 2011 ) .", "It has thus been speculated that bRG expansion is responsible for the emergence and evolution of cortical convolutions ( Fietz and Huttner , 2011; Lui et al . , 2011; Reillo et al . , 2011; Reillo and Borrell , 2012; Geschwind and Rakic , 2013; Nonaka-Kinoshita et al . , 2013; Borrell and Götz , 2014; Dehay et al . , 2015; Fernández et al . , 2016 ) .", "However , the direct link of bRG to cortical gyrification remains unclear because bRG abundance does not correlate with either gyrification or the phylogeny of the neocortex ( García-Moreno et al . , 2012; Hevner and Haydar , 2012; Kelava et al . , 2012 ) .", "In mice , genetic manipulations of a number of intrinsic factors ( Stahl et al . , 2013; Florio et al . , 2015; Wong et al . , 2015; Ju et al . , 2016b ) or signaling pathways ( Lui et al . , 2014; Pollen et al . , 2015; Wang et al . , 2016 ) enhance the generation of bRG as well as IPC .", "In some of these experiments , expansion of these basal progenitors resulted in the folding of the mouse neocortex ( Stahl et al . , 2013; Florio et al . , 2015; Wong et al . , 2015; Wang et al . , 2016 ) , highlighting the importance of bRG and IPC in cortical convolution .", "Yet little is known about the molecular events that specifically control the transition of aRG into bRG .", "This information is crucial to our understanding of the molecular basis for the greater abundance of bRG in gyrencephalic than in lissencephalic cortices .", "It is also important to determine the precise contribution of bRG , relative to IPC , to cortical gyrification .", "In amniotes , the folding of the cerebellar cortex results in the formation of an elaborate set of folia similar to neocortical gyri .", "More extensive folding of the cerebellar cortex correlates with more complex behaviors ( Iwaniuk et al . , 2006; Lisney et al . , 2008; Hall et al . , 2013 ) .", "From sharks to primates , the cerebellum and neocortex grow regularly and disproportionately to the rest of the brain , with the extent of gyrification reflecting the size of these structures ( Yopak et al . , 2010 ) .", "These observations suggest that folding of the cerebral and cerebellar cortex is an evolutionary adaptation that allowed the enlargement of brain surface area and thereby the accommodation of more complex functions .", "Expansion of the granule cell precursors that reside in the external granule layer ( EGL ) is thought to be the primary driver of cerebellar foliation ( Corrales et al . , 2004 , 2006; Sudarov and Joyner , 2007 ) .", "However , emerging evidence suggests that the interaction between BG and basement membrane is important for cerebellar foliation ( Belvindrah et al . , 2006; Mills et al . , 2006; Qiu et al . , 2010; Ma et al . , 2012 ) .", "We recently discovered that the deletion of Ptpn11 , which codes for the protein tyrosine phosphatase Shp2 , blocks BG formation and cerebellar foliation , whereas EGL proliferation remains relatively normal in perinatal mouse cerebella ( Li et al . , 2014 ) .", "The expression of a constitutively active Mek1 ( Map2k1 ) , Mek1DD ( Cowley et al . , 1994 ) , which specifically activates ERK , rescues both cerebellar foliation and BG generation ( Li et al . , 2011 ) , demonstrating the critical role of ERK signaling in BG induction and the essential role of BG in cerebellar foliation .", "Similar to the cytogenesis of bRG , nascent BG are derived from cerebellar aRG between embryonic day ( E ) 13 . 5 and E17 . 5 , again by selectively losing their apical processes and relocating their soma to a basal position ( but in this case to the prospective Purkinje cell layer ) ( Yuasa , 1996; Yamada and Watanabe , 2002 ) .", "These nascent BG continue to proliferate until postnatal day ( P ) seven when they exit the cell cycle and become mature BG ( Parmigiani et al . , 2015 ) .", "After their induction , BG express neural stem cell markers , such as Sox2 , Sox9 , and Tnc through adulthood ( Sottile et al . , 2006; Alcock and Sottile , 2009; Koirala and Corfas , 2010 ) .", "The increase and rearrangement of BG basal fibers are associated with dramatic expansion of the cerebellar cortex and fissure formation in perinatal stages ( Sudarov and Joyner , 2007 ) .", "Several bRG-signature genes , such as TNC , FABP7 , and PTPRZ1 ( Pollen et al . , 2015; Thomsen et al . , 2016 ) , are also well-known BG markers ( Feng et al . , 1994; Yuasa , 1996; Tanaka et al . , 2003 ) .", "These observations raise the question whether the formation of bRG and BG is controlled by related mechanisms involving ERK signaling .", "In the current study , we investigated the hypothesis that conserved mechanisms regulate the transition of aRG to BG in the cerebellum , and to bRG in the neocortex .", "We first established the transcriptomic profile of nascent BG in the mouse cerebellum , and then compared the molecular features of BG to those of human bRG that were recently identified by single-cell RNA sequencing ( seq ) ( Pollen et al . , 2015; Thomsen et al . , 2016 ) .", "Given the crucial role of ERK signaling in the aRG-to-BG transition in the mouse cerebellum ( Li et al . , 2014 ) , we analyzed and compared the ERK signaling activity in cortical aRG during human and mouse corticogenesis .", "Finally , we tested whether activation of ERK induced bRG in the mouse neocortex .", "Our findings demonstrate that BG and bRG not only share similar molecular features , but also related molecular mechanisms in their generation ." ], [ "Using single-cell RNA-seq , two groups have independently identified specific molecular markers of bRG in human fetal neocortex ( Pollen et al . , 2015; Thomsen et al . , 2016 ) .", "Using a new computational pipeline ( Guo et al . , 2015 ) , we identified the consensus signatures for bRG based on the published single-cell RNA-seq datasets ( Figure 1A ) .", "Our analysis confirmed most of the reported bRG markers ( Pollen et al . , 2015; Thomsen et al . , 2016 ) and identified additional ones ( Supplementary file 1A ) .", "Inspection of in situ hybridization data of the Allen Mouse Brain Atlas revealed that more than half ( 51 . 5% , n = 66 ) of these bRG-signature gene orthologs were specifically or highly expressed in BG of mouse cerebellum at P56 ( Figure 1B , and Supplementary file 1B ) .", "By contrast , only 2 . 2% and 4 . 5% of two randomly selected gene groups were detected in BG ( n = 44 and 46 , Fisher's exact test , p<0 . 001 , see Method section for details of unbiased expression analysis ) .", "Inspection of the Allen Mouse Developmental Brain revealed that many bRG markers appeared to be expressed in BG of the mouse cerebellum at P4 when immature BG were relatively easy to be identified ( Figure 1—figure supplement 1A ) .", "In agreement with RNA in situ hybridization data , immunofluorescence revealed that Hopx , a specific bRG marker ( Pollen et al . , 2015; Thomsen et al . , 2016 ) , was co-localized with the BG marker Fabp7 ( also known as BLBP ) and Sox2 , in the mouse cerebellum at E14 . 5 and E18 . 5 ( Figure 1C ) .", "As Hopx , Fabp7 and Sox2 , like many other BG markers , are also expressed in the VZ , we define BG as those triple labeled cells that have delaminated from the VZ at E14 . 5 or those reside in the Purkinje cell layer with radial fibers in the molecular layer .", "Moreover , antibody staining for Hopx and Fabp7 and in situ hybridization for Etv4 , Etv5 , Ptprz1 , Tnc1 , and Slc1a3 showed that these bRG markers were absent from the cerebellar cortex of Ptpn11-deficient cerebella , where BG generation is blocked ( Li et al . , 2014 ) , at E16 . 5 ( Figure 1—figure supplement 1B and C ) .", "Collectively , our data suggest that bRG and BG have similar molecular features . 10 . 7554/eLife . 23253 . 003Figure 1 . Mouse orthologs of human basal radial glia ( bRG ) -signature genes are specifically or highly expressed in Bergmann glia ( BG ) of mouse cerebella .", "( A ) Heatmap showing the consensus signature gene sets for apical radial glia ( aRG , top bracket to the left ) and bRG ( lower bracket to the left ) in the single-cell RNA-seq datasets of Pollen et al . and Thomsen et al . Genes of the aRG and bRG gene sets are mostly not expressed in intermediate progenitor cells ( IPC ) .", "( B ) In situ hybridization of selected bRG-specific genes in BG of P56 mouse cerebella .", "The images were generated by the Allen Institute for Brain Science ( Lein et al . , 2007 ) .", "( C ) Immunofluorescent staining of Hopx , Fabp7 , and Sox2 shows that Hopx is expressed in BG in E14 . 5 and E18 . 5 mouse cerebella .", "The boxed areas are enlarged and shown in separate channels below; arrowheads point to BG triple-labeled with Hopx , Fabp7 , and Sox2 .", "Scale bar: 40 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 23253 . 00310 . 7554/eLife . 23253 . 004Figure 1—figure supplement 1 . bRG-specific genes are expressed in wild-type but not Ptpn11-cKO cerebella .", "( A ) In situ hybridization of selected bRG-specific genes in BG of P4 mouse cerebellum .", "The images were obtained from the Allen Developmental Mouse Brain ( Thompson et al . , 2014 ) .", "( B and C ) .", "Immunohistochemistry ( B ) and in situ hybridization ( C ) on sagittal sections of E16 . 5 wild-type and Ptpn11-cKO cerebella .", "The arrows indicate the transcript in presumptive BG in the cerebellar cortex; asterisks show the absence of transcripts; arrowheads denote sporadic Hopx and Fabp7 expression in the ventricular zone . DOI: http://dx . doi . org/10 . 7554/eLife . 23253 . 00410 . 7554/eLife . 23253 . 005Figure 1—figure supplement 2 . Inspection of gene expression in P56 mouse cerebella . Examples of validation of BG markers based on ISH on sagittal section of P56 mouse cerebella ( from Allen Brain Atlas ) .", "Arrows indicate the Purkinje cell layer; arrowheads denote signals in radial fibers in the molecular layer .", "Note that the soma of Purkinje cells are noticeably larger than those of BG . DOI: http://dx . doi . org/10 . 7554/eLife . 23253 . 005 We next sought to investigate whether nascent BG also have similar molecular features as the human fetal bRG through genome-wide expression analysis .", "Previous studies have demonstrated that nascent BG are initially formed at E13 . 5 ( Yuasa , 1996 ) , and their generation is blocked by Ptpn11 deletion but rescued by Mek1DD expression ( Li et al . , 2011 ) .", "Therefore , we reasoned that the level of BG-enriched transcripts would increase from E12 . 5 to E14 . 5 in wild-type ( WT ) cerebella , decrease in Ptpn11 conditional knock-out ( cKO ) cerebella , and become normal in Ptpn11-cKO cerebella with Mek1DD expression ( Ptpn11-cKO;Map2k1 ) .", "Through RNA-seq , differentially expressed genes were identified by pairwise comparisons among the cerebella of these three genotypes at E12 . 5 , E13 . 5 , and E14 . 5 ( Supplementary file 2 ) .", "Validation with qPCR and in situ hybridization confirmed the overall accuracy of the RNA-seq data ( Figure 2—figure supplement 1 ) .", "As expected , markers for BG , but not other major cerebellar cell types were significantly decreased in Ptpn11-cKO cerebella compared to the control at E13 . 5 and E14 . 5 ( Figure 2—figure supplement 2A and B ) .", "By using an intersection-of-list approach based on the above-mentioned logic , we identified 117 putative BG-specific genes ( Figure 2A ) , 35 . 5% of which were apparently expressed in BG in P56 mouse cerebella ( Fisher's exact test to compare random gene sets , p<0 . 001; Supplementary file 1C ) according to Allen Brain Atlas ( Lein et al . , 2007 ) . 10 . 7554/eLife . 23253 . 006Figure 2 . The molecular features of newly generated BG are similar to those of human bRG .", "( A ) Identification of BG candidate genes by intersecting significantly up- ( red ) and down- ( blue ) regulated genes between different embryonic stages and/or genotypes as indicated .", "( B ) Dendrograms showing average linkage hierarchical clustering of genes on the basis of topological overlap .", "Modules of coexpressed genes are assigned in color blocks , as indicated by the horizontal bar beneath the dendrograms; the second bar shows BG-specific genes , indicated by individual vertical lines; the arrow indicates the aggregation of vertical lines – enrichment of BG-specific genes – corresponding to the BG module .", "( C ) Boxplots showing module eigengene expression of the BG-module in different genotypes and embryonic stages .", "**p<0 . 01 ( ANOVA with a post-hoc Turkey-Kramer multiple comparison test ) ; ns , not significant ( p>0 . 05 ) .", "( D ) Functional enrichment of BG-module hub genes .", "( E ) PAGODA analyses of Pollen’s ( left ) and Thomsen’s ( right ) single-cell RNA-seq datasets show that human cortical bRG are significantly enriched for the BG-module genes detected by PAGODA .", "The question mark indicates an undefined cell type .", "Adjusted Z-scores ( a Z-score >1 . 96 is equivalent to p<0 . 05 ) are shown to the left of the heatmap . DOI: http://dx . doi . org/10 . 7554/eLife . 23253 . 00610 . 7554/eLife . 23253 . 007Figure 2—figure supplement 1 . Validation of RNA-seq data .", "( A ) Correlation between qPCR and RNA-seq data .", "Pearson correlation test .", "( B ) In situ hybridization on sagittal sections of wild-type and Ptpn11-cKO cerebella at E14 . 5 .", "Arrowheads indicate transcripts in the VZ . DOI: http://dx . doi . org/10 . 7554/eLife . 23253 . 00710 . 7554/eLife . 23253 . 008Figure 2—figure supplement 2 . The loss of Ptpn11 greatly reduces the expression of BG , but not non-BG , marker genes .", "( A and B )", "Bar plots of log2 fold changes of molecular markers for different cerebellar cell types at E13 . 5 ( A ) and E14 . 5 ( B ) .", "Note that RNA-seq data suggest no significant change in Slc1a3 , a known BG marker , in agreement with our ISH data that show abundant Slc1a3 transcripts in aRG in Ptpn11-cKO cerebella ( Figure 2— figure supplementary 1B ) .", "Furthermore , the slight increase in the mRNA of Atoh1 and Pax6 in Ptpn11-cKO cerebella at E14 . 5 compared to the control are in agreement with the thickened EGL found in Ptpn11-cKO mutants as described previously ( Li et al . , 2014 ) .", "( C and D )", "Gene set enrichment analysis plot ( C ) and heatmap ( D ) show the significant downregulation of mouse homologues of human bRG markers in Ptpn11-cKO cerebella at E13 . 5 and E14 . 5 compared to the control .", "The color blocks in the horizontal bar beneath the dendrograms indicate the sample genotype . DOI: http://dx . doi . org/10 . 7554/eLife . 23253 . 008 Because the intersection-of-list approach is difficult to control for a type I error ( Natarajan et al . , 2012 ) , we conducted an unsupervised weighted gene coexpression network analysis ( WGCNA ) .", "WGCNA can elucidate the higher-order relationships between genes based on their coexpression relationships and thus allows the delineation of modules – groups of genes with highly correlated expression patterns , which represent biologically related genes or cell-type-specific markers ( Oldham et al . , 2008; Lui et al . , 2014 ) .", "A coexpression module that was enriched for the 117 BG-specific genes was identified ( Figure 2B ) .", "In agreement with our prediction , the BG-module eigengene values , which summarize the overall expression profile of genes in a module ( the first principal component ) ( Langfelder and Horvath , 2008 ) , gradually increased from E12 . 5 to E14 . 5 , was significantly lower in Ptpn11-cKO cerebella than the control at each stage , and became comparable between Ptpn11-cKO; Map2k1 and WT cerebella ( Figure 2C ) .", "BG-module hub genes ( those most highly connected within the module ) were enriched for functions in stem cell development and Map2k1-ERK signaling ( Figure 2D ) , in agreement with the critical role of this signaling pathway in the generation of BG as presumptive neural progenitors in the cerebellum .", "98 of the 117 BG-specific genes identified by the intersection-of-list approach were included in the 334 BG-module hub genes , suggesting that WGCNA discovers additional BG-enriched genes that are missed by the intersection-of-list method .", "Indeed , we identified additional 29 genes that are apparently expressed in BG at P56 according to the Allen Mouse Brain ( Supplementary file 1D ) .", "We then compare the high-confidence BG-specific genes with bRG-signature markers with three statistical methods .", "Hypergeometric distribution revealed significantly overlap between BG module hub genes and bRG markers ( p=1 . 39 × 10−7 ) .", "Moreover , gene set enrichment analysis ( Subramanian et al . , 2005 ) showed that bRG marker genes were significantly down-regulated in Ptpn11-cKO cerebella at E13 . 5 ( Figure 2—figure supplement 1C and D ) .", "Finally , a pathway and gene set over dispersion analysis ( PAGODA ) was performed on the single-cell RNA-seq datasets ( Pollen et al . , 2015; Thomsen et al . , 2016 ) using extensive gene sets , including the newly identified putative BG and bRG markers , to identify cells that exhibit a statistically significant excess of coordinated variability ( Fan et al . , 2016 ) .", "Notably , both the consensus bRG signature gene ( Figure 1A ) and BG-specific gene sets identified the same population of human cortical progenitor cells ( presumably bRG , Figure 2E ) .", "Collectively , these data show that the gene expression signatures of neocortical bRG and nascent murine BG are remarkably similar .", "We also identified 15 human cortical aRG-specific markers based on the published single-cell RNA-seq datasets ( Figure 1A and Supplementary file 1A ) .", "Among them , FOS and EGR1 , two early-response targets of FGF-ERK signaling ( Kang et al . , 2005 ) , are expressed in human but not mouse aRG ( Pollen et al . , 2014 ) .", "To identify ERK early-response gene , we mined published microarray data profiling the transcriptional responses to ERK activation in mouse embryonic stem cells ( Hamilton and Brickman , 2014 ) .", "We identified genes that were significantly up-regulated ( at least 2-fold; adjusted p<0 . 05 ) within 4 hr after ERK activation ( Supplementary file 3 ) .", "Out of the 15 aRG-specific markers , seven genes , EGR1 , FOS , ANXA1 , CLU , CRYAB , CYR61 , and FBXO32 , were among the early-response genes induced by ERK signaling ( Figure 3A ) .", "Remarkably , orthologs of these human aRG-specific genes , with the exception of Cy61 and Clu , were transcriptionally silent in the VZ of mouse neocortex at E14 . 5 ( Figure 3B ) .", "These data suggest the presence of robust ERK signaling activity in human , but not mouse , aRG during mid-neurogenesis of the developing cortex . 10 . 7554/eLife . 23253 . 009Figure 3 . Heightened ERK signaling activity in human aRG .", "( A ) Line-graph showing the fold increases ( log2 scale ) in ERK-responding genes between time zero and the indicated time points after ERK activation , based on a microarray time course analysis ( Hamilton and Brickman , 2014 ) .", "The fold changes above the dashed line are statistically significant ( adjusted p<0 . 05 ) .", "( B ) In situ hybridization of mouse cortical sections at E14 . 5 .", "Images were obtained from GenePaint ( Visel et al . , 2004 ) .", "( C ) Principal component plot showing the relationship among samples: GSE30765 ( Ayoub et al . , 2011 ) , GSE38805 ( Fietz et al . , 2012 ) , GSE65000 ( Florio et al . , 2015 ) , and GSE66217 ( Johnson et al . , 2015 ) .", "( D ) RNA-seq analysis of FGF-ERK read-out genes in aRG from human and mouse neocortex .", "**p<0 . 005; ***p<0 . 001; ns , not significant .", "( E ) Gene Set Enrichment Analysis plot shows the significant enrichment of early-response ( within 2 hr ) genes induced by ERK activation in human versus mouse aRG .", "( F ) Preservation of pathways and gene sets in human and mouse cortical coexpression networks .", "The blue and red dashed lines indicate Zsummary at 2 and 10 , which are the cutoff for not significant and highly significant , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 23253 . 00910 . 7554/eLife . 23253 . 010Figure 3—figure supplement 1 . Comparison of gene expression in aRG/VZ between human and mouse neocortex .", "( A ) Heatmap showing the relationship among samples , GSE30765 ( Ayoub et al . , 2011 ) , GSE38805 ( Fietz et al . , 2012 ) , GSE65000 ( Florio et al . , 2015 ) , and GSE66217 ( Johnson et al . , 2015 ) , as calculated from the variance stabilizing transformation of the gene counts .", "( B ) Scatterplot of ranked average gene expression between human and mouse .", "( C ) MAplot showing log2 fold changes attributable to a given variable over the mean of normalized counts .", "Red points indicate the adjusted p<0 . 05 , triangles indicate points outside the range .", "( D ) Histogram of p value distribution .", "Note the anti-conservative p value distribution as expected .", "( E ) Barplots showing human versus mouse fold changes ( log2 ) in levels of pan radial glial markers identified previously ( Lui et al . , 2014 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 23253 . 010 To systematically compare FGF-ERK signaling activities in the neocortical aRG between humans and mice , we performed differential expression analyses based on six published RNA-seq datasets generated from laser-capture microdissected VZ ( Ayoub et al . , 2011; Fietz et al . , 2012 ) or sorted aRG ( Florio et al . , 2015; Johnson et al . , 2015 ) .", "Among the 21 E14 . 5 mouse and 13 GW13–18 human aRG samples included in these independent studies , there was a close similarity among samples of the same species ( Figure 3C and Figure 3—figure supplement 1A ) .", "On the other hand , there was a significant positive correlation ( Spearman's rank correlation coefficient R = 0 . 85 , p=1 × 10−200 ) between the mean expression levels in mouse and human aRG ( Figure 3—figure supplement 1B ) , demonstrating the compatibility of the mouse and human datasets .", "The percentages of up and down regulated genes in the human and mouse samples were similar ( Figure 3—figure supplement 1C ) .", "Across a panel ( n = 56 ) of pan-aRG markers ( Lui et al . , 2014 ) , 37 . 9% were up-regulated ( 17 . 3% down ) in human aRG whereas 44 . 8% were unchanged ( Figure 3D and Figure 3—figure supplement 1E ) .", "Collectively , these results demonstrated the absence of systematic bias between the two species in gene expression measurements by RNA-seq .", "The mRNA levels of FGF-readout genes ( SPRY1 , SPRY2 , SPRY4 , and ETV5 ) and of all seven ERK early-response genes were significantly higher in humans than those in mice ( Figure 3D ) .", "In agreement with a recent report ( Wang et al . , 2016 ) , the hedgehog readout gene Gli1 ( but not Ptch1 ) was moderately increased in human versus mouse aRG ( Figure 3D ) .", "To analyze pathways differentially enriched in either human or mouse aRG , we performed a gene set enrichment analysis ( GSEA ) ( Subramanian et al . , 2005 ) .", "In agreement with earlier findings ( Fietz et al . , 2012; Florio et al . , 2015 ) , GSEA showed that the gene sets significantly enriched in human aRG contain genes encoding hallmarks of epithelial-mesenchymal transition , extracellular matrix components , extracellular matrix receptors , and integrin signaling , whereas those enriched in mouse aRG are involved in transcription , translation , DNA replication , and the citric acid cycle ( Supplementary file 4A ) .", "Among the most highly enriched genes in human aRG were early-response genes induced by ERK ( NES = 2 . 06 , FDR = 0 . 001 ) and BG-module genes ( NES = 1 . 91 , FDR = 0 . 01 ) ( Figure 3E and Supplementary file 4A ) .", "These findings indicate elevated FGF-ERK signaling activity in human cortical aRG compared to the mouse cortical aRG .", "To further study the difference between mouse and human corticogenesis , we used a complementary approach in which the higher-order relationships between genes were compared based on their coexpression relationships in separate human and mouse networks .", "Changes in network position between species can reveal divergent regulation or a novel function that contributes to human-specific phenotypes ( Oldham et al . , 2006; Miller et al . , 2010; Lui et al . , 2014 ) .", "We used WGCNA , which had been extensively applied to compare corticogenesis across species ( Oldham et al . , 2006; Miller et al . , 2010; Konopka et al . , 2012; Lui et al . , 2014 ) and under pathological conditions ( Horvath et al . , 2006; Chen et al . , 2008; Oldham et al . , 2012; Shirasaki et al . , 2012 ) , to study the preservation of signaling pathways ( KEGG and Reactome ) , pan-RG signature genes ( Lui et al . , 2014 ) , early-response genes induced by ERK , and signature genes for aRG , bRG , and IPC ( Figure 1A ) .", "As expected , the strongly preserved gene sets ( Zsummary sore >10 ) were those related to pan-aRG , IPC , and cell cycle ( Figure 3F ) .", "Of note , the gene sets related to aRG , ERK-early-response genes , FGF and BMP signaling pathway were weakly preserved between the human and mouse coexpression networks ( Figure 3F ) .", "This suggests that the transcriptional regulation of aRG and FGF-ERK pathway genes greatly differs between human and mouse corticogenesis .", "To corroborate our bioinformatic analysis , we performed immunohistochemistry of human and mouse embryonic cortices using an anti-phospho-ERK ( pERK ) antibody that detects both phosphorylated ERK1 and ERK2 .", "We detected robust pERK immunoreactivity in the soma and radial fibers of radial glia marked by Pax6 in the VZ and outer SVZ of human fetal brain gestation week ( GW ) 19 , when bRG are present in abundance ( Hansen et al . , 2010; Pollen et al . , 2015 ) ( Figure 4A and B ) .", "Furthermore , abundant pERK/HOPX double-labeled cells were found in the outer SVZ ( Figure 4A and C ) .", "In mouse embryonic brains , in contrast to the strong pERK immunoreactivity seen in the VZ of the ventral telencephalon , ventral mid-hindbrain , and cerebellum , only weak and diffuse pERK signals were detected in the dorsal telencephalon between E13 . 5 and E16 . 5 ( Figure 4D and F ) .", "The cortical pERK expression pattern was similar to that described in the previous publications ( Faedo et al . , 2010; Pucilowska et al . , 2012 ) .", "Remarkably , by E17 . 5 strong pERK signal was detected in aRG and some basally located cells in the neocortex ( Figure 4E and F ) .", "Although weak Hopx expression was detected in the VZ in the E16 . 5 cortex , few Hopx-positive ( Hopx+ ) cells were found in the SVZ until E17 . 5 ( Figure 4G , H , and data not shown ) .", "Between E17 . 5 and E18 . 5 , scattered Hopx+ cells were present in the SVZ and cortical plate; the colocalization for Fabp7 and Sox2 ( Figure 4G ) suggested that these cells were mouse bRG .", "Notably , pERK and Hopx colocalized in basally located cells , with about half of them displaying unipolar processes that were extending to the pial basement membrane ( unipolar , 50 . 21 ± 4 . 76%; bipolar , 7 . 55 ± 9 . 67%; the rest were ambiguous or multipolar; n = 1 , 246; Figure 4H and I ) .", "Our data suggest that the development of presumptive bRG correlates closely with ERK signaling in both human and mouse cortex , albeit the delayed appearance and reduced number of bRG in the mouse neocortex . 10 . 7554/eLife . 23253 . 011Figure 4 . Activation of ERK signaling is associated with bRG formation .", "( A–H )", "Immunofluorescence on sections of human ( A–C , 19 gestational week ) and mouse ( D–H ) fetal brains .", "Arrowheads indicate the regions that are enlarged in B and C and show pERK immunoreactivity in Pax6-positive aRG ( B ) and Hopx/Sox2 doubled-labeled bRG ( C ) ; the boxed areas in D are enlarged in the inset; the dashed line demarcates the ventricle; arrowheads point to robust pERK staining in the ventricular zone ( VZ ) ; the unfilled arrowhead in E indicates the area enlarged in G and H . Note that pERK signals are increased at the apical surface of VZ from E16 . 5 to E17 . 5 ( F ) .", "Arrows indicate Hopx/Fabp7/Sox2 triple-positive cells ( G ) and Hopx/pERK double-labeled cells ( H ) on E18 . 5 cortical sections .", "Nuclei are stained with Hoechst 33342 .", "( I ) Bar charts showing the percentage of Hopx/pERK double-labeled cells relative to the total number of counted pERK ( n = 642 ) or Hopx ( n = 488 ) cells .", "Abbreviations: cp , cortical plate; ISVZ , inner subventricular zone; OSVZ , outer subventricular zone; Pia , pial surface; SP , cortical subplate .", "Scale bars: 20 µm ( B , C , G , H and F ) , 200 µm ( D ) , and 100 µm ( E ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 23253 . 011 The function of the ERK pathway in bRG formation was tested by expressing the ERK activator Mek1DD in the E14 . 5 mouse cortex through in utero electroporation ( Shimogori and Ogawa , 2008 ) .", "The electroporation of GFP had little effect on the expression of bRG markers; however , bRG markers including Hopx , Fabp7 , Tnc , Slc1a3 , and Ptprz1 were robustly induced following the electroporation of Mek1DD 48 hr after electroporation ( Figure 5A–C , Figure 5—figure supplement 1 , and Video 1 ) .", "As reported previously ( Maiorano and Mallamaci , 2009 ) , electroporation occasionally caused the basal dispersion of aRG in the VZ ( Figure 5—figure supplement 2 ) , but only a small fraction of the dispersed GFP-expressing cells ( 3 . 3 ± 1 . 5% ) expressed Hopx , whereas 31 . 8 ± 4 . 7% of the Mek1DD-expressing cells ( marked by GFP ) simultaneously expressed Hopx and Sox2 at 48 hr after electroporation ( Figure 5B , C , Figure 5—figure supplement 1 , and Video 1 ) .", "This showed that Mek1DD cell-autonomously induces bRG-like cells .", "Similar to the characteristic morphology of bRG in primate cortices ( Fietz et al . , 2010; Hansen et al . , 2010; Reillo et al . , 2011; Kelava et al . , 2012; Betizeau et al . , 2013; Gertz et al . , 2014 ) , the Mek1DD-expressing cells that expressed bRG markers displayed long basal processes that reached the pial surface ( 78 . 2 ± 3 . 1% , n = 384 ) and occasionally also an apical process ( 21 . 8 ± 3 . 1%; Figure 5C and Video 1 ) .", "In agreement with previous reports of an association between bRG and persistent Pax6 expression ( Fietz et al . , 2012; Wong et al . , 2015 ) , 57 . 4 ± 3 . 8% of the Mek1DD-expressing cells were positive for Pax6 ( n = 1787 cells counted ) in 48–96 hr after electroporation ( Figure 5D ) .", "Notably , only 5 . 24 ± 1 . 75% Mek1DD-expressing cells were positive for the IPC marker Eomes ( n = 1 , 083; Figure 5E ) .", "Collectively , our data show that Mek1DD expression induces bRG , but not IPC , in the mouse neocortex . 10 . 7554/eLife . 23253 . 012Figure 5 . Hyperactivation of ERK signaling induces bRG in the mouse neocortex .", "( A ) Schematic of the electroporation experiment .", "The box with the dotted lines indicates the approximate cortical area shown in the rest of the figure .", "( B–E )", "Immunofluorescence and in situ hybridization images of adjacent coronal sections of mouse cortices 48 hr ( B , C , and E ) and 96 hr ( D ) after the in utero electroporation of the indicated transgene .", "The thin dotted lines demarcate the pial surface; and the thick dotted lines separate the areas with and without electroporated cells; asterisks indicate the induction of bRG; the arrowheads in D indicate the heterotopia above the VZ; the inset in B is a low-magnification image showing Tnc expression in the cortical VZ on both the transfected and untransfected sides .", "A three-dimensional rendering of the induced bRG is shown in Video 1 .", "Nuclei are stained with Hoechst 33342 .", "Scale bars: 200 µm ( B ) , 20 µm ( C–E ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 23253 . 01210 . 7554/eLife . 23253 . 013Figure 5—figure supplement 1 . Mek1DD-induced bRG form heterotopia in the lower layer of the cortex . Immunofluorescence on coronal section of mouse cortices at E16 . 5 , P3 , and P7 as indicated after in utero electroporation at E14 . 5 .", "The dashed line demarcates the ventricular surface; arrowheads indicate the cluster of Mek1DD-transfected cells in the lower layer of the cortex; the arrow shows that a cluster of Sabt2+ neurons derived from Mek1DD-transfected cells .", "Note that Mek1DD-transfected cells in the heterotopia mostly express Sabt2 but not Gfap .", "Scale bars: 40 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 23253 . 01310 . 7554/eLife . 23253 . 014Figure 5—figure supplement 2 . Electroporation of EGFP does not induce bRG . Immunofluorescence on coronal sections of E16 . 5 neocortex following in utero electroporation of EGFP at E14 . 5 .", "The boxed area is enlarged; the dashed line outlines the pial surface .", "Arrows indicate the perturbed VZ due to electroporation .", "Arrowheads show bRG that are positive for Hopx and Sox2 , but lack GFP expression . DOI: http://dx . doi . org/10 . 7554/eLife . 23253 . 01410 . 7554/eLife . 23253 . 015Video 1 . Movie of three-dimension rendering of Mek1DD-induced bRG in the mouse cortex . The red , green and blue channels represent antibody-staining for Hopx , GFP , and Sox2 , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 23253 . 015 The proliferation and differentiation potential of the induced bRG in the mouse neocortex was studied using immunofluorescence .", "48 hr after electroporation , most Mek1DD-induced Hopx+ cells were positive for Ki67 and a fraction of them was also positive for phospho-vimentin ( pVim ) , a marker of M-phase cells ( Figure 6A ) .", "This result demonstrated that most Mek1DD-induced bRG were highly proliferative .", "To determine whether the induced bRG were capable to undergo multiple rounds of self-renewing divisions , a hallmark of basal progenitors in primate neocortex , embryos were electroporated at E14 . 5 and then treated with the thymidine analogues 5-bromodeoxyuridine ( BrdU ) and 5-ethynyldeoxyuridine ( EdU ) at E16 . 5 and E17 . 5 , respectively ( Figure 6B ) .", "In embryos electroporated with GFP , few GFP+ cells were co-labeled with BrdU , EdU , or Ki67 at E18 . 5 ( type 4 cells , Figure 6B and C ) .", "By contrast , most Mek1DD-expressing cells were positive for at least two of the three labeling at E18 . 5 ( types 1 and 2 cells , Figure 6B and C ) , consistent with their having undergone at least two rounds of divisions and/or continuing to divide 96 hr after electroporation .", "The latter conclusion was supported by the notably weakened GFP immunoreactivity in BrdU+/EdU+ cells ( Figure 6C ) .", "Moreover , the clustering of Mek1DD–expressing cells that were positive for Hopx , BrdU , and EdU between the SVZ and cortical plate ( Figure 6D ) suggested that Mek1DD induced bRG underwent clonal expansion and extensive self renewal . 10 . 7554/eLife . 23253 . 016Figure 6 . Mek1DD-induced bRG undergo multiple rounds of self-renewing divisions .", "( A ) Immunofluorescence of phosphorylated vimentin ( pVim ) , GFP , and Ki67 on coronal sections of E14 . 5 mouse cortex 48 hr after their electroporation .", "Dashed lines demarcate the pial surface; arrowheads indicate pVim-positive transfected cells ( marked by GFP ) ; arrows indicate a doublet of bRG at the end of mitosis .", "( B ) Illustrations showing the boxed area that is corresponding to the images in this figure ( upper ) and the protocol used to study the proliferation of transfected cells ( lower ) .", "BrdU/EdU/Ki67 triple labeling identifies four types of cells with distinct proliferation history .", "( C and D )", "Immunofluorescence on coronal sections of E18 . 5 mouse cortex electroporated in utero with EGFP or Mek1DD at E14 . 5 .", "The boxed areas are enlarged and shown in individual channels .", "Arrowheads denote clusters of Mek1DD-transfected cells ( C ) and the induced Hopx-positive cells ( D ) , which are mostly BrdU/EdU double ( type 2 ) and BrdU/EdU/Ki67 triple ( type 1 ) positive .", "( E ) Illustration of the cell-pair assay procedure .", "The red dashed line indicates separation of the upper and lower parts of the cortex transfected with Mek1DD .", "( F ) Immunocytochemistry of transfected cells ( GFP+ ) and daughter-cell pairs .", "( G ) Quantification of the percentage of transfected cells marked by GFP that underwent different modes of divisions .", "P values were calculated using a χ2 test .", "Scale bars: 20 µm ( A ) , 200 µm ( C ) , 50 µm ( D ) , and 5 µm ( F ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 23253 . 016 To examine the mode of cell division of the induced bRG , we conducted clonal cell-pair assays ( Figure 6E ) .", "This method has been extensively used to analyze symmetric versus asymmetric cell division in neural progenitor cells ( Shen et al . , 2002 ) .", "We found that in transfected cells isolated from the cortex 48 hr after electroporation most Mek1DD-expressing cells underwent symmetric division to generate two Fabp7+ daughter cells ( data not shown ) .", "At 72 hr after electroporation , all three possible cell division types were present among the GFP+ Mek1DD-expressing doublets: bRG-bRG ( Fabp7-Fabp7 ) , bRG-neuron ( Fabp7-Tubb3 ) , and neuron-neuron ( Tubb3-Tubb3 ) ( Figure 6F and G ) .", "Most of the Mek1DD-expressing doublets underwent proliferative division ( Fabp7-Fabp7 ) either from the upper ( 80 . 0% ) or the lower ( 70 . 2% , including the SVZ and VZ ) part of the cortex , in contrast to the predominantly neurogenic division ( Tubb3-Tubb3 , 64 . 5% ) of the GFP-only-expressing doublets ( Figure 6G ) .", "Collectively , our data show that expression of Mek1DD induces bRG-like cells that have extensive self-renewing and neurogenic potential .", "To follow the fate of the induced bRG , markers of cortical neurons were examined in P3 cortices .", "In brains transfected with GFP alone , GFP+ cells were arranged uniformly in the upper layer and expressed Satb2 ( layer II-IV neurons ) , but not the deep-layer neuronal markers Tbr1 and Ctip2 ( Figure 7A and data not shown ) .", "Many Mek1DD-transfected cells with strong GFP also expressed Satb2 but they were arranged instead in clusters ( Figure 7A ) , in agreement with the clonal expansion of the induced bRG observed at the earlier stages and the subsequent formation of Satb2+ cells from the bRG .", "Although increased numbers of astrocytes marked by Gfap were detected in the area containing Mek1DD-transfected cells compared to non-transfected area ( Figure 7A and B ) , GFP-labeled Mek1DD-tranfected cells in the P3 neocortex were mostly devoid of Gfap and the oligoglial markers Olig2 and Sox10 ( Figure 7B and data not shown ) .", "At P7 , however , some Mek1DD-expressing cells with low-level GFP expression were positive for Gfap ( Figure 7C ) , suggesting that the induced bRG are multipotent .", "Despite the ectopic bRG induction , in utero electroporation of Mek1DD did not cause folding of the cortex ( n = 10 ) .", "Together , our data show that Mek1DD-induced bRG have extensive proliferation capacity and give rise to both neurons and astrocytes .", "However , at least to the extent of increased number of bRG obtained with the electroporation folding of the mouse cortex is not induced by expansion of bRG in the absence of parallel expansion of IPCs . 10 . 7554/eLife . 23253 . 017Figure 7 . Mek1DD-induced bRG form neurons and astrocytes .", "( A ) Immunofluorescence of Satb2 , GFP , and Gfap on coronal sections of mouse cortex 8 days after electroporation .", "The bracket indicates layers of Satb2-positive neurons derived from GFP-transfected cells; the arrows show clusters of Satb2-positive neurons formed by Mek1DD-transfected cells; the asterisk denotes the accumulation of astrocytes; arrowheads indicate scattered Mek1DD -transfected cells with weak GFP and negative for Gfap .", "( B and C )", "Immunofluorescence of Gfap and GFP on coronal sections of P3 ( B ) and P7 ( C ) brains electroporated at E14 . 5 .", "Arrowheads and arrows point to absence and presence , respectively , of GFP in Gfap+ cells .", "Nuclei were stained with Hoechst 33342 and are shown in the blue channel . DOI: http://dx . doi . org/10 . 7554/eLife . 23253 . 017 We found that ETV5 and , to a lesser degree , Etv4 were increased in human cortical aRG compared to mouse aRG ( Figure 3C ) .", "Etv4 and Etv5 are well-known effectors of the FGF-Ras-ERK signaling cascade ( Mao et al . , 2009; Zhang et al . , 2009; Hollenhorst et al . , 2011; Li et al . , 2012; Breunig et al . , 2015 ) .", "Moreover , ETV5 is specifically expressed in bRG among human cortical progenitor cells ( Pollen et al . , 2015; Thomsen et al . , 2016 ) .", "Finally , the transcripts of Etv4 and Etv5 were missing in Ptpn11-cKO cerebella but were restored in Ptpn11-cKO;Map2k1 cerebella at E14 . 5 ( Figure 8A ) .", "These observations raised the question if an FGF-ERK-ETV cascade is involved in the genesis of bRG and BG . 10 . 7554/eLife . 23253 . 018Figure 8 . Etv4 and Etv5 are important for BG formation .", "( A ) In situ hybridization for Etv4 and Etv5 on sagittal sections of E14 . 5 cerebella .", "The presence and absence of Etv4 and Etv5 transcripts are indicated by arrows and arrowheads , respectively .", "( B and C )", "Immunofluorescence on sagittal sections of E16 . 5 Gbx2+/creER;R26RFP/+ ( B ) and Gbx2+/creER;R26Etv4DN/+ cerebella ( C ) treated with tamoxifen at E9 . 5 .", "( D ) Quantification of RFP and Etv4DN-expressing cells that display Fabp7 immunoreactivity in combined ( all ) or individual areas 1–3 , as indicated in the illustration in the lower right corner .", "Unpaired Student’s t-test , p=0 . 008268 , t ( 4 ) = 8 . 059 ( all ) ; p=0 . 004507 , t ( 4 ) = 5 . 760 ( area 1 ) ; p=0 . 006968 , t ( 4 ) = 5 . 103 ( area 2 ) ; p=0 . 01634 , t ( 4 ) = 3 . 985 ( area 3 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 23253 . 01810 . 7554/eLife . 23253 . 019Figure 8—figure supplement 1 . Contribution of Gbx2-expressing cells at E9 . 5 to different cerebellar cell types . Immunofluorescence on sagittal sections of E16 . 5 Gbx2+/creER;R26RFP/+ cerebella treated with tamoxifen at E9 . 5 .", "Arrows indicate that colocalization of RFP and different cerebellar markers , Calb1 ( marking Purkinje cells ) , Pax2 ( GABAergic interneurons ) , and Pax6 ( granule cell precursors ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 23253 . 01910 . 7554/eLife . 23253 . 020Figure 8—figure supplement 2 . Inactivation of Etv4 and Etv5 does not affect the activation of ERK , neurogenesis , or cell survival .", "( A and B )", "Immunofluorescence of GFP together with pERK ( A ) or Olig2 ( B ) on sagittal sections of E13 . 5 Gbx2+/creER;R26Etv4DN/+ embryos treated with tamoxifen at E9 . 5 .", "Arrowheads point to pERK and Olig2 immunoreactivity in GFP+ Etv4DN-expressing cells; the bracket demarcates the VZ .", "( C ) Immunofluorescence of activated caspase three on sagittal cerebellar sections of E16 . 5 wild-type and Gbx2+/creER;R26Etv4DN/+ embryos treated with tamoxifen at E9 . 5 .", "The few Casp3+ cells are indicated by arrows .", "Note that the Casp3 is not colocalized with GFP , which marks Etv4DN-expressing cells . DOI: http://dx . doi . org/10 . 7554/eLife . 23253 . 02010 . 7554/eLife . 23253 . 021Figure 8—figure supplement 3 . Simultaneous deletion of Fgfr1 , Fgfr2 , and Fgfr3 results in the similar phenotype as that found in Ptpn11-cKO mice . Immunofluorescence for Gfap on sagittal sections P25 cerebellum of indicated genotypes .", "The boxes indicate the area that is enlarged in the lower panel; the arrow and asterisk show the presence and absence , respectively , of the parallel radial fibers of BG in the molecular layer .", "The nuclei were stained with Hoechst 33342 . DOI: http://dx . doi . org/10 . 7554/eLife . 23253 . 021 To test this hypothesis , we first investigate the role of Etv4 and Etv5 in BG formation .", "Through tamoxifen-induced Cre-mediated recombination in Gbx2creER/+;R26Etv4DN/+ embryos at E9 . 5 , we expressed a dominant negative Etv4 ( Etv4DN ) , a fusion between the Etv4 DNA-binding domain and the engrailed repressor domain ( Mao et al . , 2009 ) , to block the redundant function of Etv4 and Etv5 in cerebellar aRG and their progeny .", "Fate-mapping study using a tdTomato red fluorescent protein ( RFP ) reporter R26RFP mouse line ( Madisen et al . , 2010 ) , we found that the Gbx2-expressing cells at E9 . 5 gave rise to BG , Purkinje cells , GABAergic interneurons , and granule cell precursors ( Figure 8B and Figure 8—figure supplement 1 ) .", "Compared to cells that expressed RFP in Gbx2creER/+;R26RFP/+ embryos , significantly fewer Etv4DN-expressing cells ( marked by GFP ) formed BG ( Figure 8B–D ) .", "pERK immunoreactivity was detected in Etv4DN-expressing cells in the cerebellar VZ at E13 . 5 ( Figure 8—figure supplement 2A ) , indicating that ERK signaling acts upstream of Etv4 and Etv5 .", "No difference in the expression of Olig2 ( cerebellar GABAergic precursors ) ( Seto et al . , 2014; Ju et al . , 2016a ) was detected between Etv4DN-positive and Etv4DN-negative cells inside or near the cerebellar VZ in E13 . 5 Gbx2creER/+;R26Etv4DN/+ embryos ( Figure 8—figure supplement 2B ) , suggesting that Etv4DN expression does not overtly alter neurogenesis from cerebellar aRG .", "Finally , we ruled out that Etv4DN caused cell death of developing BG by immunohistochemistry for activated caspase 3 ( Figure 8—figure supplement 2C ) .", "Collectively , our data suggest that Etv4 and Etv5 are essential for BG formation .", "Whether the forced expression of Etv4 or Etv5 restores BG in Ptpn11-cKO cerebella was investigated in a novel ex vivo electroporation procedure ( Figure 9A ) .", "With this procedure , only cerebellar aRG , which line the ventricular zone surface and contact with DNA solution injected into the fourth ventricle , are transfected .", "In WT cerebellar slices after 24 or 48 hr of in vitro culture , aRG transfected with GFP formed BG , which were identified by markers ( Fabp7 and Sox9 ) and the unipolar morphology of the cells , and non-BG cells ( Figure 9B ) .", "In Ptpn11-cKO cerebellar slices , GFP-transfected cells were mostly restricted to the VZ , displaying long basal processes and immunoreactivity for Sox9 but not Fabp7 ( Figure 9B ) .", "This result demonstrated that the inactivation of Ptpn11 blocks the aRG-to-BG transition , as previously described ( Li et al . , 2014 ) .", "Remarkably , electroporation of Mek1DD robustly rescued BG in Ptpn11-cKO cerebella ( Figure 9C ) .", "Electroporation of Etv4 or Etv5 also rescued the formation of BG but to less extent compared to that of Mek1DD ( Figure 9D and data not shown ) .", "The BG marker Fabp7 was selectively expressed in Mek1DD- or Etv-rescued BG in the cerebellar cortex , suggesting that Map2k1 and ETV act cell-autonomously to promote BG formation .", "Furthermore , inactivation of Fgf9 or the three FGFR genes ( Fgfr1 , 2 , and 3 ) depletes BG in the mouse cerebellum as found in Ptpn11-cKO mutants ( Lin et al . , 2009 ) ( Figure 8—figure supplement 3 ) , demonstrating the essential role of FGF signaling in BG formation .", "We did not attempt to rescue BG by FGF because Ptpn11 is essential to mediate FGF receptor ( FGFR ) signaling to ERK ( Hadari et al . , 2001 ) .", "Collectively , these observations demonstrate that Ptpn11 regulates the FGF-ERK-ETV cascade in the control of the aRG-to-BG transition . 10 . 7554/eLife . 23253 . 022Figure 9 . Activation of the ERK-ETV cascade in cerebellar aRG at E13 . 5 is critical to BG generation .", "( A ) The procedure for ex vivo electroporation .", "( B–D )", "Immunofluorescence of Fabp7 , GFP , and Sox9 on sections of cerebellar slices 48 hr ( 2 day in vitro , 2 DIV ) after the electroporation of EGFP ( B ) , Mek1DD ( C ) , or Etv4 ( D ) .", "Arrows point to the rescued BG; boxed areas are enlarged and shown in individual channels .", "Scale bar: 50 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 23253 . 022 Next , we examined whether the FGF-ERK-ETV cascade is important for bRG formation by in utero electroporation of constitutively active FGFR1 , FGFR1K656E ( Olsen et al . , 2006 ) , or Etv4 in mouse cortex at E14 . 5 .", "Similar to Mek1DD , FGFR1K656E and Etv4 induced cells that expressed Hopx , Sox2 , Tnc , Slc1a3 , and Ptprz1 48 hr after electroporation ( Figure 10A ) .", "Similar percentages of Hopx/Sox2 double labeling were found among FGFR1K656E- , Mek1DD- , and Etv4-expressing cells , and they were significantly higher than that in GFP- expressing cells ( Figure 10B ) .", "These findings suggest that FGFR , Map2k1 , and Etv4 act in the same pathway to induce bRG .", "Notable , in utero electroporation of Mek1DD , FGFR1K656E , or Etv4 caused slightly different phenotypes , despite their similar function in inducing bRG-like cells .", "For example , the Hopx+ cells induced by Etv4 occupied a more apical position than those induced by FGFR1K656E and Mek1DD ( Figure 10C ) .", "Furthermore , Mek1DD , but not FGFR1K656E and Etv4 , induced heterotopia near the VZ , and these heterotopia were composed of Sabt2+ cortical neurons with few Gfap+ astrocytes after birth ( Figure 5B and Figure 5—figure supplement 1 ) .", "Taken together , our results show that activation of the FGF-ERK-ETV axis is involved in inducing the transition of aRG to BG in the cerebellum and to bRG in the neocortex . 10 . 7554/eLife . 23253 . 023Figure 10 . Expression of activated FGFR or Etv4 induces bRG in the mouse neocortex .", "( A ) Immunofluorescence and in situ hybridization images of adjacent coronal sections of mouse cortices 48 hr after the in utero electroporation of the indicated transgene .", "( B ) Quantification of Hopx/GFP double-labeled cells relative to the total number of transfected cells .", "Each data point represents one embryo in which three or more adjacent sections were examined .", "Data are presented as the mean ± SEM .", "P values were calculated using a one-way ANOVA followed by a post-hoc Turkey-Kramer multiple comparison test , F ( 9 ) =21 . 76 .", "( C ) Distribution of Hopx-positive cells induced by Mek1DD , FGFR1K656E , and Etv4 in the upper ( U ) , middle ( M ) , and lower ( L ) parts of the cortex .", "Scale bars: 200 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 23253 . 023" ], [ "In the present study , we investigated the similarities between bRG and BG in their genome-wide transcriptional profiles and developmental programs .", "Using differential gene expression and coexpression studies based on RNA-seq , we establish the molecular features of mouse nascent BG , and show the remarkably similarity between human bRG and mouse BG in their molecular characteristics .", "Through transcriptome analysis and immunohistochemistry , we demonstrate that heightened ERK signaling is associated with the timely formation of endogenous bRG in the mouse cortex , while stronger ERK activity in cortical aRG is linked to the abundance of bRG in the human compared to mouse cortex .", "We show that the activation of the FGF-ERK-ETV axis that is important for BG formation induces multipotent bRG , which display canonical human bRG gene markers and extensive proliferative capacity , in the mouse neocortex .", "Our results have revealed surprising parallels in the transition of aRG to bRG in the neocortex , and aRG to BG in the cerebellum .", "By taking advantage of the specific loss of BG in Ptpn11-cKO and the rescue of BG in Ptpn11-cKO;Map2k1 cerebella , we used RNA-seq to examine the transcriptome of the cerebella with and without BG at the stages before and after BG induction .", "Using conventional differential expression analysis and WGCNA , we identified putative markers for nascent BG .", "The validation of these markers was mostly carried out by inspection of expression data of Allen Mouse Brain .", "Because of the inherent difficulty to assign cell-type specific expression based on in situ hybridization , the gene expression analysis was performed by an examiner who was blinded to the gene symbol and the origin of the gene list .", "We showed that candidate gene lists that were produced by the two statistical methods were both significantly enriched in BG in P56 mouse cerebellum compared to randomly generated gene lists .", "One potential caveat is that BG gene expression may be dynamic during development .", "However , interrogation of BG-specific expression in the Allen Mouse Developmental Brain and GenePaint has revealed that many BG markers , such as Fabp7 , Hopx , Etv5 , Ptprz1 , Slc1a3 and Tnc are expressed in both nascent and mature BG ( Figure 1—figure supplement 1A ) .", "Another caveat is that our strategy would likely identify downstream targets of the Ptpn11-ERK pathway , which may also be involved in development of non-BG cells in the cerebellum .", "Single-cell RNA-seq analysis will help clarify the definite markers for nascent BG .", "Ptpn11 and other components of the FGF-ERK pathway play a multifaceted roles in the progression of neural progenitors , including the timely transition from neuroepithelial cells to aRG ( Sahara and O'Leary , 2009 ) , from aRG to IPC ( Kang et al . , 2009 ) , from proliferative to neurogenic self-renewing aRG ( Dee et al . , 2016 ) , and from neurogenic to gliogenic aRG ( Gauthier et al . , 2007; Ke et al . , 2007 ) .", "After their broad expression in the cerebellar primordium at E10 . 5 , the transcripts of Etv4 and Etv5 disappear , except for the region near the mid-hindbrain junction , by E12 . 5 ( Li et al . , 2014 ) , but re-appear in cerebellar aRG at E14 . 5 ( Figure 8A ) , following the robust expression of pERK in these cells at E13 . 5 ( Figure 4D ) .", "While Ptpn11 deletion does not change the transcription of Etv4 and Etv5 at E10 . 5 ( Li et al . , 2014 ) , neither gene is expressed in the cerebellar anlage of Ptpn11-cKO embryos after E12 . 5 ( Figure 8A ) .", "This suggests that activation of ERK-ETV in cerebellar aRG at E13 . 5 but not before is essential for BG induction .", "In the current study , we have extended our previous findings by showing that the expression of Mek1DD , Etv4 , or Etv5 cell-autonomously rescues BG in Ptpn11-cKO cerebella at E13 . 5 .", "Coincidentally , strong ERK signaling is present in the VZ starting at E13 . 5 to at least E16 . 5 , matching closely with the onset and duration of BG generation from the VZ .", "These results demonstrate that heightened ERK signaling activity in cerebellar aRG controls the timely aRG-to-BG transition .", "In parallel with the BG induction , intensified pERK in cortical aRG correlates with the formation of endogenous bRG in the mouse cortex at E17 . 5 ( Figure 4E–I ) , whereas the hyperactivation of the ERK signaling induces ectopic bRG at E14 . 5 ( Figures 5 and 10 ) .", "Therefore , the intensification of ERK signaling controls the timely transition of aRG to BG in the cerebellum , and to bRG in the neocortex .", "How ERK signaling controls the formation of BG and bRG is currently unknown .", "Previous studies have shown that increasing the horizontal division , in which the cleavage furrow is parallel to the ventricular surface , contributes to the generation of bRG in both the human and mouse cortex ( LaMonica et al . , 2013; Martínez-Martínez et al . , 2016 ) .", "As it has been shown that the ERK pathway determines mitotic spindle orientation epithelial cells ( Tang et al . , 2011 ) , it is possible that ERK signaling controls the generation of BG and bRG by regulating the spindle orientation .", "On the other hand , the transformation of aRG into bRG resembles the epithelial-to-mesenchymal transition ( Itoh et al . , 2013; Pollen et al . , 2015 ) , a developmental process that is regulated by the FGF-ERK pathway ( Lamouille et al . , 2014 ) .", "Future studies will be needed to determine whether FGF-ERK signaling promotes the transition from aRG to BG or bRG similarly to the regulation of the epithelial-to-mesenchymal transition , and/or by regulating spindle orientation .", "A previous study showed that in utero electroporation of Mek1DD or Etv5 in E15 . 5 mouse neocortex enhances the generation of proliferative Fabp7+ cells , but no other bRG markers were examined ( Li et al . , 2012 ) .", "The authors concluded that these Fabp7+ cells are astrocyte precursors because 20–25% of the transfectants become astrocytes at P22 and P60 ( Li et al . , 2012 ) .", "Although we detected supernumerary Gfap+ cells in cortical region with Mek1DD transfection , many of the Gfap+ astrocytes were not colocalized with EGFP , which marked Mek1DD transfection , at P3 ( Figure 7B , C , and Figure 5—figure supplement 1 ) .", "Because EGFP expression was rapidly diminished during the successive division of the induced bRG , we were uncertain whether the transfection of Mek1DD cell autonomously or non-autonomously induced astrocytes .", "In the current study , we showed that forced expression of Mek1DD induced neurogenic progenitor with remarkably high proliferation and self-renewing potential ( Figure 6 ) .", "Furthermore , these progenitors express many canonical human bRG markers ( Pollen et al . , 2015; Thomsen et al . , 2016 ) ( Figures 5 and 10 ) .", "Importantly , Pax6 , which is not linked to astroglial lineage , was maintained in Mek1DD-induced bRG at least to E18 . 5 ( Figure 5D ) .", "In basal progenitors ( including IPCs and bRG ) , persistent Pax6 expression is a hallmark of the primate neocortex ( Mo and Zecevic , 2008; Fietz et al . , 2010; Hansen et al . , 2010; Reillo et al . , 2011; Betizeau et al . , 2013 ) .", "Indeed , sustained Pax6 expression is sufficient to expand IPC and bRG-like cells in the mouse neocortex ( Wong et al . , 2015 ) .", "Importantly , we show that the expression of Mek1DD primarily induced bRG , whereas IPC marker Eomes was mostly absent from Mek1DD-expressing cells ( Figure 5 ) .", "Although the expansion of bRG-like cells and IPC through various genetic manipulations has previously been reported , to the best of our knowledge , this is the first report of the specific induction of primate-like bRG in the mouse neocortex .", "Together , our data strongly suggest that activation of the FGF-ERK-ETV cascade is involved in the induction of bRG in the mouse neocortex .", "In any given species , the relative abundance of bRG can be attributed , at least partially , to the bRG-forming competence of aRG and/or to the self-expansion potential of bRG .", "We demonstrated that FGF-ERK-ETV activity is lower in the mouse than in the human neocortical aRG ( Figures 3 and 4 ) , and that activation of this axis is sufficient to induce bRG ( Figures 5–7 and 10 ) .", "These observations indicate that a higher level of FGF-ERK-ETV activity in aRG contributes to the greater abundance of bRG in humans than in mice .", "Comparative epigenetic profiling of human , rhesus macaque , and mouse corticogenesis has shown that cis-regulatory elements with activity in humans are enriched in the modules of coexpressed genes belonging to the FGF and TGFβ pathways ( Reilly et al . , 2015 ) .", "In agreement with these findings , our coexpression network analysis revealed significant divergence in FGF-ERK and BMP signaling pathways in human and mouse corticogenesis ( Figure 3F ) .", "Therefore , gene regulation changes that are specific to the human lineage modify corticogenesis in humans , in part by enhancing FGF-ERR signaling activity in aRG leading to expansion of bRG .", "Gain-of-function mutations of FGFR2 and FGFR3 cause Apert syndrome and thanatophoric dysplasia , which are characterized by unique and complex malformation of the cortex , including megalencephaly and polymicrogyria ( Hevner , 2005 ) .", "Moreover , mutations of the genes of the ERK signaling cascade have been implicated in neuro-cardio-facial-cutaneous ( NCFC ) syndromes that include an abnormal size and gyrification of the neocortex and cerebellum ( Samuels et al . , 2009 ) .", "Our new findings imply that abnormal bRG development may contribute to the pathologies of the human neocortex that are caused by aberrant FGF-ERK function .", "Despite the similarity in their generation , BG do not produce neurons during normal development ( Parmigiani et al . , 2015 ) , unlike bRG in the human cortex .", "Although the Mek1DD-induced bRG possess extensive proliferative self-renewing potential , no dramatic increase in cortical neurons was observed in neocortex transfected with Mek1DD .", "Furthermore , both the endogenous and Mek1DD-induced bRG are distributed in both the SVZ and the cortical plate in the mouse cortex rather than being restricted to the outer SVZ as described in the primate cortex .", "Therefore , additional intrinsic and/or extrinsic factors may be required for the neurogenic potential and localization of human bRG .", "Comparative studies in human , ferret , and mouse have shown that the neocortical basal progenitors of humans exhibit a greater neuronal lineage commitment and degree of differentiation , partially through the activity of proneural genes ( Johnson et al . , 2009 ) .", "In vitro and in vivo studies have shown that the forced expression of proneural genes , such as Ascl1 , Neurog2 , and NeuroD1 , can reprogram glial cells to neocortical neurons ( Guo et al . , 2014; Masserdotti et al . , 2015; Zhang et al . , 2015 ) .", "Further studies are required to determine whether the expression of proneural genes enhances the neurogenic potential of BG in the cerebellum or Mek1DD-induced bRG in the mouse neocortex .", "The expression of Mek1DD specifically expands bRG but fails to induce folding of the mouse neocortex .", "These findings are in agreement with the notion that an abundance of bRG is necessary , but insufficient , for gyrencephaly ( Hevner and Haydar , 2012; Kelava et al . , 2012 ) .", "Rather , increased neuronal output through the expansion of other basal progenitors together with bRG may be necessary for folding of the neocortex .", "A notable parallel may be cerebellar foliation , in which the important role played by the proliferation of granule cell precursors has been well documented .", "For example , functional alterations of Shh , which promotes granule cell precursor proliferation in the EGL ( Dahmane and Ruiz i Altaba , 1999; Wallace , 1999; Wechsler-Reya and Scott , 1999 ) , have been linked to the extent of cerebellar foliation ( Corrales et al . , 2004 , 2006 ) .", "Interestingly , hedgehog signaling also promotes the development of BG ( Dahmane and Ruiz i Altaba , 1999; Fleming et al . , 2013 ) and bRG ( Wang et al . , 2016 ) .", "These observations suggest that the hedgehog and FGF-ERK signaling pathways act in concert to promote neuronal output via IPC or granule cell precursors as well as to expand bRG and BG populations in the evolution of a convoluted neocortical and cerebellar cortex , respectively ." ], [ "Husbandry of mice was carried out according to guidelines approved by the University of Connecticut .", "Light/dark cycle in the vivarium was 12 hr light on and 12 hr light off .", "All mouse strains were maintained on CD-1 outbred genetic background .", "The day of vaginal plug detection was considered embryonic day ( E ) 0 . 5 .", "For tamoxifen administration , 4–6 milligrams of tamoxifen ( Sigma , St . Louis , MI ) in corn oil were administered to pregnant females through oral gavage as described ( Li and Joyner , 2001 ) .", "Generation and characterization of the En1cre ( En1tm2 ( cre ) Wrst/J; #007916 ) ( Li et al . , 2002 ) , Gbx2creER ( Gbx2tm1 . 1 ( cre/ERT2 ) Jyhl/J; #022135 ) ( Chen et al . , 2009 ) , Ptpn11floxed ( Yang et al . , 2013 ) , R26Etv4DN ( Mao et al . , 2009 ) , and R26Mek1DD ( Gt ( ROSA ) 26Sortm8 ( Map2k1* , EGFP ) Rsky/J; #012352 ) ( Srinivasan et al . , 2009 ) alleles have been previously reported .", "The R26Etv4DN allele contained the Etv4DN-ires-YFP bicistronic sequence downstream of a loxP-flanked Neo-STOP cassette .", "Therefore , Etv4DN-expressing cells and their progeny were permanently marked by YFP ( recognized by anti-GFP antibodies ) after tamoxifen-induced creER activation .", "Embryonic mouse brains were dissected in cold phosphate buffered saline and fixed in 4% paraformaldehyde for 40 min to overnight .", "Brains were cryoprotected , frozen in Tissue-Plus ( ThermoFisher Scientific , Carlsbad , CA ) , and sectioned in a cryostat ( Leica , Germany , CM3050S ) .", "Standard protocols were used for immunofluorescence and in situ hybridization as described ( Chen et al . , 2009 ) .", "Detailed protocols are available on the Li Laboratory website ( http://lilab . uchc . edu/protocols/index . html ) .", "As the antibodies for Hopx and pERK were both raised in rabbits , we used a two-step technique as described previously ( Shindler and Roth , 1996 ) .", "First we used the sensitive Tyramide Signal Amplification Kits ( ThermoFisher Scientific ) to detect a highly diluted anti-pERK antibody .", "Subsequently , conventional immunostaining was performed to detect Hopx .", "We confirmed that the conventional method could not detect the bound anti-pERK antibodies in the earlier steps .", "Primary and secondary antibodies used in the study are listed in the Supplementary file 5A .", "To generate riboprobes for RNA in situ hybridization , PCR primers were designed using Primer 3 ( Untergasser et al . , 2012 ) to amplify a 500–700 bp region of the open reading frame for a given gene; a T7 promoter sequence appended to the reverse primer enabled direct generation of antisense riboprobes from T7-mediated transcription of PCR products .", "Primer sequences are listed in Supplementary file 5B and Supplementary file 5C .", "To generate cDNA , total RNA was extracted from E13 . 5 mouse brain tissues using Trizol extraction kit ( Invitrogen , Carlsbad , CA ) and reverse transcribed with Superscript III First Strand Synthesis System using random hexamers ( Invitrogen ) .", "The cerebellar anlage was microdissected from E12 . 5 and E13 . 5 brains .", "For E14 . 5 cerebellar anlage , microdissection was performed on brain slices ( 300 µm thickness ) that were prepared with a vibratome ( Leica , VT1000S ) .", "Total RNA was isolated with TRIzol ( Invitrogen ) or Maxwell 16 LEV RNA FFPE Kit for automated RNA isolation ( Promega , Madison , WI ) .", "Approximately 500 ng of total RNA , with a RNA integrity number of at least 7 . 5 ( mostly above 9 . 0 ) , was used for library preparation with Illumina TruSeq RNA Sample Prep Kit v2 ( for E12 . 5 and E14 . 5 samples ) or TrueSeq Stranded mRNA LT ( E13 . 5 ) .", "For the E12 . 5 and E14 . 5 samples , eight libraries were sequenced in two lanes with HiSeq2000 ( Illumina , San Diego , CA ) using 50-base single end sequencing chemistry .", "For the E13 . 5 samples , 21 of them were pooled and run on NextSeq500 ( Illumina ) high output flow cell using 75-cycle single end sequencing chemistry .", "RNA-seq raw data have been deposited in the Gene Expression Omnibus under accession codes GSE87104 .", "For expression quantification , RNA-seq data were mapped to reference genome sequences of mouse ( GRCm38 ) and human ( GRCh38 ) with STAR version 2 . 42a ( Dobin et al . , 2013 ) .", "Gene annotation files from GENCODE ( Harrow et al . , 2006 ) were used for mouse ( vM5 ) and human ( V22 ) .", "Resulted BAM files were used to generate gene counts with featureCount ( Liao et al . , 2014 ) using the uniq-counting mode .", "Differential expression analysis was performed by DESeq2 ( Love et al . , 2014 ) .", "Batch effects from library preparation and sequencing were removed using the ruv R package ( Risso et al . , 2014 ) .", "Pathway and functional analysis of gene lists were performed using the Go-Elite ( Zambon et al . , 2012 ) .", "To examine gene expression in aRG in human and mouse neocortex , FASTQ files were obtained using the fastq-dump program of the SRA toolkit from the Gene Expression Omnibus , under the accession numbers GSE30765 , GSE38805 , GSE65000 , and GSE66217 .", "These datasets contained 13 human and 21 mouse RNA-seq samples of the VZ or aRG .", "Sequencing reads mapping , expression quantification and differentiation analysis were performed as described above .", "Orthologous genes were downloaded from Ensembl release 85 .", "Human-mouse orthologs were defined as single-copy genes conserved in human and mouse .", "Because of different efficiency of mRNA enrichment among the different studies , rRNA and mitochondria genes were excluded resulting in a total 16 , 036 features ( Ensembl ID ) .", "To assess the overall similarity between samples , we first transformed the count data into gene expression matrix using the varianceStabilizingTransformationby function of DESeq2 ( Love et al . , 2014 ) , and used the R function dist to determine the Euclidean distance between samples ( Figure 3—figure supplement 1A ) and the plotPCA function of DESeq2 to show the sample relationship ( Figure 3—figure supplement 1B ) .", "To perform GSEA ( Subramanian et al . , 2005 ) , expression matrix was generated by variance stabilizing transformation of the RNA-seq count data using the DESeq2 package ( Love et al . , 2014 ) .", "A comprehensive and monthly-updated gene-set containing all mouse pathways was downloaded from Bader lab ( http://download . baderlab . org/EM_Genesets/current_release/ ) ( October_01_2016 version ) .", "We added the BG-specific genes and ERK responding genes to the gene lists .", "GSEA was done using gene shuffling for P value estimated with 1000 permutations .", "Results of GSEA are listed in in Supplementary file 4A .", "Microarray data of time course study of ERK activation ( Hamilton and Brickman , 2014 ) were retrieved from the Gene Expression Omnibus using GEOquery ( Davis and Meltzer , 2007 ) under the accession number GSE59755 .", "Technical sources of variation were removed with ComBat function of the sva package ( Leek and Storey , 2007 ) .", "Differentially expressed genes with two fold changes at different time points relating to time 0 was identified with the limma package ( Ritchie et al . , 2015 ) .", "Results of differential gene expression analysis are shown in Supplementary file 3 .", "Expression data from E13 . 5 to P56 were automatically batch downloaded from the Image Download Service of Allen Brain Institute .", "BG-specific expression was manually confirmed using the following criteria .", "As BG are interlocked with Purkinje cells in the Purkinje cell layer ( PCL ) , genes without any detectable signals in the PCL were scored as 0 .", "For those with signals in PCL , genes that are specific to Purkinje cells , which were identified by their bigger , round and distinct soma , were scored as 1 , those with indistinguishable between BG and Purkinje cells as 2 , those that are specific to BG , which were identified by their smaller and irregular soma , as well as their radial projections around the Purkinje cells into the molecular cell layer , as 3 ( Figure 1—figure supplement 2 ) .", "Scoring was performed by an examiner blinded to gene symbols .", "Fisher's exact test was performed to determine if BG-specific genes were significantly enriched in the bRG- and BG-specific gene lists compared with the randomly selected gene of comparable numbers .", "Gene expression matrix was generated by variance stabilizing transformation of the RNA-seq count data using the DESeq2 package ( Love et al . , 2014 ) .", "Expression data were normalized for batch effect ( Leek , 2014 ) and outlier removal ( Z . K less than –2 ) using SampleNetwork R function ( Oldham et al . , 2008 ) .", "From these processed expression data , we followed the protocols of WGCNA ( Zhang and Horvath , 2005 ) to create a gene co-expression network .", "Modules were defined as branches of a hierarchical cluster tree using the top-down dynamic tree cut method ( Langfelder et al . , 2008 ) .", "For each module , the expression patterns were summarized by the module eigengene ( ME ) , defined as the singular vector of the standardized expression patterns .", "Pairs of modules with high module eigengene correlations ( R > 0 . 85 ) were merged .", "The module membership ( kME ) for each gene with respect to each module was then defined as the Pearson correlation between the expression level of the gene and the module eigengene ( Oldham et al . , 2008 ) .", "To study the preservation of human and mouse cortical coexpression networks , we combined available RNA-seq data from all cortical cell types ( Ayoub et al . , 2011; Fietz et al . , 2012; Florio et al . , 2015; Johnson et al . , 2015 ) .", "After careful filtering and preprocessing of the data to remove batch effects and outliers ( Oldham et al . , 2012 ) , 37 human ( WG13-18 ) and 40 mouse ( E14 . 5 ) samples were included .", "Between the human and mouse datasets , both gene expression and connectivity were highly preserved ( R = 0 . 78 , p<1 × 10−200 for expression; R = 0 . 33 , p<1 × 10−200 for connectivity ) , indicating that the cross-species datasets are well matched .", "We used the modulePreservation function in R ( Langfelder et al . , 2011 ) to study the preservation of 11 signaling pathways ( KEGG and Reactome ) , pan-RG signature genes ( Lui et al . , 2014 ) , early-response genes induced by ERK , and signature genes for aRG , bRG , and IPC .", "The complete results are shown in Supplementary file 4B .", "In utero electroporation was performed as described previously ( Shimogori and Ogawa , 2008 ) .", "Briefly , 1–2 µl of plasmid DNA ( 1 . 5 µg/µl ) were injected into the lateral ventricles of E14 . 5 brains and electroporated using four pulses at 40 V for 50 ms at 100 ms intervals through the uterine wall using a BTX ElectroSquarePorator ( BTX , Holliston , MA , ECM 830 ) .", "Ex vivo electroporation was performed in E13 . 5 embryos .", "Briefly , embryos were placed in ice cold Krebs buffer containing 126 mM NaCl , 2 . 5 mM KCl , 1 . 2 mM NaH2PO4 , 1 . 2 mM MgCl2 , 2 . 5 mM CaCl2 , 11 mM glucose , and 25 mM NaHCO4 .", "Ptpn11-cKO embryos were identified based on truncation of the tectum ( Li et al . , 2014 ) , and DNA solution was injected into the VI ventricle .", "To increase the efficiency of the experiment , both sides of the cerebellar anlagen were electroporated by rotating the orientation of the electrode using five pulses at 60 V for 50 ms at 100 ms intervals .", "After electroporation , brains were dissected and embedded in 4% low-melting agarose ( Seakem , VWR International , Radnor , PA ) .", "Sagittal brain slices in 300 µm thickness were prepared on a vibratome ( Leica , VT1000S ) , and kept in cold Krebs buffer on ice , and the sections were transferred to serum medium ( Invitrogen , MEM with glutamine , 10% fetal calf serum , 0 . 5% glucose and penicillin/streptomycin antibiotics ) .", "After 15 min , the sections were transferred to polycarbonate culture membranes ( Whatman™ 13mm Nuclepore™ , Fisher Scientific ) in Falcon organ tissue culture dishes containing 1 ml of Neurobasal/B-27 medium ( Neurobasal with 1x glutamine , 1% B-27 , 0 . 5% glucose and penicillin/streptomycin antibiotics ) .", "They were subsequently incubated at 5% CO2 and 37°C for 48 hr .", "After incubation , slices were fixed in 4% paraformaldehyde/phosphate buffered saline , washed in phosphate buffered saline , embedded in Tissue-Plus ( ThermoFisher Scientific ) , and sectioned in a Cryostat ( Leica , CM3050S ) .", "The sections were subjected to standard in situ hybridization and immunofluorescence procedures .", "The full length cDNA for Mek1DD , Etv4 , Etv5 , and FGFR1K656E was cloned into the pMES expression vector ( Xiong et al . , 2009 ) , placing upstream of an internal ribosomal entry site ( ires ) and the cDNA encoding enhance green fluorescent protein ( EGFP ) .", "The expression cassette is under the control of cytomegalovirus early enhancer/chicken β-actin ( CAG ) promoters .", "Pair-cell analysis was performed as described ( Shen et al . , 2002 ) .", "Following electroporation at E14 . 5 as described above , electroporated brains at E16 . 5 and E17 . 5 were sectioned using a vibratome ( Leica , VT1000S ) .", "Cortical tissues that were enriched for transferred cells ( marked by GFP ) were dissected with tungsten needles from cortical sections .", "For the cortex transfected with Mek1DD , the upper and lower half of the cortex were further separated .", "To dissociate cells , dissected tissues were incubated in a protease solution containing 10 unit/ml papain ( Fluka , Japan ) , 1000 units/ml DNAse I ( Roche , Switzerland ) and 5 mM L-cysteine in DMEM ( Invitrogen ) , and triturated using a fire-polished Pasteur pipette to create a single-cell suspension .", "Cells were resuspended in culture medium containing DMEM , glutamine , penicillin/streptomycin , sodium pyruvate ( Invitrogen ) , 1 mM N-acetyl-L-cysteine ( Sigma ) , B27 , N2 and 10 ng/ml bFGF2 ( Invitrogen ) and plated onto coverslips coated with poly-L-lysine ( Sigma ) at clonal density .", "The cultures were maintained in a humidified incubator at 37°C with constant 5% CO2 supply .", "In 24 or 48 hr later , the cultures were fixed and immunostained for GFP , Fabp7 , and Tubb3 and counterstained with the DNA dye Hoechst 33342 solution ( Invitrogen ) .", "To determine the co-localization of markers , we divided the dorsolateral cortex into six bins paralleling to the ventricular surface with the sixth bin immediate above the ventricular zone .", "Images of each bin were automatically produced by ImageJ software , and the images were randomized and coded .", "The number of Sox2+ nuclei in each bin was counted with ImageJ .", "The numbers of GFP+ and GFP+/Hopx+ cells in each bin were counted manually with the examiner blinded to relevant variables , such as DNA constructs and bin numbers .", "Data processing , statistical analysis and plotting were performed in R version 3 . 2 . 5 .", "An unpaired two-tailed t-test with Welch’s correction or Student's t-test was used for analysis of experiments involved two groups .", "One-way ANOVA followed by Turkey-Kramer multiple comparison test was used for analysis of experiments involving more than two groups with one comparison .", "Bartlett's test was performed to verify equal variance assumption before ANOVA and Student's t-test .", "Reproducible results were obtained from three or more samples , and quantitative data are expressed as means ± standard error of the mean ( SEM ) ." ] ]

[ "Neocortical basal radial glia ( bRG ) and cerebellar Bergmann glia ( BG ) are basal progenitors derived from ventricular apical radial glia ( aRG ) that selectively lose their apical processes .", "bRG and BG have been implicated in the expansion and folding of the cerebrum and cerebellum , respectively .", "Here , we analyzed the molecular characteristics and development of bRG and BG .", "Transcriptomic comparison revealed striking similarity of the molecular features of bRG and BG .", "We found that heightened ERK signaling activity in aRG is tightly linked to the temporal formation and the relative abundance of bRG in human and mouse cortices .", "Forced activation of an FGF-ERK-ETV axis that is crucial to BG induction specifically induced bRG with canonical human bRG features in mice .", "Therefore , our data point to a common mechanism of bRG and BG generation , bearing implications to the role for these basal progenitors in the evolution of cortical folding of the cerebrum and cerebellum ." ]

[ "The outer layer of the brain of a mammal , called the cortex , helps support mental abilities such as memory , attention and thought .", "In rodents , the cortex is smooth whereas in primates it is organized into folds .", "These folds increase the surface area of the brain and thus the number of neurons it can contain , which may in turn increase its processing power .", "Folding occurs as the brain develops in the womb .", "Specialized cells called basal or outer radial glia , which are more abundant in humans than in rodents , are believed to trigger the folding process .", "Another area of the brain , called the cerebellum , is intricately folded in both rodents and humans .", "As the brain develops , cells within the cerebellum called Bergmann glia cause the tissue to fold .", "Bergmann glia and basal radial glia share a number of similarities , but it was not known whether the same molecular pathway might regulate both types of cell .", "Now , Heng et al . show that Bergmann glia in the cerebellums of mice and basal radial glia in human cortex contain similar sets of active genes .", "Moreover , the molecular pathway that gives rise to Bergmann glia in mice is also active in the cortex of both mice and humans .", "However , it is much more active in humans , leading Heng et al . to speculate that high levels of activity in this pathway might give rise to basal radial glia .", "Consistent with this prediction , artificially activating the pathway at high levels in mouse cortex triggered the formation of basal radial glia in mice too .", "These results thus suggest that a common mechanism generates both types of glial cells involved in brain folding .", "The work of Heng et al . lays the foundations for further studies into how these cells fold the brain and thus how they contribute to more complex mental abilities .", "Remaining questions to address are whether other species with Bergmann glia also have folded cerebellums , and whether incorrect development of basal radial glia in humans leads to disorders in which the cortex folds abnormally ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "stem cells and regenerative medicine", "cell biology" ]

[ [ "Cellular senescence was previously identified as a process that permanently halts the proliferation of normal cells in culture following replicative exhaustion ( Hayflick and Moorhead , 1961 ) .", "It subsequently became clear that cellular senescence is a stress response that acts both in culture and in vivo to prevent proliferation of cells exposed to oncogenic stress , such as telomere attrition , and various types of DNA damage and oncogene insertions ( Serrano et al . , 1997; Bodnar et al . , 1998; d'Adda di Fagagna , 2008 ) .", "It therefore acts as an effective anti-tumourigenic mechanism ( Braig et al . , 2005; Chen et al . , 2005; Collado et al . , 2005 ) .", "However , senescent cells can also have detrimental effects on biological processes .", "Long-term accumulation of senescent cells leads to disruption of tissue structure and function , possibly through the acquisition of a senescence-associated secretory phenotype ( Campisi , 2005; Campisi et al . , 2011 ) .", "This is particularly relevant in the context of ageing , as in most species there is a marked accumulation of senescent cells with time ( Herbig et al . , 2006; Wang et al . , 2009; van Deursen , 2014 ) .", "Indeed , recent studies have uncovered a causative link between cellular senescence and age-related deterioration ( Baker et al . , 2008 , 2011 , 2013 ) , underscoring the therapeutic benefits of targeting senescent cells ( Naylor et al . , 2013; van Deursen , 2014 ) , and establishing cellular senescence as a hallmark of ageing ( Lopez-Otin et al . , 2013 ) .", "In mammals , the ability to regenerate tissues declines with age ( Sousa-Victor et al . , 2014 ) .", "The decline in muscle regeneration with age has recently been linked to the loss of quiescent stem cells through senescence ( Sousa-Victor et al . , 2014 ) .", "In contrast , other vertebrates , such as zebrafish and salamanders , are able to accomplish perfect regeneration of a wide range of complex structures throughout their lifespan ( Margotta et al . , 2002; Azevedo et al . , 2011; Eguchi et al . , 2011; Itou et al . , 2012 ) .", "It is therefore possible that these organisms exhibit mechanisms to curtail cellular senescence , thus allowing them to undergo indefinite rounds of regeneration .", "However , the occurrence and regulation of cellular senescence during regeneration have not been addressed in such regeneration-competent species .", "Here , we analysed the process of cellular senescence in salamanders .", "Our results demonstrate that cellular senescence is a recurrent process during salamander limb regeneration , and is subject to a highly efficient mechanism of macrophage-dependent elimination ." ], [ "In order to determine whether cellular senescence occurs in vivo within normal and regenerating salamander tissues , we adapted the SA-βgal staining to detect senescent cells within salamander tissues and addressed whether senescence occurs during limb regeneration in an adult salamander , the newt ( Notophthalmus viridescens ) .", "We found that there is a significant induction of cellular senescence during the intermediate stages of regeneration ( mid-blastema and palette ) , but that the number of senescent cells diminishes subsequently ( Figure 2A , B ) .", "Within the regenerating limb , senescent cells are found at the level of the amputation plane , including cartilage and muscle , and within the blastema , including fibroblast , monocyte-like cells , and epidermal glands ( Figure 2—figure supplement 1 ) .", "To corroborate that SA-βgal+ cells exhibit other hallmarks of cellular senescence in vivo , we performed SA-βgal/EdU stainings in regenerating or normal tissues following systemic EdU injections .", "We were unable to detect SA-βgal+/EdU+ cells under any condition examined ( Figure 2C ) , suggesting that SA-βgal+ cells are withdrawn from the cell cycle , a characteristic of senescent cells . 10 . 7554/eLife . 05505 . 004Figure 2 . Induction of cellular senescence during salamander limb regeneration .", "( A ) Induction and disappearance of senescent cells during salamander limb regeneration as indicated by SA-β-gal staining of newt tissues .", "dpa: days post amputation .", "( B ) Quantification of senescent cells during key stages of limb regeneration following SA-β-gal/Hoescht staining ( n = 8; *p < 0 . 05 , **p < 0 . 01 , ***p < 0 . 005 ) .", "( C ) Representative image of SA-β-gal/EdU co-staining of newt blastemas at 14 dpa , 48 hr after EdU administration .", "No SA-β-gal+/EdU+ cells are found ( n = 6 ) .", "Scale bar: 40 µM .", "( D ) Induction and disappearance of senescent cells during axolotl limb regeneration as indicated by SA-β-gal staining ( blue ) .", "dpa: days post amputation .", "Scale bar: 200 µm .", "( E ) Cellular senescence is specifically induced during salamander limb regeneration , but not limb development .", "Representative SA-β-gal staining ( n = 5 ) of a developing limb ( left ) and its contralateral counterpart following amputation and subsequent regeneration of the limb bud ( right ) .", "Scale bar: 100 µm .", "( F ) Repetitive amputation rounds do not lead to an increase in cellular senescence in regenerated limbs .", "Representative images SA-β-gal staining in a newt limb following 5 regeneration rounds compared to its contralateral intact limb ( n = 6 ) .", "Scale bar: 100 µm .", "( G ) The percentage of senescent cells does not increase with ageing in salamander tissues .", "Quantification of senescent cells following SA-β-gal/Hoescht staining ( n = 6 ) .", "The percentage of senescent cells in tissues of mature axolotls ( 1 and 3 year-old ) and adult newts is not significantly different ( NS ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 05505 . 00410 . 7554/eLife . 05505 . 005Figure 2—figure supplement 1 . Distribution of senescent cells during limb regeneration .", "( A ) Representative images of different structures of a regenerating limb ( wound epidermis and blastema mesenchyme , muscle and cartilage ) following SA-β-gal staining .", "Scale bar: 400 µM . DOI: http://dx . doi . org/10 . 7554/eLife . 05505 . 00510 . 7554/eLife . 05505 . 006Figure 2—figure supplement 2 . Senescence is induced specifically during regeneration but not development of the salamander limb .", "( A , B )", "Representative SA-β-gal staining of axolotl limb sections during different stages of development ( A ) or regeneration during development ( B ) .", "Scale bar: 200 µM . DOI: http://dx . doi . org/10 . 7554/eLife . 05505 . 00610 . 7554/eLife . 05505 . 007Figure 2—figure supplement 3 . Cellular senescence does not increase with ageing in salamanders .", "( A ) Representative images of different axolotl tissues upon ageing following SA-β-gal staining ( blue ) .", "Scale bar: 200 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 05505 . 007 Similar observations were made in another salamander species , the axolotl ( Ambystoma mexicanum ) , both in mature animals as well as at early stages of development ( Figure 2D , E ) .", "Interestingly , the induction and subsequent disappearance of senescent cells appear to be characteristic of regeneration , as no significant induction of senescence takes place during normal limb development ( Figure 2E , Figure 2—figure supplement 2 ) , in contrast to previous findings in amniotic limb development ( Banito and Lowe , 2013; Munoz-Espin et al . , 2013; Storer et al . , 2013 ) .", "These observations suggest that cellular senescence is a normal process during salamander limb regeneration , and is subject to dynamic regulation .", "Salamanders have the remarkable ability to undergo multiple rounds of regeneration through their lives ( Eguchi et al . , 2011 ) .", "Given the proportion of senescent cells induced during each regeneration cycle ( 7% of total cells at the mid-blastema stage ) , it seemed possible that senescent cells could accumulate following multiple rounds .", "However , we found that the percentage of senescent cells does not increase following sequential amputation–regeneration cycles .", "The tissues of adult newts that had been subjected to five regeneration cycles over 1 . 5 years did not show any accumulation of senescent cells compared to intact limbs ( Figure 2F ) .", "This data suggests that elimination of senescent cells occurs during each round of limb regeneration .", "The proportion of senescent cells within tissues is known to increase as the organism matures in most species studied so far , including mammals ( Herbig et al . , 2006; van Deursen , 2014 ) .", "Remarkably , we found that the percentage of senescent cells in heart , spleen and liver does not increase in older salamanders ( Figure 2G , Figure 2—figure supplement 3 ) ; the latter two organs are known to accumulate senescent cells in mammals as they age ( Herbig et al . , 2006; van Deursen , 2014 ) .", "Together , these findings raise the possibility that active mechanisms of senescent cell clearance operate within normal and regenerating salamander tissues .", "To test this possibility , we implanted one thousand senescent or normal cells ( labelled with nuclear GFP expressed from an integrated retrovirus ) within newt limb tissues and analysed their persistence over time ( Figure 3A ) .", "After an initial non-specific loss of cells following implantation , the normal cells ( A1 GFP+ ) persist for at least 40 days and contribute to structures within the limb tissues ( Figure 3A , B ) .", "In contrast , 80% of the implanted senescent cell population is cleared from the limb tissues within 2 weeks post-implantation ( Figure 3A , B ) , consistent with a half-life of 9 days ( calculated from the exponential equation corresponding to the senescent dataset , after subtraction of the initial loss observed in normal cells ) , comparable to the rate of clearance of senescent cells during regeneration ( Figure 2 ) .", "Although this experiment suggests that senescent cells are being cleared from salamander tissues , it cannot exclude the possibility that some cells may reverse their senescence state .", "To distinguish between these two possibilities , we performed the same experiment using retrovirally labelled nGFP senescent cells ( Figure 3B and Figure 3—figure supplement 1 ) .", "The rate of clearance of nGFP+ senescent cells corresponds to the rate of clearance of unlabelled senescent cells , supporting the clearance hypothesis .", "In contrast , the implantation of a population of nGFP cells exposed to a UV dose of 1J/m2 , 10 times lower than the dose required for senescence induction ( Figure 3—figure supplement 1 ) , results in persistence of the implanted cells ( Figure 3B ) .", "These data suggest that salamander tissues have a significant capacity for senescent cell clearance . 10 . 7554/eLife . 05505 . 008Figure 3 . Active mechanisms of senescent cell clearance operate in salamander tissues .", "( A ) Schematic representation of the implantation experiment .", "1000 UV-induced senescent or nGFP+ non-senescent cells were implanted within the left or right newt forelimbs , respectively , and analysed at different weeks post-implantation ( wpi ) by SA-b-gal staining or immunofluorescence ( below ) .", "Note the complete clearance of senescent cells at 4wpi .", "Scale bar: 100 µm .", "( B ) Dynamics of senescent cell clearance within adult newt limbs as described in A as shown by a quantification of total cells remaining within entire limbs at different days post implantation ( dpi , n = 12 ) .", "In addition to senescent and nGFP+ control cells , the dynamic of cell clearance was evaluated following implantation of nGFP+ senescent cells , nGFP+ UV-irradiated ( 1J/m2 ) cells and a 1:1 mixture of nGFP+ control and senescent cells ( nGFP:sen ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 05505 . 00810 . 7554/eLife . 05505 . 009Figure 3—figure supplement 1 . Distribution of senescent cells during limb regeneration .", "( A ) Quantification of SA-β-gal positive cells after staining of control and UV ( 10J/m2 or 1J/m2 ) irradiated nGFP+ cells ( 12 days post irradiation ) .", "( B ) Quantification of DHR123 ( ROS ) positive cells after staining of control and UV ( 10J/m2 or 1J/m2 ) irradiated nGFP+ cells ( 12 days post irradiation ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 05505 . 009 Interestingly , the implantation of a 1:1 mixture of senescent and normal cells does not result in the persistence of the normal cells , but in the clearance of both populations ( Figure 3B , Figure 4A , B ) , albeit with a delay when compared to the clearance of purely senescent cells .", "It has been recently shown that senescent cells are able to induce senescence in their neighbours , a phenomenon termed ‘senescence bystander effect’ ( Nelson et al . , 2012; Acosta et al . , 2013 ) .", "We found that this property is also present in senescent salamander cells .", "Cellular senescence can be induced in normal cells by co-culture with senescent cells ( Figure 4C ) , or following incubation with purified media from senescent populations ( Figure 4D ) , supporting the idea that senescence can be transmitted in a paracrine fashion .", "In addition , implantation of the 1:1 mixture of senescent + normal cells results in the induction of senescence in normal cells ( Figure 4E , F ) , providing an explanation for their eventual clearance .", "Together , these results suggest that active mechanisms identify and target senescent cells for clearance within salamander tissues . 10 . 7554/eLife . 05505 . 010Figure 4 . A senescence bystander effect in salamander cells and tissues .", "( A ) Schematic representation: implantation of a 1:1 mixture of senescent and non-senescent ( nGFP+ ) salamander cells within limb tissues .", "Scale bar: 50 µM .", "( B ) Both senescent and non-senescent cell populations are cleared from newt limb tissues following implantation .", "Scale bar: 100 µM .", "( C ) Co-culturing of senescent and non-senescent ( GFP+ ) cells promotes cellular senescence in the non-senescent population -circled areas- .", "Scale bar: 50 µM .", "( D ) Incubation of proliferating salamander cells with growth media derived from senescent ( blue ) but not normal ( red ) cells induces cellular senescence .", "Scale bar: 100 µM .", "( E ) Co-implantation of normal and senescent cells leads to the induction of senescence in the non-senescent ( GFP+ ) cell population -circled areas- .", "( F ) Percentage of SAβgal+ nGFP+ cells when co-implanted with ( nGFP- ) senescent or normal cells at different times post implantation .", "( n = 3 , **p < 0 . 01 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 05505 . 010 A series of elegant experiments have recently described a role for the immune system in the surveillance and clearance of p53 or oncogene-induced senescent hepatocytes in mice ( Xue et al . , 2007; Kang et al . , 2011; Lujambio et al . , 2013 ) .", "In addition , recent evidence suggests that there is an extensive recruitment of immune cells , and in particular macrophages , to the regenerating limb in salamanders ( Godwin et al . , 2013 ) .", "Hence , we decided to investigate if the macrophage was part of the senescence clearance mechanism .", "In vivo labelling using TMR-dextran , which is specifically internalised by macrophages ( Figure 5A–C ) , as well as monocyte specific α-naphthyl acetate esterase staining ( Figure 5D ) , reveal that macrophages and senescent cells are found in close proximity within regenerating limbs , suggesting cell-to-cell contact and engulfment in some cases .", "To determine whether macrophages mediate the clearance of senescent cells , we took advantage of an efficient method for macrophage depletion , based on the intravenous administration of selectively toxic , clodronate salt-filled liposomes ( clodrosomes ) .", "These are internalised specifically by macrophages via phagocytosis ( van Rooijen and Hendrikx , 2010 ) and have been shown to operate effectively in salamanders ( Godwin et al . , 2013 ) .", "We found that treatment with clodrosomes is able to reduce the macrophage population by fivefold , compared with control DiI liposomes ( Figure 6A ) .", "As with the TMR-dextran , these liposomes are specifically internalised by macrophages , as shown by their incorporation into cells expressing F4/80 , a specific macrophage marker ( Morris et al . , 1991 ) ( Figure 6B ) .", "We therefore used this system to induce systemic macrophage depletion in salamanders prior to the implantation of senescent or normal nGFP+ cells in contralateral regenerating limbs , and followed the fate of the implanted cells with time ( Figure 6C ) .", "At 20 hr post implantation , we observed a strong accumulation of DiI-labelled macrophages in the vicinity of senescent cells ( Figure 6D–F ) .", "In contrast , macrophage recruitment to areas of normal cells ( control ) was not significantly different from their recruitment to other areas of the limb mesenchyme ( Figure 6D , F ) .", "At 2 weeks post implantation , the normal cells remained within the regenerating limbs ( Figure 6G ) .", "However , the senescent cells were cleared in animals injected with control DiI liposomes , but remained in animals whose macrophages had been depleted ( Figure 6G ) .", "This demonstrates that the macrophage is critical for clearance of senescent cells , and constitutes the first evidence for senescence surveillance mechanisms that operate during normal regeneration . 10 . 7554/eLife . 05505 . 011Figure 5 . Macrophages are recruited to senescent cells within salamander tissues .", "( A ) Representative images of axolotl limbs and gills , 24 hr after i . v . injection of TMR-dextran -left panels- or PBS -right panels- .", "Scale bar: 200 µM .", "( B ) TMR-dextran incorporating cells stain positive for the macrophage marker α-napthtyl esterase ( NAE ) .", "( C , D )", "Macrophages ( TMR+ -red- in A; NAE+ , brown in B ) are recruited to sites of senescent cells ( Sa-β-gal+ , blue ) within regenerating limb tissues .", "Scale bar: µm . DOI: http://dx . doi . org/10 . 7554/eLife . 05505 . 01110 . 7554/eLife . 05505 . 012Figure 6 . Macrophages mediate the efficient clearance of senescent cells during salamander limb regeneration .", "( A ) A clodronate-dependent system for effective macrophage depletion in salamanders .", "Representative images of axolotl limb tissues 36 hr post i . v . injection with fluorescent DiI-liposomes ( red ) or DiI + clodronate-liposomes .", "Scale bar: 1000 µm .", "( B ) DiI-liposomes are incorporated specifically by macrophages .", "Blood cells were extracted from treated animals and stained with antibodies against the specific macrophage marker F4/80 .", "Scale bar: 50 µM .", "( C ) Schematic representation of the implantation experiment .", "Axolotls with limbs at the midbud stage of regeneration were injected i . v . with either DiI-liposomes or DiI + clodronate ( clodro ) - liposomes , 36 hr prior to implantation of normal or senescent nGFP+ cells into contralateral regenerating limbs .", "Liposome treatments were maintained for 2 weeks .", "( D ) Macrophage ( DiI , red ) recruitment to areas of senescent cells 12 hr following implantation of normal and senescent GFP+ cells within regenerating axolotl limbs .", "Scale bar: 1000 µm .", "( E ) DiI-labelled macrophages ( red ) are recruited to sites of implanted nGFP+ senescent cells ( green ) within regenerating limbs at 16 hs post implantation .", "Scale bar: 100 µm .", "Note that in the DiI-liposome treated animals macrophages are recruited to sited of senescent but not normal cells .", "( F ) Quantification of macrophage ( DiI-labelled ) recruitment to implantation sites at 16 hpi .", "Note macrophage depletion induced by clodronate-liposome treatment .", "( G ) Clearance of senescent cells is impaired upon macrophage depletion .", "Quantification of GFP+ cells after 2wpi in DiI-liposome or clodronate-liposome treated animals . DOI: http://dx . doi . org/10 . 7554/eLife . 05505 . 012" ], [ "Adult newts ( N . viridescens ) and axolotls ( A . mexicanum ) were used in this study , in compliance with the Animals ( Scientific Procedures ) Act 1986 , approved by the United Kingdom Home Office .", "SA-β-gal activity was determined in cultured cells or tissues fixed with 0 . 5% glutaraldehyde , using the SA-βgal kit ( Cell Signalling ) according to the manufacturer's instructions .", "Immunofluorescence staining , EdU and BrdU labelling of tissues or cultured cells were performed according to standard protocols .", "Fluorescence and bright-field imaging was performed using a Zeiss Axioscope ( Zeiss ) ." ], [ "Procedures for care and manipulation of all animals used in this study were performed in compliance with the Animals ( Scientific Procedures ) Act 1986 , approved by the United Kingdom Home Office .", "Adult newts ( N . viridescens ) were obtained from Charles Sullivan and Co . ( Tennessee , USA ) and maintained as described elsewhere ( Ferretti and Brockes , 1988 ) .", "Axolotls ( A . mexicanum ) were obtained from Neil Hardy Aquatica ( Croydon , UK ) and maintained in individual aquaria at approximately 18°C .", "Newts and axolotls were anesthetised in 0 . 1% tricaine prior to amputation at the mid-humerus level .", "Animals were allowed to regenerate at 20°C .", "For repetitive amputation–regeneration cycles , animals were allowed to achieve full limb regeneration prior to re-amputation .", "Axolotl and newt regeneration stages were defined as previously described ( Iten and Bryant , 1973; Tank et al . , 1976 ) .", "A1 cells were previously derived from newt limb mesenchyme ( Ferretti and Brockes , 1988 ) .", "A1 nGFP+ cells were obtained by stable transfection with a pseudotyped retroviral VSV-G vector as described below .", "Cells were grown on 0 . 75% gelatin-coated plastic dishes in MEM ( Gibco , UK ) supplemented with 10% heat-inactivated foetal calf serum ( FCS , Gibco ) , 25% H2O , 2 nM L-Glutamine ( Gibco ) , 10 µg/ml insulin ( Sigma , St Louis , MO ) and 100U/ml penicillin/streptomycin ( Gibco ) in a humidified atmosphere of 2 . 5% CO2 at 25°C .", "Cell subculture was performed as previously ( Lo et al . , 1993 ) .", "Human 293GP cells ( Burns et al . , 1993 ) were propagated in DMEM ( Gibco ) supplemented with 10% heat-inactivated foetal calf serum ( FCS , Gibco ) .", "Pseudotyped retroviral vectors were generated essentially as described ( Yee et al . , 1994 ) .", "293GP cells containing the gag and pol MoMLV genes were cotransfected by standard calcium precipitation with the retroviral vector LZRSpBMN-Z—nGFP ( encoding an eGFP protein with a nuclear localisation signal between the BglII and BamHI sites ) and pCMV-VSV-G ( an expression plasmid for Vesicular Stomatitis Virus protein G ) in order to generate pseudotyped retrovirus .", "Medium was collected at 48 , 60 and 72 hr post transfection , filtered through a 0 . 45 mm filter and concentrated 100 fold by ultracentrifugation at 25 , 000 rpm , 1 . 5 hr .", "Stock titers were determined by infecting QT6 cells .", "A1 cells were grown to 30% confluence and infected with pseudotyped retrovirus stocks ( 1–2 × 106 PFU ) in the presence of 8 µg/ml polybrene ( Sigma ) .", "After overnight infection , cells were incubated in fresh culture medium with 2 mg/ml G418 until reaching confluence .", "In order to induce cellular senescence in cultures , cells were washed in amphibian PBS ( A-PBS , PBS plus 25% dH2O ) and exposed to 1 , 3 or 10J/m2 of UV irradiation ( UV Stratalinker ) in 1 ml A-PBS and incubated in growth medium , supplemented with 1 µM ( − ) nutlin3a ( Cayman , Ann Arbor , MI ) in a humidified atmosphere of 2 . 5% CO2 at 25°C .", "Nutlin3a was added to the growth medium in 1 µM increments every 24 hr for the duration of the experiment .", "Growth media was changed after 1 week .", "Cells were then subcultured and senescence induction evaluated using various methods described throughout this manuscript .", "SA-β-gal activity was determined in 0 . 5% glutaraldehyde fixed cell culture cells using the SA-βgal kit ( Cell Signalling , Danvers , MA ) according to manufacturer's instructions .", "For detection of SA-β-gal in animal tissues , salamander embryos or adult tissues were fixed for 30 min or 1 hr respectively in 0 . 5% glutaraldehyde , washed 3 times in PBS .", "For whole mount staining , salamander embryos or adult tissues ( heart , spleen , liver ) were stained O/N using the SA-β-gal kit ( Cell Signalling ) according to manufacturer's instructions , washed in PBS , fixed in 4%PFA for 4 hr and embedded in Tissue Tek-II .", "Whole-mount samples were then cryosectioned , washed in PBS and mounted in glycerol for imaging .", "Adult intact or regenerating limbs were cryosectioned in Tissue Tek-II , washed in PBS for 15 min and stained O/N using the SA-β-gal kit ( Cell Signalling ) according to manufacturer's instructions .", "Culture medium was cleared by centrifugation and concentrated using Ultracel-3K Amicon Ultra centrifugal tubes ( Merck Millipore Ltd , Germany ) .", "The corresponding cell extracts were prepared by washing cells in PBS and resuspending them in ice-cold 1% NP40 , 150 mM NaCl , 50 mM Tris–HCl , pH 7 . 5 , 1 mM NaVO4 and Protease Inhibitor Cocktail ( Sigma ) , incubating for 30 min at 4°C and clearing the debris by centrifugation .", "Concentrated media and their corresponding cell extracts were analysed using the Proteome Profiler Human Protease and Cytokine Array Kits ( R&D systems , Minneapolis , MN ) as per manufacturer's instructions .", "Antibody arrays were developed in an Odyssey scanner ( LI-COR , Lincoln , NE ) , and protein levels quantified using Image Studio software .", "One Matrix CIM heat maps were built using CIMminer ( NCI-NIH , USA ) .", "For the analysis of DiI liposome incorporation and NAE or NacdE staining of salamander limbs following cell implantation , regenerating or intact limbs were collected by amputation , fixed in 4% ice-cold paraformaldehyde ( PFA ) for 16–18 hr at 4°C , washed twice in PBS and embedded in Tissue Tek-II .", "The blocks were serially sectioned longitudinally in a cryostat ( Leica , UK ) at 12 µm .", "Sections were collected in Superfrost slides and stored at −30°C until use .", "To determine the percentage of cells in S-phase , proliferating , quiescent or senescent A1 cells were sub-cultured and incubated in growth medium supplemented with 5 µM 5-ethynyl-2′-deoxyuridine ( EdU ) for 24 hr .", "Cells were then fixed in glutaraldehyde and SA-β-gal staining performed as described .", "Next , cells were fixed in 4%PFA for 10 min and stained using Click-iT Edu Alexa Fluor 594 Imaging kit ( Life Technologies , UK ) according to manufacturer's instructions .", "To detect EdU incorporation in salamander tissues , 10 mM EdU ( 20 µl per animal ) were administered to regenerating newts by i . p . injection .", "After 48 hr , blastemas were collected , cryosectioned and stained to determine SA-β-gal activity as described .", "Sections were fixed in 4% PFA for 10 min and EdU incorporation determined using Click-iT Edu Alexa Fluor 594 Imaging kit ( Life Technologies ) .", "Cellular senescence was induced in cultured A1 cells by UV irradiation as described above .", "Following verification by O/N SA-β-gal staining , senescent cells were trypsinised , spun at 850 rpm for 10 min and the resulting pellet resuspended in A-PBS .", "Cells were then transferred to a 10 µl Hamilton syringe ( Hamilton , Reno , NV ) with a 301/2 g , 45° tip needle ( Hamilton ) attached to a micromanipulator .", "1000 cells were injected within the mesenchymal tissues of intact or regenerating ( midbud stage ) limbs of newts or axolotls .", "The animals were allowed to recover on a wet tissue for 10 min before transfer to water .", "Successful implantation was verified by fluorescence stereomicroscopy using a Zeiss Axioscope ( Zeiss , Germany ) .", "17 cm axolotls ( 3 year old animals at a reproductive stage ) were anaesthetised in 0 . 1% Tricaine for 20 min , prior to i . v . microinjection of 20 µl DiI or clodronate liposome solutions ( Encapsula Nano Sciences , Brentwood , TN ) as previously described ( Godwin et al . , 2013 ) .", "For long-term depletion experiments , animals were injected every other day 3 times , with a resting period of 3 days before the next injection rounds .", "The same procedure was applied for macrophage labelling using 20 µl 2MDa tetra-methylrhodamine ( TMR ) labelled dextran ( Invitrogen , UK ) .", "All injections were performed 36–48 hr before cell implantation or analysis .", "Successful compound uptake by macrophages was verified by fluorescence stereomicroscopy using a Zeiss Axioscope ( Zeiss ) .", "Granulocytes -including neutrophils- were detected by specific staining of tissue cryosections with the naphtol AS-D chloroacetate esterase kit ( Sigma–Aldrich ) , while monocytes/macrophages were identified using the α-naphtyl acetate esterase ( NAE ) kit ( Sigma–Aldrich ) , as previously described18 .", "For staining of cultured cells , these were fixed in 2% PFA for 1 min , followed by a 5-min incubation in cold 100% methanol and processed as described elsewhere ( Yun et al . , 2014 ) .", "Fixed cell samples were incubated overnight with the following primary antibodies: anti-BrdU ( Sigma; 1:3000 ) , anti-γH2AX ( Upstate , UK; 1:1000 ) , anti-laminB1 ( Abcam , UK; 1:500 ) , and anti-F4/80 ( ABD Serotec , UK; 1:500 ) .", "In all cases , anti-mouse or anti-rabbit AlexaFluor488 and AlexFluor594 antibodies ( Invitrogen; 1:1000 ) were used for secondary staining .", "Hoechst 33258 ( 2 µg/ml ) was used for nuclei counterstaining .", "For detection of ROS production , cells were incubated with 10 µM DHR123 ( Life Technologies , Invitrogen ) for 2 hr at 25°C , fixed and mounted in DAKO fluorescent mounting media .", "Mitochondrial and Lysosomal networks were visualised following incubation of cells with 200 nM MitoTracker red FM and 500 nM LysoTracker red DD99 ( Life Technologies , Invitrogen ) respectively for 1 hr at 25°C .", "Samples were observed under a Zeiss Axiskop2 microscope and images were acquired with a Hamamatsu Orca camera using Openlab ( Improvision ) software .", "Whenever comparative analyses between samples were performed , all images were acquired with identical camera settings and illumination control .", "Image processing ( contrast enhancement ) was equally applied to all matched experimental and control samples using Openlab software .", "Cells were labelled for 2 hr by adding 1 µl/ml 5-bromo-2′-deoxyuridine ( BrdU ) to the growth media .", "Following the corresponding incubation period , cells were fixed in 4% paraformaldehyde for 1 min at RT followed by 100% methanol for 5 min , and stained for bromodeoxyuridine as previously described ( Yun et al . , 2013 ) .", "RNA was isolated from salamander tissue culture cells using Tri Reagent ( Sigma ) and random primed cDNA synthesised using Superscript II ( Invitrogen ) .", "Gene expression was determined by quantitative real time PCR using the following primers: Newt Gadd45-β fwd ( AGGGCACAGGAAAGAAGATG ) ; Newt Gadd45-β rev ( TCATTGTCGCAGCAGAAGG ) ; Newt L-27 fwd ( TACAACCACTTGATGCCA ) ; Newt L-27 rev ( CAGTCTTGTATCGTTCCTCA ) .", "Rt-PCR was carried out using iQ SYBR Green supermix ( Bio-rad , Hercules , CA ) on a Chromo 4 instrument running Opticon 3 software ( Bio-rad ) .", "All reactions were run in triplicate and at least 2 independent RNA preparations were analysed for each sample .", "Newts and axolotls in each sample group were randomly selected .", "Sample group size ( n ) is indicated in each figure legend , while all experiments were carried out in at least three biological replicates .", "Statistical analyses were performed with Prism 4 . 0 software and unpaired two-tailed t tests were applied unless otherwise stated .", "Paired two-tailed t tests were carried out to analyse RT-PCR experiments ." ] ]

[ "Cellular senescence has been recently linked to the promotion of age-related pathologies , including a decline in regenerative capacity .", "While such capacity deteriorates with age in mammals , it remains intact in species such as salamanders , which have an extensive repertoire of regeneration and can undergo multiple episodes through their lifespan .", "Here we show that , surprisingly , there is a significant induction of cellular senescence during salamander limb regeneration , but that rapid and effective mechanisms of senescent cell clearance operate in normal and regenerating tissues .", "Furthermore , the number of senescent cells does not increase upon repetitive amputation or ageing , in contrast to mammals .", "Finally , we identify the macrophage as a critical player in this efficient senescent cell clearance mechanism .", "We propose that effective immunosurveillance of senescent cells in salamanders supports their ability to undergo regeneration throughout their lifespan ." ]

[ "As humans and other mammals get older , they become less able to recover from injury or repair damage to their tissues .", "This happens because mammalian cells gradually lose the ability to divide to produce new cells .", "This process is called senescence and it helps to prevent cancer by stopping old cells that are more likely to carry harmful mutations from replicating .", "However the link between senescence and many age-related declines in human health has led scientists to ask whether targeting senescent cells might be one way to treat age-related conditions .", "Some organisms can regenerate their tissues throughout their lives; and creatures like salamanders are even able to re-grow limbs and organs if they are lost .", "Scientists are eager to learn how these animals are able to do this when humans are not , and answering this and related questions might help us to develop therapies that boost our ability to recover from injury or age-related diseases .", "Yun et al . took a closer look at senescence in salamanders and unexpectedly found that a large number of senescent cells appeared in a salamander limb as it regenerates .", "But , by the time the limb had completely regrown , these senescent cells had disappeared .", "Further experiments revealed that when normal and senescent cells are implanted into a salamander the senescent cells also quickly disappear .", "These findings suggest that senescent cells may possibly play a role in the regeneration process , and that salamanders have a system that can efficiently destroy these cells .", "Previous research had suggested that parts of the immune system , in particular cells called macrophages , help to eliminate senescent cells in some tissues .", "Yun et al . found that macrophages did accumulate around senescent cells in the regenerating limbs of living salamanders .", "And when a toxin was used to destroy the macrophages in some salamanders , the senescent cells were not cleared in the way they were in salamanders with active macrophages .", "Hence , macrophages are an essential part of the mechanism that eliminates senescent cells from salamander tissues .", "This efficient mechanism for the elimination of senescent cells could explain how salamanders are able to maintain their ability to regenerate in spite of ageing .", "These findings also reveal the salamander as a model system that could be used to find new ways to target senescent cells , which could be eventually used in anti-ageing therapies ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "structural biology and molecular biophysics" ]

[ [ "Mitochondria are adaptable cellular organelles critical for a spectrum of essential functions including ATP generation , cell signaling , metabolism , proliferation , and death ( Vyas et al . , 2016 ) .", "This central function causes mitochondria to fulfill a crucial role as mediators of tumorigenesis .", "Mitochondria sense changes in energetic , biosynthesis , and cellular stress , and adapt to the surrounding tumor environment to modulate cancer progression and drug resistance ( Tosatto et al . , 2016; Vyas et al . , 2016 ) .", "One critical function of mitochondria that is poorly understood in cancer cells is the role of these organelles as a major hub for cellular Ca2+ signaling ( De Stefani et al . , 2016; Pathak and Trebak , 2018 ) .", "Essentially all mitochondrial functions are controlled by changes in mitochondrial Ca2+ ( mtCa2+ ) levels .", "Increased mitochondrial Ca2+ uptake stimulates mitochondrial bioenergetics through the activation of Ca2+-dependent dehydrogenases of the tricarboxylic acid ( TCA ) cycle in the mitochondrial matrix ( Hansford , 1994; McCormack et al . , 1990; Montero et al . , 2000 ) .", "In turn , mtCa2+-mediated changes in mitochondrial metabolism can alter the generation of mitochondrial reactive oxygen species ( mtROS ) .", "In addition , through changes in Ca2+ uptake and extrusion , mitochondria shape the spatial and temporal nature of cytosolic Ca2+ signals to regulate downstream gene expression programs ( De Stefani et al . , 2016; Pathak and Trebak , 2018 ) .", "Mitochondria form very close contact sites with the endoplasmic reticulum ( ER ) known as mitochondria-associated membranes ( Booth et al . , 2016 ) .", "These contact sites are hotspots of communication through which Ca2+ release from the ER via inositol-1 , 4 , 5-trisphosphate receptors ( IP3R ) is efficiently transferred to the mitochondrial matrix through the mitochondrial Ca2+ uniporter ( MCU ) channel complex ( Baughman et al . , 2011; Booth et al . , 2016; De Stefani et al . , 2011; Wu et al . , 2018 ) .", "This ensures that the bioenergetic output of the cell is tailored to the strength of cell stimulation by growth factors ( Pathak and Trebak , 2018 ) .", "The major mtCa2+ extrusion route is mediated by the mitochondrial Na+/Ca2+/ Li+ exchanger ( NCLX ) ( Palty et al . , 2010 ) .", "The balance between Ca2+ uptake by MCU and Ca2+ extrusion by NCLX is critical for maintaining mtCa2+ homeostasis , which in turn regulates metabolism and cell fate .", "Perturbations in mtCa2+ homeostasis have been linked to a multitude of diseases including cancer ( De Stefani et al . , 2016; Pathak and Trebak , 2018 ) , and altered mtCa2+ homeostasis has recently emerged as a novel hallmark of cancer cells ( Danese et al . , 2017; Kerkhofs et al . , 2018; Paupe and Prudent , 2018 ) .", "However , the underlying molecular mechanisms by which mtCa2+ regulate cancer progression are still poorly understood .", "In recent studies , altered MCU expression has been reported in cancer cells , and increased expression of MCU and alterations in proteins that regulate MCU channel activity have been associated with increased mtCa2+ and downstream effects that contribute to proliferation and tumor progression ( Koval et al . , 2019; Marchi et al . , 2013; Ren et al . , 2017; Tosatto et al . , 2016; Vultur et al . , 2018 ) .", "In comparison to MCU , NCLX activity is ~100 fold slower , thus NCLX operation is the rate-limiting factor in mtCa2+ homeostasis ( Ben-Kasus Nissim et al . , 2017; Palty et al . , 2010 ) .", "For instance , cardiomyocyte-specific MCU knockout mice have unaltered levels of mitochondrial matrix Ca2+ and no obvious phenotype , whereas cardiomyocyte-specific NCLX knockout mice display mtCa2+ overload and die from sudden cardiac arrest ( Luongo et al . , 2017 ) .", "This suggests that although lack of MCU is compensated for by an alternative pathway , the lack of NCLX is not ( Pathak and Trebak , 2018 ) .", "Given the importance of NCLX as the major extrusion mediator of mitochondrial matrix Ca2+ , it is imperative to understand how aberrant expression of this protein influences mitochondrial and cellular function in cancer .", "However , to date , the role of NCLX in tumor biology has not been directly investigated .", "Colorectal cancer ( CRC ) is the third most commonly diagnosed cancer type and the third leading cause of cancer deaths in both men and women in the United States ( Siegel et al . , 2020 ) .", "Patient diagnosis at an advanced stage with significant metastatic spread is associated with a 5 year overall survival rate in less than 15% of these CRC patients , demonstrating a need to further understand the underlying mechanisms driving CRC metastasis , recurrence , and development of chemoresistance ( Siegel et al . , 2020 ) .", "In order to better classify and inform therapeutic interventions the CRC Subtyping Consortium derived four CRC consensus molecular subtypes ( CMS ) based on comprehensive molecular signatures including mutation , DNA copy number alteration , DNA methylation , microRNA , and proteomics data .", "These four subtypes differ in their metastatic potential and in survival outcomes of patients ( Guinney et al . , 2015 ) .", "For example , CMS4 , which is characterized as mesenchymal , is associated with an epithelial-to-mesenchymal transition ( EMT ) phenotype and poor patient survival statistics .", "In addition , colorectal cancer cell-intrinsic transcriptional signatures ( CRIS ) , which exclude the contribution of tumor-associated stroma , similarly demonstrate the existence of distinct subtypes based on transcriptional profiling ( Dunne et al . , 2017; Isella et al . , 2017 ) .", "Here , we show that the expression of NCLX is significantly downregulated in human colorectal adenocarcinomas .", "We demonstrate that a loss of NCLX decreases mtCa2+ extrusion in CRC cells and that this increase in mtCa2+ has important consequences on colorectal tumor cells: NCLX loss ( 1 ) inhibits proliferation and primary tumor growth , while ( 2 ) enhances metastasis , and drug resistance , suggesting that a loss of NCLX contributes to CRC metastatic progression .", "Importantly , decreased NCLX expression leads to transcriptional changes reminiscent of highly metastatic mesenchymal CMS4 CRC subtype , including increased expression of genes regulating EMT and cancer stemness , and decreased expression of cell-cycle progression mediators .", "Mechanistically , decreased expression or loss of NCLX results in mtCa2+ overload , causing depolarization of mitochondria , increased mtROS production , which drives ROS-dependent HIF1α protein stabilization and pro-metastatic phenotypes of NCLX-low CRC cells .", "Thus , we show a novel dichotomous role of NCLX in cancer , where reduced NCLX function lead to reduced tumor growth , while driving a mesenchymal phenotype that leads to increased metastasis and drug resistance ." ], [ "Using the publicly available Cancer Genome Atlas ( TCGA ) database , we found that NCLX ( SLC8B1 ) mRNA levels were significantly downregulated in both colon and rectal adenocarcinoma ( COADREAD ) tumors as compared to the adjacent normal tissue ( Figure 1A ) .", "Consistent with the TCGA data , we observed a substantial reduction to a near loss in SLC8B1 mRNA in colorectal tumor samples isolated from patients undergoing surgery at Penn State University Medical Center as compared to the paired normal adjacent tissues ( Figure 1B ) .", "There was no difference in SLC8B1 mRNA levels between male and female tumor tissues ( Figure 1—figure supplement 1A ) and between patients bearing mutated and normal proto-oncogene KRAS and phosphoinositide 3-kinases ( PI3K ) ( Figure 1C ) .", "However , low NCLX expression was associated with TP53 mutations and wild type BRAF tumors ( Figure 1D ) .", "TCGA data analysis from UALCAN ( Chandrashekar et al . , 2017 ) revealed that SLC8B1 mRNA was appreciably reduced in CRC patients of all age groups ( Figure 1—figure supplement 1B ) .", "Both adenocarcinoma and mucinous adenocarcinoma had a significant reduction in SLC8B1 mRNA levels as compared to the normal tissue ( Figure 1—figure supplement 1C ) .", "Subsequent analysis revealed a significant loss of NCLX in adenomas with malignant transformation from stage I through stage IV ( Figure 1E ) .", "There was a significant reduction in SLC8B1 mRNA level in late-stage ( stage III and IV ) colorectal tumors as compared to early-stage ( stages I and II ) tumors from the TCGA database ( Figure 1E , F ) , with similar results when we analyzed the patient samples obtained from Penn State University Medical Center ( Figure 1G ) .", "Together , these results show that NCLX expression is significantly downregulated in CRC specimens , and that NCLX loss correlates with late-stage colorectal adenocarcinomas .", "To determine the functional link between loss of NCLX expression and the development of CRC in vivo , we first used global NCLX ( Slc8b1 ) KO ( NCLX KO ) mice that were generated using CRISPR/Cas9 , where 13 nucleotides ( 120513241–120513253 on chromosome 13 , a region coding for five amino acids ) were deleted from the first exon of Slc8b1 , resulting in a frameshift mutation and an early stop codon in exon 2 at nucleotide 248 of the coding sequence ( Figure 1—figure supplement 1D ) .", "This deletion was confirmed by genotyping of genomic DNA using specific primers for wildtype and knockout alleles ( Figure 1—figure supplement 1E ) .", "We then used the NCLX KO mice and their wildtype littermate controls to determine the contribution of NCLX to the development of colorectal tumors in the colitis-associated colorectal cancer model .", "We subjected NCLX KO mice ( n ≥ 30 ) and littermate control mice ( n ≥ 30 ) to one intraperitoneal injection of azoxymethane ( AOM; 100 µl of 1 mg/ml ) and three cycles of dextran sodium sulfate ( DSS; 1 . 5% ) in drinking water with two weeks of normal water between each DSS cycle ( Figure 1H ) .", "At day 78 , mice were sacrificed , and their colorectal tracts were harvested .", "Colorectal tissues from five representative mice from each experimental group are shown in ( Figure 1I ) , and the tissues from fifteen additional mice are depicted in ( Figure 1—figure supplement 1F , G ) .", "Although there is a clear association between NCLX loss and CRC in TCGA data , the colons of NCLX KO mice displayed approximately 50% less tumors than those of littermate control mice ( Figure 1I , J and Figure 1—figure supplement 1F , G ) .", "Further , the tumors that developed in the colons of NCLX KO mice were markedly smaller than those in the colons of littermate control mice , as determined by measurements of tumor size ( Figure 1K ) .", "Histological analyses of colon tissues revealed significantly reduced dysplasia in the colons of NCLX KO mice compared with colons from littermate control mice ( Figure 1L , M ) .", "The DSS/AOM administration in mice is an excellent tumor growth model that is not associated with tumor metastases .", "Therefore , to further investigate the role of NCLX on CRC tumor growth and metastatic spread in vivo , we utilized the human CRC parental cell line HCT116 and its NCLX knockout counterpart in an intrasplenic xenograft model .", "While the overall efficiency of liver metastasis and colonization is low during intrasplenic injection , this model remains nonetheless is a well established method to study metastasis to distant organs such as the colon and liver ( Heijstek et al . , 2005 ) .", "For in vivo studies NCLX KO clone #33 was used , which was generated using a guide RNA ( g1 ) resulting in a single cut at nucleotide 150 in exon one causing a frameshift mutation and introduction of a stop codon at position 180 in the NCLX open reading frame ( Figure 2A ) .", "The HCT116 NCLX KO cells and their control HCT116 counterparts were tagged with luciferase and injected ( 5 × 105 cells/mouse ) in the spleens of two groups of NOD-SCID mice for a total of 15 mice per experimental group ( Figure 2A ) .", "In vivo metastasis to the colon and liver was assessed by monitoring luciferase bioluminescence .", "The mice were injected IP with 100 µl luciferin , and the total flux was measured using the In Vivo Imaging System ( IVIS ) by exposing the mice for 2 min .", "There was a significant reduction in the total luciferase bioluminescence flux in mice xenografted with HCT116 NCLX KO cells by comparison to the HCT116 control-injected mice ( Figure 2B , C ) , indicating that the loss of NCLX in CRC cells caused reduced tumor growth .", "The luciferase flux at 2 , 4 , and 6 weeks from five representative mice per experimental group are shown in ( Figure 2B ) with the remaining 10 mice represented in ( Figure 2—figure supplement 1 ) .", "Similarly , the primary tumor volumes ( at the time of sacrifice , at six weeks ) in the spleens of HCT116 NCLX KO-injected mice were significantly reduced compared to control HCT116-injected mice ( Figure 2D , E ) , consistent with the in vivo tumor growth in the AOM-DSS model .", "Interestingly , SCID mice injected with the HCT116 NCLX KO cells showed strikingly increased metastasis ( Figure 2B ) , specifically to the liver and colon as compared to control HCT116-injected mice at the time of sacrifice ( Week 6; Figure 2F–H ) .", "We did not observe any metastasis to the lung , heart or brain of mice of both groups in this xenograft model .", "Significantly , the SCID mice with intra-splenic injection of HCT116 NCLX KO cells had reduced overall survival compared to mice injected with control HCT116 cells ( Figure 2I ) , suggesting that increased CRC metastasis is the primary cause of lethality in the HCT116 NCLX KO xenograft model .", "Altogether , our results show that loss of NCLX causes reduced primary tumor growth with increased metastatic progression of colorectal cancer .", "To elucidate the mechanisms by which loss of NCLX elicits these seemingly dichotomous functions on CRC , we investigated the effects of CRISPR/Cas9-mediated knockout , and si/shRNA-driven decreases in NCLX expression in HCT116 and DLD1 CRC cell lines ( see Methods and Figure 3—figure supplement 1A–H ) .", "To alleviate potential off-target effects of the CRISPR/Cas9 system , we generated several independent clones obtained with three independent guide RNAs ( gRNAs; see methods ) ( Figure 3—figure supplement 1A–H ) .", "Genome sequencing and PCR on genomic DNA confirmed NCLX KO ( Figure 3—figure supplement 1G ) .", "With the exception of one commercially available polyclonal NCLX antibody ( Ben-Kasus Nissim et al . , 2017 ) that is now discontinued , there are currently no reliable NCLX antibodies .", "All commercially available NCLX antibodies have failed our validation assays and our own attempts to generate a monoclonal antibody against NCLX have not yet produced a reliable clone that detects native levels of NCLX expression .", "We thus resorted to mRNA quantification , and in all three clones of HCT116 NCLX KO cells , RT-qPCR showed complete absence of SLC8B1 mRNA ( Figure 3—figure supplement 1D ) .", "Similarly , NCLX KO #06 , 24 , and 32 of DLD1 cells all had an almost complete deletion of the NCLX open reading frame ( Figure 3—figure supplement 1E ) , which was confirmed by PCR on genomic DNA ( Figure 3—figure supplement 1F , G ) and RT-qPCR quantifying mRNA ( Figure 3—figure supplement 1H ) .", "Since the loss of NCLX reduced tumor size in both AOM-DSS and xenograft models ( Figures 1 and 2 ) , we assessed the effect of reduced NCLX function on the proliferation of CRC cells by CyQUANT proliferation assays .", "A significant reduction in proliferation was observed in NCLX KO clones of both HCT116 and DLD1 cells ( Figure 3A , B ) .", "To rule out the possibility of long-term compensation in NCLX KO clones , we downregulated NCLX in HCT116 cells using two independent shRNAs and validated the downregulation by qPCR , showing around 60% reduction in SLC8B1 mRNA levels in HCT116 cells ( Figure 3—figure supplement 1I ) .", "Similarly , transient knockdown of NCLX ( NCLX KD ) using shRNA reduced HCT116 cell proliferation ( Figure 3—figure supplement 1J ) .", "The decrease in NCLX KO cell proliferation was accompanied by appreciable changes in apoptotic cells .", "We observed significant increase in cleaved caspase-3 protein using immunofluorescence staining in HCT116 NCLX KO clones as compared to control HCT116 cells , suggesting an increase in apoptosis of NCLX KO CRC cells ( Figure 3—figure supplement 1K , L ) .", "These data suggest that the reduced tumor sizes observed in vivo ( Figures 1 and 2 ) due to NCLX knockout are likely a consequence of reduced CRC cell proliferation and increased apoptosis .", "Given that NCLX knockout increased metastatic spread in xenograft models ( Figure 2 ) , we investigated the effect of NCLX knockout and knockdown on the migration pattern of CRC cells .", "A gap closure assay revealed that all NCLX KO clones of both HCT116 cells ( Figure 3C , D ) and DLD1 cells ( Figure 7—figure supplement 1J light colors ) had a marked increase in migration at 24 hr .", "Similar to knockout , shRNA-mediated knockdown of NCLX in HCT116 cells also caused a significant increase in cell migration at 12 and 24 hr time points ( Figure 3E , F ) .", "Although we showed above that the proliferation of NCLX KO clones of HCT116 cells was inhibited compared to control HCT116 cells ( Figure 3A ) , we ruled out any potential contribution from proliferation in the cell migration assays by analyzing the migration of HCT116 cells and the HCT116 NCLX KO #33 cells at the 6 hr time point .", "At 6 hr , we observed a significant increase in migration of NCLX KO #33 cells as compared to HCT116 control cells ( Figure 3—figure supplement 1M ) .", "Effects of proliferation on cell migration were further ruled out by documenting that the increased cell migration of HCT116 NCLX KO cells is preserved in the presence of the cytostatic compound , mitomycin C at 12 hr and 24 hr time points ( Figure 3—figure supplement 1N ) .", "Further , the invasive behavior of NCLX KO cells was determined using a Matrigel-coated Boyden chamber assay .", "A marked increase in invasion was observed in all NCLX KO clones of both HCT116 and DLD1 as compared to their respective controls ( Figure 3G , H , Figure 3—figure supplement 1O ) .", "Supporting this invasive phenotype are our observations that mRNA levels of the matrix metalloproteinases MMP1 , 2 , and 9 were significantly upregulated in all the HCT116 NCLX KO clones , as compared to control HCT116 cells ( Figure 3I–K ) .", "Similarly , the protein levels of MMP1 , 2 , and 9 were also significantly increased in NCLX KO clones of both HCT116 and DLD1 cells ( Figure 3L , M , Figure 3—figure supplement 1P , Q ) .", "MMP9 and MMP1 protein levels were slightly increased in HCT116 cells in which NCLX was knocked down by shRNA ( NCLX KD ) , although for MMP1 , this increase was not statistically significant ( Figure 3—figure supplement 1R , S ) .", "We then tested the MMPs activity using zymography and revealed that the activity of MMP2 and 9 were markedly increased in all the NCLX KO clones of HCT116 cells compared to control HCT116 cells ( Figure 3N , O ) .", "This was more prominent than changes observed at the mRNA and protein levels , suggesting that the loss of NCLX expression in CRC cells mostly contributes to post-transcriptional regulation of MMPs .", "Collectively , these results suggest that the loss of NCLX in CRC cells causes an increase in migration and invasion of CRC cells through increased MMP1 , 2 , and 9 protein levels and activity .", "The data also confirm the dichotomous role of NCLX knockout observed in vivo , demonstrating that NCLX loss results in decreased cell proliferation and an increase in migratory and invasive phenotypes .", "We and others have previously shown that NCLX is the major molecular mediator of mtCa2+ extrusion and that inhibiting NCLX expression and function prevents mtCa2+ extrusion ( Ben-Kasus Nissim et al . , 2017; Palty et al . , 2010 ) .", "We measured mtCa2+ extrusion in all NCLX KO clones by loading the cells with the mitochondrial Ca2+ sensitive dye Rhod-2 AM .", "Cells were co-loaded with the mitochondrial specific dye MitoTracker Green FM as a control .", "Cells were then stimulated with ATP , a purinergic G protein-coupled receptor ( P2Y ) agonist that couples to phospholipase Cβ ( PLCβ ) activation and subsequent inositol-1 , 4 , 5-trisphosphate ( IP3 ) -dependent release of Ca2+ from the ER through IP3 receptors ( Buvinic et al . , 2009; Gonzalez et al . , 1989 ) .", "Upon stimulation with 300 µM ATP in the presence of extracellular Ca2+ , a portion of Ca2+ released from the ER through IP3 receptors is transferred to mitochondria .", "Hence , cells showed a biphasic response with an increase in Rhod-2 fluorescence followed by a decrease in fluorescence , corresponding to mtCa2+ uptake and mtCa2+ extrusion , respectively .", "All NCLX KO clones showed a significant reduction in mtCa2+ extrusion ( Figure 4—figure supplement 1A–L ) with no significant change in the rate of mtCa2+ uptake .", "To rule out the possibility of long-term compensation in NCLX KO clones , we transiently downregulated NCLX expression in HCT116 and DLD1 cells using siRNA and validated NCLX knockdown by RT-qPCR .", "We observed around ~60% reduction in SLC8B1 mRNA levels in both HCT116 , DLD1 , and HT29 cells ( Figure 4—figure supplement 1M ) .", "Similar to the NCLX KO cells , HCT116 cells , DLD1 cells , and HT29 cells transfected with siRNA against NCLX ( siNCLX ) exhibited a significant reduction in mtCa2+ extrusion with no significant change in mtCa2+ uptake ( Figure 4—figure supplement 1N–V ) .", "We previously showed that inhibition of mtCa2+ extrusion through NCLX knockdown in several cell types leads to the inhibition of plasma membrane ORAI1 channels , reduced Ca2+ entry from the extracellular space , and decreased cytosolic Ca2+ ( Ben-Kasus Nissim et al . , 2017 ) .", "Therefore , we measured cytosolic Ca2+ in response to stimulation with ATP in HCT116 cells and their NCLX KO #33 counterparts using the dye Fura-2 and showed a reduction in Ca2+ entry in NCLX KO cells with no effect on Ca2+ release from the ER ( Figure 4—figure supplement 1W , X ) .", "Collectively , these results show that inhibition of NCLX function in CRC cell lines leads to enhanced mitochondrial matrix Ca2+ concentration due to reduced mtCa2+ extrusion and to decreased cytosolic Ca2+ due to reduced Ca2+ entry across the plasma membrane .", "The decrease in mtCa2+ extrusion in HCT116 and DLD1 cell clones in which NCLX expression was either reduced by siRNA knockdown or ablated by CRISPR/Cas9 knockout suggested that these clones may be experiencing mitochondrial Ca2+ overload .", "In normal cells , mitochondrial Ca2+ overload alters bioenergetics and causes drastic cellular dysfunction leading to cell death ( Celsi et al . , 2009; Santulli et al . , 2015 ) .", "This occurs mainly through the opening of the mitochondrial permeability transition pore ( mPTP ) ( Bernardi and Di Lisa , 2015; Halestrap , 2009 ) and subsequent mitochondrial membrane depolarization .", "Therefore , the effects of NCLX knockout on mitochondrial membrane potential were measured using the tetramethylrhodamine methyl ester ( TMRE ) dye .", "We observed a significant decrease in the accumulation of TMRE in NCLX KO cells , indicating that mitochondria of NCLX KO cells are more depolarized than control HCT116 and DLD1 cells ( Figure 4A , B ) .", "Mitochondrial depolarization is a sign of mitochondrial damage and a major driver of mitophagy , by mediating Pink1 accumulation at the outer mitochondrial membrane ( Jin et al . , 2010 ) .", "Examining mitochondrial structure using transmission electron microscopy ( TEM ) imaging revealed that 60–70% of mitochondria in NCLX KO CRC clones showed altered shape and disrupted cristae compared to control cells ( Figure 4C and Figure 4—figure supplement 2A–C ) .", "We discovered that mitochondrial membranes and cristae of the NCLX KO clones of HCT116 and DLD1 cells were disrupted ( Figure 4C , Figure 4—figure supplement 2A–C ) .", "Specifically , while overall mitochondrial area was not changed in NCLX KO clones ( Figure 4D ) , we observed a greater number of mitochondria with disordered cristae ( Figure 4E ) and a decrease in the number of intact cristae per mitochondrion ( Figure 4F ) .", "We also observed a significant increase in autophagic vesicles in NCLX KO cells compared to control HCT116 cells ( Figure 4—figure supplement 2D ) .", "The number of mitophagic vesicles were slightly increased in NCLX KO cells compared to control HCT116 cells ( e . g . , see images in Figure 4—figure supplement 2C ) , although this increase was not statistically significant ( Figure 4—figure supplement 2E ) .", "The autophagy/mitophagy vesicle marker LC3BII was significantly increased in all NCLX KO clones of HCT116 and DLD1 cells ( Figure 4G , H , and Figure 4—figure supplement 2F , G ) .", "Interestingly , the levels of the p62 protein , a signaling molecule that is downstream of LC3BII that is degraded on induction of autophagy ( Liu et al . , 2016; Youle and Narendra , 2011 ) , were also increased in all the NCLX KO clones of HCT116 and DLD1 cells ( Figure 4G , I and Figure 4—figure supplement 2F , G ) .", "Similarly , the shRNA-mediated knockdown of NCLX in HCT116 ( see Figure 3—figure supplement 1I for evidence of SLC8B1 mRNA knockdown using shRNA ) resulted in increased LC3BII and p62 protein levels ( Figure 4—figure supplement 2H , I ) .", "To assess if these mitochondrial perturbations have consequences on mitochondrial electron transport chain function , we measured mitochondrial oxygen consumption rate ( OCR ) of NCLX KO HCT116 and DLD1 cells using Seahorse extracellular flux assays .", "We observed a significant reduction in maximal respiration and spare respiratory capacity in NCLX KO clones of both HCT116 and DLD1 cell ( Figure 4J–M , and Figure 4—figure supplement 2J–M ) .", "Interestingly , basal respiration and ATP generation were significantly reduced in DLD1 NCLX KO clones , and remained mostly unaltered in HCT116 NCLX KO clones as compared to their respective controls ( Figure 4—figure supplement 2J–M , and Figure 4J–M ) .", "We determined whether reduced OCR in NCLX KO clones was due to altered protein expression of the mitochondrial respiratory complexes I-V .", "To access the changes in mitochondrial respiratory complexes , we measured the protein levels of one component from each of the five complexes including NADH dehydrogenase [ubiquinone] 1 β subcomplex subunit 8 ( NDUFB8 , Complex-I ) , Succinate dehydrogenase [ubiquinone] iron-sulfur subunit ( SDHB , Complex-II ) , Cytochrome b-c1 complex subunit 2 ( UQCRC2 , Complex-III ) , Cytochrome c oxidase subunit 1 ( MTCO1 , Complex IV ) , and ATP synthase subunit α ( ATP5A , Complex-V ) .", "Interestingly , we did not observe any significant change in the expression of any of the above described components of the mitochondrial respiratory Complexes I–V between HCT116 cells and their NCLX KO clones ( Figure 4—figure supplement 2N , O ) .", "These data suggest that lack of NCLX does not completely abrogate basal mitochondrial respiration , but negatively affects respiratory reserve , which is an indication of the cells ability to enhance mitochondrial respiration in response to higher energy demands .", "A further consequence of mtCa2+ overload is the generation of mitochondrial reactive oxygen species ( mtROS ) ( Bertero and Maack , 2018; Brookes et al . , 2004 ) .", "In normal cells , enhanced mtROS can lead to mitochondrial dysfunction and cell death; however , many tumor cells utilize mitochondria-derived ROS as cellular signals to drive pro-survival adaptations , including changes in downstream transcription ( Vyas et al . , 2016 ) .", "Hence , we measured mtROS in HCT116 and DLD1 CRC cells and their respective NCLX KO clones using the dye MitoSOX and flow cytometry .", "MitoSOX dye intensity was significantly increased in all the NCLX KO clones of both HCT116 and DLD1 cells ( Figure 4N , O ) , indicating increased mtROS in these cells .", "Using fluorescence microscopy , we also show that downregulation of NCLX with siRNA in HCT116 and DLD1 cells ( and in another CRC cell line , HT29; See Figure 4—figure supplement 1M for evidence of SLC8B1 mRNA knockdown in HT29 ) results in a significant increase in mtROS levels ( Figure 4—figure supplement 2P , Q ) .", "The above data demonstrate that loss of NCLX decreases mtCa2+ extrusion , which affects mitochondrial cristae morphology and depolarization of the mitochondrial membrane , leading to formation of autophagic vesicles and possibly mitophagy , decreased respiratory reserve capacity , and enhanced mtROS production .", "To further delineate the role of NCLX loss in CRC , we performed transcriptional profiling of HCT116 cells and their NCLX KO counterparts using RNA sequencing .", "Gene Set Enrichment Analysis ( GSEA ) revealed that NCLX knockout drives the positive enrichment of pathways involved in hypoxia , epithelial-to-mesenchymal transition ( EMT ) , TGF-β , pro-inflammatory , glycolysis , apoptosis and angiogenesis pathways , while negatively influencing gene expression of Myc targets , cell-cycle regulation , and oxidative phosphorylation ( Figure 5A–G , and Figure 5—figure supplement 1A–F ) .", "Thus , GSEA analysis revealed that NCLX loss drives gene expression signatures associated with metastatic progression and inhibition of proliferation , mirroring the phenotypic changes we observed following NCLX knockout and knockdown .", "Interestingly , these gene expression signatures are shared by the mesenchymal CMS4 CRC subtype , which is characterized by high rates of recurrence , and predictive of poor patient outcome ( Guinney et al . , 2015 ) .", "A phenotype of mesenchymal CRC is the enrichment of cancer stem cell traits ( Guinney et al . , 2015; Polyak and Weinberg , 2009 ) .", "In agreement with these findings , we found that transcript levels of stem cell markers NANOG , Oct4 , Sox2 , and Fox3 , as well as regulators of the glutathione synthesis pathway implicated in regulating these transcription factors in breast cancer , SLC7A11 and GCLM ( Lu et al . , 2015 ) , were significantly upregulated in NCLX KO cells ( Figure 5H–J , Figure 5—figure supplement 1G–O ) .", "One exception was the mRNA levels of Oct4 in DLD1 NCLX KO clones , which were not significantly different from those of control DLD1 cells ( Figure 5—figure supplement 1N ) .", "These data suggest that NCLX KO clones acquire stem cell-like properties .", "In most cancer types , a stem cell-like phenotype is associated with enhanced invasion and chemoresistance ( Blank et al . , 2018; Munro et al . , 2018; Reya et al . , 2001; Touil et al . , 2014 ) .", "The chemoresistance properties of the NCLX KO clones and respective control HCT116 and DLD1 cells were tested in response to treatment with the antimetabolite agent 5-Fluorouracil ( 5-FU ) , which is widely used in the treatment of CRC .", "First , we performed titration experiments with doses of 5-FU ranging from 1 to 25 µM .", "The control HCT116 cells and the HCT116 NCLX KO cells showed a dose-dependent reduction in proliferation in response to 5-FU treatment ( Figure 6—figure supplement 1A ) .", "The IC50 of 5-FU for HCT116 NCLX KO cells ( 15 ± 1 . 2 µM ) was significantly higher than the control HCT116 cells ( 8 . 2 ± 1 . 2 µM ) ( Figure 6—figure supplement 1B ) .", "Therefore , subsequent experiments were performed with 10 µM 5-FU .", "The treatment with 10 µM 5-FU caused a significant reduction in proliferation of control HCT116 and DLD1 cells at 24 and 72 hr ( Figure 5K , Figure 6—figure supplement 1C ) .", "Treatment of the NCLX KO clones of HCT116 ( Figure 5K ) and DLD1 ( Figure 6—figure supplement 1C ) cells with 10 µM 5-FU yielded only a marginal reduction in proliferation at 72 hr as compared to non-treated NCLX KO clones , although this reduction was statistically significant for two NCLX KO clones of DLD1 cells at 72 hr time point ( Clone#24 and #32; Figure 6—figure supplement 1C ) .", "5-FU also caused a significant inhibition in migration of control HCT116 cells ( Figure 6—figure supplement 1D , E ) , but did not affect the migratory capabilities of the NCLX KO clones of both HCT116 and DLD1 cells ( Figure 6—figure supplement 1D , E ) .", "The mesenchymal , invasive , and chemoresistance phenotypes of NCLX KO cells , as well the majority of enriched pathways identified by GSEA , including hypoxia , EMT , glycolysis , angiogenesis , and the suppression of OXPHOS , share a common regulator , the hypoxia-inducible factor HIF1α ( Figure 5A–G and Figure 5—figure supplement 1A-F ) .", "This was intriguing in light of our observations that NCLX knockdown decreases respiration and increases mitochondrial ROS production in CRC ( Figure 4J–O ) , as mtROS is a known activator of HIF1α ( Bell et al . , 2007; Dan Dunn et al . , 2015; Hamanaka and Chandel , 2010; Pan et al . , 2007 ) .", "Hence , we measured HIF1α protein levels and found that HIF1α protein levels were strikingly increased in the NCLX KO clones of both HCT116 and DLD1 cells in the absence of a hypoxic stimulus ( Figure 6A–D ) .", "Similarly , HIF1α protein levels were also increased in HCT116 cells in which NCLX was knocked down with shRNA ( NCLX KD; Figure 6E , F ) .", "Importantly , we were able to demonstrate that HIF1α protein stabilization was significantly abrogated in NCLX KO cells when mtROS were scavenged using the mitochondria-targeted antioxidant mitoTEMPO ( Figure 6G , H ) .", "Both AMPK and mTORC1 are known regulators of HIF1α ( Dodd et al . , 2015; Hudson et al . , 2002; Tandon et al . , 2011 ) .", "Therefore , we assessed the phosphorylation levels of AMPK and the ribosomal protein S6K ( a readout of mTORC1 activation ) in HCT116 and clones of HCT116 NCLX KO cells .", "Assessment of AMPK and S6K1 phosphorylation revealed no difference between control HCT116 and clones of HCT116 NCLX KO cells ( Figure 6—figure supplement 1F–I ) , suggesting that mTORC1 does not contribute to the regulation of HIF1α in response to NCLX ablation .", "Taken together , these results suggest that the major regulator of HIF1α protein levels in NCLX KO CRC cells is mtROS .", "Further , a significant reduction in migration was observed when HCT116 NCLX KO cells were treated with mitoTEMPO ( Figure 6I , J ) , while this had no effect on the proliferation of either control HCT116 cells or their NCLX KO counterparts ( Figure 6K ) .", "HIF1α is an important regulator of glycolysis .", "The transcriptomic analysis ( Figure 5A and Figure 5—figure supplement 1A ) , GSEA pathway , and enrichment analysis ( Figure 5B , C ) showed that glycolysis-related genes were upregulated in HCT116 NCLX KO cells .", "RT-qPCR results confirmed that the glycolysis-related genes GLUT1 ( glucose transporter one or SLC2A1 ) , HK2 ( hexokinase 2 ) , ALDOA ( aldolase A ) , ENO1 ( enolase 1 ) , and LDHA ( lactate dehydrogenase A ) were distinctly upregulated in all the NCLX KO HCT116 clones ( Figure 7A ) .", "Loss of NCLX in either HCT116 or DLD1 increased the protein levels of HK2 , ALDOA , and LDHA ( Figure 7B , C , and Figure 7—figure supplement 1A–D ) .", "We also validated this further using shRNA-mediated knockdown of NCLX ( NCLX KD ) in HCT116 cells .", "SLC8B1 mRNA levels were reduced by 60–70% in NCLX KD cells compared to cells transfected with shRNA scramble control ( Figure 3—figure supplement 1I ) .", "Similar to NCLX KO , the HCT116 NCLX KD cells showed a modest but significant increase of HK2 , ALDOA , and LDHA protein levels ( Figure 7—figure supplement 1E , F ) .", "Interestingly , we observed a significant downregulation of the pentose phosphate pathway genes in HCT116 NCLX KO cells , including decreased mRNA expression of the enzymes glucose 6-phosphate dehydrogenase ( G6PD ) , 6-phosphogluconate dehydrogenase ( PGD ) and Transketolase ( TKT ) ( Figure 7—figure supplement 1G ) .", "Since inhibition of NCLX function caused upregulation of glycolytic genes , we used Seahorse extracellular flux analysis to determine whether NCLX knockout affects the extracellular acidification rate ( ECAR ) of HCT116 and DLD1 cells .", "Consistent with the upregulation of glycolytic genes , ECAR , and glucose dependency were increased in all NCLX KO clones ( Figure 7D–F and Figure 7—figure supplement 1H ) .", "Furthermore , direct measurements of glucose and lactate in growth media using the YSI system also revealed that the glucose consumption and lactate production were markedly increased in NCLX KO clones of both HCT116 and DLD1 cells ( Figure 7G–J ) .", "These data suggest that NCLX KO cells compensate for their decreased respiratory reserve capacity ( Figure 4J–M , and Figure 4—figure supplement 2J–M ) by enhancing glycolytic pathways to meet their energy demands .", "Considering these findings , the glycolytic inhibitor 2-deoxy-D-glucose ( 2-DG ) was used to test if CRC cells that lack NCLX expression are more vulnerable to glycolysis inhibition .", "While 2-DG caused a significant reduction in proliferation of both DLD1 control and NCLX KO cells ( Figure 7—figure supplement 1I ) , it did not affect the proliferation of NCLX KO clones of HCT116 ( Figure 7K ) .", "However , the enhanced migration of NCLX KO clones was abrogated when glycolysis was inhibited using 2-DG ( Figure 7L–M; Figure 7—figure supplement 1J ) , suggesting that increased glycolysis is supporting migration of NCLX KO clones and that targeting this metabolic adaptation may be an avenue to block metastatic progression of CRC cells with low NCLX expression ." ], [ "In addition to the critical role mitochondria play in cellular bioenergetics , lipid metabolism , and cell death , recent attention has focused on their direct and indirect contributions to mediating crucial signal transduction pathways that control the shift in cellular metabolic activity and changes in gene transcription ( Tennant et al . , 2009; Tennant et al . , 2010 ) .", "Mitochondria are generators and active participants in Ca2+ and ROS signaling ( Hempel and Trebak , 2017; Vyas et al . , 2016 ) .", "Growing evidence has shown a critical role of mtCa2+ homeostasis in mitochondrial function and cell survival ( Celsi et al . , 2009; Santulli et al . , 2015 ) and reported a strong correlation between altered mitochondrial function and disease including cancer progression ( Porporato et al . , 2018 ) .", "Mitochondria are major mediators of the shift in metabolic activity of cancer cells .", "This metabolic shift is a mechanism of adaptation to stressors within the tumor microenvironment that is required for cancer cell survival ( Vyas et al . , 2016 ) .", "Nevertheless , the mechanisms of mtCa2+ in driving mitochondrial signaling and metabolic shifts have largely remained unknown .", "Previous studies on the mitochondrial Ca2+ uniporter ( MCU ) complex indicated that the level of mtCa2+ is an important determinant in the progression of different cancer types ( Marchi et al . , 2019a; Marchi et al . , 2013; Marchi et al . , 2019b; Tosatto et al . , 2016 ) .", "The downregulation of MCU and subsequent reduction of mtCa2+ uptake correlated with resistance to apoptosis of colorectal cancer cell lines ( Marchi et al . , 2013 ) .", "Conversely , in triple-negative breast cancer cell lines , the downregulation of MCU caused a reduction in mtROS , HIF1α levels , cell migration , invasion and inhibited metastasis to the lung in xenograft experiments ( Tosatto et al . , 2016 ) , suggesting that although mtCa2+ overload is pro-apoptotic , it is likely beneficial for invasion and metastasis of cancer cell clones that have evaded apoptosis .", "Similarly , our TCGA data analysis of colorectal cancer patients showed that SLC8B1 mRNA levels are significantly reduced in both early and late-stage tumors with a more pronounced reduction in late-stage tumors , suggesting that mtCa2+ overload through reduced NCLX function has a critical role in CRC progression .", "Nevertheless , we cannot rule out possible contributions of cytosolic Ca2+ to the phenotype of NCLX KO cells .", "Furthermore , future studies investigating the role of MCU in CRC progression are required to support the notion that mtCa2+per se is the critical driver of CRC progression .", "It is well established that mtCa2+ overload is detrimental to mitochondrial function and is a precursor of death in normal cells through the opening of the mitochondrial permeability transition pore , cytochrome C release from mitochondria and subsequent activation of the caspase family of pro-apoptotic proteases ( Giorgi et al . , 2012; Pinton et al . , 2008 ) .", "However , the exact mechanisms by which cancer cells adapt and survive under these conditions are not fully understood .", "Our data show that downregulation or complete loss of NCLX in CRC cells causes mtCa2+ overload , an increase in mtROS production and mitochondria depolarization .", "Our transmission electron microscopy data show that loss of NCLX in CRC cells causes altered mitochondria shape and morphology with disrupted cristae and inner mitochondrial membrane structures .", "We also show that this coincides with increased LC3BII- and p62-dependent autophagosome formation and decreased cell-cycle-related gene expression .", "This phenotype is consistent with the reduced proliferation of NCLX KO and NCLX KD CRC cells , and the reduced tumor burden and tumor size in NCLX KO mice subjected to the colitis-associated CRC model .", "Similarly , our xenograft model yielded smaller primary tumors when HCT116 NCLX KO cells were injected in SCID mice by comparison to xenografts of control HCT116 cells .", "Thus , the reduction in NCLX expression likely limits proliferation and primary tumor growth .", "Yet , it is also apparent that clones of cancer cells survive this loss of NCLX and coopt the downregulation of NCLX to undergo metabolic reprogramming and gene expression changes that support tumor migration , invasion and metastasis , by initiating a mitochondrial Ca2+/ROS signaling axis to drive HIF1α activation .", "Increased p62 is a marker for autophagosome formation but upon autophagy the levels of p62 decrease ( Liu et al . , 2016 ) .", "Interestingly , our NCLX KO cells have maintained an increase in p62 levels , suggesting possible accumulation of autophagosomes .", "Future studies are required to determine whether increased autophagosome formation is accompanied by increased mitophagy or whether accumulation of autophagosomes in NCLX KO cells causes cytotoxicity and apoptosis ( Button et al . , 2017 ) .", "We have shown that cleaved caspase three staining was increased in HCT116 NCLX KO cells , suggesting enhanced apoptosis in NCLX KO cells .", "However , additional experiments including more direct approaches are required to determine the effect of NCLX deletion on apoptosis of CRC cells .", "Herein , we have considered studying the effects of NCLX overexpression on metastasis of CRC to complement out NCLX knockout and knockdown studies .", "However , these studies are not feasible because overexpressed NCLX localizes not only to mitochondria but also to other organelles , making the interpretation of results from these experiments tenuous .", "While it is generally thought that mtCa2+ induces mtROS production through increased activation of TCA cycle proteins and consequential increases in ETC function , it is also evident that defective electron transport chain function results in mtROS production ( Starkov , 2008 ) .", "Our data clearly demonstrate that increased mtROS production is accompanied by mitochondrial structural perturbations , decreased OXPHOS , and mitochondrial membrane depolarization .", "These phenotypes of mitochondrial dysfunction are commonly associated with apoptosis initiation .", "However , NCLX knockout resulted in the selection of clones that initiate pro-survival and pro-metastatic adaptations .", "Mitochondrial depolarization and mtROS production are known initiators of mitophagy ( Ashrafi and Schwarz , 2013; Fan et al . , 2019; Frank et al . , 2012; Schofield and Schafer , 2020; Twig and Shirihai , 2011 ) .", "Moreover , HIF1α , which is regulated in a mtCa2+/mtROS-dependent manner in response to NCLX loss in CRC , also contributes to mitophagy , by upregulating the pro-mitophagy proteins BNIP3 and NIX ( Bellot et al . , 2009; Chourasia et al . , 2015; Zhang et al . , 2008; Zhang and Ney , 2009 ) .", "The gene expression signatures and phenotypes observed in response to NCLX downregulation largely mirror those of the mesenchymal CRC subtype labeled as CMS4 .", "Shared pathways include the increase in EMT , TGF- β , matrix remodeling , stemness , and decreases in Myc and cell-cycle progression ( Guinney et al . , 2015 ) .", "No single mutation is solely associated with one of the CMS or CRIS subtypes , and at present , the underlying drivers of colorectal cancer molecular subtypes still remain to be fully elucidated ( Guinney et al . , 2015 ) .", "However , it is interesting to note that ~ 50% of CMS4 tumors display TP53 mutations and generally lack mutations in BRAF , and that loss of NCLX is statistically linked to TP53 mutant tumors and wild type BRAF CRC tumors .", "We used two different CRC cell lines HCT116 and DLD1; both of these cell lines are derived from male CRC patients carrying a mutation in KRAS and PIK3CA .", "TCGA data analysis showed no difference in NCLX expression between CRC patients bearing the wildtype KRAS and PIK3CA and their respective mutations .", "Interestingly , we also saw lower basal NCLX expression in DLD1 cells that have mutated TP53 ( S241F ) , compared to HCT116 cells , which are wildtype for TP53 .", "Whether increased mtCa2+ and subsequent mtROS signaling is one of the underlying drivers of CMS4 tumors remains to be determined .", "A metabolic shift towards aerobic glycolysis is a hallmark of cancer progression ( Burns and Manda , 2017; Liberti and Locasale , 2016 ) .", "Our data demonstrated that the loss of NCLX leads to reduced oxygen consumption , increased glycolysis , and increased transcription of major glycolytic enzymes .", "This increased transcription of glycolytic enzymes is commonly controlled by HIF1α , which is upregulated in response to increased mtROS resulting from loss of NCLX and subsequent increase in mtCa2+ .", "Furthermore , we show that inhibiting glycolysis of NCLX KO CRC cells partially normalizes the increased migration of these cells , consistent with previous findings that showed a critical role of glycolysis in cancer metastasis ( Bu et al . , 2018; Gillies et al . , 2008; Han et al . , 2013 ) .", "Although the increase in HIF1α protein levels was shown to be critical for the migration and metastasis of cancer cells ( Lehmann et al . , 2017; Masoud and Li , 2015; Semenza , 2003; Wigerup et al . , 2016 ) , the mechanisms by which mtCa2+ regulates HIF1α protein levels are not clear .", "Here , we provide evidence that reduced NCLX function , causing reduced mtCa2+ extrusion and resulting in mtCa2+ overload , enhances HIF1α levels through an increase in mtROS .", "Our xenograft model shows that despite the reduced tumor burden and tumor size in SCID mice injected with HCT116 NCLX KO cells compared to SCID mice injected with control HCT116 cells , the survival of the former mice was significantly reduced compared to the latter .", "This result can be explained by the enhanced metastasis to the liver and colon in SCID mice injected with HCT116 NCLX KO cells .", "Indeed , we were able to show that the increased invasive properties of NCLX KO CRC cells were associated with increased MMP1 , MMP2 , and MMP9 activities , which are known to be regulated by HIF1α ( Ben-Yosef et al . , 2002; Choudhry and Harris , 2018; Muñoz-Nájar et al . , 2006; Shin et al . , 2015; Tsai et al . , 2016 ) .", "We showed that NCLX KO CRC cells are chemoresistant to treatment by 5-FU , with both proliferation and migration of NCLX KO CRC cells not significantly altered by 5-FU treatment .", "It is reasonable to suspect that the decrease in proliferation contributes to the chemoresistance phenotype of cells lacking NCLX .", "In previous work , we demonstrated that decreased mtCa2+ extrusion in HEK293 cells leads to a decrease in cytosolic Ca2+ and reduced store-operated Ca2+ entry ( SOCE ) mediated by ORAI1 channels ( Ben-Kasus Nissim et al . , 2017 ) .", "Similarly , we see a decrease in cytosolic Ca2+ and SOCE when NCLX is knocked out in CRC cell lines .", "A consequence of reducing SOCE and cytosolic Ca2+ is decreased activation of the Ca2+ sensitive transcription factor NFAT ( Trebak and Kinet , 2019 ) .", "NFAT is a known stimulator of MYC activation ( Buchholz et al . , 2006; Mognol et al . , 2012; Singh et al . , 2010 ) , suggesting that reduced NFAT signaling as a consequence of NCLX downregulation might be contributing to the decrease in MYC pathway activation , cell-cycle progression , and proliferation .", "The EMT phenotype and decreases in proliferation are hallmarks of cancer stem cells and the presence of cancer stem cells within tumors is one of the major causes of chemotherapy resistance ( Izumiya et al . , 2012; Munro et al . , 2018; Ohata et al . , 2019; Zhao , 2016 ) .", "Studies have shown that CSCs are enriched on treatment with 5-FU through different molecular mechanisms ( Abdullah and Chow , 2013; Lu et al . , 2015; Touil et al . , 2014 ) .", "Recent work showed that increased chemoresistance of breast cancer was mediated by HIF1α-mediated glutathione biosynthesis and glutathione-mediated enrichment with cancer stem cells ( Lu et al . , 2015 ) .", "Chemotherapy in breast cancer induces HIF1α-dependent glutathione biosynthesis by enhancing the expression of the cystine transporter xCT ( SLC7A11 ) and the regulatory subunit of glutamate-cysteine ligase ( GCLM ) ( Lu et al . , 2015 ) .", "The glutathione thus generated inhibits the MEK/ERK pathway through copper chelation resulting in enhanced expression of the stem cell markers NANOG , Oct4 and Sox2 .", "We observed enhanced expression of SLC7A11 and GCLM in NCLX KO clones from both HCT116 and DLD1 cells .", "In addition to contributing to stem cell regulation , an increase in enzymes responsible for glutathione synthesis potentially provides additional survival advantages to NCLX knockout cells , by enhancing the ROS scavenging ability of CRC cells and preventing any lethal build-up of ROS as a consequence of mtCa2+ elevation .", "Throughout this study , HIF1α emerged as major regulator of metastasis , chemoresistance and stemness in NCLX KO CRC cells .", "HIF1α might represent a novel marker or target of anti-CRC drugs .", "One could speculate that the concomitant inhibition of HIF1α and chemotherapy might be more effective against CRC cancer .", "In summary , we have identified a novel mitochondrial Ca2+/ROS signaling axis in colorectal cancer that is initiated in response to decreased NCLX expression , as commonly observed in CRC patient tumors .", "In one hand , reduced mtCa2+ extrusion causes mitochondrial depolarization , mitochondrial dysfunction , reduced cell-cycle-related gene expression , increased autophagosome formation , and reduced proliferation , leading to smaller tumors both in the colitis-associated CRC model and the xenograft CRC model .", "On the other hand , mtCa2+ overload induced by NCLX loss enhances mtROS , which in turn enhances CRC metastasis through HIF1α-dependent increases in glycolysis , chemoresistance and pro-metastatic gene expression signatures .", "This contributes to increased metastatic burden and enhanced lethality of SCID mice injected with NCLX KO CRC cells ( summarized in Figure 7N ) .", "These changes are reminiscent of the highly metastatic mesenchymal CMS4 CRC subtype , and our work suggests that restoring mtCa2+ homeostasis in CRC tumors might be beneficial in limiting or preventing CRC progression and metastasis ." ], [ "See article appendix for key resources table .", "The Pennsylvania State University College of Medicine institutional review board approved this study ( IRB Protocol number HY98-057EP-A ) .", "Prior to surgery , patients are consented to have resected tissues collected and banked into the Penn State Hershey Colorectal Disease Biobank .", "As outlined in the consent form and IRB Protocol , de-identified tissues are made available to study on a per-request basis after such requests are considered by an internal review panel and deemed to be collaborative in nature .", "The CRD biobank has been in operation since 1998 and substantial efforts are in place to protect patient information including storage of data behind firewalls and limited access to electronic medical records .", "Methods regarding tissue procurement , mRNA isolation and processing were previously described ( Schieffer et al . , 2017 ) .", "A breeding pair of global NCLX KO and wildtype C57BL/6 mice were obtained from the Jackson laboratory .", "NOD/SCID ( NOD . CB17-Prkdcscid/J ) were also obtained from the Jackson laboratory .", "All mice were maintained in 12:12 hr light-dark cycle ( 22°C to 25°C ) in a pathogen-free barrier facility at The Pennsylvania State University animal facility .", "The mice were sacrificed ( age at the time of sacrifice is mentioned in the figure legends ) at the end of the experiment , and organs were collected for histopathology , protein , and mRNA analysis .", "All experiments with mice were approved and conducted in accordance with The Pennsylvania State Institutional Animal Care and Research Advisory Committee .", "To investigate the role of NCLX in human CRC cells , we chose two widely used CRC cell lines HCT116 and DLD1 derived from the colon of male CRC patients with colorectal carcinoma and colorectal adenocarcinoma , respectively ( Ahmed et al . , 2013 ) .", "HCT116 and HT29 cells were cultured in McCoy’s 5A media supplemented with 10% FBS and 1X antibiotic-antimycotic agents .", "DLD1 cells were cultured in RPMI-1640 media supplemented with 10% FBS and 1X antibiotic-antimycotic agents .", "All cells were kept at 37°C in a 5% CO2 incubator .", "The cells were transfected with siRNA or plasmids using the Lipofectamine 2000 reagent , according to manufacturer protocol ( Invitrogen ) .", "In experiments with 2-DG and 5-FU , cells were cultured in complete growth media McCoy’s or RPMI media with 10% FBS and 1X antibiotic-antimycotic agents and treated with different concentrations of 2-DG or 5-FU for the specified time .", "Our cell lines were purchased from ATCC immediately before use and the identities of the cell lines were further confirmed by RNA sequencing .", "Further , we routinely perform mycoplasma tests on all our cell lines , including the cell lines used herein .", "These mycoplasma tests were negative .", "The cells were seeded at 80–90% confluency at the time of transfection .", "The plasmids or siRNAs and Lipofectamine 2000 were diluted in Opti-MEM and mixed .", "The mixture was incubated for 5–10 min at room temperature and then added to the cells cultured in media with 10% serum without antibiotic-antimycotic agents .", "The medium of transfected cells was changed to normal media with 10% FBS and 1X antibiotic-antimycotic agents after 4–6 hr .", "The expression of plasmid and siRNA was confirmed after 24 hr and 72 hr of transfection , respectively .", "To generate stable NCLX knockdown cells , shScramble , shNCLX #2 , and shNCLX #3 cloned in pLKO .", "The lentiviral construct was packaged into HEK293FT using the ViraPower kit ( Invitrogen ) and Lipofectamine 2000 ( Thermofisher Scientific ) .", "HCT116 cells were infected with respective lentivirus for 72 hr and selected with puromycin ( 1 . 5 μg/ml ) for 72 hr .", "The knockdown of mRNA was confirmed through RT-qPCR .", "Stable knockdown cells were used within two weeks , then discarded .", "We generated several clones of NCLX knockout ( NCLX KO ) in both HCT116 and DLD1 cells using the CRISPR/Cas9 system .", "To alleviate potential off-target effects of the CRISPR/Cas9 system , we generated several independent clones obtained with three independent guide RNAs ( gRNAs; see methods ) ( Figure 3—figure supplement 1A–H ) .", "Genome sequencing and PCR on genomic DNA confirmed NCLX KO .", "For the case of HCT116 cells , which we generated first , NCLX KO #33 was generated using a guide RNA ( g1 ) which resulted in a single cut at nucleotide 150 in exon one causing a frameshift mutation and introduction of a stop codon at position 180 in the NCLX open reading frame ( Figure 3—figure supplement 1A ) .", "NCLX KO #37 and #59 were generated using two guide RNAs ( g1 and g2 ) , which resulted in a double-strand cut and introduction of a stop codon in the NCLX open reading frame ( Figure 3—figure supplement 1B , C; method table ) .", "For the NCLX KO clones of DLD1 generated later , we employed an advanced strategy with each knockout condition using simultaneously two guide RNAs flanking a region starting from exon three to the end of the SLC8B1 gene ( Figure 3—figure supplement 1E ) to completely excise most of NCLX open reading frame .", "PCR on genomic DNA ( Figure 3—figure supplement 1G ) confirmed NCLX knockout in DLD1 clones .", "We used RT-qPCR to document the absence of mRNA in NCLX KO clones of HCT116 ( Figure 3—figure supplement 1D ) and DLD1 cells ( Figure 3—figure supplement 1H ) .", "The positions of the primers used for qPCR are shown in Figure 3—figure supplement 1A and Figure 3—figure supplement 1E for HCT116 and DLD1 cells , respectively , and labeled as ‘qNCLX’ .", "The mRNA was isolated ( minimum 3 µg ) using the RNeasy Mini Kit .", "A fragmentation buffer ( Ambion ) was used to short fragment the mRNA .", "These short-fragmented mRNA were used as a template to synthesize double-stranded cDNA .", "The cDNA was end-repaired and ligated to Illumina adapters .", "Further , to generate the library , these cDNAs were size selected ( ~250 bp ) on an agarose gel , and PCR amplified .", "To sequence the cDNA , the prepared cDNA library was fed to the Illumina HiSeq 2500 sequencing platform ( Berry Genomics ) .", "The expression level of each transcript of a gene was quantified using read counts with HTseq .", "After removing gene identifiers with zero read counts in each sample , the biomaRt R package ( Durinck et al . , 2009 ) was used to convert Ensemble gene identifiers to gene symbols using the January 2019 archive ( http://jan2019 . archive . ensembl . org ) .", "Identifiers corresponding to multiple gene symbols were removed .", "The edgeR R package ( McCarthy et al . , 2012; Robinson et al . , 2010 ) was used to identify lowly expressed genes based on thresholds defined using counts per million reads mapped .", "This yielded expression data for a total of 10 , 179 genes .", "Exploratory analyses were performed after utilizing the variance stabilizing transformation ( VST ) function in the DESeq2 R package ( Love et al . , 2014 ) to apply VST to the read count data .", "The results strongly suggested the presence of a batch effect .", "After applying the voom transformation ( Law et al . , 2014 ) to the read counts , the limma R package ( Ritchie et al . , 2015 ) was used to identify differentially expressed genes based on a q-value threshold of 0 . 05 .", "The limma design matrix included batch information as a factor .", "The gene set enrichment analysis ( GSEA ) software ( Subramanian et al . , 2005 ) was used to perform pathway analyses based on the limma output .", "Briefly , genes in the limma output were ordered according to the t test statistic .", "Then a GSEA pre-ranked analysis was performed using the Hallmark Gene Sets in the Molecular Signatures Database ( http://software . broadinstitute . org/gsea/msigdb/index . jsp ) .", "RNA-sequencing ( RNA-seq ) -based gene expression data for both tumor and normal samples , as well as clinical data for the combined TCGA COADREAD cohort were downloaded from the Broad Institute’s Firehose GDAC ( https://gdac . broadinstitute . org/ ) .", "Gene expression was quantified as log2 ( normalized RSEM + 1 ) .", "Somatic gene mutation data was obtained from Ellrott et al . , 2018 after restricting to samples in the RNA-seq cohort .", "Wilcoxon rank-sum tests and Kruskal-Wallis tests were used to compare expression values of SLC8B1 across groups defined by the clinical and mutation data .", "Human colorectal cancer tissues utilized for mRNA extraction were obtained from the Penn State Hershey medical center with IRB approval .", "Briefly , mRNA were isolated from either tissues or cells .", "The cells were washed with cold PBS , and RNA was isolated using the RNeasy Mini Kit .", "The quality and quantity of RNA were analyzed using NanoDrop2000 ( Thermo Scientific , Wilmington , DE , USA ) .", "High-Capacity cDNA Reverse Transcription Kit ( Applied Biosystems , Foster City , CA ) was used to make cDNA .", "The cDNA was further used for quantitative real-time PCR using the SYBR select master mix ( Thermo Scientific ) according to the manufacturer protocol .", "The reactions were run in technical duplicates using the Quant Studio three system ( Applied Biosystems ) .", "The expression levels of genes were normalized to at least two housekeeping genes , GAPDH or Tubulin or NONO ( specified in the figure legends ) using the 2-ΔCt method .", "The cells were cultured with 80–90% confluency and were washed with chilled PBS and lysed in 100 µl RIPA buffer ( Sigma ) containing Halt protease and phosphatase inhibitor .", "30–50 µg of the protein was loaded on a 4–12% gel ( NuPAGE Bis-Tris precast gels , Life Technologies ) and transferred to a polyvinylidene difluoride membrane .", "The membrane was incubated in Odyssey blocking buffer for 1 hr at room temperature for blocking and was incubated overnight at 4°C in primary antibody diluted in Odyssey blocking buffer .", "The primary antibodies were used are mentioned in the key resource table , and their dilutions are as follows: anti-HIF1α ( 1:500 ) , all other antibodies were used at 1:1000 dilution .", "The next day , the membrane was washed with 0 . 1% TBS-T for 5 min ( three times ) at room temperature , followed by incubation in IRDye for 2 hr at room temperature .", "The IRDyes used are mentioned in the key resource table , and they were diluted in Odyssey blocking buffer ( IRDye 680RD Goat anti-Mouse at 1:10 , 000 dilution and IRDye 800CW Donkey anti-Rabbit at 1:5000 ) .", "The membrane was washed with 0 . 1% TBS-T for 5 min ( three times ) at room temperature .", "Then the blot was imaged in Odyssey CLx Imaging System ( LI-COR , NE , USA ) .", "Densiometric analysis of the bands on the membrane was performed using ImageJ software .", "The cells were cultured in six well plates on glass coverslip at 60–70% confluency .", "The next day , the cells were washed with cold PBS and fixed with chilled 100% Methanol for 30 min .", "The cells were washed with chilled PBS ( 3X for 5 min ) .", "The cells were incubated in an anti-cleaved caspase-3 antibody ( 1:250 ) .", "Secondary Alexa Fluor 594 ( 1:5000 ) was used to stain the cells .", "For live-cell imaging and mitochondrial staining , the cells were stained with MitoTracker Green FM ( 50 nM in complete media for 30 min at 37°C ) , TMRM ( 100 nM in complete media for 30 min at 37°C ) or MitoSOX Red ( 2 . 5 µM in HBSS for 30 min at 37°C ) and Hoechst ( 1 µM in HBSS for 5 min ) .", "The images were acquired using the Leica SP8 confocal microscope .", "The fluorescence intensity was quantified using LAS X ( Leica Microsystems ) software .", "To measure mitochondrial Ca2+ using Rhod-2 AM ( 543 nm/580–650 nm ) dye , the cells were cultured at 50–60% confluency .", "The cells were washed with media without FBS and antibiotic-antimycotic agents .", "Then the cells were incubated in media containing 1 µM Rhod-2 AM ( without FBS and antibiotic-antimycotic agents ) at 37°C for 30 min .", "The cells were washed with media and were loaded with MitoTracker Green FM ( 50 nM in media for 30 min at 37°C ) for photobleaching and focus corrections .", "After loading with MitoTracker Green , cells were washed and kept in HBSS ( HEPES-buffered saline solution ( 140 mM NaCl , 1 . 13 mM MgCl2 , 4 . 7 mM KCl , 2 mM CaCl2 , 10 mM D-glucose , and 10 mM HEPES , adjusted to pH 7 . 4 with NaOH ) ) containing 5 mM CaCl2 for imaging .", "The cells were stimulated with 300 µM ATP in HBSS containing 5 mM CaCl2 .", "The time-lapse images were acquired using the Leica TCS SP8 confocal microscope equipped with a 63X oil objective .", "The images were acquired every 5 s intervals for 10–15 min .", "The fluorescence intensity was quantified using LAS X ( Leica Microsystems ) software .", "Intracellular Ca2+ imaging was performed described previously ( Emrich et al . , 2019; Zhang et al . , 2019 ) .", "Briefly , the cells were cultured on glass coverslips with 50–60% confluency .", "Next day the cells were washed with fresh media , and the glass coverslip with cells were mounted on a Teflon chamber .", "Further , the cells were incubated at 37°C for 45 min in culture media containing 4 μM Fura-2/acetoxymethyl ester ( Molecular Probes , Eugene , OR , USA ) .", "Then the cells were washed and kept in HEPES-buffered saline solution ( 140 mM NaCl , 1 . 13 mM MgCl2 , 4 . 7 mM KCl , 2 mM CaCl2 , 10 mM D-glucose , and 10 mM HEPES , adjusted to pH 7 . 4 with NaOH ) for at least 10 min before Ca2+ measurements were made .", "Time-lapse imaging was performed , and the change in fluorescence of Fura-2 was recorded and analyzed with a digital fluorescence imaging system ( InCyt Im2; Intracellular Imaging Inc , Cincinnati , OH , USA ) .", "An ROI was drawn around each cell , and 340/380 ratio of each cell was calculated .", "The HCT116 , DLD1 , NCLX KO HCT116 , and NCLX KO DLD1 cells were cultured at 80–90% confluency and fixed with 1% glutaraldehyde in 0 . 1 M sodium phosphate buffer , pH 7 . 3 .", "After fixation , the cells were washed with 100 mM Tris ( pH 7 . 2 ) and 160 mM sucrose for 30 min .", "The cells were washed again twice with phosphate buffer ( 150 mM NaCl , 5 mM KCl , 10 mM Na3PO4 , pH 7 . 3 ) for 30 min , followed by treatment with 1% OsO4 in 140 mM Na3PO4 ( pH 7 . 3 ) for 1 hr .", "The cells were washed twice with water and stained with saturated uranyl acetate for 1 hr , dehydrated in ethanol , and embedded in Epon ( Electron Microscopy Sciences , Hatfield , PA ) .", "Roughly 60 nm sections were cut and stained with uranyl acetate and lead nitrate .", "Further , the stained grids were analyzed using a Philips CM-12 electron microscope ( FEI; Eindhoven , The Netherlands ) and photographed with a Gatan Erlangshen ES1000W digital camera ( Model 785 , 4 k 3 2 . 7 k; Gatan , Pleasanton , CA ) .", "Morphometric analysis of mitochondrial was analyzed by double-blinded independent observers in at least 10 different micrographs per condition .", "The mitochondria without intact inner mitochondrial membrane and cristae were classified as damaged , and their number was counted in each cell and divided by the total number of mitochondria in that cell to calculate % damaged mitochondria .", "The mitochondrial area was measured by manually tracing the outer mitochondrial membrane and using the measure function of NIH ImageJ software .", "Cristae per mitochondria were calculated by counting the number of intact cristae in each mitochondrion .", "Cells were cultured in 6-well plates at 50–60% confluency .", "The next day , 1 × 106 cells were harvested and loaded with Tetramethyl rhodamine ( TMRM ) dye .", "The cells were stained with 100 nM TMRM dye in complete growth media and kept at 37°C in 5% CO2 for 20–30 min .", "CCCP ( 50 µM ) was used as a positive control .", "To measure mitochondrial ROS , 1 × 106 cells were stained with MitoSOX ( 2 . 5 µM in HBSS at 37°C for 30 min ) .", "Antimycin ( 50 µM ) was used as a positive control .", "The intensity of staining was measured on an LSRII flow cytometer using FACSDiva software ( BD Biosciences ) and analyzed with FlowJo software ( Tree Star ) .", "The cells were harvested , and 3000–5000 cells were plated in each well of 96 well plates .", "The cells were kept at 37°C in 5% CO2 for 4 hr to allow the cells to adhere to the plate .", "The CyQUANT-NF dye was diluted in HBSS buffer , and 100 µl of the mixture was added in each well .", "The plate was kept at 37°C in 5% CO2 for 1 hr .", "The fluorescence intensity ( ~485 nm/~530 nm ) was measured using FlexStation 3 Multimode Plate Reader ( VWR ) .", "To analyze the effect of 2-DG and 5-FU on proliferation , different doses of the drugs were added in the culture media and cells were grown for the indicated amount of time .", "The fluorescence intensity of the dye was recorded after the drug treatment .", "The normalized intensity was calculated using the following formula: Briefly , the cells were harvested and cultured in 6-well plates .", "Once the culture becomes 80–90% confluent , 20 µl of the media was taken and mixed with Tris-Glycine-SDS sample buffer and loaded in a Novex Zymogram gel and Tris-glycine SDS running buffer was used to run the gel .", "After electrophoresis , the gel was kept in Novex Zymogram renaturing buffer for 30 min and transferred to Novex Zymogram Developing buffer and kept at 37°C overnight .", "The next day the gel was stained with SimplyBlue Safe Stain and imaged using FluorChem M imager .", "The images were analyzed using ImageJ .", "Migration was measured using two different methods a transwell migration assay and wound healing assay .", "For the transwell migration assay using FluoroBlok , the cells were serum-starved for 24 hr , and 1 , 000 cells were plated on the chamber in 200 µl media without FBS .", "The cells were stimulated to migrate towards preconditioned media with 10% FBS .", "After 8 hr of migration , the upper part of the membrane was cleaned , and the lower part was stained with DAPI , and the intensity of DAPI was measured using FlexStation 3 Multimode Plate Reader ( VWR ) .", "For the wound healing ( gap closure ) assay , cells were serum-starved for 24 hr to synchronize the cell cycle .", "Then 50 , 000 cells were plated in ibidi-silicone insert with a defined cell-free gap .", "After 24 hr , the inserts were lifted , and the gap between two migrating fronts was measured at 0 hr , 12 hr , and 24 hr using Zeiss inverted fluorescence microscope equipped with 5X air objective .", "The area between the two migrating fronts was measured using ImageJ ( NIH , Bethesda ) .", "To analyze the effect of 2-DG and 5-FU on migration , a specific dose of the drugs was added in the culture media at the time of removal of the silicon insert , and cells were grown for the indicated amounts of time .", "Fresh media with the drug was added every 12 hr .", "The following formula was used to calculate % gap closure: Cell invasion was measured using the BioCoat Tumor Invasion Plate with 8 µm pore size inserts , which were coated with growth factor-reduced Matrigel .", "The cells were serum-starved for 24 hr , and a total of 5 , 000 cells were plated on the chamber in 200 µl media without FBS .", "The cells were stimulated to invade towards preconditioned media with 10% FBS .", "After 24 hr invasion , the upper part of the membrane was cleaned , and the lower part was stained with DAPI , and the intensity of DAPI was measured using FlexStation 3 Multimode Plate Reader ( VWR ) .", "The percentage of invasion was calculated using the following formula-%invasion= ( Fluorescenceofinvadingcells−Fluorescenceofblankchamber ) / ( Fluorescenceofmigratingcells−Fluorescenceofblankchamber ) X100 The ECAR , OCR , and % metabolite dependency was measured using the Seahorse XFp Extracellular Flux Analyzer ( Seahorse Bioscience ) .", "All experiments were performed according to the manufacturer’s protocol .", "Seahorse XFp Glycolysis Stress Test Kit , Seahorse XFp Cell Mito Stress Test Kit , and Seahorse XFp Mito Fuel Flex Test Kit ( Agilent Technologies ) were used to measure ECAR , OCR , and % dependency respectively .", "Briefly , cells were harvested , and 30 , 000 cells were plated per well and cultured in a Seahorse XFp cell culture microplate with complete growth media for 10–12 hr .", "The next day the cells were washed with Seahorse XF DMEM media ( pH 7 . 4 ) .", "The plate with cultured cells was placed in the Seahorse XFp Extracellular Flux Analyzer , and baseline measurements were recorded .", "For ECAR measurements , oligomycin , and 2-DG were sequentially injected into each well at the indicated time points .", "Similarly , for OCR measurements , oligomycin , FCCP ( p-trifluoromethoxy carbonyl cyanide phenylhydrazone ) , and antimycin A ( Rote/AA ) were sequentially injected .", "For % dependency measurements , UK5099 , BPTES , and Etomoxir were sequentially injected .", "The results obtained for ECAR , OCR , and Mito Fuel Flex Test were normalized to cell number .", "Data were analyzed using Seahorse XFp Wave software .", "The results for OCR are reported in pmols/minute , ECAR in mpH/minute , and Mito Fuel Flex Test in % dependency .", "To measure glucose and lactate in media , 100 , 000 cells were plated in each well of 6-well plates with 2 ml of complete growth media .", "After 24 hr , 400 µl of the media were taken from each well of the 6-well plates and divided into four wells in a 96-well plate ( quadruplicate for each NCLX KO clone and respective control ) .", "Then the plate was kept in YSI 7100 multichannel biochemistry analyzer ( YSI Life Sciences ) to measure glucose and lactate levels in the media .", "Fresh media was used to measure the basal level of glucose and lactate .", "After measurements were completed , cells were harvested , and the protein content was measured using the BCA assay .", "This experiment was performed at least three times independently .", "The following formulas were used to calculate glucose consumption and lactate generation: Mice were anesthetized using isoflurane , and after proper sterile preparation of the abdomen , a small ( 4–8 mm ) incision was made by sharp sterile blade over the left upper quadrant of the abdomen .", "The peritoneal cavity was carefully exposed , and the spleen was located .", "Further , the spleen was carefully exteriorized on the sterile field around the incision area , and 500 , 000 cells suspended in 50 µl McCoy’s ( 10 or 20% FBS ) were then injected into the spleen via a 1 ml insulin syringe .", "A small bleb was observed while injecting the cells in the spleen , confirming that the cells were successfully injected .", "The spleen was carefully placed back into the peritoneal cavity .", "Both the peritoneal cavity and skin were closed with sterile absorbable suture .", "The mice injected with luciferin ( 100 µg/ml ) and imaged in the IVIS system every week till the time of sacrifice .", "6–8 week-old mice were given a single azoxymethane ( AOM ) injection via IP .", "One week after AOM injection , the mice were given three cycles of dextran sodium sulfate ( DSS ) , which was supplemented in drinking water ( 1 . 5 % m/v , MW 36–50 kDa , MP Biochemicals ) ad libitum for five days .", "The DSS cycles were intermixed with two weeks of normal autoclaved drinking water .", "Mice were sacrificed , and the colons were collected ten weeks after the last DSS cycle .", "The collected colons were flushed with PBS to clear feces and photographed .", "Before dissection , tumors were scored according to their number and size .", "PBS-rinsed colons were either snap-frozen for further analysis or fixed in 10% neutral buffered formalin and sectioned for histological analysis .", "Data are represented as mean ± SEM and analyzed using Origin pro 2019b ( Origin lab ) .", "In the box plot , the box represents the 25th to 75th interquartile range , midline in the box represents the median , and the solid square box represents mean data points .", "To test single variables between two groups , paired t-test or Kruskal-Wallis ANOVA was performed ( specified in each figure legend ) .", "One-way ANOVA followed by post-hoc Tukey’s test was used for comparison between multiple conditions and the control group .", "The p-value<0 . 05 was considered to be significant and is presented as *p<0 . 05 , **p<0 . 01 , or ***p<0 . 001 ." ] ]

[ "Despite the established role of mitochondria in cancer , the mechanisms by which mitochondrial Ca2+ ( mtCa2+ ) regulates tumorigenesis remain incompletely understood .", "The crucial role of mtCa2+ in tumorigenesis is highlighted by altered expression of proteins mediating mtCa2+ uptake and extrusion in cancer .", "Here , we demonstrate decreased expression of the mitochondrial Na+/Ca2+/Li+ exchanger NCLX ( SLC8B1 ) in human colorectal tumors and its association with advanced-stage disease in patients .", "Downregulation of NCLX causes mtCa2+ overload , mitochondrial depolarization , decreased expression of cell-cycle genes and reduced tumor size in xenograft and spontaneous colorectal cancer mouse models .", "Concomitantly , NCLX downregulation drives metastatic spread , chemoresistance , and expression of epithelial-to-mesenchymal , hypoxia , and stem cell pathways .", "Mechanistically , mtCa2+ overload leads to increased mitochondrial reactive oxygen species , which activate HIF1α signaling supporting metastasis of NCLX-null tumor cells .", "Thus , loss of NCLX is a novel driver of metastasis , indicating that regulation of mtCa2+ is a novel therapeutic approach in metastatic colorectal cancer ." ]

[ "Colorectal cancer is the second largest cause of cancer deaths worldwide .", "Even in cases where the cancer is diagnosed and treated early , cells can sometimes survive treatment and spread to other organs .", "Once the cancer has spread , the survival rate is less than 15% .", "Mitochondria are compartments in the cell that produce energy , and they play an important role in supporting the rapid growth of cancer cells .", "The levels of calcium ions in mitochondria control how they produce energy , a process that is altered in cancer cells .", "To better understand how calcium ions influence colorectal cancer growth , Pathak , Gueguinou et al . studied a protein called NCLX , which controls calcium levels by pumping them out of the mitochondria .", "Two mouse strains that were used to study what happens if NCLX is missing .", "The first strain was genetically modified to disable the gene for NCLX and then exposed to carcinogens .", "The second strain was injected with colorectal cancer cells from a human tumor that were lacking NCLX .", "In both strains , the tumors that formed were smaller than in mice with NCLX .", "However , the human cancer cells in the second model were more likely to spread to other organs .", "This is likely because the build-up of calcium ions in the mitochondria of mice lacking NCLX led to an increase in the production of hypoxia-inducible factor-1a , a protein that is a common driver of cancer spread .", "Pathak , Gueguinou et al . demonstrated how NCLX can affect colorectal cancer progression .", "It suggests that it may have opposing effects during early and late-stage colorectal cancer , encouraging tumor growth but also decreasing the spread to other organs .", "Further research could help refine treatments at different stages of the disease ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "stem cells and regenerative medicine", "developmental biology" ]

[ [ "The cardiac vascular system is comprised of blood and lymphatic vessels .", "Arteries and connected capillaries transport oxygenated , nutrient-rich blood to the myocardium .", "Cardiac veins drain the blood back into the systemic circulation , and excess fluid and nutrients from the blood are hydrostatically released into tissue during this process .", "This interstitial fluid , immune cells , debris and waste products are then drained via the cardiac lymphatic vessels .", "The heart critically requires this continuous cycle through the myocardium for optimal function and interruption has pathological results including myocardial infarction ( MI ) or cardiac lymphedema ( Aspelund et al . , 2016; Karunamuni , 2013 ) .", "Not only are the cardiac vessel systems required for heart function , but they are also likely to have active roles in disease resolution , at least helping to provide a permissive environment for regeneration ( Das et al . , 2019; Klotz et al . , 2015; Marín-Juez et al . , 2016 ) .", "This has yet to be successfully leveraged clinically in part due to the lack of effective therapeutic strategies ( Taimeh et al . , 2013 ) .", "A more detailed understanding of these systems , their formation , regeneration and promotion of positive disease environments are critically required .", "Zebrafish represent a simple coronary vessel system , many features of which are conserved in the mammalian system .", "However , unlike mammals , the coronary structure in zebrafish starts to develop relatively late at juvenile stages when endocardial cells that give rise to coronary vessels sprout from the atrioventricular ( AV ) canal region of the heart to vascularize the ventricle ( Harrison et al . , 2015 ) .", "The existence and development of a cardiac lymphatic system to complement this coronary system has yet to be described in zebrafish .", "Extensive work using zebrafish embryonic models has yielded much of our understanding of embryonic lymphatic development ( Hogan and Schulte-Merker , 2017 ) .", "During trunk angiogenesis , lateral plate mesoderm-derived angioblasts from the ventral side of the posterior cardinal vein ( PCV ) migrate to the dorsal myoseptum , lose their connection with the PCV and become lymphangioblasts known as parachordal cells ( Hogan et al . , 2009a; Nicenboim et al . , 2015 ) .", "These cells then migrate from the midline along arteries under the guidance of Cxcl12b-Cxcr4a/b chemokine signaling to form lymphatic vessels , including the thoracic duct between the dorsal aorta and PCV ( Cha et al . , 2012 ) .", "This thoracic duct is connected to a continuous dorsal longitudinal lymphatic vessel via intersegmental lymphatic vessels , which align along the same intersegmental arteries that provided guidance during their formation ( Bussmann et al . , 2010 ) .", "This process of trunk lymphangiogenesis is completely blocked in embryos with mutations in genes encoding the secreted vascular endothelial growth factor C ( vegfc ) or its receptor flt4/vegfr3 ( Hogan et al . , 2009b; Villefranc et al . , 2013 ) .", "Vegfc is similarly required for the sprouting of lymphatic endothelial cells in mice ( Karkkainen et al . , 2004 ) and , as in zebrafish embryos , these lymphatic endothelial cells are derived primarily from venous endothelium ( Hägerling et al . , 2013; Yang et al . , 2012 ) with some contribution from non-venous sources to specific lymphatic vasculature beds ( Ulvmar and Mäkinen , 2016; Eng et al . , 2019 ) .", "In the case of mouse cardiac lymphatic system , there is an additional contribution to the lymphatic vasculature from yolk sac endothelial cells ( Klotz et al . , 2015 ) .", "Together with venous-derived lymphatic endothelial cells , they invade the embryonic heart at embryonic day ( E ) 12 . 5 from an extra-cardiac source to populate its base via the sinus venosus and outflow tract ( Flaht et al . , 2012; Klotz et al . , 2015 ) .", "This lymphatic endothelium then continues to sprout over the surface of the ventricle along Emcn-expressing cardiac veins .", "Vessels expressing lymphatic endothelial marker genes Lyve-1 , Prox1 and Flt4 are identifiable specifically along the side of the cardiac veins by birth and continue expanding over the ventricle until postnatal ( P ) day 15 ( Klotz et al . , 2015 ) .", "After a myocardial injury , the cardiac lymphatic vasculature is reactivated .", "Despite this expansion of the lymphatic network after myocardial infarction , the mammalian heart still scars rather than regenerates functional tissue .", "However , a reduction in this scarring can be observed when Vegfc is induced to further enhance lymphangiogenesis in injured adult mice ( Klotz et al . , 2015 ) .", "The cardiac lymphatic system is thought to regulate fluid homeostasis and provide immune system surveillance and clearance which may have important implications for cardiac tissue recovery after insult ( Vieira et al . , 2018 ) .", "In contrast to the mammals , the zebrafish has retained a remarkable capacity to regenerate cardiac tissue after tissue damage or resection ( Gamba et al . , 2014; Poss et al . , 2002 ) .", "After resection or moderate cryoinjury to the apex or ventral wall of the ventricle , fully functional cardiac tissue is regenerated within 30–90 days and there is little or no detectable scar in the majority of injured zebrafish ( Chablais et al . , 2011; González-Rosa and Mercader , 2012; Poss et al . , 2002 ) .", "The regenerated tissue is also vascularized by blood vessels by this time and this vascularization supports the function of this regenerated tissue as well as the repair process itself ( Marín-Juez et al . , 2016 ) .", "Historical anatomical descriptions of lymphatic vessels surrounding the fish heart ( Hewson and Hunter , 1769 ) suggest that these vessels constitute an ancient feature of jawed vertebrates , but the functional overlap of this vasculature with that of mammalian lymphatic vessels has been questioned ( Vogel and Claviez , 1981 ) .", "As such the existence and function of the cardiac lymphatic vasculature in zebrafish and its potential implication for normal tissue homeostasis as well as natural regeneration remains an open question of significant clinical interest .", "We here characterize of the development of zebrafish cardiac lymphatic vasculature , describe its functions and analyze lymphangiogenesis during heart regeneration .", "Our results suggest that cardiac lymphatic vessels should be a promising therapeutic target for the treatment of heart disease and may help modulate a pro-regenerative immune response ." ], [ "To begin to investigate the subtype identity of the cardiac lymphatic vasculature in the adult zebrafish , we used the flt4:mCitrine transgenic line that is expressed in both venous and lymphatic endothelial cells ( Gordon et al . , 2013; van Impel et al . , 2014 ) .", "flt4:mCitrine endothelial expression is detected at either systemic pole of the heart in the sinus venosus ( SV ) and on the surface of the bulbus arteriosus ( BA ) ( Figure 1A ) .", "The population of endothelial cells on the outside of the BA was of particular interest given that they cover the entire BA , but do not extend down on the ventricle until adult stages ( after 90dpf; Figure 1A–C ) .", "In young adult fish , flt4:mCitrine-positive cells bud from this BA population and appear to migrate down on the ventricle to form a vessel that extends to the apex of the ventricle ( Figure 1B–D ) .", "To determine if this vasculature is venous or lymphatic we used the prox1:Gal4-UAS:RFP transgenic line that is not expressed in venous endothelium , but in lymphatic endothelial cells ( LECs ) and neurons ( van Impel et al . , 2014 ) .", "Expression in the BA population suggests that this is lymphatic endothelium unlike the flt4:mCitrine-positive , prox1:Gal4-UAS:RFP-negative cells of the sinus venous , ( Figure 1E , F , Figure 1—figure supplement 1A , B ) .", "The lack of markers exclusively specific to lymphatic endothelial cells has been one of the precluding factors for their visualization and study ( Jung et al . , 2017 ) .", "We confirmed the lymphatic identity of this endothelium with four additional reporter lines known to be expressed in lymphatic endothelial cells: lyve1b:GFP , lyve1b:DsRed ( Okuda et al . , 2012 ) , stab1:YFP ( Hogan et al . , 2009a ) and mrc1a:GFP ( Jung et al . , 2017 ) ( Figure 1—figure supplement 1C-F ) .", "As such the cardiac lymphatic system is identifiable in adult zebrafish and appears to extend from a BA population in older zebrafish .", "We next addressed the stage at which the BA population develops relative to coronary vessel development at juvenile stages .", "The formation of a cardiac lymphatic vessel on the ventricle of adult zebrafish from a pre-existing population on the BA occurs very late and after the formation of the coronary vasculature in juvenile zebrafish .", "In rodent models , cardiac lymphatic vasculature on the ventricle is observed in embryos by E14 . 5 ( Klotz et al . , 2015 ) .", "However in zebrafish we found that the lymphatic endothelium is first established on the BA post-embryonically , before the development of the coronary vasculature ( Figure 1G–I ) .", "The lymphatic endothelium extends to the base of the BA into the cleft between the BA and the ventricle ( Figure 1J ) .", "Within this cleft the lymphatic endothelial cells sprout along coronary vessels to extend back up out on the ventricle side ( Figure 1J ) .", "The sprouting lymphatic cells extend along the coronary arteries and track them as they progress down the ventricle towards the apex of the heart ( Figure 2A–F ) .", "As the cells extend towards the apex they expand and flatten around the artery to form a vessel .", "This vessel is in close proximity to the arterial endothelium that expresses higher levels of kdrl:mTurquoise at this stage and shows more specific expression of flt1enh:tdTomato , dll4:GFP and cxcr4a:mCitrine ( Figure 2D–G ) .", "This association between an artery and lymphatic vessel is also observed as the lymphatic vasculature extends along the aorta and branches off to the brachial aches .", "Here the lymphatic vessels associate with the brachial arteries and the arterial side of the gill filaments ( Figure 2—figure supplement 1A–D ) .", "In both zebrafish and mammals , there is a sequential development of the coronary vasculature and the cardiac lymphatic system suggesting that the coronary vessels provide a scaffold or guidance for the lymphatic system .", "To test this hypothesis , we analyzed the formation of cardiac lymphatic vessels in cxcr4a mutant zebrafish that lack coronary arteries .", "Unlike intersegmental lymphatic vessels , cxcr4a:mCitrine expression in the adult heart is restricted to the coronary arteries and is absent in migrating LECs and lymphatic vessels that form along them ( Figure 3A–C ) .", "Zebrafish with mutations in cxcr4a present with disrupted coronary vessel development and largely lack formed coronary vessels in young adult fish ( Figure 3—figure supplement 1B ) .", "These mutants fail to regenerate after ventricular injury ( Harrison et al . , 2015 ) .", "In older adult cxcr4a mutant zebrafish there is some formation of lumenized vessels , but the coronary vasculature structure remains highly disorganized over the ventricle .", "Often this coronary vasculature presents as a conglomerate of enlarged or dense interconnected by sparse intermediary non-luminized vessels or isolated cells ( Figure 3E and F , Figure 3—figure supplement 1A–F ) .", "The density of this vasculature increases with time , as observed in wildtype transgenic zebrafish , however it lacks any identifiable coronary arteries ( Figure 3—figure supplement 1G–H , Figure 3—figure supplement 2C-M ) .", "Analysis of lymphatic markers in cxcr4a mutant zebrafish demonstrated that cardiac lymphatic vessels do not extend down the ventricle in the absence of coronary arteries , but that the pre-forming BA population is unaffected ( Figure 3D–H , Figure 3—figure supplement 1A–H , Figure 3—figure supplement 2A-M ) .", "There is a significant correlation between the extent of the coronary arterial tree over the ventricle and coverage of the ventricle by the cardiac lymphatic vessels ( Figure 3G , Figure 3—figure supplement 2M ) , suggesting that the later uses the coronary vessels to migrate down the ventricle and that coronary artery derived signals may promote this process during development .", "The zebrafish ventricle shows an amazing capacity to regenerate following insult or injury and previous work suggested that vegfc expression is elevated during this regeneration process ( Lien et al . , 2006 ) .", "In adult hearts vegfc expression is observed on the BA in its junction with the ventricle base , but little detectable signal is observed on the ventricle itself ( Figure 4A , Figure 4—figure supplement 1A ) .", "After amputation elevated vegfc levels are detectable at the wound site at 3dpa and 7dpa , but expression is reduced by 14dpa and absent thereafter ( Figure 4B , Figure 4—figure supplement 1B-G ) .", "To test the effect of having remaining necrotic tissue and a pre-existing extracellular matrix after injury , we used the more severe cyroinjury model .", "Cryoinjury also results in vegfc expression at 3dpc also , however subsequent expression appears to be more prolonged and remains elevated after 42dpc ( Figure 4C and D , Figure 4—figure supplement 1H-L and G ) .", "Following resection of the ventricle apex there is little expansion of the cardiac lymphatic vasculature , with only some hearts showing extension of the lymphatic vessels into the wound site after 14dpa ( Figure 4E–H ) .", "If present these lymphatic vessels appear to largely be extensions of the existing lymphatic vessel into the apex and do not appear to be enlarged or expanded ( Figure 4E–H , Q and R; 4/7 hearts at 60dpa show wound site lymphatic vessels ) .", "In contrast , after cryoinjury migration of LECs into the wound site is observed at 14dpc , but also enlargement and expansion of the vessel on the ventricle ( Figure 4I–P , S–V ) .", "Particularly at later stages , a highly branched network of cardiac lymphatic vasculature is observed within the wound site ( Figure 4L–P , S–V ) .", "These regenerated lymphatic vessels express flt4:mCitrine , prox1:Gal4-UAS:RFP , lyve1:RFP , mrc1a:eGFP ( Figure 4L , N–P ) .", "Quantification of lymphatic vessel coverage within a set area of the ventricle shows significantly more lymphatic vessels in or around the wound site after cryoinjury when compared to the resection model ( Figure 4U ) .", "In addition post-cryo-injured hearts have a more branched cardiac lymphatic structure over the ventricle ( Figure 4V ) .", "This suggests that the presence of necrotic tissue and scar formation promotes the formation of more lymphatic vessels after cardiac injury .", "We next wanted to address if this was related to the function of the cardiac lymphatic system and its potential utilization in a disease setting .", "The lymphatic system in mammals is known to maintain fluid homeostasis and modulate immune surveillance and clearance ( Alitalo , 2011 ) .", "Given these functions it has long been considered an integral apparatus in the maintenance of heart function and postulated to encourage a regenerative response to injury ( Aspelund et al . , 2016; Karunamuni , 2013; Klotz et al . , 2015; Vieira et al . , 2018 ) .", "In order to ascertain the function of the zebrafish cardiac lymphatic vasculature and in light of the discontinuous nature of LECs sometimes observed in these vessels ( Figures 2 and 4 ) , we established an intra-myocardial injection assay of microspheres ( MS ) and quantum dots ( Qdots ) .", "Within 1 hr both were found at the injection site ( Figure 5A ) , however the smaller Qdots ( <10 nm diameter ) were more dispersed from the injection site as well as being concentrated within the lymphatic lumen ( Figure 5A–C , Figure 5—video 1 ) .", "This demonstrates that , despite the discontinuations in lyve1:RFP marker expression , the ventricular cardiac lymphatic vessel forms a blunt ended contiguous tube alongside the blood vessels .", "In addition these vessels are pervious to the uptake of fluid and small , but not large , debris as the larger microspheres ( 200 nm diameter ) remained at the injection site , but the smaller Qdots become dispersed within the interstitium and are taken up into the lymphatic vessels .", "In the uninjured heart , resident macrophages ( labeled with IB4 ) were observed near the vasculature and there were very few neutrophils ( Mpx-positive ) present on the ventricle ( Figure 5—figure supplement 1A ) .", "Given the strong lymphangiogenic response to cryoinjury ( Figure 4 ) , we decided to investigate the immunological role of the cardiac lymphatic system after a mild cryoinjury .", "Following injury , macrophages migrate to the wound site and are joined by a large number of neutrophil cells ( Figure 5—figure supplement 1B ) .", "Clearance of these cells is known to be a key step in the process of regenerating heart tissue ( Lai et al . , 2017; Vieira et al . , 2018 ) .", "We find lymphatic vessels may provide a conduit for this clearance during zebrafish heart regeneration .", "After 1 and 7dpc ( mild cryoinjury ) mpx-positive neutrophil cells and debris became highly enriched within or on the cardiac lymphatic vessels on the BA , a location relatively distant from the more apex ventricular wound site ( Figure 5D–H , Figure 5—figure supplement 1C–G ) .", "Despite weak expression on the ventricle of lyve1:GFP that precluded antibody detection above background , Mpx-positive cells were observed to align up along the optically dense blood vessels on the ventricle ( Figure 5E insert ) consistent with their association and uptake into lymphatic vessels .", "Immune cells were observed within the labeled BA cardiac lymphatic vessels after injury ( Figure 5G , Figure 5—figure supplement 1E and F , Figure 5—videos 2–5 ) .", "This suggests these cells can be taken up by the cardiac lymphatic vasculature at or near the wound site after injury and then transported away from the heart via the lymphatic vasculature on the aorta .", "Given Vegfc’s well established role in lymphatic vasculature development ( Aspelund et al . , 2016; Joukov et al . , 1996; Kaipainen et al . , 1995 ) and its potential role after injury ( Figure 4 ) we decided to test if it was utilized during cardiac lymphatic system development .", "We inactivated the Vegfc ligands by induced expression of a soluble version of the Flt4/Vegfr3 receptor ( serving as a ligand trap; sFlt4 ) in transgenic juvenile zebrafish during the formation of the ventricular cardiac vessels ( Matsuoka et al . , 2017 ) .", "Unexpectedly , in heat shocked non-transgenic control zebrafish we observed an increase in lymphatic vessels on the heart relative to non-heat shocked controls ( Figure 6A , B , Figure 6—figure supplement 1A , B , D ) .", "This increase in cardiac lymphatic vessels is not attributable to an increase in body size , in fact , heat shocked zebrafish showed a slight reduction in the overall length compared to non-heat shocked zebrafish at the same stage ( Figure 6—figure supplement 1E ) .", "In stark contrast , the induced transgenic zebrafish that expressed sFlt4 from 35dpf had few or no LECs on the ventricle and no cardiac lymphatic vessels on the ventricle ( Figure 6C and D , Figure 6—figure supplement 1C , D ) .", "This was also the case with sFlt4 induction during and after coronary vessel induction in older juvenile ( from 71dpf ) and adult zebrafish respectively ( from 91dpf; Figure 6—figure supplement 1G–J ) .", "Induction of sFlt4 from 35 , 71 or 92dpf did not overtly affect the development of the coronary vasculature , nor did we observe overt edema or malformation ( Figure 6B , C , Figure 6—figure supplement 1B , C , F , G-N ) .", "Ventricle tissue appears to be normal with sFlt4 induction in juvenile zebrafish and does not significantly affect cardiomyocyte ( CM ) or non-CM numbers or the overall size of the ventricle ( Figure 6—figure supplement 1O-S ) .", "This suggests that functional post-embryonic Vegfc signaling is specifically critical for the formation of this relatively late developing structure .", "We next used these zebrafish that lacked cardiac lymphatic vessels in the presence of coronary arteries to specifically test the effect of this system on cardiac regeneration .", "To test if the zebrafish cardiac lymphatic system has the potential to influence the varying levels of fibrosis observed after cardiac injury ( Lai et al . , 2017 ) , we used zebrafish with differing levels of cardiac lymphatic vasculature due to the developmental heat shock and induction of a sFlt4 receptor ( Figure 6A–D ) , and performed cryoinjury and assayed scar size 60 days later without post injury induction of sFlt4 ( 60dpc , Figure 6D ) .", "Scoring these scars on the basis of severity we found large deposits of scar tissue in the transgenic fish ( 5/5 , large scar , >0 . 005 mm3 ) , in comparison to that of non-transgenic siblings , where the majority of scarring was relatively small ( 3/5 , minimal scar , <0 . 005 mm3 ) ( Figure 6E and F ) .", "Comparison of heart tissue scaring levels shows a significant increase in scarring severity with a reduction in the cardiac lymphatic vasculature ( Figure 6H ) .", "Hearts lacking ventricular cardiac lymphatic vessels have a significantly larger scar volume ( Figure 6—figure supplement 2A ) .", "This increase in scar volume could not be attributed to an observable defect in the coronary vasculature or a loss of coronary vessel angiogenesis at the wound site ( Figure 6—figure supplement 1A-C , G-J; Figure 6—figure supplement 2B-E ) .", "To test if the induction of vegfc after injury supported this function we inactivated the Vegfc ligands by induced expression of sFlt4/Vegfr3 receptor 2 days prior to and then after cryoinjury ( Figure 6—figure supplement 3A ) .", "As is the case with induction of sFlt4 during cardiac lymphatic development , we observed only moderate non-significant effects on contrary vessel regeneration ( Figure 6—figure supplement 3B-G ) and no relative difference in post-injury heart morphology or size with sFlt4 post-injury induction ( Figure 6—figure supplement 3H-K ) .", "Despite the lack of significant extension into the wound site at this stage , an increase in Mpx-positive neutrophils was observed at the 14dpc wound site with post injury induction of sFlt4 ( Figure 6I–K ) , suggesting that blocking Vegfc-signal could prolong the inflammatory response , potentially by limiting the removal of Mpx-positive cells and debris from the wound site .", "Indeed , some sFlt4-induced hearts still showed elevated neutrophil levels at later stages of regeneration ( Developmental induction , Figure 6—figure supplement 2F-H; Post-injury induction , Figure 6—figure supplement 4A-C ) .", "Post-injury induction to block the response of the cardiac lymphatic vasculature did result in a reduction in the regeneration of the cardiac wall and resulting internalization of the scar tissue ( Figure 6—figure supplement 4D-J ) .", "However the scar area itself showed no significant increase in these zebrafish ( Figure 6—figure supplement 4D-J ) .", "There appears to be no inhibition of the induction of cardiomyocyte proliferation or epicaridal activation following injury with overexpression of sFlt4 ( Figure 6—figure supplement 4K-P ) , suggesting instead that during regeneration the lack of ventricular cardiac lymphatic vessels , or perturbing their response to injury , shifts the balance of the wound site from pro-regenerative to pro-fibrotic .", "Consistent with this hypothesis we observed an inability of zebrafish that lack cardiac lymphatic vessels to modulate the wound environment and to resolve scar specifically after cryoinjury ( Figure 6H ) .", "The majority of amputation-injured heat shocked transgenic hsp70l:sflt4 zebrafish had only minimal scaring after 30 days ( 30dpa , Figure 6G and H ) .", "The amount of scar tissue in those zebrafish with detectable scars at 30dpa was relatively small and also observed in amputated control zebrafish , suggesting the role of cardiac lymphatic vessels is less critical for regeneration following resection ( Figure 6—figure supplement 5A–G ) ." ], [ "We here show that during zebrafish post-embryonic stages , there is a sequential development of blood vessel network and the cardiac lymphatic system as occurs embryonically in mice ( Flaht et al . , 2012; Klotz et al . , 2015 ) .", "The juvenile zebrafish heart undergoes significant morphological change and expansion due to the physiological demands of increasing body size on heart output ( Gupta and Poss , 2012; Harrison et al . , 2015 ) .", "This expanded myocardium likely requires auxiliary oxygenated blood supply and lymph clearance to function optimally ( Harrison et al . , 2015; Figure 1 ) .", "By contrast , in mammals the evolution of the pulmonary system has permitted a much elevated systemic blood pressure and metabolic rate that requires a greater cardiac output from birth ( Bettex et al . , 2014 ) .", "As such , the needs of the post-natal compact myocardium have driven the timing of cardiac blood and lymphatic vasculature development embryonically .", "The sequential development of cardiac blood and lymphatic vasculature is conserved between mammals and fish and has also been reported within the embryonic zebrafish trunk ( Bussmann et al . , 2010 ) .", "However , as in the zebrafish heart , intersegmental arteries , not veins , provide a scaffold for the extension of lymphatic vessel sprouts and are required for their formation ( Bussmann et al . , 2010 ) .", "We have identified a population of LECs that migrate down the arteries in the zebrafish heart ( Figures 1–3 ) and a similar interaction of developing cardiac lymphatic vessels with arteries has been described in the human fetus ( Kampmeier , 1928 ) .", "This is in contrast to mice in which the cardiac lymphatic vessels track cardiac veins rather than coronary arteries ( Klotz et al . , 2015 ) .", "In humans , like zebrafish , large arteries ( and to lesser extent veins ) are located subepicardially; in contrast the mouse heart has subepicardial veins , but deeper intramyocardial arteries ( Sharma et al . , 2017 ) .", "Zebrafish lymphatic development may represent the ancestral mechanism of lymphatic guidance which has been entirely conserved or diverged to different degrees across the mammalian class ( Ratajska et al . , 2014 ) .", "In each case , the cardiac lymphatic vessels appear to follow the more superficial blood vasculature on the ventricle and our data suggest this is required for or promotes their extension over the heart .", "We have demonstrated that loss of the coronary artery scaffold results in a failure of these vessels to extend down the ventricle in cxcr4a mutants that lack coronary arteries .", "Although Cxcr4-Cxcl12 signaling has been implicated in the arterial association of LECs in the zebrafish trunk ( Cha et al . , 2012 ) , we do not observe cxcr4a:mCitrine expression in lymphatic endothelial cells , but do not rule out that a similar direct signaling pathway , for example through Cxcr4b , could also occur in the cardiac system and that this may be compromised in cxcr4a mutants .", "We have however identified a signaling pathway required for the development of the cardiac lymphatic system in zebrafish .", "Vegfc/Flt4 signaling has been shown to have important roles in embryonic and perinatal angiogenesis ( Hogan et al . , 2009a ) , lymphangiogenesis , ( Küchler et al . , 2006; Nurmi et al . , 2015; Yaniv et al . , 2006 ) and maintenance of mature intestinal lymphatic vessels ( Nurmi et al . , 2015; Tammela et al . , 2008 ) .", "We here show that Vegfc is critical for the outgrowth of cardiac lymphatic vessels along the arteries of the ventricles in adult zebrafish ( Figure 6 ) .", "Our experiments also demonstrate that regular heat shock treatment of zebrafish actually promotes cardiac lymphatic development in non-transgenic control zebrafish ( Figure 6 ) .", "Such positive effect on lymphatic development is likely due to the induced stress response and associated demand on cardiac output that is observed during heat shock ( Sallin and Jaźwińska , 2016 ) .", "It also suggests that the natural expansion in adult zebrafish is regulated in part by physiological demands of the cardiac tissue to respond to the increasing body size of the fish .", "In much the same way , in juvenile hearts we see a requirement for a second source of blood vasculature as the myocardium expands ( Harrison et al . , 2015 ) .", "With increased vasculature and heart mass comes increased extravascular fluid and cellular waste products such that the increased interstitial fluid may need an auxiliary conduit from the heart back to the circulation .", "The cardiac lymphatic system in zebrafish appears to have the capacity to clear fluid and debris from the myocardium ( Figure 5 ) .", "Only moderate lymphangiogenesis is observed after amputation and the majority of the vasculature in the wound site is blood vasculature ( Figure 4 ) .", "Our analyses have demonstrated that there is a marked effect on the vascular response injury after extensive tissue damage ( cyroinjury ) .", "Upon cryoinjury , there is a significant amount of lymphangiogenesis in and around the regeneration site that is not observed after amputation ( Figure 4 ) .", "Both injury models lead to increased vegfc levels , although this expression is more prolonged after cyroinjury ( Figure 4 ) .", "Coupled with a strong immune response to cryoinjury that could utilize the cardiac lymphatic system for the removal of cell debris and immune clearance ( Figure 5 ) we postulate that this response can avoid a prolonged inflamed wound site and aid regeneration ( Lai et al . , 2017; Vieira et al . , 2018 ) .", "Consistent with this we see large scar tissue volume following severe cryoinjury of zebrafish ventricles that lack lymphatic vessels ( Figure 6 ) .", "Scar tissue is detectable in control ventricles at 60dpc , but the size and severity of this scar tissue is less than that observed in hsp70l:flt4 zebrafish without cardiac lymphatic vasculature ( Figure 6 ) .", "We also observe significantly more neutrophils at the 14dpc wound site with post-injury induction sFlt4 ( Figure 6 ) .", "This is early in the response of the lymphatic vessels to injury ( Figure 4 ) , but suggests compromised ability of the ventricular cardiac lymphatic vessels to remove the influx of neutrophils to the woundsite ( Lai et al . , 2017 ) with blocking Vegfc-signaling .", "We cannot rule out a direct effect on elevating the immune response with blockade of Vegfc-signal , but the resulting shift has a potent negative effect on the continuing regenerative environment of the tissue , while not directly effecting cardiomyocyte proliferation , epicardial activation , coronary vessel revascularization or heart structure and morphology ( Figure 6 ) .", "Consistent with our studies using sFlt4 , recently it has been reported that a vegfd-mutation in a vegfc-hypermorphic background can also limit the extension of the cardiac lymphatic vessels down the ventricle while not affecting coronary vessels ( Vivien et al . , 2019 ) .", "Further , almost half of the vegfc/d mutant zebrafish surviving to adulthood fail to complete regeneration by 180dpc ( Vivien et al . , 2019 ) .", "Interestingly , these zebrafish also show cardiac hypertrophy , which we did not observe after induction of sflt4 at juvenile stages ( Figure 6—figure supplement 1 ) .", "It remains to be determined if sflt4 induction during embryonic stages results in cardiac hypertrophy .", "Nonetheless our results suggest that disruption of the cardiac lymphatic system impacts the hearts response to injury , rather than the enlargement of the myocardium .", "Shifting the regenerative balance in human hearts for patients that have suffered acute myocardial infarct or insult and related sequelae will likely require the priming of a number of component systems in the heart including that which supplies blood to the injured tissue and removes fluid and debris .", "Key for this is a detailed understanding of the development of these systems and their interaction in a regenerative environment , which will ultimately help us understand what changes to modulate in patients and how this might be done .", "Zebrafish has provided an excellent system to understand the development of these systems from a deeper evolutionary understanding through to the ability of these systems to regenerate and play important roles in promoting the regenerative response of adult cardiac tissue to injury ." ], [ "The following zebrafish lines were raised and maintained at Children’s Hospital Los Angeles ( CHLA ) under standard conditions of care ( Aleström et al . , 2019 ) and with CHLA IACUC oversight .", "IACUC vetted and prior approved all experimental procedures used in this study .", "Tg ( fli1a:EGFP ) y1 ( Lawson and Weinstein , 2002 ) , Tg ( −5 . 1myl7:DsRed2-NLS ) f2 ( Mably et al . , 2003 ) , Tg ( −6 . 5kdrl:mCherry ) ci5 ( Proulx et al . , 2010 ) , Tg ( −0 . 8flt1:RFP ) hu5333 ( referred to as flt1enh:tdtomato ) ( Bussmann et al . , 2010 ) , TgBAC ( flt4:Citrine ) hu7135 ( referred to as flt4:YFP ) ( Gordon et al . , 2013 ) , TgBAC ( prox1aBAC:KalTA4-4xUAS-ADV . E1b:TagRFP ) nim5 ( referred to as prox1a:Gal4-UAS:RFP ) ( van Impel et al . , 2014 ) , Tg ( ubb:LOX2272-LOXP-RFP-LOX2272-CFP-LOXP-YFP ) a132 ( referred to as ubb:zebrabow ) ( Pan et al . , 2013 ) , Tg ( kdrl:Cre-ERT2 ) fb13 ( Zhao et al . , 2014 ) , Tg ( flk1:EGFP ) s843 ( referred to as kdrl:GFP ) ( Jin et al . , 2005 ) , Tg ( gata1a:DsRed ) sd2 ( Traver et al . , 2003 ) , Tg ( hsp70l:flt4 , cryaa:Cerulean ) ( Matsuoka et al . , 2016 ) , Tg ( −5 . 2lyve1b:DsRed ) nz101 , Tg ( −5 . 2lyve1b:EGFP ) nz151 ( Okuda et al . , 2012 ) , TgBAC ( cxcr4a:Citrine ) mu104 ( Harrison et al . , 2015 ) , Tg ( stab1:YFP ) hu4453 ( Hogan et al . , 2009a ) , Tg ( mrc1a:EGFP ) y251 ( Jung et al . , 2017 ) , Tg ( ubb:LOXP-AmCyan-LOXP-ZsYellow ) fb5 ( referred to as ubb:CSY ) ( Zhou et al . , 2011 ) , Tg ( dll4:EGFP ) lcr1 ( Sacilotto et al . , 2013 ) transgenic lines have been described previously , as has the cxcr4aum20 mutation ( Bussmann et al . , 2011 ) .", "Tg ( hsp70l:sflt4 , cryaa:Cerulean ) juvenile zebrafish were heat-shocked at 39°C for 1 hr 30 min , three times a week from 35d/1mpf to >6 mpf , or four times a week from 71dpf and 92dpf to >6 mpf ( or up to the day prior to surgery , with then no heat-shock 21 , 60dpc or 30dpa ) .", "Sibling zebrafish ( both transgenic and non transgenic ) were raised at the same density and heat-shocked at the same time .", "Non-heat-shocked controls born on the same day were maintained at the same density , but not subjected to heat-shock .", "Post-injury induction was carried out in a similar fashion .", "For studies performed at 14 and 56dpc fish were first heat-shocked initially for 2 days prior to injury then four times per week after injury .", "For studies performed at 3dpc , fish were heat-shocked daily for 10 days ( seven prior to injury ) .", "Tg ( kdrl:mTurquoise ) was generated as outlined ( Bussmann and Schulte-Merker , 2011 and unpublished ) .", "Standard confocal imaging was carried out as described previously ( Harrison et al . , 2015 ) .", "Whole mount antibody and cleared native fluorescent zebrafish heart tissue was isolated in the same manner but fixed for 2 hr at room temperature in 4% PFA/PBS before being immunolabeled or mounted in 1% agarose for clearing; further details of which included in a forthcoming publication .", "Whole-mount immunolabeling was carried out on zebrafish hearts following fixation as follows .", "Hearts were bleached in Dent’s bleach ( DMSO:H2O2:methanol , 1:2 . 5:40 ) overnight , rinsed in methanol and then fixed overnight in Dent’s fixative ( DMSO:Methanol 1:4 ) at 4°C and washed in PBS + 0 . 1%Tween ( PBST ) for 3 to 8 hr the next day .", "Hearts were then incubated in primary antibody ( see Table 1 ) in blocking solution ( HIHS:DMSO:PBS , 1:4:15 ) for 3–5 days .", "After which they were washed with PBST for 6 hr .", "Hearts were then incubated secondary antibody ( see Table 1 ) in blocking solution overnight , then washed in PBST for 5 hr .", "Hearts were then mounted in low melting point agarose and imaging was carried out as before ( Harrison et al . , 2015 ) .", "Section immunohistochemistry was carried out on de-waxed paraffin sections ( Toluene 5 min x2 , 100% ethanol 5 min x2 , 80% ethanol 5 min , PBS 5 min x2 ) , after antigen retrieval in Unmasking Solution ( Vector Laboratories , H-3300 ) .", "Primary antibody ( see Table 1 ) was applied overnight at 4°C in blocking solution ( 5% goat serum , 2 . 5% BSA , 0 . 3% Triton , 1% DMSO , 0 . 1% Tween20 in TBS ) , after incubation for 1 hr at room temperature in this blocking solution .", "Slides were rinsed in PBST ( 5 min x3 ) before and after application of secondary antibody ( 1 hr room temperature ) then imaged using lsm710 and lambda/spectral imaging .", "Section in situ hybridization was carried out on de-waxed paraffin sections using RNAscope Multiplex Fluorescent Assay v2 as per manufacturer's instruction ( Cat No . 323100 , Advanced Cell Diagnostics ) , vegfc probe ( Cat No . 528701 , Advanced Cell Diagnostics ) was visualized with Opal 690 Reagent Pack ( PN: FP1497001KT , Akoya Biosciences ) .", "Quantification of Mpx or IB4-positive cell numbers was carried out in ImageJ though unprojected z-planes , only cells within the lymphatic vessel endothelium were counted .", "ImageJ was used for quantification of Mpx and IB4-cells in and within 100 μm of the woundsite and averaged across at least three images through woundsite .", "Similarly quantification of percentage PCNA/Mef2c-positive cells was carried by counting double positive and Mef2c-only cells within a 500 μm2 window proximal to the woundsite , and then averaged across at least three images .", "Quantification of all double positive and Raldh2-only cells within an entire 708 μm2 imaging window proximal to the wound site was carried out to calculate percentage of Raldh2 cells ( epicardium ) that are proliferative .", "Quantification of post-injury angiogenic sprouts was carried out by counting kdrl:GFP-positive sprouts within the wound site and then dividing this by woundsite area .", "For lyve1b:DsRed-positive , flt4:mCitrine-positive lymphatic vessel coverage and kdrl:GFP-positive coronary vessel coverage intensity thresholds of a projected z-stack were used to determine the area of the lyve1-positive , flt4:mCitrine-positive or kdrl:GFP-positive vasculature on the ventricle in comparison to the total/partial area of the ventricle or woundsite as defined in text .", "For ventricle size quantification , the largest AFOG/immune-labeled heart section of each zebrafish was imaged and area measured using threshold function in ImageJ .", "This maximum area was then plotted against , or normalized to , standard body length of the zebrafish .", "Raw Mef2c/DAPI counts we made using ImageJ cell counter .", "Chi-squared statistical analysis of counts was completed in Prism 6 ( GraphPad ) .", "Visualization and analysis of confocal data were carried out in Zen ( Zeiss ) and ImageJ , with the exception of lymphatic-neutrophil/macrophage analysis in Vision4D ( Arivis ) .", "A 10 μl volume of filtered microspheres ( 0 . 2 μm Fluoro-Max , B200 , Thermo Fisher Scientific ) and Qdots ( Qtracker705 vascular label , Q21061MP , Thermo Fisher Scientific ) was prepared in 50:50 ratio and loaded into a micro-injector pulled-capillary syringe .", "Zebrafish were anesthetized in Tricaine and the chest opened and held opened with forceps .", "The injection was made using a FemtoJet microinjector ( Eppendorf ) , 1 μl was injected into the ventral myocardium tissue at an acute angle to the plane of the heart .", "Resection surgery and AFOG histology carried out as described previously ( Poss et al . , 2002 ) .", "Cryoinjury was carried out as described ( González-Rosa and Mercader , 2012 ) , but with the modification for two injuries: a severe cryoinjury using a 0 . 8 mm diameter spherical probe and mild cryoinjury using a 0 . 6 mm dia spherical probe ( 10160–13 , Fine Scientific Tools ) .", "Both are cooled in LN2 prior to application to the ventricle for 7–10 s after which the probe was warmed with water and removed , the post-surgery fish were returned to system water to recover .", "At least five zebrafish were injured per observation/group to allow for potential variability of the wound response within a single experiment .", "For comparative studies sibling zebrafish were used when possible and randomly assigned into different injury groups .", "Estimation of lymphatic vessel coverage after the injury was calculated in a fixed 600 μm2 square section centered on the wound site or uninjured apical region .", "Using thresholding of the 514 nm channel in ImageJ area of the lymphatic vessel was calculate and results presented as percentage coverage of the fixed area of measurement .", "To quantify the relative level of branching after injury , vessels were traced and total length calculated in ImageJ , then total number of bifurcations over the ventricle counted to give normalized bifurcations per unit length of vessel .", "For cyroinjury scar volume calculations , whole ventricle and scar ( collagen and fibrin ) regions were traced as regions of interest in imageJ .", "Intensity thresholding was used to select tissue to calculate the area without the inclusion of intertrabecular space .", "This area measurement was repeated on every 9th 7 μm consecutive section through the entire heart .", "The individual area measurements were then multiplied by 63 μm and subsequently added together to give a total estimated volume .", "Hearts were grouped based on the volume of this scar tissue with a cut off set at 0 . 05 μm3 above which was defined as large scar .", "For amputation , hearts were grouped on the basis of collagen and fibrin levels .", "Hearts with or less than a thin layer of collagen ( blue ) on the inside of the thickened regenerated tissue were classed as ‘minimally scared’ .", "Fibrin deposition at the wound site ( red ) was considered indicative of scar tissue presence .", "Level of regeneration of the cardiac tissue or level of myocardial wall thickening at the wound site was not considered in scar severity assessments .", "For quantification of regenerated tissue , thickened myocardial wall proximal to the scar was traced and area calculated for the section though the middle of the scar ( largest scar area ) .", "For estimation of scar internalization the minimal distance of scar tissue from the edge of the tissue ( epicaridum ) was measured in ImageJ .", "Scar area was calculated as volume , but in a single section with the largest scar area ." ] ]

[ "The cardiac lymphatic vascular system and its potentially critical functions in heart patients have been largely underappreciated , in part due to a lack of experimentally accessible systems .", "We here demonstrate that cardiac lymphatic vessels develop in young adult zebrafish , using coronary arteries to guide their expansion down the ventricle .", "Mechanistically , we show that in cxcr4a mutants with defective coronary artery development , cardiac lymphatic vessels fail to expand onto the ventricle .", "In regenerating adult zebrafish hearts the lymphatic vasculature undergoes extensive lymphangiogenesis in response to a cryoinjury .", "A significant defect in reducing the scar size after cryoinjury is observed in zebrafish with impaired Vegfc/Vegfr3 signaling that fail to develop intact cardiac lymphatic vessels .", "These results suggest that the cardiac lymphatic system can influence the regenerative potential of the myocardium ." ]

[ "Human hearts have coronary vessels that supply oxygen and essential nutrients to the heart .", "When this supply is interrupted , a heart attack can occur .", "After a heart attack , scar tissue forms that impairs the heart’s ability to pump blood around the body .", "The heart also has lymphatic vessels that drain excess fluid and remove waste products and damaged cells from the heart .", "Less is known about the lymphatic vessels and their role in heart disease .", "Unlike human hearts , which scar easily , the zebrafish heart can regenerate after injury .", "Because of this , scientists often study zebrafish to try to find ways to improve healing of heart injuries in humans .", "However , it is not yet known whether lymphatic vessels contribute to regeneration of zebrafish hearts .", "Now , Harrison et al . show that , in the zebrafish heart , lymphatic vessels develop after the coronary arteries .", "In fact , the coronary arteries provide a scaffold that the lymphatic vessels grow along .", "When the zebrafish are genetically modified so that they lack coronary arteries , the lymphatic vessels fail to grow .", "Further experiments showed that , when the heart was injured by briefly freezing part of it , extra lymphatic vessels grew , but this did not happen when a part of the heart was removed via surgery .", "This may be because the cold-induced injury causes inflammation , which can trigger the growth of lymphatic vessels .", "The lymphatic vessels then help battle inflammation , allowing regeneration to proceed .", "Using genetically engineered zebrafish , Harrison et al . were then able to turn the genes that control lymphatic vessel growth on and off .", "They showed that zebrafish lacking lymphatic vessels in the heart are less efficient at regenerating heart tissue and develop more scar tissue after injury .", "This result is supported by the findings of a separate study conducted by Gancz et al . The results suggest that stimulating the growth of lymphatic vessels or enhancing their activity in the injured heart may aid recovery .", "More studies may help scientists understand exactly how lymphatic vessels aid regeneration in zebrafish and whether promoting lymphatic vessel growth or activity may aid heart attack recovery in humans ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "chromosomes and gene expression", "immunology and inflammation" ]

[ [ "Inflammation is an innate immune response to tissue injury or infection .", "It relies on macrophages , which recognize pathogen-associated molecular patterns and other ‘danger’ signals via their toll-like receptors ( TLRs ) ( Glass and Saijo , 2010 ) .", "This initiates a signaling cascade that leads to the activation and DNA binding of the effector transcription factors NF-kB and AP1 ( O'Neill et al . , 2013 ) which recruit coregulators , and , ultimately , the basal transcription machinery that together alter the chromatin state in the vicinity of many pro-inflammatory genes and enable their transcription ( Smale and Natoli , 2014; Glass and Natoli , 2015 ) .", "Acute transcriptional activation of pro-inflammatory genes is , therefore , critical for overriding the homeostatic set-point and producing a robust immune response that helps to resolve infection or tissue injury ( Kotas and Medzhitov , 2015 ) .", "Although the magnitude and dynamics of inflammation is affected at multiple levels , the temporal coordination of cytokine gene transcription by RNA Polymerase ( Pol ) 2 is a key mechanism that defines acute inflammatory response .", "The Pol 2 transcription cycle has been divided into three phases: initiation , elongation and termination .", "Initiation involves the recruitment of Pol 2 to the promoter , histone modifications and changes in histone occupancy .", "In addition , the C-terminal domain ( CTD ) of Pol 2 , which contains multiple heptad repeats ( YS2PTS5PS ) , is phosphorylated at S5 , and Pol 2 synthesizes short ( 20–60 nt ) RNA transcripts .", "During the elongation step , Pol 2 is further phosphorylated at S2 by the cyclin T1/CDK9 positive transcription elongation factor ( P-TEFb ) and synthesizes the full length RNA transcript , which is followed by the termination step and RNA transcript dissociation from the DNA ( Nechaev and Adelman , 2011 ) .", "Although Pol 2 recruitment and initiation has been historically considered the rate-limiting step in signal-dependent transcription , numerous recent studies revealed that transcriptionally engaged Pol 2 often remains paused near promoters in the absence of activating signal and that entry into productive elongation is rate-limiting for activation of up to 40% of inducible genes ( Core et al . , 2012 ) .", "The paused Pol 2 is in a complex with the 4-subunit negative elongation factor ( NELF ) ; NELF phosphorylation by P-TEFb leads to its release and Pol 2 entry into productive elongation ( Chiba et al . , 2010; Nechaev and Adelman , 2011 ) .", "A subset of cytokine genes in macrophages is controlled at the level of Pol 2 pausing .", "Indeed , while for genes such as Il1a and Il1b , signal-dependent Pol 2 recruitment to their transcription start sites ( TSS ) and transcription initiation are rate-limiting , other genes , exemplified by Tnf , are occupied by Pol 2 even under resting conditions ( Adelman et al . , 2009; Hargreaves et al . , 2009; Gupte et al . , 2013 ) .", "At Tnf , Pol 2 is S5-phosphorylated , bound by NELF and paused ~50 bp downstream of the TSS .", "Pause release following S2 and NELF phosphorylation by P-TEFb occurs in response to inflammatory signal .", "Aside from Pol 2 occupancy , the chromatin state plays an integral part in the regulation of transcription ( Smale et al . , 2014 ) .", "In particular , histone code ‘writers’ such as acetyltransferases ( HATs ) GCN5 and p300 have been implicated in modifying H3K9/14 and H4K5/8/12 at inflammatory genes in macrophages following treatment with TLR4 ligands ( Hargreaves et al . , 2009; Escoubet-Lozach et al . , 2011 ) .", "Both HATs are recruited by the NF-kB subunit p65 to regulatory regions in a stimulus-dependent manner ( Hargreaves et al . , 2009; Ghisletti et al . , 2010 ) .", "Histone modifications are then bound by ‘readers’ such as BRD4 , a protein containing two conserved N-terminal bromodomains ( BD1 and BD2 ) , which associates with most active promoters and some active enhancers , and has been proposed to couple the acetylation state at enhancers and promoters with Pol 2 elongation ( Lovén et al . , 2013; Brown et al . , 2014 ) .", "BRD4 occupancy correlates with acetylation marks at H4K5/8/12 , H3K9/27 ( Lovén et al . , 2013; Kanno et al . , 2014; Nagarajan et al . , 2014 ) and with gene activation , whereas chemical inhibition of BRD4 binding abrogates the induction of a subset of genes ( Nicodeme et al . , 2010 ) .", "Furthermore , BRD4 has been shown to associate with P-TEFb , affecting Pol 2 CTD phosphorylation , and hence , transcription elongation ( Itzen et al . , 2014 ) .", "These events coalesce ensuring a rapid remodeling of the inflammatory transcriptome , with hundreds of genes undergoing a dramatic upregulation ( Escoubet-Lozach et al . , 2011; Chinenov et al . , 2012; Gupte et al . , 2013; Uhlenhaut et al . , 2013; Tong et al . , 2016 ) .", "Although essential for host defense , unabated inflammation imposes a threat to the host and can result in tissue damage and autoimmunity .", "One systemic mechanism that controls acute inflammatory response is a feedback loop whereby inflammatory cytokines trigger the production of steroid hormones known as glucocorticoids ( GCs ) ( reviewed in [Sacta et al . , 2016] ) .", "Lipophilic GCs diffuse through the cell membrane and bind the intracellular glucocorticoid receptor ( GR ) , a transcription factor ( TF ) , which then translocates to the nucleus and regulates gene expression .", "The transcriptional outcomes of GR activation are context-specific and are determined by the genomic GC response elements ( GRE ) to which the receptor binds .", "GR can bind directly to specific , usually pseudopalindromic , DNA sequences either as a homodimer or complexed with other TFs such as AP1 and STAT3 ( Biddie et al . , 2011; Langlais et al . , 2012 ) .", "In this context , GR recruits various coregulators such as members of the p160 family , HATs , the Mediator complex and ATP-dependent chromatin remodelers ( Weikum et al . , 2017b ) , ultimately leading to the activation of numerous genes including the anti-inflammatory genes , such as Dusp1 and Tsc22d3 ( GILZ ) .", "At other sites , known as ‘tethering’ GREs , GR does not directly bind DNA but interacts with other DNA-bound TFs such as pro-inflammatory AP1 and NF-kB and usually represses their activity ( reviewed in [Chinenov et al . , 2013] ) – a property fundamental to the ability of GCs to dramatically attenuate inflammation .", "In contrast to GR-mediated activation , the mechanisms of transcriptional repression by GR remain poorly understood .", "Strikingly , however , in a few cases analyzed , genes activated through Pol 2 recruitment and those induced by signal-dependent Pol 2 pause release were both susceptible to GR-mediated repression ( Gupte et al . , 2013 ) .", "Here , we use a combination of cell-based and genome-wide approaches to elucidate the mechanisms by which GR represses pro-inflammatory genes in primary macrophages challenged acutely with the TLR4 agonist lipopolysaccharide ( LPS ) and GCs .", "We present evidence of ‘tethering’ as a prevalent mechanism of repression among p65/GR co-regulated genes .", "We further demonstrate a widespread yet gene class-specific role of NELF in glucocorticoid-mediated repression of early elongation .", "Conversely , at other genes , GR precludes the ordered assembly of HATs , Brd4 and the Mediator complex which ultimately blocks Pol 2 recruitment and transcription initiation ." ], [ "To understand the mechanisms by which GR elicits its repressive effects , we first assessed by RNA-seq the global transcriptional changes upon acute activation of primary mouse bone-marrow-derived macrophages ( BMDM ) with LPS or LPS together with a synthetic GC dexamethasone ( Dex ) for 1 hr .", "At FDR < 0 . 1 we found that , compared to vehicle-treated BMDM , 597 genes were induced by LPS >1 . 5 fold .", "Of these , the induction of 201 genes was attenuated >1 . 3 fold by Dex co-treatment ( Figure 1A and Supplementary file 1 ) .", "As expected , GO analysis of acutely GR-repressed genes revealed predominantly those involved in cytokine signaling ( Figure 1A ) .", "Despite rapid remodeling of the macrophage LPS-induced transcriptome in response to Dex observed by us and others ( Figure 1 , [Rao et al . , 2011; Chinenov et al . , 2012; Uhlenhaut et al . , 2013; Chinenov et al . , 2014] ) , no comprehensive analysis of the GR and p65 genome-wide occupancy under acutely repressing conditions has been reported .", "Therefore , we determined the distribution of p65 and GR genomic binding sites in BMDM pulsed with LPS , Dex or LPS + Dex for 45 min ( see Figure 1—figure supplements 1–2 and Supplementary file 2 for quality metrics and comparison of replicates ) .", "Following LPS + Dex exposure , we detected 9987 GR peaks ( union of two replicates ) 5397 ( 54 . 1% ) of which did not overlap with p65 peaks at the same conditions ( Figure 1B , top , Figure 1—figure supplement 1A ) .", "Motif overrepresentation analysis in these GR unique peaks revealed predominance of centrally enriched NR3C-binding motifs , which represent GREs and highly related NR-binding sites , those for ETS family members , such as the macrophage lineage-determining TF SPI1 ( PU . 1 and SPIB ) , and AP1 family members ( Figure 1B , Figure 1—figure supplement 1B , left panel ) .", "The analysis of p65 binding after LPS + Dex treatment revealed 7052 peaks ( union of two replicates ) of which 2344 ( 33 . 8% ) were uniquely bound by p65 ( Figure 1B , Figure 1—figure supplement 2A ) .", "Motif analysis indicated an enrichment of NF-kB/Rela binding motifs , as well as ETS and AP1 motifs ( Figure 1B ) .", "Importantly , the GR and p65 cistromes shared 4589 peaks , which corresponds to nearly half of all GR- and 2/3 of all p65-binding peaks .", "Motif analysis of these peaks showed a predominance for NR3C/GRE , ETS family , NF-kB/Rela and AP1 binding motifs that were enriched near the peak summits ( Figure 1B , bottom , Figure 1—figure supplement 1B , middle panel ) .", "Because of the significant enrichment of peaks with NF-kB elements ( especially among those overlapping p65-binding peaks ) in the GR cistrome under repressing conditions , we performed GR ChIP-seq in BMDM treated with Dex only for 45 min to compare the two GR cistromes .", "In Dex-treated BMDM , GR-binding sites formed 3377 peaks .", "Of those , 3165 also appeared in the GR LPS + Dex cistrome ( with only 212 peaks unique to Dex-treated BMDM ) , whereas 6817 were gained in the GR LPS + Dex cistrome ( Figure 1—figure supplement 1A , right panel ) .", "ETS and NR3C-binding motifs were over-represented in both Dex-unique and Dex – LPS + Dex shared subsets of GR peaks and trended toward the peak summit ( Figure 1C , Figure 1—figure supplement 1B , right panel ) .", "We did not detect NF-kB/Rela motif enrichment in these two subsets of GR-binding peaks .", "However , among 6817 peaks unique to the GR LPS + Dex cistrome we readily observed an overrepresentation of NF-kB and AP1 motifs while NR3C motifs were no longer enriched ( Figure 1C , compare top/middle vs . bottom motif enrichment panels ) indicating that inflammatory signaling and p65/NF-kB activation was driving GR recruitment to such sites specifically under repressing LPS + Dex conditions .", "The majority of GR and p65-binding sites were located in distal intergenic ( ~39–47% of peaks ) and intronic ( ~40% on average ) regions ( Figure 1D ) , similar to previously reported GR and p65 cistromes in various cell lines ( Reddy et al . , 2009; Barish et al . , 2010 ) .", "To correlate GR binding with transcriptional outcomes , we focused on our subset of 201 LPS-induced Dex-repressed genes as determined by RNA-seq ( Figure 1A , Supplementary file 1 ) and evaluated GR peak localization within these genes and 100 Kb of their 5’- and 3’-flanking regions in Dex- and LPS + Dex-treated BMDM .", "In this subset , a somewhat larger fraction ( ~52% , compared to 39–47% genome-wide ) of GR-binding peaks were located in distal intergenic regions , whereas the fraction of peaks in the introns dropped from 40% to 24% compared to whole-genome GR cistrome ( Figure 1D ) .", "This shift was not due to a preponderance of shorter introns or genes in Dex-repressed subset ( Figure 1—figure supplement 1C ) .", "Comparison of GR binding near the 201 Dex-repressed genes with an entire GR cistrome shows that a greater fraction of binding sites was unique to the LPS + Dex condition ( 81% vs . 68% , Figure 1E ) consistent with a disproportional increase in unique binding site utilization among this functionally constrained set of genes .", "Several representative examples of GR and p65 co-binding near GR-sensitive genes are shown in Figure 1F: at each gene , GR binding occurred at sites matching those of p65 , but only in LPS + Dex and not LPS- or Dex-alone treated BMDM .", "Importantly , LPS-dependent p65 binding fully persisted in the presence of Dex .", "In fact , the total number of p65 binding peaks in the presence of LPS and LPS + Dex was comparable both genome-wide , and in the vicinity of our GR-repressed genes ( Figure 1—figure supplement 2A , right and 2B ) .", "In each case , ~2/3 of the LPS-induced p65 peaks persisted in LPS + Dex-treated BMDM .", "Moreover , among p65 LPS + Dex peaks functionally constrained to Dex-repressed genes , 80% ( up from 68% genome-wide ) overlapped LPS-induced peaks ( Figure 1—figure supplement 2C ) .", "Interestingly , of the 201 genes repressed by Dex in the context of LPS-mediated macrophage activation , only 56 were repressed ≥1 . 3 fold ( and only 16 of those ≥2 fold ) upon treatment with Dex alone ( Supplementary file 1; RNA-seq dataset from [Chinenov et al . , 2014] ) – further supporting a requirement for NF-kB activation for GR recruitment to the majority of genes Dex-sensitive genes .", "Combined , these results further corroborate a tethering model in which p65 is a central component of repression complexes in GC-treated BMDM .", "We have reported that at several pro-inflammatory genes in unstimulated BMDM , promoter-proximally paused Pol 2 is in a complex with NELF and enters productive elongation following LPS treatment ( Adelman et al . , 2009; Gupte et al . , 2013 ) .", "To assess how common this type of Pol 2 dynamics is among inflammatory genes , we performed Pol 2 ChIP-seq in untreated , LPS- or LPS + Dex treated BMDM .", "Figure 2A shows Pol 2 tracks for six genes all of which were among 201 that were rapidly induced by LPS and repressed by Dex as established by RNA-seq ( Figure 1A ) .", "Of those , Tnf , Hilpda and Btg2 , all display accumulation of Pol 2 near the TSS in untreated BMDM .", "Upon a 45-min LPS treatment , we detect additional Pol 2 loading and , notably , its redistribution into the body of the gene; conversely , upon LPS + Dex treatment , Pol 2 largely remains near the TSS resembling a ‘paused’ pattern seen in the unstimulated BMDM ( Figure 2A , left ) .", "In contrast , non-paused genes Il1a , Il1b and Cd83 display no substantial Pol 2 occupancy in the unstimulated BMDM , and a dramatic and uniform increase in Pol 2 occupancy throughout the gene in response to LPS , which is nearly abrogated by co-treatment with Dex ( Figure 2A , right ) .", "These findings raised the possibility that GR mediates its repressive effects genome-wide by regulating distinct steps of Pol 2 transcription cycle depending on the rate-limiting step for gene activation .", "To address this possibility , we first calculated Pol 2 pausing indexes ( PI ) for approximately 300 transcripts corresponding to our 198 LPS-induced Dex-repressed genes ( three genes were excluded due to the conflict of annotation ) .", "As described in Nechaev et al . ( 2010 ) , we defined PI as the ratio of log-transformed normalized Pol 2 counts around the promoter ( −200/+500 bp relative to the annotated TSS ) to those within the gene body downstream of +500 bp ( Figure 2B , Supplementary file 3 ) .", "Based on the PI in untreated BMDM , we classified GC-repressed genes into two groups: 61 transcripts had a PI >1 and were considered to be paused ( twice as much of Pol2 at the promoter region versus gene body ) , whereas 82 had a PI <0 . 8 and were considered non-paused ( see Materials and methods and [Nechaev et al . , 2010] ) .", "Figure 2C shows Pol 2 distribution within the −200/+1500 region for individual transcripts of both classes in unstimulated BMDM , as well as BMDM exposed for 45 min to LPS or LPS + Dex .", "The read density distribution for 61 paused and 82 non-paused transcripts in differentially treated BMDM ( Figure 2D ) reveals a peak of Pol 2 occupancy in the promoters of the paused genes , additional Pol 2 loading , and , importantly , its entry into gene bodies in response to LPS .", "Co-treatment with Dex decreases Pol 2 occupancy in gene body with most Pol 2 remaining near the TSS ( Figure 2C and D ) .", "Conversely , little Pol 2 is seen in the non-paused genes in untreated BMDM; Pol 2 occupancy increases dramatically throughout the genes in LPS-treated BMDM and this loading is largely abrogated by Dex ( Figure 2C and D ) , consistent with the pattern shown in Figure 2A for representative genes .", "Because Pol 2 pausing within the first 100 nt of a gene is mediated by NELF ( Adelman and Lis , 2012 ) , we assessed genome-wide NELF distribution by ChIP-seq using antibodies to the NELF-E subunit of the complex .", "Aligned with Pol 2 PI heat maps , NELF-E occupancy matched closely Pol 2 distribution in untreated BMDM with striking accumulation immediately downstream of TSS of paused genes and little to no NELF-E seen in non-paused genes ( Figure 2C and D , far right ) .", "Indeed , read density distribution in NELF-E ChIP-seq shows highly gene class-specific NELF-E occupancy at paused ( PI >1 ) promoters ( Figure 2D , right ) .", "As reported previously for a few individual genes ( Adelman et al . , 2009; Schaukowitch et al . , 2014 ) , following LPS stimulation , NELF-E was broadly evicted from promoters of LPS-induced genes with little occupancy detected at 1 hr ( Figure 3A ) .", "Interestingly , however , this dismissal was transient , as despite continued LPS exposure , NELF reloaded onto promoters reaching widespread occupancy by 3 hr ( Figure 3A , also see average occupancy graphed for all paused transcripts ) .", "This release and reloading can be seen at specific paused GC-sensitive genes , Tnf , Hilpda , and Btg2 ( Figure 3A , right ) , which show substantial NELF-E occupancy at the TSS co-localizing with Pol 2 peaks in resting BMDM , its dissociation following a 1 hr LPS induction , and re-establishment of the TSS-associated NELF-E peaks by 3 hr .", "To directly assess whether NELF occupancy in GC-sensitive genes in BMDM correlates with Pol 2 pausing in early elongation , we compared the NELF-E and Pol 2 cistromes in the unstimulated BMDM .", "Among the LPS-induced Dex-sensitive genes with PI >1 ( approximately 24% of 300 Dex-repressed transcripts ) , 86 . 3% displayed promoter-associated NELF-E peaks , compared to only 31 . 7% in genes with PI <0 . 8 ( which comprised approximately 66% of 300 transcripts; Figure 3B , left ) .", "Importantly , similar relative numbers of paused and non-paused genes ( 23% and 70% , respectively; Figure 3—figure supplement 1B ) were found among LPS-induced Dex-insensitive genes from RNA-seq ( Figure 1A ) .", "In this group , NELF-E occupancy in untreated BMDM was again much more prevalent in paused genes ( 81 . 1% ) than in non-paused ones ( 44 . 2% ) .", "Thus , GR does not preferentially repress genes in one class vs . the other , and high levels of TSS-associated NELF in a basal state is a common feature of paused genes irrespective of their sensitivity to GC .", "Given that NELF and Pol 2 co-localize at the TSS of the paused genes in unstimulated BMDM , that activation of such genes by LPS coincides with NELF dismissal , and that Pol 2 remains near the promoters of these genes under repressing conditions consistent with their early elongation arrest , we questioned whether GR-mediated repression was globally mediated by NELF .", "We first evaluated NELF-E occupancy in BMDM co-treated with LPS + Dex by ChIP-seq and found the relative distribution of peaks among paused ( PI >1 ) and non-paused ( PI <0 . 8 ) repressed genes to be indistinguishable from NELF-E distribution in resting BMDM ( 83 . 6% and 33 . 2% , respectively; Figure 3B , right – compare to left ) .", "We then evaluated NELF-E distribution across several of our target genes in the presence of LPS + Dex and detected striking promoter-proximal NELF peaks in paused Tnf , Myc , Errfi1 and Ccl2 , but not in non-paused Il1b or Lif ( Figure 3B ) .", "To address directly whether NELF is necessary for GR-mediated repression , we used a new mouse strain conditionally lacking the NELF-B subunit and , hence , the functional NELF complex in myeloid cells ( see Materials and methods ) .", "BMDM from NELF-B LysM-Cre mice ( NELF-B KO ) show a dramatic reduction in NELF-B mRNA and protein ( Figure 3C , top ) .", "Importantly , as the NELF complex requires all four subunits for stability and the loss of a single subunit leads to the proteolytic degradation of the complex ( Gilchrist et al . , 2008 ) , immunoblot also reveals a near complete loss of the NELF-E protein in the BMDM of the NELF-B KO ( Figure 3C , top ) .", "Using WT and NELF-B KO BMDM , we then compared GR-mediated repression of our candidate GC-sensitive genes .", "Consistent with the lack of overt phenotype in these mice , RNA-seq of resting BMDM of the two genotypes revealed no significant differences in the expression levels of LPS-induced Dex-repressed genes at baseline ( Figure 3—figure supplement 1C ) .", "Moreover , at the time-frame examined , LPS challenge led to a similar induction of Tnf , Myc , Errfi1 , Ccl2 , Il1b and Lif transcripts irrespective of the genotype ( Figure 3C , bottom left ) .", "Interestingly , for all genes classified as ‘paused’ , repression by Dex was significantly attenuated in the NELF-B KO BMDM , but not in non-paused genes Il1b and Lif ( Figure 3C , bottom right ) .", "Collectively , these findings strongly suggest that NELF-mediated block in productive elongation is an integral part of GR-mediated repression of paused genes .", "To extend these observations to a whole-genome level , we analyzed transcriptomes from the WT and NELF-B KO BMDM treated with LPS + Dex for 1 hr by RNA-seq which identified 393 differentially expressed genes ( fold change = 1 . 5 , FDR p<0 . 05 ) .", "Out of 201 genes that were repressed by Dex in the WT BMDM ( Figure 1A ) , 23 were expressed at higher level in the LPS + Dex-treated NELF-B KO BMDM; notably , 21 of them had PI >0 . 8 ( Figure 3D ) .", "Conversely , out of 396 LPS-induced Dex-insensitive genes , only nine were upregulated in the LPS + Dex-treated NELF-B KO BMDM , 7 of which had PI >0 . 8 ( Figure 3—figure supplement 1D , left ) .", "These observations indicate that NELF ablation disproportionally affects paused LPS-induced Dex-repressed genes .", "Because NELF release is triggered by CDK9-mediated phosphorylation , we evaluated the recruitment of CDK9 to the TSS of paused and non-paused genes .", "Consistent with earlier observations ( Luecke and Yamamoto , 2005 ) , GR inhibited LPS-induced CDK9 recruitment but did so irrespective of the gene class ( Figure 3E ) suggesting that NELF retention rather than CDK9 occupancy serves as a defining class-specific feature of glucocorticoid repression of paused genes .", "The dynamics of Pol 2 binding at non-paused genes , as shown in Figure 2 , suggested that the major barrier to activation at these genes is the loading of Pol 2 .", "BMDM surpass this barrier by recruiting NF-kB and AP1 to enhancer regions ( Glass and Natoli , 2015 ) that in turn assemble histone-modifying proteins , which help create a more permissive chromatin environment for the binding of basal transcriptional machinery and Pol 2 .", "In particular , histone tail modifications , which are associated with both enhancers and promoters are H3K9Ac and H4K5/8/12Ac ( Smale et al . , 2014 ) .", "Because these marks correlate with gene transcriptional status , we evaluated the histone acetylation at a subset of our GC-repressed genes of each class .", "We noted striking differences in histone tail modifications between representatives of the two gene classes .", "In particular , paused genes - Tnf and Ccl2 - contained high basal levels of H4PanAc and , specifically , H4K5Ac , at both TSS- and kB-binding sites which were unaffected by LPS or LPS + Dex treatment ( Figure 4A , bottom row ) .", "In contrast , non-paused genes - Il1b and Il1a - showed a significant increase in H4Ac levels only after LPS treatment , especially at the Il1b TSS and two Il1a kB enhancers at −10 Kb and −20 Kb , and this increase was fully attenuated by Dex ( Figure 4A , top row ) .", "The change in acetylation seen preferentially at our non-paused genes , appeared to denote a specific ‘histone code’ for histone binding proteins that could potentially affect the transcription of these genes .", "In particular , BRD4 , the Bromodomain and Extra-Terminal domain ( BET ) histone binding protein , affects inflammatory cytokine transcription both in vitro and in vivo through direct binding to acetylated H3 and H4 ( Shi and Vakoc , 2014 ) .", "The changes in H4PanAc including H4K5/K12Ac in several GC-sensitive genes , suggested a possible role for BRD4 in transcriptional repression by GR .", "To test this hypothesis , we first assessed activation of pro-inflammatory genes by LPS in the presence of increasing concentrations of I-BET , an inhibitor of BRD4 binding .", "The induction of Il1b and Il1a transcripts was significantly attenuated by I-BET in a dose-dependent manner , whereas Tnf and Ccl2 induction persisted ( Figure 4B ) .", "In agreement with gene expression results , ChIP-qPCR experiments revealed that BRD4 was recruited to promoters of non-paused genes Il1b and Il1a upon LPS treatment and , interestingly , this recruitment was attenuated by Dex ( Figure 4C ) .", "Conversely , at the paused genes , Tnf and Ccl2 , BRD4 was readily detectable at the TSS in unstimulated BMDM and this association did not significantly change after either LPS or LPS + Dex treatment .", "Thus , BRD4 occupancy patterns at the promoters of these genes resembled signal-responsive H4Ac profiles suggesting that loss of BRD4 in response to Dex may underlie GR-mediated repression of , specifically , the non-paused genes .", "We then assessed genome-wide distribution of BRD4 by ChIP-seq .", "Not surprisingly , we observed frequent BRD4 binding across the genome in untreated BMDM ( ~3700 peaks , Figure 4—figure supplement 1A , left panel ) .", "There was a 1 . 8-fold increase in the number of BRD4 peaks in response to LPS relative to that in untreated BMDM ( 4345 new peaks , Figure 4—figure supplement 1A , left panel ) .", "The increase in the total peak number was even more apparent when limited to LPS-induced genes: 2 . 9-fold for Dex-insensitive or -repressed genes ( Figure 4—figure supplement 1A , middle and right panel , respectively ) .", "Furthermore , BRD4 loading density specifically at our Dex-sensitive genes increased dramatically in response to LPS which , interestingly , was nearly abrogated by Dex - a trend very apparent at promoters , but also significant at BRD4:p65 shared binding sites ( Figure 4—figure supplement 1B ) .", "BRD4 read distribution at individual non-paused genes of interest reflected this dynamics .", "For example , the Il1b TSS and −2 . 3 Kb and −10 Kb p65 enhancers acquired strong BRD4 binding in response to LPS which was significantly attenuated by Dex , concomitantly with GR loading ( Figure 4D , left top , purple arrows ) .", "Il1a also displayed increased LPS-induced BRD4 loading at kB-associated upstream enhancers ( −10 Kb and −20 Kb ) with a dramatic reduction in occupancy upon Dex co-treatment corresponding to GR binding at both regions ( Figure 4D , left bottom , purple arrows ) .", "Consistent with our ChIP-PCR data , paused genes , Ccl2 ( −12 . 5 kB enhancer ) and especially Tnf ( TSS ) exhibited modest yet detectable BRD4 occupancy in untreated BMDM that was potentiated by LPS but only minimally affected by Dex ( Figure 4D , right ) .", "Moreover , our analysis of BRD4 occupancy at Dex-sensitive genes of the two classes revealed that in paused genes , 45% of the BRD4-binding sites seen in LPS-treated BMDM were already pre-bound in untreated cells and 55% were LPS-induced; in non-paused genes , however , only 38% of the sites were pre-occupied in untreated BMDM , whereas 62% were LPS-dependent ( Figure 4E ) .", "Thus , our functional studies together with occupancy data suggest that the activation of non-paused genes is more dependent on BRD4 recruitment , and therefore , its dismissal may have a greater impact on genes of this class .", "Initial BRD4 characterization revealed its interaction with the Mediator complex subunits MED1 and MED12 ( Jang et al . , 2005; Lovén et al . , 2013 ) .", "Mediator is an evolutionarily conserved multi-protein co-activator complex that facilitates transcriptional activation of many genes in part by linking physically and functionally effector TFs and Pol 2 .", "In the context of LPS-induced activation of pro-inflammatory genes , MED1 is reportedly recruited to both the TSS and p65 enhancers ( Hargreaves et al . , 2009; Brown et al . , 2014 ) , occupying similar sites across the genome as BRD4 , and the two appear to stabilize each other’s occupancy at enhancer regions ( Lovén et al . , 2013 ) .", "We therefore assessed MED1 and MED12 occupancy at the promoters and p65 enhancers of GR-sensitive genes and found that both were recruited to TSS and p65 sites in response to LPS treatment and their recruitment was attenuated by Dex ( Figure 4F ) .", "Thus , by inhibiting BRD4 binding to the TSS and certain enhancer regions at non-paused genes , GR destabilizes MED1 and MED12 occupancy ultimately affecting Pol 2 recruitment .", "Of note , MED1 and MED12 loss in response to Dex occurred at paused genes as well ( Figure 4F ) , suggesting that GR may antagonize the Mediator complex binding irrespective of its effects on BRD4 .", "GR activation disrupted histone acetylation and subsequent BRD4 and Mediator complex assembly at our analyzed non-paused genes , suggesting a central role for LPS-induced histone acetylation as a potential target for GR .", "Various HATs , including GCN5 and p300 , have been implicated in altering modifications at the histone H3 and H4 tails ( Smale et al . , 2014 ) Furthermore , p300 has been shown to also interact with and acetylate p65 , which contributes to the activation of NF-kB-dependent genes ( Huang et al . , 2009; Nagarajan et al . , 2014; Roe et al . , 2015 ) .", "Thus , p300 appeared as a plausible HAT to execute H3/H4 acetylation , thereby dictating the recruitment of BRD4 to the promoters and kB sites of our genes of interest .", "ChIP-qPCR experiments revealed LPS-dependent recruitment of p300 to the TSS- and p65-binding sites of non-paused genes Il1a and Il1b , which was blocked by Dex .", "Interestingly , at paused genes , p300 occupancy varied , showing some LPS-potentiated yet Dex-insensitive recruitment to Tnf , but a strong constitutive occupancy at Ccl2 ( Figure 5A ) .", "Notably , loss of p300 from these genes did not reflect a simple ‘titration’ of p300 by the activated GR potentially broadly sequestering it away from kB enhancers , as p300 occupancy at the p65-binding sites of LPS-induced Dex-insensitive genes identified by our RNA-seq analysis - Cxcl10 , Cd40 , Tnfsf9 , Trim13 - was fully resistant to Dex ( Figure 5B ) .", "We reasoned that p300 catalytic activity rather than its occupancy is a better indicator of whether or not this HAT is involved in regulating target GR-sensitive genes .", "Therefore , a selective and competitive inhibitor of the p300 HAT activity , C646 , was used to determine whether p300-mediated acetylation of histones was necessary for transcriptional activation of candidate pro-inflammatory genes .", "C646 attenuated in a dose-dependent manner LPS-mediated induction of non-paused genes Il1b and Il1a , whereas activation of paused genes Tnf and Ccl2 was unaffected ( Figure 5C ) , consistent with a selective requirement for p300 at the non-paused genes .", "Furthermore , if GR represses Il1a and Il1b specifically by precluding p300 recruitment , its ectopic introduction into cells should rescue LPS-mediated induction irrespective of GC treatment .", "Figure 5D shows that overexpression of wild-type p300 but not its ΔHAT mutant devoid of the catalytic activity in macrophage-like RAW264 . 7 cells dramatically and specifically reversed GR-mediated repression of non-paused genes .", "This suggests that GR represses these genes by precluding p300 recruitment , H3/H4 acetylation and the assembly of the BRD4-Mediator complex , ultimately blocking Pol 2 loading ." ], [ "Despite the unmatched therapeutic utility of GCs stemming in large part from rapid and direct transcriptional repression of the key inflammatory genes , our knowledge of the overall architecture , dynamics , stability and distribution of such repressive GR complexes in inflammatory cells has been lacking .", "Given fundamental differences in the rate-limiting events for inflammatory gene activation , we sought to dissect the mechanisms by which GR elicits repression in such distinct gene classes and use genome-wide approaches to assess the generality of our findings .", "Numerous studies in cell culture and cell-free systems implicated physical interactions between GR , NF-kB and AP1 family members in the inhibition of pro-inflammatory gene transcription ( reviewed in [Sacta et al . , 2016] ) and indeed , we observe extensive co-localization of GR and the NF-kB subunit , p65 , genome-wide and especially nearby Dex-repressed genes following short-term LPS + Dex co-treatment – conditions under which we observe rapid glucocorticoid repression .", "GCs did not cause global displacement of p65; in fact , the number on p65-binding sites in the presence of LPS vs . LPS + Dex is comparable .", "Moreover , 80% of p65 peaks associated with our Dex-repressed genes overlap in LPS- and LPS + Dex-treated BMDM .", "Interaction with p65 is further corroborated by the persistence of p65 peaks near our candidate Dex-repressed genes of both classes .", "With respect to GR binding , both globally and restricted to Dex-repressed genes , several observations point to a tethering mechanism .", "First , the predominant motifs enriched in GR peaks present uniquely under LPS + Dex conditions are those of NF-kB and AP1 and not the NR3C motif overrepresented in Dex-treated BMDM or peaks shared between the two cistromes .", "Second , when compared between an entire genome and restricted to Dex-repressed genes , the fraction of LPS + Dex unique GR-binding sites is increasing substantially from 68% to 81% .", "Third , the majority of the 201 Dex-sensitive genes are only repressed in LPS-activated macrophages , pointing to a requirement for NF-kB activation for GR recruitment .", "Indeed , the analysis of GR occupancy nearby our candidate Dex-sensitive genes of both classes reveals co-localized GR and p65 peaks associated with NF-kB enhancers under repressing LPS + Dex conditions and no GR binding in Dex-only - treated macrophages .", "Thus , although this is certainly not the only mechanism by which GR affects inflammatory gene expression ( Rao et al . , 2011; Uhlenhaut et al . , 2013; Oh et al . , 2017; Weikum et al . , 2017a ) , tethering to p65 is a widespread regulatory mechanism that GR relies upon to elicit acute repression of pro-inflammatory genes in macrophages .", "How GR enacts repression appears to depend on the state of the target promoters prior to activation .", "At paused genes , Pol 2 is pre-loaded , bound by NELF and ‘stalled’ nearby the TSS , ( Gilchrist et al . , 2012 ) .", "These genes have elevated levels of histone acetylation at the TSS , suggestive of an open chromatin state , which would favor constitutive Pol 2 loading and transcription initiation .", "Conversely , non-paused genes show little Pol 2 occupancy in unstimulated BMDM .", "Among our Dex-repressed genes , both classes were well represented: in a set of transcripts filtered for Pol 2 occupancy and used to calculate PI , 61 were paused and 82 were not; in a total pool of transcripts corresponding to LPS-induced Dex-repressed 198 genes , approximately 24% were paused ( PI > 1 ) and 66% non-paused ( PI < 0 . 8 ) .", "This distribution matched closely that of genes that were LPS-induced but insensitive to Dex ( 23% and 70% , respectively ) , suggesting that GR does not display a preference for a specific gene type for repression .", "Given a critical role of NELF in establishing Pol 2 pausing ( Gilchrist et al . , 2008; Core et al . , 2012 ) , we evaluated the genomic distribution of NELF at our LPS-induced Dex-repressed genes in basal , activated and repressed state .", "This analysis revealed a striking correlation between Pol 2 promoter-proximal pausing and NELF occupancy .", "Indeed 81% of the paused genes had TSS-associated NELF peaks compared to only 44% on non-paused genes .", "As expected , NELF dissociated from Pol 2 after LPS treatment , presumably due to P-TEFb-mediated phosphorylation , enabling productive elongation .", "Although the rate of NELF dismissal varies depending on culture conditions and in our experience takes 30–60 min , this loss is consistently transient as NELF ‘re-loads’ onto the TSS of these genes despite continuous presence of LPS .", "We previously reported a highly dynamic occupancy of NELF at the Tnf promoter ( Adelman et al . , 2009 ) , but a global synchronous reloading of NELF onto promoters of activated pro-inflammatory genes was unexpected .", "We envision that NELF re-loading may provide a tonic control of the inflammatory response by limiting further entry of Pol 2 into productive elongation ( Aida et al . , 2006 ) , yet maintain genes poised for induction by preserving a nucleosome-depleted environment ( Gilchrist et al . , 2008; Core et al . , 2012 ) .", "A distinct mechanism of ‘tonic control’ of inflammatory gene expression was recently described for a transcriptional repressor Hes1 which limits the recruitment of P-TEFb and hence , NELF release and Pol 2 elongation ( Shang et al . , 2016 ) .", "In that regard , it would be informative to examine the dynamics of P-TEFb and phosphorylation of the Pol 2 CTD at the promoters of these genes over the time frame of NELF recycling .", "Interestingly , paused genes were originally proposed to be fast and transient responders to inducing signals ( Adelman et al . , 2009; Rogatsky and Adelman , 2014 ) ; NELF reloading despite prolonged LPS exposure could potentially contribute to cessation of activation and establishing a ‘tolerant’ LPS-unresponsive state .", "More generally , our finding illustrates that the transcriptional landscape of macrophages during a sustained exposure to a signal , even in a course of a few hours , undergoes a significant remodeling and a secondary stimulus is likely to elicit variable responses depending on the exact timing of stimulation .", "Furthermore , given intrinsic macrophage plasticity , whereby a 12 hr treatment with a relevant signal ( e . g . LPS or Dex ) is sufficient to ‘polarize’ them to a distinct myeloid cell population – caution needs to be taken in interpreting results of ‘sequential’ treatments , which may document a response of a reprogrammed macrophage to a new signal rather that simple transcriptional antagonism or synergy .", "Under conditions of GC repression , we observed a broad failure of paused genes to release NELF concomitantly with inhibition of Pol 2 entry into productive elongation .", "Moreover , genetic disruption of NELF resulted in GC resistance of genes in this class establishing a causal relationship between NELF accumulation and GR-mediated repression .", "Interestingly , NELF was previously shown to participate in estrogen receptor ( ER ) alpha-mediated gene expression .", "ERa primarily affects Pol 2 post-initiation steps , whereby pausing is alleviated via hormone-induced recruitment of CDK9 to Pol 2 and NELF and their phosphorylation ( Kininis et al . , 2009 ) .", "Given that NRs can dynamically affect P-TEFb occupancy and that P-TEFb recruitment to GC-sensitive genes is attenuated after GC treatment in this and earlier studies ( Luecke and Yamamoto , 2005; Gupte et al . , 2013 ) , GR may block elongation by preventing P-TEFb recruitment , possibly through direct steric hindrance .", "Interestingly , in addition to phosphorylation , NELF has recently been shown to undergo ADP-ribosylation which also facilitates its release ( Gibson et al . , 2016 ) .", "It would be informative to assess whether , similar to P-TEFb , ADP-ribosyl transferases that modify NELF are susceptible to regulation by GCs .", "Finally , a physical interaction between ERa and NELF has been documented at promoters of certain estrogen-activated genes , where NELF recruitment limits the response to hormone ( Aiyar et al . , 2004 ) .", "Conceivably , NELF could also serve as a non-conventional ‘co-repressor’ recruited by GR to the promoter-proximal regions of pro-inflammatory genes in a gene-specific manner .", "Once recruited , NELF may no longer require GR and assume its known function in Pol 2 pausing .", "Whether GR-mediated repression involves either of these mechanisms remains to be elucidated .", "Interestingly , non-paused genes , such as Il1a and Il1b , exhibit low CpG content , stable nucleosome assembly at promoters , low levels of H3K9/14Ac in the basal state and low TBP occupancy ( Ramirez-Carrozzi et al . , 2009 ) .", "This suggests that histone acetylation marks are required for chromatin remodeling which may pose a major barrier to the recruitment of Pol 2 at these genes .", "We show that an increase in H4Ac at promoters and kB sites in response to LPS correlated with Pol 2 recruitment , and GC attenuated these effects , suggesting that GR may repress these genes by acting upon factors that ‘write’ and ‘read’ histone marks .", "Among many HATs that modify H3 and H4 , p300 is recruited by p65 to the TSS and NF-kB sites and has been shown to acetylate histones that are then bound by BRD4 ( Huang et al . , 2009; Brown et al . , 2014; Nagarajan et al . , 2014; Roe et al . , 2015 ) .", "Conceivably , GR attenuates p300 loading by competing for a tethering site on p65 as has been previously documented for IRF3 ( Ogawa et al . , 2005 ) .", "We cannot exclude the possibility that additional HATs , that is , GCN5 , contribute to writing H3/H4Ac at our GC-sensitive pro-inflammatory genes .", "Given its role as a histone binding protein that reportedly contributes to recruiting P-TEFb and couples the acetylation state at promoters and enhancers with Pol 2 elongation , a clear bias for LPS-induced novel sites of BRD4 recruitment and their sensitivity to Dex specifically at non-paused genes was unexpected .", "BRD4 binding at promoters broadly correlates with gene activation ( Nicodeme et al . , 2010; Lovén et al . , 2013; Brown et al . , 2014; Kanno et al . , 2014 ) .", "We now show that similar to I-BETs , GR inhibits , albeit indirectly , loading of BRD4 particularly at non-paused genes and , by exploiting their dependency on histone acetylation , disrupts interactions with Mediator , ultimately antagonizing Pol 2 recruitment and transcription initiation .", "Because this effect is far from uniform , and some p65/BRD4-bound LPS-induced enhancers are more sensitive to the effects of Dex than others , we speculate that a subset of p65-binding sites has greater functional consequences for gene activity .", "Identifying a subpopulation of ‘dominant’ enhancers whose BRD4 occupancy is a definitive predictor of transcriptional state , and correlating those with sites of GR recruitment would likely sharpen the differences in BRD4 behavior between the two gene classes .", "Finally , although the two classes of genes are activated and repressed through distinct mechanisms , the consequences of GR activation share commonalities including a failure to recruit P-TEFb and the Mediator complex .", "P-TEFb is required for gene activation post Pol 2 loading , so at non-paused genes failing to recruit Pol 2 , P-TEFb loss would have little functional consequences .", "Conversely , a block in Mediator recruitment at both the TSS and kB sites could potentially contribute to repression of both classes of genes .", "Mediator is a multi-subunit complex that interacts with numerous activators and components of basal transcription machinery including Pol 2 ( Malik and Roeder , 2010 ) .", "With respect to non-paused genes , Mediator interacts directly with both BRD4 and p300 , with Mediator and BRD4 stabilizing each other’s occupancy ( Jang et al . , 2005; Malik and Roeder , 2010; Shi and Vakoc , 2014 ) .", "Furthermore , Mediator and p300 can act cooperatively to alter the chromatin landscape and facilitate PIC formation ( Malik and Roeder , 2010 ) .", "Although the contribution of Mediator to activation of pro-inflammatory paused genes needs further study , it has been suggested that Mediator may help recruit P-TEFb indirectly promoting pause release ( Lu et al . , 2016 ) .", "Additionally , because kB sites are typically distant from promoters , and pro-inflammatory genes were proposed to be activated through DNA looping ( Tong et al . , 2016 ) , Mediator ( perhaps together with Brd4 ) may contribute to bridging promoters with NF-kB enhancers .", "Thus , it is tempting to speculate that by hindering Mediator assembly , GR globally disrupts promoter-enhancer communication thereby attenuating pro-inflammatory gene expression ." ], [ "BMDM were prepared from 8-to-10 week old mice as in Gupte et al . ( 2013 ) .", "RAW264 . 7 cells were cultured in DMEM media ( Corning , cat# 10–013-CV ) supplemented with 10% fetal bovine serum ( Atlanta Biologicals cat# S11550 ) .", "Dex and LPS were purchased from Sigma .", "C57BL/6 mice ( NCI , Charles River Laboratories ) , C57BL/6 LysM-Cre mice -/-:Nelfb fl/fl mice and their derivatives were maintained in the Weill Cornell Animal Facility in compliance with guidelines from the Weill Cornell Animal Care and Use Committee .", "8-to-10-week-old male mice were used for bone marrow isolation .", "To create the NELF-B conditional KO strain , Nelfb fl/fl mice ( with Nelfb exon 4 floxed [Amleh et al . , 2009] ) were bred to C57BL/6-derived LysM-Cre mice ( Jackson Laboratories , 004781 ) to obtain double heterozygous LysM-Cre/wt:Nelb fl/wt ( LysM-Cre-HET ) animals .", "To create homozygous ( LysM-Cre:Nelfb fl/fl ) animals , we self-crossed LysM-Cre-HET mice .", "The genotype of the progeny was determined using PCR primers described in Amleh et al . ( 2009 ) .", "LysM-Cre primers were obtained from Jackson Laboratories .", "BMDM were plated in 6-well plates at 2*106 cells/well .", "For BRD inhibitor experiments , cells were pretreated with I-BET ( Calbiochem , 401010 ) for 30 min , followed by co-treatment with LPS ( 10 ng/ml ) .", "For p300 inhibitor experiments , cells were treated with LPS for 30 min , followed by co-treatment with C646 ( Abcam , ab142163 ) for 1 hr .", "Concentrations of inhibitors are shown in Figure Legends .", "RAW264 . 7 cells were plated at 2*105 cells/well in 24-well plates and transfected ON using Turbofect ( Thermo Scientific , R0531 ) as per manufacturer’s instructions .", "Cells were treated the following day as described in Figure Legends .", "Plasmids used are pcDNA3 . 1-p300 , pcDNA3 . 1-300 ( HAT- ) ( Addgene , Plasmid #23252 and #23254 , respectively ) and pcDNA3 . 1 to equalize total amount of transfected DNA .", "Total RNA isolation from BMDM ( Qiagen RNAeasy Kit ) , random-primed cDNA synthesis , and qPCR with Maxima Sybr Green/ROX/2x master mix ( Fermentas ) on StepOne Plus real time PCR system were performed using standard protocols .", "Data analysis was performed using the ddCT method .", "All data were normalized to Actb as housekeeping control .", "Primers are listed in Supplemental file 4 .", "Whole cell extracts were prepared in RIPA buffer ( 10 mM Tris-HCl pH 8 . 0 , 1 mM EDTA , 0 . 5 mM EGTA , 140 mM NaCl , 5% glycerol , 0 . 1% Na deoxycholate , 0 . 1% SDS , 1% Triton X-100 ) .", "Immunoblotting was performed with rabbit polyclonal antibodies to NELF-B ( Cell Signaling , 1:2000 , 1489S ) , NELF-E ( Proteintech , 1:2000 , 10705–1-AP ) , HSP90 ( Cell Signaling 1:200 , 4874S ) .", "BMDM were treated for 45 min as specified in Figure Legends and single cross-linked in 1% methanol-free formaldehyde for 10 min at RT ( AcH4 ) or double cross-linked using 2 mM disuccinimidyl glutarate ( Proteochem , c1104 ) for 30 min followed by 1% methanol-free formaldehyde for 10 min at RT ( CDK9 , BRD4 , MED1 , MED12 , p300 ) .", "The reaction was quenched by 0 . 125 M glycine for 5 min .", "Cells were then washed with PBS , scraped and lysed for 10 min at 4°C in lysis buffer with protease inhibitor cocktail .", "The nuclear extracts were collected by centrifugation at 600*g for 10 min .", "Nuclei were then washed for 10 min at 4°C in wash buffer with protease inhibitors and collected as described above .", "Nuclei were lysed in lysis buffer for 10 min and sonicated to fragment chromatin using 15–18 cycles ( 30 s ‘on’ , 30 s ‘off’ ) in a Bioruptor at 4°C .", "For CDK9 , nuclei were sonicated with Covaris S220 Ultrasonicator according to manufacturer’s instructions ( 130 μl shearing buffer , 200 cycles/burst , 120 s , DF 10 ) .", "Lysates were cleared by centrifugation at 14 , 000*g , 20 min , 4°C , and then incubated with normal rabbit IgG ( Santa Cruz Biotech , sc-2027x ) , BRD4 ( Abcam , ab84776 and Bethyl Laboratories , A3001-985A100 ) , MED12 ( Bethyl Laboratories , A300-774A ) , MED1 ( Bethyl Laboratories , A300-793 ) , p300 ( Santa Cruz Biotech , sc-585X ) , Anti-AcH4 ( Millipore , 06–866 ) , Anti-AcH4K12 ( Millipore , 07–595 ) , Anti-AcH4K5 ( Millipore , 07–327 ) and 40 μl of 50% protein A/G plus agarose ( Santa Cruz Biotech , sc-2003 ) per reaction at 4°C ON .", "Beads were washed 4x with RIPA buffer and once with TE buffer .", "For CDK9 , 5 μg of antibodies ( Santa Cruz Biotech , sc-8338X or sc-13130X ) were pre-bound to 40 μl of Dynabeads Protein A ( Invitrogen ) , washed 2x with beads blocking buffer and incubated with lysates at 4°C ON; IPs were washed 6x with modified RIPA buffer containing 100 mM LiCl on a magnetic stand and once with TE buffer +50 mM NaCl . Each reaction was then incubated in TE + 0 . 5% SDS +200 μg/ml proteinase K ( Invitrogen , 25530049 ) for 2 hr at 55°C , followed by 6 hr at 65°C to reverse crosslinks .", "DNA was purified using phenol-chloroform extraction and ethanol precipitation or using Qiagen PCR purification kit .", "Recruitment at binding sites was assessed by qPCR .", "All data at putative binding sites were normalized to 28S ribosomal RNA as control .", "Primers are listed in Supplemental file 4 .", "For GR ( Santa Cruz Biotech , sc-1004X ) , BRD4 ( Abcam , ab84776 ) and p65 ( Santa Cruz Biotech , sc-372X ) ChIP-seq , nuclei were sonicated with Covaris S220 sonicator according to manufacturer’s instructions ( 130 μl shearing buffer , 200 cycles/burst , 120 s , DF 10 ) .", "For Pol 2 ( Santa Cruz Biotech , sc-9001X ) and NELF-E ( Proteintech , 10705–1-AP ) ChIP-seq , cells were formaldehyde cross-linked and nuclei were sonicated as above to obtain fragments in 150–500 bp range .", "Input DNA was prepared from sonicated material saved prior to IP .", "Lysates were cleared by centrifugation at 14 , 000 rpm , 20 min , 4°C , and then incubated with respective antibodies using 40 μl of 50% protein A/G PLUS agarose beads ( for GR , BRD4 and Pol 2 ) or 60 μl of Dynabeads ( Invitrogen ) ( for p65 ) per reaction at 4°C ON .", "GR , BRD4 and Pol 2 IPs were then processed as described for ChIP-qPCR above .", "p65 IPs were washed 6x with modified RIPA buffer containing 100 mM LiCl on a magnetic stand and once with TE buffer +50 mM NaCl and processed as described for ChIP-qPCR above .", "The efficiency of ChIP was assessed by qPCR .", "The integrity and quality of DNA was evaluated with Bionalyzer 2100 ( Agilent Technologies ) before using 10 ng of material to prepare Illumina-compatible sequencing libraries using Illumina Truseq ChIP sample prep kit .", "Library preparation and sequencing was performed by Weill Cornell Epigenomics Core .", "Libraries were sequenced by a HiSeq 2500 ( 50 bp , single-end ) .", "BMDM from LysM-Cre:NELF-B wt/wt ( WT ) and/or LysM-Cre:NELF-B fl/fl ( NELF-B KO ) mice were treated as indicated in individual figure legends ( vehicle , LPS , LPS + Dex for 1 hr ) and RNA was isolated using Qiagen RNA-easy kit .", "Total RNA was polyA enriched and converted into Illumina-compatible sequencing library with TruSeq mRNA-Seq sample preparation kit ( Illumina ) .", "Quality control of RNA and libraries was performed using the BioAnalyzer 2100 .", "Pair-end sequencing was performed at the Weill Cornell Epigenomics Core using HiSeq2500 .", "Two-tailed Student’s t-test was used to ascertain the differences between means as detailed in Figure Legends .", "Sequencing quality control was performed using FASTQC; adapters , when needed , where trimmed using trimmomatic .", "50 bp single-end reads were aligned to the mouse genome ( mm10 ) using CLC Bio Genomic Workbench ( GR , Pol 2 ) or bowtie2 ( p65 , NELF-E , BRD4 ) .", "Aligned BAM files were converted into bigwig format for data visualization purposes .", "The quality of Chip-seq experiments was assessed using ChIPQC package ( Carroll et al . , 2014 ) ( Supplemental file 2 ) .", "Cross-correlation analysis , Relative Strand Correlation ( RSC ) and Normalized Strand Cross-correlation coefficient ( NSC ) for all ChIP-seq datasets used in this study were calculated with CLC BIO genomics workbench ( Figure 1—figure supplements 1D and 2C; Figure 3—figure supplement 1A and Figure 4—figure supplement 1C , Supplemental file 2 ) as described in Marinov et al . , 2014 .", "RSC reflects the ratio of the fragment-size peaks and the read-size peak in cross-correlation plot .", "For all experiments with the exception of one NELF-E condition , the RSC is larger than 0 . 8 as per ENCODE recommendations ( Landt et al . , 2012 ) .", "Peak calling was performed with CLC Bio Genomics Workbench ( Pol 2 ) or MACS2 ( Zhang et al . , 2008 ) ( --gsize 2150570000 --bw=300 ratio 1 . 0 --slocal 1000 --llocal 10000 --keep-dup 1 --bdg --qvalue 0 . 05 ) with a matching input file to estimate background read distribution .", "Peak annotation relative to known genomics features was performed using ChIPpeakAnno package ( R , Bioconductor ) ( Zhu et al . , 2010 ) with TxDb . Mmusculus . UCSC . mm10 . knownGene annotation ( 2016-09-29 04:05:09 + 000 ) .", "Peak overlaps between datasets were determined using subsetByOverlap function from GenomicRanges package ( R , Bioconductor ) with the minimum overlap of 1 nt and visualized with makeVennDiagram function from ChIPpeakAnno ( Zhu et al . , 2010 ) package .", "Ab initio analysis of overrepresented sequences in ChIP-seq peaks was performed using MEME-ChIP suite with MEME ( long sequences ) , DREME ( short sequences ) and CentriMO ( centrally-enriched sequences ) .", "E-values estimate the expected number of motifs in an experimental set of sequences compared to random sequences of the similar size .", "Sequencing motifs with E-values under 0 . 0001 were considered statistically significant .", "Pol 2 pausing indexes ( PI ) were calculated as previously described ( Nechaev et al . , 2010 ) .", "All transcripts for LPS-induced Dex-sensitive genes present in TxDb . Mmusculus . UCSC .", "mm10 . knownGene annotation were filtered to collapse all annotated transcripts with identical 5’ ends to a single gene model .", "For remaining transcripts , the PI was calculated as the ratio of log-transformed , length-normalized read counts at the 5’ end flanking region ( −200:+500 ) and transcript ‘body’ ( +500: end of a transcript ) .", "To compare between replicates , the PI were normalized to respective library sizes ( as in Figure 2B ) .", "Read distributions in the region of interest ( ‘promoters’ and gene ‘bodies’ ) were visualized in the form of ‘heat’ maps that show scores ( coverage ) at a given sequence position or bin using genomation package ( R , Bioconductor ) ( Akalin et al . , 2015 ) .", "For heat maps visualization , paused and non-paused transcripts were further filtered by selecting only those that had overlapping Pol 2 peaks in the ‘promoter’ area in LPS-treated BMDM .", "To summarize read distributions , we plotted mean coverages ( plotMeta , genomation ) over regions of interest ( Figures 2D and 3A and Figure 4—figure supplement 1B ) with the standard error and the 95% confidence interval bands .", "RNA-seq analysis has been performed as previously described ( Coppo et al . , 2016 ) .", "50 bp paired reads were mapped to annotated mouse genome ( mm10 ) with CLC Bio Genomic Workbench ( Qiagen ) .", "Read count table containing unique exon reads was analyzed using EdgeR ( Robinson et al . , 2010 ) package to determine differentially expressed genes .", "Read counts were scale normalized using the weighted trimmed mean method and expressed as log-transformed counts per million ( cpm ) .", "All genes with unadjusted p-value<0 . 01 ( p<0 . 05 for NELF-B KO experiment ) and fold change >1 . 5 in at least one pairwise comparison were considered to be differentially expressed and were selected for further analysis .", "All raw sequence data generated in this study are deposited to NCBI GEO: GSE110279 https://www . ncbi . nlm . nih . gov/geo/query/acc . cgi ?", "acc=GSE110279" ] ]

[ "The glucocorticoid receptor ( GR ) potently represses macrophage-elicited inflammation , however , the underlying mechanisms remain obscure .", "Our genome-wide analysis in mouse macrophages reveals that pro-inflammatory paused genes , activated via global negative elongation factor ( NELF ) dissociation and RNA Polymerase ( Pol ) 2 release from early elongation arrest , and non-paused genes , induced by de novo Pol2 recruitment , are equally susceptible to acute glucocorticoid repression .", "Moreover , in both cases the dominant mechanism involves rapid GR tethering to p65 at NF-kB-binding sites .", "Yet , specifically at paused genes , GR activation triggers widespread promoter accumulation of NELF , with myeloid cell-specific NELF deletion conferring glucocorticoid resistance .", "Conversely , at non-paused genes , GR attenuates the recruitment of p300 and histone acetylation , leading to a failure to assemble BRD4 and Mediator at promoters and enhancers , ultimately blocking Pol2 initiation .", "Thus , GR displays no preference for a specific pro-inflammatory gene class; however , it effects repression by targeting distinct temporal events and components of transcriptional machinery ." ]

[ "Inflammation is one of the body’s responses to fight infection and heal tissue damage .", "The response is controlled by hundreds of genes , which fall into two classes .", "In the first class , an injury or infection triggers the enzyme RNA Polymerase to bind to and transcribe the gene into long RNA strands , which are then translated into the proteins that play a role in the inflammation response .", "The second class has a more quick-fire response .", "RNA Polymerase binds to these genes even without an injury or infection to serve as a trigger .", "But most of the time the enzyme only transcribes the beginning of these genes .", "This is because it is inhibited by a so-called negative elongation factor , which acts like a brake .", "For this second class of genes , an infection or injury triggers the release of the negative elongation factor from the enzyme , and allows RNA Polymerase to transcribe the full RNA strand .", "In excess , inflammation can be dangerous .", "The body’s way of limiting or controlling inflammation is via steroid hormones called glucocorticoids .", "These bind to the glucocorticoid receptor , which acts to switch off the inflammatory genes .", "But exactly how the receptor does this has not been fully understood .", "Sacta et al . investigated how the glucocorticoid receptor turns off these gene complexes .", "Experiments looking at white blood cells in mice found that the receptor can switch off both groups of inflammatory genes , but by a different mechanism for each class .", "Sacta et al . discovered that in the first gene class , the receptor blocks proteins that open up the DNA for RNA Polymerase , so it could not bind to the gene .", "In the second class , the receptor stops the release of the brake-like negative elongation factor from RNA Polymerase .", "As a result , the enzyme stalls at the beginning of the gene and fails to make a full-length transcript required to make the necessary protein .", "Glucocorticoids are often used as drugs to treat chronic inflammation , but they can have debilitating side effects .", "Understanding how the glucocorticoid receptor switches off inflammatory genes could help to design drugs with fewer side effects to treat chronic inflammation , and diseases caused by specific inflammatory genes ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "tools and resources", "neuroscience" ]

[ [ "A core goal of modern neuroscience is to map neural circuits across the entire brain , as long-range connectivity dictates how information flows between brain regions ( Bohland et al . , 2009 ) .", "Brain-wide inter-areal connectivity has been probed using bulk injections of anterograde and retrograde tracers ( Gerfen and Sawchenko , 1984; Hunnicutt et al . , 2014; Luppi et al . , 1990; Markov et al . , 2014; Oh et al . , 2014; Veenman et al . , 1992; Zingg et al . , 2014 ) and functional imaging methods with millimeter-scale spatial resolution ( van den Heuvel and Hulshoff Pol , 2010 ) .", "However , intermingled neurons within a brain region have heterogeneous projection patterns ( Nassi and Callaway , 2009; Shepherd , 2013 ) and carry diverse messages in the behaving brain ( Li et al . , 2015; Movshon and Newsome , 1996; Sato and Svoboda , 2010; Turner and DeLong , 2000 ) .", "Projection maps based on bulk tracer injections are thus comprised of multiple functionally and structurally distinct groups of neurons .", "Reconstructions of single neurons therefore provide vital information about how neural signals are organized and transmitted across the brain to target regions .", "Single-neuron reconstructions are also critical for defining the fundamental cell types in the brain .", "The nervous system has long been viewed as a collection of discrete cell types defined by their developmental origins , gene expression , morphology , and function .", "Axonal structure has been particularly useful in classifying projection neurons ( Cowan and Wilson , 1994; Kawaguchi et al . , 1990 ) but this information is currently lacking for most cell types .", "Visualization of the brain-wide projection maps of the individual neurons comprising a projection pathway would enable the isolation of well-defined classes that target similar structures and may otherwise be impossible to distinguish .", "Single-cell and sparse-labeling techniques ( Furuta et al . , 2001; Horikawa and Armstrong , 1988; Pinault , 1996; Reiner et al . , 2000; Rotolo et al . , 2008 ) have facilitated the reconstruction of individual axonal projections over long distances in the basal ganglia , hippocampus , neocortex , thalamus , olfactory cortex , and neuromodulatory systems ( Ghosh et al . , 2011; Kawaguchi et al . , 1990; Kita and Kita , 2012; Kuramoto et al . , 2009; Oberlaender et al . , 2011; Ohno et al . , 2012; Wittner et al . , 2007; Wu et al . , 2014 ) .", "However , the reliability and throughput of axonal reconstruction have remained limited by the necessity to restrict labeling to one or a few neurons in a single brain and to manually track individual segments between serial sections , which are often deformed and may be damaged by standard histological processing techniques .", "Visualization of neurons in continuous whole-brain image volumes may overcome these limitations and allow reconstruction of long-range axonal projections in a reliable and efficient manner .", "This requires an approach capable of imaging every location in a three-dimensional space within a large tissue volume with high resolution so that fine axonal collaterals may be unambiguously tracked to their targets .", "Here , building on serial two-photon ( STP ) tomography ( Portera-Cailliau et al . , 2005; Ragan et al . , 2012; Tsai et al . , 2009 ) , we describe a system for fast volumetric microscopy that can be used to image the entire mouse brain at high resolution .", "Combining this technique with high-intensity , sparse neuronal labeling , a novel technique for clearing tissue , and an informatics pipeline for processing and visualizing large imaging datasets , we present a platform for efficient reconstruction of axonal morphology .", "We demonstrate the utility of this system by reconstructing the extensive , brain-wide axonal arborizations of diverse projection neurons in the motor cortex within a single mouse brain ." ], [ "To image the axonal arbors of individual neurons , we constructed a platform for fast , automated , volumetric fluorescence imaging at sub-micron resolution .", "This system is based on a two-photon laser scanning microscope integrated with a vibrating microtome ( Figure 1a ) , and was optimized for high-fidelity mosaic imaging in three dimensions . 10 . 7554/eLife . 10566 . 003Figure 1 . Schematic of imaging system .", "( a ) Schematic of apparatus for automated volumetric two-photon tomography .", "( b ) To image large volumes of tissue , a collection of three-dimensional image stacks ( tiles ) covering the full volume of the tissue sample was acquired serially .", "Tiles overlapped in all three dimensions to aid in image registration and ensure coverage of the full volume .", "( c ) The active imaging region is determined for each section by first tracing the perimeter of the tissue block and then filling in any tiles internal to the traced region .", "( d ) Flow chart illustrating the tasks performed during data acquisition . DOI: http://dx . doi . org/10 . 7554/eLife . 10566 . 00310 . 7554/eLife . 10566 . 004Figure 1—figure supplement 1 . Point spread function measurement .", "( a ) Empirically-measured point spread function measured using 200-nm fluorescent polystyrene beads . DOI: http://dx . doi . org/10 . 7554/eLife . 10566 . 004 To achieve high imaging speeds , we used a resonant scanning galvanometer , a piezoelectric motor-mounted objective for fast Z-scanning , and high excitation power .", "This approach allowed us to image up to 16 million voxels/s ( >50 mm3/day ) with signal-to-noise ratios ( SNRs ) sufficient for axonal reconstruction .", "This fast-scanning microscope represents a 16–48× increase in speed over conventional laser-scanning microscopy systems ( Pologruto et al . , 2003 ) .", "The system is comprised primarily of commercially-available hardware and custom control software ( freely available at github . com/TeravoxelTwoPhotonTomography ) and operates as follows: ( 1 ) the entire volume of space addressable by the stage system is partitioned into overlapping 3D tiles , each corresponding to a single image stack ( Figure 1b ) ; ( 2 ) tiles intersecting the exposed surface of the sample are marked for imaging ( Figure 1c ) ; ( 3 ) an image stack corresponding to each marked tile is acquired; ( 4 ) the integrated vibratome removes a portion of the imaged volume; ( 5 ) the precise surface plane of the remaining tissue is determined; and ( 6 ) this sequence of events is automatically repeated until the entire tissue sample has been processed ( Figure 1d ) .", "In the past , STP tomography has been limited to acquiring two-dimensional ( 2D ) snapshots of tissue volumes separated by >50 µm in the axial direction .", "Following small structures through highly scattering brain tissue in three dimensions requires a strategy for clearing fixed tissue that is compatible with long-term imaging .", "Specifically , we sought an approach that preserved the native fluorescence of fluorescent proteins , introduced little background autofluorescence , effectively cleared gray and white matter , was compatible with serial sectioning , and that could be used as a stable , long-term immersion medium during extended imaging sessions .", "We evaluated the compatibility of a number of recently described techniques ( Chung and Deisseroth , 2013; Ertürk et al . , 2012; Hama et al . , 2011; Ke et al . , 2013; Renier et al . , 2014; Susaki et al . , 2014 ) .", "Strategies employing organic solvents ( i . e . 3Disco , iDisco ) quenched fluorescent proteins produced hard , brittle tissue that could not be sectioned .", "Clearing agents requiring high-index saturated solutions ( i . e . SeeDB , Clarity , CUBIC ) were not robust to evaporation and solutes precipitated during extended imaging experiments .", "Scale expanded tissue , attenuated fluorescence , and resulted in unstable tissue geometry .", "Due to these incompatibilities , we sought an alternative method meeting these requirements .", "We devised a clearing medium comprised of a ternary mixture of dimethyl sulfoxide ( DMSO ) , d-sorbitol , and aqueous buffer ( see Methods ) .", "DMSO was chosen due to its high refractive index ( 1 . 479 ) and extensive use in mounting medium formulations for fluorescence microscopy .", "The sugar alcohol d-sorbitol was found to be highly soluble in DMSO and , unlike d-fructose ( Ke et al . , 2013 ) , may be obtained at high purity without autofluorescent contaminants and does not cause non-enzymatic browning of biological tissue .", "Aqueous buffer was added to preserve green fluorescent protein ( eGFP ) fluorescence ( Figure 2b , c ) .", "The high refractive index of the resulting solution ( 1 . 468 ) reduced light scattering in tissue by minimizing index mismatches with lipid- and protein-rich brain structures ( Figure 2a ) .", "The excellent tissue penetrance of these reagents further contributed to a high degree of clearing of white and gray matter in whole brains .", "We note that the use of subsaturating d-sorbitol solutions was important for preventing precipitation or gellation of the mixture following the evaporation and mechanical agitation that accompanies long ( >1 week ) imaging sessions .", "Another key advantage of this medium over other clearing agents is its low viscosity; serial sections with consistent thickness were difficult to obtain in viscous media .", "This approach allowed small structures , including fine axons , to be imaged with minimal signal attenuation at imaging depths >200 μm ( Figure 2d–f ) .", "Samples could be reliably sectioned and imaged over the course of many days and stored for many months without adverse effects on fluorescent labels or gross changes in brain volume ( Figure 2a ) .", "Hence , this medium met the requirements for high-resolution volumetric imaging and serial sectioning . 10 . 7554/eLife . 10566 . 005Figure 2 . Sample preparation and clearing .", "( a ) Whole brains ( left ) and 1 mm-thick tissue sections ( right ) cleared using dimethyl sulfoxide ( DMSO ) and d-sorbitol .", "( b ) Fluorescence of purified eGFP as a function of DMSO concentration ( v/v ) in 10 mM HEPES , pH 7 . 3 ( open circles ) and 10 mM HEPES , pH 7 . 3 containing 44% ( w/v ) d-sorbitol ( filled circles ) .", "( c ) eGFP and tdTomato fluorescence in a cleared hemi-brain demonstrates preservation of native fluorescence .", "( d ) Maximum intensity projections of side views of image tiles of histone 2B-eGFP labeled nuclei from a DMSO/d-sorbitol cleared section ( right ) and from a matched section in phosphate buffered saline ( PBS; left ) from the contralateral hemisphere of the same brain .", "( e ) Quantification of intensities of histone 2B-eGFP labeled nuclei as a function of depth ( PBS: n=204 detected nuclei in two tiles; DMSO/d-sorbitol: n=815 detected nuclei in three tiles ) .", "( f ) Representative images of a single axonal collateral as it passes through the anterior commissure ( white ) and surrounding olfactory cortex ( gray ) .", "Images were acquired at a depth >180 μm in each case and scaled in the same manner .", "( g ) Axons labeled with multiple fluorophores could be simultaneously imaged with high signal to noise within cleared tissue .", "Note that imaging fine axons through the axially oriented anterior commissure in this example represent a stringent test of clearing due to the high degree of optical scattering observed in white matter tracts .", "DMSO , dimethyl sulfoxide; eGFP , enhanced green fluorescent protein . DOI: http://dx . doi . org/10 . 7554/eLife . 10566 . 005 To reduce distortion of the tissue during sectioning , samples were embedded in gelatin and fixed a second time .", "This procedure ensured adhesion of the tissue to the surrounding gelatin .", "As our clearing solution quickly diffused through the cross-linked gelatin matrix , tissue samples—including whole mouse brains—could be effectively cleared following gelatin embedding .", "Treatment with CUBIC-1 ( Susaki et al . , 2014 ) for lipid removal prior to embedding was found to further improve clearing ( data not shown ) .", "The complete protocol for preparation of cleared tissue samples for long-term imaging is detailed in Materials and methods and Table 1 . 10 . 7554/eLife . 10566 . 006Table 1 . Clearing solutions . DOI: http://dx . doi . org/10 . 7554/eLife . 10566 . 006Solution ( # ) Dimethyl sulfoxide", "( g ) PB", "( g ) D-sorbitol", "( g ) Refractive index126 . 8373 . 170 . 001 . 373252 . 3847 . 620 . 001 . 412344 . 5240 . 4815 . 001 . 425436 . 6733 . 3330 . 001 . 440534 . 2520 . 7545 . 001 . 468625 . 5611 . 4463 . 001 . 489 The diameters of fine axonal processes are often less than 100 nm ( De Paola et al . , 2006; Shepherd and Harris , 1998 ) .", "A complete volumetric representation of axonal arborizations thus requires diffraction-limited , high-numerical aperture ( NA ) imaging and near-Nyquist sampling ( voxel size ~ 0 . 3 × 0 . 3 × 1 . 0 µm ) .", "At this resolution , the full volume of a mouse brain contains more than 5 trillion voxels , corresponding to more than 10 TB of image data per fluorescence channel .", "In addition , physical sectioning introduces tissue deformation at the micron scale ( Figure 3 ) .", "For these reasons , it was necessary to construct a computational pipeline for registering , visualizing and storing datasets of this magnitude . 10 . 7554/eLife . 10566 . 007Figure 3 . Registration of image tiles .", "( a ) Example registration of pairs of image tiles in the axial ( left ) and lateral ( right ) directions .", "( b ) Initial displacement of automatically-identified features as a result of sectioning ( left ) across the full extent of the exposed tissue surface .", "Displacements along each Cartesian direction are displayed in separate heat maps for a representative section .", "Right: residual displacement of the same feature set after linear interpolation of displacements across each tile .", "( c ) Distribution of the residual displacements of all features identified in a whole-brain dataset in the lateral ( top ) and axial ( bottom ) directions before ( black ) and after ( red ) image registration .", "( d ) Maximum-intensity projection through a volume containing labeled neurons before ( left ) and after ( right ) the registration procedure . DOI: http://dx . doi . org/10 . 7554/eLife . 10566 . 00710 . 7554/eLife . 10566 . 008Figure 3—figure supplement 1 . Lipofuscin imaging . Overlay of fluorescence captured in the green ( 500–550 nm ) and orange ( 580–653 nm ) spectral bands from a representative tile within the neocortex .", "Autofluorescent lipofuscin ( white puncta ) could be imaged reliably .", "These features , present throughout the brain , were utilized for post hoc registration .", "Lipofuscin was easily distinguished from enhanced green fluorescent protein ( eGFP ) -labeled structures by its broad emission spectrum . DOI: http://dx . doi . org/10 . 7554/eLife . 10566 . 008 To enable registration of tiles with submicron resolution , we ensured that they partially overlapped in all dimensions .", "Following image acquisition , a set of common features was identified in the overlap region of each pair of tiles bordering each other in the axial direction ( i . e . tiles at the same x , y location in successive sections ) .", "Autofluorescent puncta ( lipofuscin; Figure 3—figure supplement 1 ) were present throughout the mouse brain and could be reliably identified in overlapping tiles .", "Using these point correspondences , the tiles were registered ( Materials and methods; Figure 3 ) and resampled into a set of non-overlapping image volumes precisely tiling the full brain .", "The resampled volume was hierarchically downsampled to produce additional representations of the volume at progressively lower resolution .", "This multiresolution volume enabled efficient navigation at different spatial scales and neuronal structures , which appeared continuous across tile boundaries ( Video 1 ) , could be traced for over long distances . 10 . 7554/eLife . 10566 . 009Video 1 . Movie illustrating 18 serially acquired image tiles that have been registered and resampled into a continuous image volume . This volume ( 3 × 1 × 6 tiles ) spans five adjacent tissue sections .", "Fine axons are resolvable and all fibers appear continuous .", "Images were spectrally unmixed to remove autofluorescence of lipofuscin . DOI: http://dx . doi . org/10 . 7554/eLife . 10566 . 009 To test the imaging and informatics pipeline , we imaged whole mouse brains wherein a sparse subset of neurons in motor cortex was brightly labeled ( Figure 4 ) .", "Sparse labeling was achieved by expressing Cre recombinase with a highly diluted adeno-associated virus while simultaneously delivering a Cre-dependent high-titer reporter virus coding for a fluorescent reporter ( e . g . eGFP ) ( Xu et al . , 2012 ) .", "Using this labeling strategy , 10–50 motor cortical neurons could be routinely labeled with high intensity in each brain .", "Brightly-labeled neurons were likely infected with a single copy of the viral plasmid encoding Cre but with many copies of the reporter gene , thereby driving high expression in few cells .", "High-intensity labeling ( e . g . with viral vectors encoding bright fluorescent proteins like eGFP or tdTomato ) was necessary for high-contrast imaging of thin fibers .", "Using this labeling strategy , fine , long-range axonal collaterals—such as those innervating the contralateral motor cortex—were easily detected and resolved .", "These fibers were clearly discernable even in a maximum intensity projection through a large volume of tissue ( 2 . 3 × 1 . 3 × 1 . 0 mm ) corresponding to >200 individual 3D tiles ( Figure 4b ) .", "The high excitation power needed to image axons with high SNR was not found to have a detrimental effect on the resolution of our imaging system due to fluorophore saturation , as has been suggested ( Figure 4—figure supplement 1 ) ( Zipfel et al . , 2003 ) . 10 . 7554/eLife . 10566 . 010Figure 4 . Whole-brain imaging .", "( a ) Three-dimensional rendering of complete mouse brain dataset as viewed from an anterolateral ( left ) and ventral ( right ) perspective .", "( b ) Maximum intensity projection through a large tissue volume containing labeled somata and neurites in the X–Y ( top ) and X–Z ( bottom ) planes ( 2 . 3 × 1 . 3 × 1 . 0 mm ) .", "Axon collaterals are clearly discernible in the contralateral hemisphere despite the large volume of tissue depicted .", "Dotted lines represent borders between adjacent tiles .", "Insets ( top right ) represent the location and orientation of each image in relation to a coronal ( top ) and horizontal ( bottom ) section .", "Inset at top left illustrates detail in full-resolution images .", "Following registration of the full collection of tiles comprising a full dataset , individual neurites appear continuous between tiles separated in the axial direction ( bottom; separated by a physical tissue section ) while maintaining continuity in laterally adjacent tiles ( top ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 10566 . 01010 . 7554/eLife . 10566 . 011Figure 4—figure supplement 1 . High speed , high-power imaging .", "( a ) Image of an axonal collateral with low ( top ) and high ( bottom ) excitation power .", "High-power imaging improves signal-to-noise without degrading resolution due to fluorophore saturation .", "( b ) Peak-normalized fluorescence intensity along the cross sections indicated above in", "( a ) resulting from low- ( black ) and high-power imaging ( red ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 10566 . 011 Whole-brain reconstruction of axon arborizations requires that fine-caliber segments far from their associated somata remain brightly labeled .", "We examined axonal labeling at distant locations for five of the cells depicted in Figure 4b ( somatic location and dendritic morphology illustrated in Figure 5a ) .", "Axonal segments associated with brightly labeled somata remained well-labeled through white matter tracts , at branch points , and at their termini in distant structures , indicating high labeling intensity throughout each arbor ( Figure 5b ) .", "We quantified the SNR across 52 axonal cross sections selected to represent a range of axonal morphologies .", "In this sample , the z-scored median peak intensity of individual axons was consistently high compared to background ( 15 . 0 ± 2 . 9 median ± standard error of the mean; 5–95% range: 7 . 0–70 . 7; Figure 5—figure supplement 1 ) .", "Further , we found that datasets could be stored using lossy H . 264 compression while maintaining high image quality .", "At a compression ratio of 25:1 , the SNR of small axons was reduced by only 4 . 8% ( compression ratio determined in n = 996 tiles; Figure 5—figure supplement 1 ) .", "Software tools for data registration , resampling , and compression are available online at github . com/TeravoxelTwoPhotonTomography . 10 . 7554/eLife . 10566 . 012Figure 5 . Axon collaterals are labeled with high signal-to-noise across their entire length .", "( a ) Top: Virtual coronal section through whole-brain dataset .", "Boxed area denotes region containing labeled somata and is expanded in schematic below .", "Bottom: Laminar distribution and dendritic morphology of five labeled neurons spanning layers II–VI .", "( b ) Representative images of axonal segments from each of the five neurons in white matter ( left ) , at branch points ( middle; denoted by asterisks ) and at termini ( right; denoted by arrowheads ) .", "The location of each axonal segment is listed below the corresponding images , and in all cases , segments were in a different brain structure than their associated somata .", "All images are maximum intensity projections through a depth of 20 μm .", "( c ) Regions with high labeling density contain axon crossovers that introduce ambiguity into axonal reconstruction .", "Examples of crossover points ordered subjectively by approximate degree of difficulty in assignment of segment identity .", "The two-dimensional examples shown here are for illustration purposes; ambiguity arises only when segments are closely apposed in all the three dimensions . DOI: http://dx . doi . org/10 . 7554/eLife . 10566 . 01210 . 7554/eLife . 10566 . 013Figure 5—figure supplement 1 . Signal-to-noise of axonal imaging .", "( a ) Representative images before ( left ) and after ( middle ) lossy H . 264 compression .", "The intensity profile across each neurite ( along paths denoted by dotted lines in images on left ) is plotted to the right of each set of images and was minimally affected by image compression .", "( b ) Distribution of compressed file size across a random sampling of 996 tiles .", "Across the entire subset , tiles were reduced in size by 95 . 7 ± 2 . 5% percent after compression .", "( c ) SNR of axonal segments in each intensity profile ( n = 52 ) before and after compression .", "SNR along each path was reduced to 95 . 12 ± 5 . 92% of control by the compression procedure .", "SNR , signal-to-noise ratio . DOI: http://dx . doi . org/10 . 7554/eLife . 10566 . 013 Another requirement for axonal reconstruction is that single fibers must be tracked unambiguously across long distances .", "Even with sparse labeling , axonal segments of similar caliber but originating from different somata cross paths with separations smaller than the diffraction limit of light , introducing uncertainty in the assignment of axonal identity following crossover points .", "In the past , the inability to unambiguously follow single processes has precluded extensive reconstruction of fine axonal collaterals in datasets containing multiple labeled neurons in the same brain region ( Gong et al . , 2013; Zheng et al . , 2013 ) .", "Since the error rate in manual and automatic reconstruction of axonal processes from light microscopy datasets depends strongly upon the density of neurites within a local volume ( Gala et al . , 2014 ) , we assessed the feasibility of complete axonal reconstruction in this dataset .", "We attempted to reconstruct the full axonal morphology of the same cohort of neurons depicted in Figure 5 .", "The high density of processes at the site of viral injection - the region containing the highest density of labeled somata - prevented unambiguous reconstruction of many processes in this area due to axonal crossovers ( Figure 5c ) .", "However , we found that the long-distance axonal projections of these neurons and their fine collaterals in target regions could be accurately reconstructed to a high degree of completeness ( Figure 6a; Video 2 ) .", "This level of completeness was facilitated by the labeling sparsity , high resolution imaging , and lack of discontinuities in the dataset .", "Furthermore , 3–12 mm of axon could be reconstructed per hour , depending on local density—a substantial increase in throughput over classical serial-section reconstruction methods , even when samples contained only a single labeled neuron ( Wittner et al . , 2007 ) . 10 . 7554/eLife . 10566 . 014Figure 6 . Complete reconstruction of axonal morphology .", "( a ) Complete reconstruction of the same five projection neurons depicted in Figure 4 ( inset ) .", "Reconstructions are overlaid on a horizontal ( left ) and sagittal ( right ) outline of the imaged mouse brain .", "Subset includes pyramidal neurons in layer II ( blue , purple ) , layer V ( red , black ) and layer VI ( green ) .", "( b ) Illustration of axonal and dendritic reconstruction of the layer 5 pyramidal cell ( colored red in", "a ) shown in the coronal plane .", "Profile of coronal section at the rostrocaudal position of the cell body is depicted by the black dashed line .", "Segments were colored to highlight axonal arbors originating from common branch points . DOI: http://dx . doi . org/10 . 7554/eLife . 10566 . 01410 . 7554/eLife . 10566 . 015Video 2 . Single axon traced to its terminus . Depicted path represents the longest continuous axonal segment starting at the cell soma .", "The terminus is located in the anterior piriform cortex .", "White dot corresponds to the same location in both panels and corresponds approximately to the path of manually traced segment .", "The SNR of well-labeled axons remains high along their entire length and branch points are clearly visible .", "Nuclei ( magenta ) were counter-labeled using NuclearID-Red .", "Two axons , originating from distinct neurons , cross in the corpus callosum .", "Axon identity is straightforward to assign in this example on the basis of trajectory and labeling intensity when viewed in three dimensions .", "This neuron appears blue in all figures . DOI: http://dx . doi . org/10 . 7554/eLife . 10566 . 015 This group of reconstructed neurons included two intratelencephalic ( IT ) projection neurons in layer II/III , two IT neurons in layer V , and one corticothalamic projection neuron located in layer VI ( Figure 6a; Videos 3–5 ) .", "These neurons possessed extensive axon collaterals with each cell targeting a surprisingly diverse set of brain areas ( Table 2 , Figure 6a ) .", "Together , the total axon length of these five neurons exceeded 300 mm ( range: 23 . 6–121 . 2 mm ) and innervated 28 distinct brain regions ( Table 2 ) .", "The arborization of one individual layer V neuron was found to cover nearly the entire extent of the brain in the coronal plane ( Figure 6b ) and approximately half of the brain along the rostrocaudal axis , underscoring the importance of whole-brain imaging for determining axonal projection patterns .", "Interestingly , this neuron included an extensive projection to the taenia tecta ( Figure 6b ) , a region that exhibits only very sparse axonal labeling in bulk anterograde tracing experiments from motor cortex ( C . Gerfen , unpublished data; Allen Connectivity Atlas: connectivity . brain-map . org/projection/experiment/141603190 ) , further highlighting the diversity in projection patterns of similar neurons within a single brain region .", "Following our approach , we were able to reconstruct axon morphologies to a high degree of completeness .", "As a result , the ensemble of downstream targets could be precisely identified for single neurons , and , furthermore , the extent and location of collaterals in all target regions . 10 . 7554/eLife . 10566 . 016Table 2 . Axonal reconstructions .", "All lengths in mm . DOI: http://dx . doi . org/10 . 7554/eLife . 10566 . 016Soma locationLayer IILayer IILayer VLayer VLayer VIDendritic branches6657372523Dendritic length8 . 037 . 679 . 175 . 064 . 20Axonal branches796717813627Axonal length47 . 3441 . 04121 . 2075 . 4423 . 57Axonal targets ( ipsilateral ) Motor cortex Dorsal striatum Nucleus accumbensMotor cortex Orbital cortex Dorsal/medial striatum Caudal/lateral striatumMotor cortex Somatosensory cortex Auditory cortex Orbital cortex Agranular insular cortex Ectorhinal cortex Piriform cortex Dorsal striatum Nucleus accumbens Olfactory tubercle Taenia tectaMotor cortex Somatosensory cortex Auditory cortex Anterior cingulate cortex Posterior parietal cortexMotor cortex Thalamic nuclei: Ventral anterior lateral Posterior Submedial ReticularAxonal targets ( contralateral ) Motor cortex Insular cortex Piriform cortexMotor cortex Anterior cingulate cortex Agranular insular cortex Claustrum Basolateral amygdalaMotor cortex Piriform cortex Dorsal striatum Nucleus accumbens Olfactory tubercle Taenia tectaMotor cortex Anterior cingulate cortex Dorsal striatum10 . 7554/eLife . 10566 . 017Video 3 . Three-dimensional rendering of reconstructed Layer II motor cortical neurons . Displayed brain outline corresponds to the contours of the imaged brain .", "Color code is the same as in all figures . DOI: http://dx . doi . org/10 . 7554/eLife . 10566 . 01710 . 7554/eLife . 10566 . 018Video 4 . Three-dimensional rendering of reconstructed Layer V motor cortical neurons . Displayed brain outline corresponds to the contours of the imaged brain .", "Color code is the same as in all figures . DOI: http://dx . doi . org/10 . 7554/eLife . 10566 . 01810 . 7554/eLife . 10566 . 019Video 5 . Three-dimensional rendering of reconstructed Layer VI motor cortical neuron . Displayed brain outline corresponds to the contours of the imaged brain .", "Color code is the same as in all figures . DOI: http://dx . doi . org/10 . 7554/eLife . 10566 . 019 Importantly , reconstructing multiple neurons within a single brain permits fine-scale comparisons between individual neurons .", "For example , in this limited dataset , all four reconstructed IT neurons projected to the contralateral motor cortex .", "The contralateral cortical projections of the two layer V neurons spanned a similar distance along the anterior–posterior ( A–P ) axis ( red: 2152 um; black: 2096 um ) .", "However , their terminal fields were offset along this axis by an amount closely mirroring the A-P offset between their corresponding somata ( Figure 7a , b; distance between projection centroids = 490 µm , A-P distance between somata = 513 um ) .", "This relationship suggests that these projections might target a continuum of cortical locations rather than a single discrete structure .", "In contrast , the contralateral cortical projection of both layer II neurons targeted a similar location along the A-P axis more posterior than both somata ( Figure 7c , d; distance between projection centroids = 32 µm; A-P distance between somata = 271 µm ) .", "Although a detailed analysis of such topology in a larger population is beyond the scope of this study , this example illustrates how organizational principles in neural connectivity—even at the micron scale—can be investigated using our approach . 10 . 7554/eLife . 10566 . 020Figure 7 . Fine scale topology of contralateral cortical projections .", "( a ) Horizontal view of the dendrites and axons of two layer V IT neurons ( red and black ) .", "Axons projecting to locations other than the contralateral motor cortex are shown in lighter colors .", "( b ) Density of axonal projections within the contralateral motor cortex as a function of position along the anterior–posterior ( A–P ) axis .", "Each point represents the cumulative length of axon within a 200 µm bin centered at the given coordinate divided by the bin width .", "A–P coordinates are relative to the center of the injection volume .", "( c ) Horizontal view of Layer II IT neurons ( blue and purple ) .", "Same view as in", "( a ) .", "( d ) Density of axonal projections for the two neurons in", "( c ) are on the same scale as in", "( b ) .", "Color codes are the same as in Figures 5 and 6 . DOI: http://dx . doi . org/10 . 7554/eLife . 10566 . 020" ], [ "Visualization of the complete axonal morphologies of individual neurons is critical for understanding how diverse neural signals are organized and communicated to distant targets throughout the brain .", "We have developed an imaging system for whole-brain , high-resolution fluorescence imaging and demonstrate that this approach can be used to trace individual axonal fibers and fine axon collaterals across the brain to their termini ( Video", "2 ) and thereby map the long-distance projections of single neurons ( Figure 6 ) .", "Reconstruction of axonal processes with light microscopy requires 3D , diffraction-limited imaging and a high-NA optical system in order to effectively identify and disambiguate axons originating from different cells .", "At the same time , light scattering within tissue must be minimized in order to image full 3D volumes .", "To achieve high resolution with minimal scattering , we extended block-face STP tomography ( Portera-Cailliau et al . , 2005; Ragan et al . , 2012; Tsai et al . , 2003 ) to high-speed 3D imaging .", "This required the development of a compatible procedure for tissue clearing , construction of an automated imaging system that can reliably image large 3D volumes , and design and construction of novel computational tools for the registration and visualization of large ( up to 100 TB ) datasets .", "In previous studies , STP tomography has been used to image bulk axonal projections across the mouse brain ( Oh et al . , 2014 ) .", "However , this approach has been limited to the acquisition of sequences of 2D images spaced at large intervals; high-SNR imaging of complete , 3D volumes - a necessity for tracing continuous neurites - has not been previously achieved using this approach .", "A number of alternative modalities are also potentially well-suited to high-resolution whole-brain imaging , although none have been successfully applied to extensive , brain-wide reconstruction of axon collaterals .", "Selective plane illumination microscopy ( SPIM ) ( Dodt et al . , 2007; Huisken et al . , 2004 ) requires reduced acquisition times , but lacks the necessary spatial resolution ( < 0 . 5 µm ) for resolving close appositions between thin axonal collaterals within large tissue volumes .", "Knife-edge scanning microscopy ( Li et al . , 2010; Mayerich et al . , 2008 ) represents a potential alternative , but requires resin embedding of tissue samples that can quench or attenuate the fluorescence of native fluorophores and produces tears in large tissue volumes along the edges of 'ribbon' sections .", "While some axonal segments have been traced using this approach , it has been unable to identify the vast majority of fine axon collaterals innervating many target structures ( Gong et al . , 2013; Zheng et al . , 2013 ) .", "Large-scale electron microscopy ( Kleinfeld et al . , 2011 ) offers the potential for dense reconstruction of neurites and synapses , but is not yet feasible for volumes larger than 1% of the mouse brain—precluding the reconstruction of long-range axonal projections .", "The resolution and imaging speeds reported for alternative light- and electron-microscopy technologies are summarized in Table 3 . 10 . 7554/eLife . 10566 . 021Table 3 . Comparison with alternative technologies for whole-brain imaging . DOI: http://dx . doi . org/10 . 7554/eLife . 10566 . 021MethodLateral resolution ( L; µm ) Axial resolution ( A; µm ) L × L × A ( µm3 ) Speed ( × 106 µm3/s ) This study0 . 451 . 330 . 261 . 6Selective plane illumination microscopy*0 . 657 . 303 . 10160Knife-edge scanning microscopy/ micro-optical sectioning tomography ( Li et al . , 2010 ) 0 . 711 . 00 . 501 . 0Transmission electron microscopy with camera array ( Bock et al . , 2011 ) 0 . 0040 . 0457 . 2 x 10-75 . 6 x 10-6Serial block-face electron microscopy ( Helmstaedter et al . , 2013 ) 0 . 0165>0 . 025>6 . 8 x 10-61 . 1 x 10-6*Ideal resolution in fully cleared whole mouse brain ( P . Keller , personal communication ) .", "Comparison of resolution and imaging speed with other imaging modalities .", "Resolution values represent full width at half maximum except for in transmission electron microscopy with camera array and serial block-face electron microscopy , where pixel sizes are reported .", "Sparse neuronal labeling is a fundamental requirement for the reconstruction of axon collaterals using light microscopy .", "Ambiguity is introduced when axons originating from different somata are juxtaposed with separations less than the resolving limit of the optical system ( Figure 5c ) .", "This challenge can be mitigated by imaging with the highest available resolution and imposing a high degree of labeling sparsity—but cannot be eliminated entirely .", "In the example dataset presented here , excessive labeling density was largely limited to the site of virus injection and therefore only precluded the extensive reconstruction of local fibers .", "Biological questions focused on local collaterals will require alternative labeling strategies that achieve greater separation between labeled somata to mitigate this limitation .", "Sparse stochastic genetic methods ( Rotolo et al . , 2008 ) and retrograde labeling ( Mazarakis et al . , 2001; Wickersham et al . , 2007 ) provide straightforward approaches for labeling well-separated somata .", "Reconstructing multiple neurons within a single brain provides a particularly rich description of axonal organization .", "The spatial overlap - or , alternatively , segregation - of collaterals of different neurons within a single target region can be directly assessed at the micron scale .", "Comparison of projections of multiple neurons within the same population provides a means to better understand if topological organization in source areas is preserved in downstream structures .", "In addition , the fine-scale spatial relationships between axon collaterals from different sources may indicate how disparate streams of information remain isolated or are integrated in target structures .", "Such comparisons are challenging or impossible in datasets in which single neurons are reconstructed in different samples .", "Currently whole brain imaging still requires significant time ( 8–10 days ) and resources ( many TB of storage space ) .", "Labeling techniques that take advantage of multiple , spectrally separable fluorescent proteins ( Cai et al . , 2013; Shaner et al . , 2005 ) and the targeting of several populations with non-overlapping projections in a single brain should permit labeling of hundreds or even thousands of neurons in each brain while maintaining sufficient sparsity for single neuron reconstruction ( Gala et al . , 2014 ) .", "In addition , the high degree of automation employed in the acquisition procedure provides the opportunity for further scalability by taking advantage of multiple imaging systems operating in parallel .", "Although the time required for manual tracing remains substantial , this constraint is likely to be greatly reduced as a result of recent developments in automation of neuronal reconstruction ( Gala et al . , 2014; Jain et al . , 2010; Peng et al . , 2014 ) and the high SNR of axon imaging achieved with our approach .", "Recent advances in imaging techniques , genetic targeting methods , and 'big data' tools have facilitated multiple , comprehensive large-scale efforts attempting to map neural connectivity .", "In model organisms , these projects have largely focused on mapping local connectivity using electron microscopy ( Bock et al . , 2011; Helmstaedter et al . , 2013 ) and the bulk projections of neural populations using light microscopy ( Mitra , 2014; Oh et al . , 2014; Zingg et al . , 2014 ) .", "In humans , atlases of gross physical and functional connectivity have been constructed from diffusion-tensor imaging and resting-state functional MRI datasets ( Mori et al . , 2009; van den Heuvel and Hulshoff Pol , 2010 ) .", "However , techniques for atlasing the brain-wide axonal collateralization of single mammalian neurons remain unavailable , even though single-cell reconstruction has long been the gold standard for establishing the structure of long-range projections .", "The approach described here for the efficient reconstruction of single-cell axon collateral maps represents a crucial step towards filling this gap in the available tools for mapping neural connectivity , and will contribute to the ongoing renaissance in large-scale neuroanatomy ." ], [ "We constructed a microscope for fast volumetric STP tomography .", "The mouse brain , approximately 0 . 5 cm3 in volume , requires 5 × 1012 voxels for a full representation at a voxel size of 0 . 1 µm3 .", "Acquiring this volume of data with conventional galvanometric scanning at a rate of 1 megavoxel/s would require about 4 months .", "We have developed a custom resonant scanning system capable of sustaining 16 megavoxels/s for up to 4 channels ( 128 MB/s data rate ) to reduce imaging time to approximately 1 week per sample .", "The scan system is composed of a resonant scanning mirror ( CRS-8kHz , Cambridge Technology , Bedford , MA ) and a 5-mm linear scanning mirror ( 6215H , Cambridge Technology ) conjugated with a scan telescope comprised of two custom scan objectives ( Special Optics , Wharton , NJ ) to ensure flat scanning across the available scan field .", "To avoid thermally damaging the sample at the edge of the resonant mirror scan field , an adjustable slit was placed at a conjugate image plane in the excitation path to block laser light in the appropriate area .", "A Pockels Cell ( 302RM , Conoptics , Danbury , CT ) was used to modulate laser power and provide fast shuttering between frames .", "In this study , all imaging was performed using a 40×/1 . 3 NA oil-immersion objective ( Objective Plan-Apochromat 40×/1 . 3 DIC , Carl Zeiss , Oberkochen , Germany ) .", "The emitted light was passed through a primary dichroic ( FF735-Di01-25×36 , Semrock , Rochester , NY ) onto photomultiplier tubes ( H7422P-40 , Hamamatsu Photonics , Hamamatsu , Japan ) using a custom detection head ( part of the Janelia MIMMS microscope design ) ( Flickinger et al . , 2010 ) .", "Two spectral channels were collected .", "eGFP emission was further filtered through a secondary dichroic ( FF01-750/SP-25 , Semrock ) and an emission filter ( FF03-525/50-30-D , Semrock ) .", "A second channel ( FF02-617/73-30-D , Semrock ) was collected for identification of red-labeled nuclei and/or spectral disambiguation of eGFP and broad-spectrum autofluorescence .", "High-fidelity synchronization of the digitizer and scan system ( Pockels cell , galvanometric scanner , and objective positioner ) with the resonant mirror scan period was accomplished by reflecting a probe laser ( 635 nm , S1FC635PM , ThorLabs , Newton , NJ ) from the resonant mirror onto a photodiode ( PDA100A , Thorlabs ) .", "The photodiode was positioned 1 m away at one edge of the arc swept out by the resonant mirror .", "The voltage pulse generated by the incident probe laser was converted into a square wave using a custom circuit .", "This circuit was designed so that the rise of the square wave corresponds with the peak of the pulse observed from the photodiode , and permits adjustment of the clock phase in 5 ns increments .", "Collected photons were converted to an analog photocurrent , which was then amplified and converted to an analog voltage using a custom transimpedance amplifier ( impulse response FWHM=25 ns ) , and digitized using a 12-bit high-speed digitizer at 125 Msamples/second ( ATS-9350 , AlazarTech , Pointe-Claire , Canada ) .", "A voltage bias was applied at the amplifier to take advantage of the digitizer’s full range .", "The digitizer generated records 16k samples wide at a rate of 8 kHz ( 256 MB/s/channel ) .", "These lines were distorted due to the spatially non-linear motion of the resonant mirror .", "Because of this , they were resampled using a cosine lookup table in a manner that is photoefficient ( discards no samples ) and avoids aliasing .", "Resampling was implemented on a GPU ( GTX 580 , NVidia , Santa Clara , CA ) in order to meet data throughput requirements .", "Scanning a large volume of tissue was achieved by serially scanning a collection of smaller 3D stacks ( tiles ) .", "Each of these tiles overlapped adjacent neighbors along each dimension , permitting post hoc registration .", "To address each tile , the sample was translated in three dimensions using a high-precision mechanical stage system ( XY: M-511 . DD , Z: M-501-DG , Controller: C843; Physike Instrumente , Karlsruhe , Germany ) .", "Fast scanning in the Z-direction was achieved using a piezo-electric motor to axially translate the objective ( P-725K . 103 and E-665 . CR , Physike Instrumente ) .", "The maximum depth of each tile - typically set to 250 μm—was limited by the working distance of the objective and the range of the objective piezo .", "Integrated tissue sectioning was implemented on a custom assembly built around the vibrating servo from a Leica 1200S ( Leica Microsystems , Wetzlar , Germany ) .", "Vibration was driven using an 86 Hz sine wave generated from a function generator and custom amplifier with vibration amplitude of approximately 1 mm .", "Tissue samples were positioned and fed across a stationary vibrating blade using the stage system at a velocity of 0 . 1 mm/sec .", "In this way , tissue sections >25 µm could be reliably removed from the tissue block once they were imaged .", "Computer aided design drawings and bill of materials are available at github . com/TeravoxelTwoPhotonTomography .", "Fully automated microscope operation was controlled by custom software ( available at github . com/TeravoxelTwoPhotonTomography ) .", "This software incorporates several features that ensure reliable operation spanning days to weeks .", "First , to guarantee an overlapping volume is reimaged after sectioning , the acquisition software applies a surface-finding procedure and adjusts the focal plane to ensure that tiles begin at the tissue surface .", "In this procedure , the surface is identified by searching for the top of the tissue by imaging autofluorescence in a subset of tiles with a coarse Z spacing around the expected surface position .", "Second , algorithms monitor the data quality of the incoming video stream to detect , for example , a photodetector fault .", "Third , to ensure that high data rates can be sustained over long acquisition times with high fidelity , data are locally cached on a solid-state hard drive ( 840 pro; Samsung ) and asynchronously transferred to a highly-redundant network file system .", "Fourth , in the event of a fault , operation is safely halted and may resume from the point at which the fault occurred .", "Volume imaging is completed in a fully automatic process .", "After the top plane of the sample is positioned under the objective , a region of interest is determined by probing tiles across a designated search area .", "A single autofluorescence image is acquired at the bottom of each tile and classified based on whether it intersects the sample with a preset intensity threshold .", "When a tile that intersects the sample is found , neighboring tiles are searched in such a way as to trace the perimeter of the tissue .", "Tiles inside the perimeter are marked for imaging without being explicitly probed for efficiency .", "Finally the region of interest is dilated by one tile to ensure that tiles only partially intersecting the tissue sample are also acquired .", "The search process is finished when all the tiles in the search area have been classified .", "The marked region of interest is then imaged by sequentially volume-scanning constituent tiles .", "After all the tiles within the marked region of interest are imaged , the vibratome is engaged and a programmed stage motion feeds the sample through the blade to remove the top layer of tissue .", "The sample is translated back under the objective such that the newly exposed tissue surface is aligned with the focal plane and the process is repeated until no further tiles are found to image .", "Data was acquired in the coronal plane with tile dimensions of 386 × 422 × 250 μm ( 1024 × 1536 × 251 pixels ) and an average dwell time of 61 ns/pixel .", "eGFP imaging was performed using a tunable ultrafast laser ( Chameleon Ultra II , Coherent , Santa Clara , CA ) at 920 nm using high excitation intensity ( 350–400 mW at the objective back aperture ) .", "Image registration was implemented using custom scripts in Matlab ( The MathWorks , Natick , MA ) .", "For laterally adjacent tiles , no registration was required; translating tiles using recorded stage positions was sufficient .", "For vertically adjacent tile pairs that spanned a sectioning plane , a set of corresponding features was determined using the descriptor-based image registration plugin in Fiji ( Preibisch et al . , 2009 ) .", "Because of the large number of tile pairs ( >15 , 000 per brain ) , this procedure was distributed across a high-performance computing cluster .", "Compute time was approximately 1 min per tile pair ( RAM 128 GB; 16 Intel E5-2680 CPUs ) .", "A set of common features , such as punctate auto-fluorescent bodies ( lipofuscin; Figure 3—figure supplement 1 ) ( Dowson and Harris , 1981; Terman and Brunk , 2004 ) , restricted to the putative overlap volume was successfully determined in approximately 2/3 of all tile pairs .", "Many of the remaining tile pairs either included little or no tissue ( e . g . around the perimeter of the sample ) , or they were from relatively featureless areas within the tissue sample ( e . g . ventricles and some white matter tracts ) .", "Next , point correspondences were aggregated across each tissue section and translated into the coordinate space defined by the stage system .", "From these point correspondences , a 3D displacement field was determined by fitting a cubic surface to the point cloud using the RegularizeData3d package for Matlab ( http://www . mathworks . com/matlabcentral/fileexchange/46223-regularizedata3d ) .", "Although only axially-adjacent tile pairs were registered , tile pairs could be displaced relative to one another in all three Cartesian coordinates , necessitating the determination of deformation in three dimensions .", "This 3D displacement field was parameterized laterally for each ( x , y ) position across the section plane ( Figure 3b ) .", "After the displacement fields between the current section and the sections above and below the current layer were determined , a 3D transform was computed to update the position of each tile within the full imaged volume .", "To solve for the transform , eight control points were determined for each tile at points offset slightly from the tile corners and located at the centroid of the overlap regions of neighboring tiles .", "Except in edge cases , there were seven overlapping tiles in the vicinity of each tile corner .", "These control points formed a regular grid across each section .", "Sampling the vector field at the centroid of these overlap regions is important for maintaining lateral continuity .", "We approximated the projection of these points from pixel space into the global coordinate space using a unique affine transform for each tile .", "Although useful for registration purposes , the presence of lipofuscin was sometimes undesirable during the tracing process even though these puncta were generally straightforward to disambiguate from labeled neurites .", "Due to the broad emission spectrum of these features , however , they could be computationally removed using standard linear spectral unmixing techniques .", "After this procedure , unmixed images - - computed from two-channel image tiles - contained only eGFP-labeled structures .", "Images in Figures 3–5 and Video 1 were unmixed according to U = G − kR , where U is the unmixed image , G is the green channel of the original image , and R is the red channel of the original image .", "k is an empirically-determined scale factor .", "For the purposes of viewing and annotating datasets , all of the tiles in each dataset ( input tiles ) were resampled into a common coordinate space according to the individual affine transforms determined during the registration procedure .", "This produced a set of axis-aligned non-overlapping image stacks ( output tiles ) spanning the imaged volume .", "Resampling was achieved by back-projecting each voxel in each output tile to one or more input tiles where the nearest voxel was sampled .", "To avoid aliasing , input tiles were pre-filtered with a Gaussian kernel ( sigma = 150 nm ) .", "In regions where two or more input tiles overlapped , the maximum intensity was used for the corresponding location in the output tile .", "The resampling task was implemented on a GPU , which ensured that execution time was dominated by the time required to read input data .", "Work was distributed output tile-wise over a GPU cluster ( 20 nodes with 7× GTX 580 1 . 5GB VRAM ) .", "Importantly , each node had parallel access to high-bandwidth network storage .", "Data were resampled to voxels 0 . 3 × 0 . 3 × 1 µm , and stored on disk along with hierarchically downsampled representations of the same volume for visualization at different spatial scales .", "In order to smoothly navigate and annotate neuronal structures across large volumes of rendered data , we used a custom plugin for the Janelia Workstation ( Murphy et al . , 2014 ) that displays and predictively pre-caches multiresolution data using a scheme similar to that employed for large imaging datasets in electron microscopy ( Anderson et al . , 2011; Cardona et al . , 2012; Saalfeld et al . , 2009 ) .", "The neuronal structures depicted in this study were reconstructed by manually placing control points approximately every 5–10 µm and were stored using the SWC file format .", "Manual reconstruction of neurons required approximately 10–30 min/mm and speed depended upon labeling density , signal intensity , and annotator experience .", "To archive datasets , raw data was compressed using the H . 264 format ( libavcodec; parameters: crf 18 , preset slow , tune film ) .", "Each color channel was compressed independently as a 16-bit grayscale volume .", "Compression of a full dataset was distributed , tile-wise , using a high-performance compute cluster .", "Compressed datasets were typically ~1 TB/channel in size .", "DMSO has a high refractive index ( 1 . 479 ) and is used extensively in mounting medium formulations for fluorescence microscopy .", "To improve the clearing properties of DMSO , we investigated solutions of DMSO containing a sugar or sugar alcohol , based on the established tissue-clearing properties of these molecules .", "We examined the solubility of several compounds: sucrose , d-fructose , d-mannitol , d-sorbitol , xylitol , maltitol , and meso-erythritol .", "We found that d-sorbitol was highly soluble in DMSO up to 1 g d-sorbitol per 1 ml DMSO ( 44% w/w ) .", "Although the saturated solution of d-sorbitol in DMSO was an effective tissue-clearing agent , this organic environment irreversibly decreased the fluorescence of eGFP in tissue slices .", "We therefore measured the fluorescence of purified eGFP as a function of DMSO concentrations ( in 10 mM HEPES , pH 7 . 3 ) with or without d-sorbitol ( Figure 2b ) .", "Surprisingly , eGFP fluorescence increased modestly at low concentrations of DMSO followed by a severe decrease at 70% v/v DMSO without d-sorbitol and at 80% v/v DMSO in solutions that included d-sorbitol .", "We then screened ternary mixtures of DMSO/H2O/d-sorbitol , to produce the clearing protocol described in Table 1 , which uses increasing amounts of d-sorbitol with a final concentration of 45% w/w d-sorbitol in a solvent made up of 60% v/v DMSO in 0 . 01 M phosphate buffer .", "Increasing the concentration of d-sorbitol to its solubility limit ( 63% w/w; Solution #6 in Table 1 ) further improved clearing , but evaporation and mechanical agitation initiated precipitation of d-sorbitol and/or gel formation during sectioning and imaging .", "Mice were deeply anesthetized with an overdose of isoflurane and transcardially perfused with PBS that included 20 U/ml heparin ( H3393; Sigma-Aldrich , St . Louis , MO ) followed by 4% paraformaldehyde in PBS .", "Brains were extracted immediately following perfusion and post-fixed for 4 hr at 4°C and washed 3× in 0 . 1 PBS for >1 hr each .", "Brains were immersed in CUBIC-1 ( Susaki et al . , 2014 ) for 3–7 days to remove lipids and subsequently washed 3× in PBS for >1 hr each .", "Nuclei of samples were optionally counter-stained with 10 µM NuclearID-Red solution ( ENZ-52406 , Enzo Life Sciences , Farmingdale , NY ) , washed , embedded in gelatin ( 12% w/v in PB ) and fixed in 4% paraformaldehyde for 12 hr .", "Cross-linking the tissue to the gelatin embedding medium enabled stable , high SNR imaging even when only a thin section of material remained .", "Embedded samples were subsequently cleared by immersion in solutions #1–5 ( Table 1 ) ( 1 day in each step ) .", "DMSO ( 472301 ) and d-sorbitol ( 85529 ) were obtained from Sigma-Aldrich .", "High-purity d-sorbitol was found to be necessary to ensure the absence of fluorescent contaminants .", "PB and PBS were used at 0 . 01 M and 0 . 1 M , respectively .", "High titer ( > 1012 GC/ml ) AAV 2/1 Syn-iCre and AAV 2/1 CAG-Flex-eGFP were obtained from the Janelia Research Campus Molecular Biology Core .", "The Cre virus was diluted 1:45 , 000 in sterile water and combined at a 1:1 ratio with the eGFP virus for injections into the motor cortex of C57/BL6 mice ( M/L +0 . 7; A/P +1 . 3; D/V −0 . 75 , −0 . 5 , −0 . 25 relative to bregma; 30 nl/depth ) .", "All experimental protocols were conducted according to the National Institutes of Health guidelines for animal research and were approved by the Institutional Animal Care and Use Committee at Howard Hughes Medical Institute Janelia Research Campus ( Protocol #14–115 ) ." ] ]

[ "The structure of axonal arbors controls how signals from individual neurons are routed within the mammalian brain .", "However , the arbors of very few long-range projection neurons have been reconstructed in their entirety , as axons with diameters as small as 100 nm arborize in target regions dispersed over many millimeters of tissue .", "We introduce a platform for high-resolution , three-dimensional fluorescence imaging of complete tissue volumes that enables the visualization and reconstruction of long-range axonal arbors .", "This platform relies on a high-speed two-photon microscope integrated with a tissue vibratome and a suite of computational tools for large-scale image data .", "We demonstrate the power of this approach by reconstructing the axonal arbors of multiple neurons in the motor cortex across a single mouse brain ." ]

[ "Nerve cells or neurons transmit electrical impulses to each other over long distances .", "These signals travel through highly branching nerve fibers called axons , which are about one hundred times thinner than a human hair , and can extend across the entire brain .", "Tracing the axon of a neuron from start to end can help to explain how individual neurons and brain areas communicate signals over long distances .", "A mouse brain contains approximately 70 million neurons , and tracing the axons of many neurons within a brain is a challenging problem .", "Tackling this problem requires a method for imaging entire brains in high enough detail to unambiguously resolve and follow axons from individual neurons across the brain .", "Economo , Clack et al . now demonstrate such a method for three-dimensional imaging of tissue samples as large as the whole mouse brain .", "This system is fully automated and works by first imaging a layer of tissue near the exposed surface of a sample , and then cutting off a slice of tissue that corresponds to the volume that has been imaged .", "These steps then repeat until the entire sample has been imaged; this takes about a week for a whole mouse brain and produces about 30 terabytes of images .", "Economo , Clack et al . ’s advance can uncover how neurons communicate over long distances with an unprecedented level of precision .", "The method can now be used to generate a comprehensive database of neurons and their long distance connections .", "Such a database would aid efforts to model the roles of neural circuits in the brain , and inform the design of experiments to study brain activity during particular behaviors ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "plant biology", "biochemistry and chemical biology" ]

[ [ "RNA silencing is a fundamental mechanism for regulating gene expression in diverse biological contexts in eukaryotes .", "The key silencing effector is a ribonucleoprotein complex ( RISC ) that is composed of AGOs and small RNAs ( sRNAs ) .", "sRNAs , including small interfering RNAs ( siRNAs ) and microRNAs ( miRNAs ) , are produced by Dicers or Dicer-like enzymes as duplexes from longer fully-complementary double-stranded ( ds ) RNAs , or from ds RNAs with imperfectly-folded hairpin structures ( Achkar et al . , 2016; Kim et al . , 2009; Sanei and Chen , 2015 ) .", "Subsequently , the sRNA duplexes are loaded into AGO proteins; and one strand ( siRNA passenger strand or miRNA* ) is ejected whereas the other ( siRNA guide strand or miRNA ) is retained in RISC .", "By using the retained sRNAs as guides , AGO proteins specify the correct target mRNAs and repress gene expression ( Chandradoss et al . , 2015; Salomon et al . , 2015 ) .", "For targets with partial complementarity , silencing is initially primed through translational repression and followed by mRNA destabilization ( Bazzini et al . , 2012; Djuranovic et al . , 2012 ) .", "Degradation of such an mRNA target is accomplished by a combination of deadenylation , decapping , and subsequently by 5'-to-3' exonucleolytic decay ( Jonas and Izaurralde , 2015 ) .", "For highly complementary targets , RISC activity generally results in 5’ and 3’ cleavage fragments ( Bartel , 2009 ) .", "Both 5’ and 3 cleavage products undergo various decay processes .", "In mammals , the 3’ cleavage fragments are canonically degraded in the 5’ to 3’ direction by an exonuclease 1 ( XRN1 ) .", "In Arabidopsis , the 3’ fragment is accumulated at a higher level in loss-of-function mutant of XRN4 compared with the amount in wild type ( Souret et al . , 2004 ) .", "Since Arabidopsis XRN4 is an ortholog of mammalian XRN1 , the 5’-to-3’ decay of RISC 3’ cleavage fragments appears to be implemented through an evolutionarily conserved mechanism .", "RISC 5’ cleavage fragments turn over rapidly reflected by the relative inability to detect them when compared with 3’ cleavage fragments .", "However , the RNA decay mechanism for 5’ cleavage fragments has remained unclear .", "Previous studies showed that 5’ cleavage fragments from miRNA-RISC activity are typically uridylated at their 3’ ends throughout eukaryotes; and that this nontemplated modification is a signature license for immediate decay ( Ren et al . , 2014; Shen and Goodman , 2004 ) .", "In human cells , the uridylation of the RISC 5’ cleavage products is fulfilled through the terminal uridylyl transferases ( TUTs ) ( Lim et al . , 2014 ) .", "The uridylation promotes decapping of RISC 5’ products , and subsequently 5’-to-3’ exonucleolytic decay by XRN1 ( Song and Kiledjian , 2007 ) .", "In Arabidopsis , HEN1 suppressor 1 ( HESO1 ) uridylates the 5’ fragments and stimulates their degradation , since a few 5’-cleavage fragments display modest overaccumulation in heso1 mutants ( Ren et al . , 2014 ) .", "Notably , HESO1 was initially recovered as a miRNA nucleotidyl transferase .", "HESO1 functions together with UTP:RNA uridylyl transferase one to promote miRNA degradation in absence of canonical miRNA methylation ( Ren et al . , 2012; Tu et al . , 2015; Wang et al . , 2015 ) .", "In Arabidopsis , different pathways might account for RNA decay of RISC 5’ cleavage fragments .", "It has been shown that 5’ cleavage fragments accumulate in xrn4 mutant in Arabidopsis; and obviously XRN4 catalyzes 5’-to-3’ degradation of the fragments in a way similar to clearing RISC 3’ fragments .", "The RNA exosome also appears to contribute to degrade the 5’ cleavage fragments because their abundance is increased in the loss-of-function mutant of SKI2/3/8 , Arabidopsis orthologs of RNA exosome subunits in yeast ( Branscheid et al . , 2015 ) .", "In contrast , accumulation of RISC 5’ fragments is not enhanced in the mutants of additional subunits and even an Rrp6 ortholog , a core 3’-to-5’ exonuclease in the RNA exosome ( Branscheid et al . , 2015 ) .", "Therefore , whether these pathways represent the totality of mechanisms for degradation of uridylated 5’ cleavage fragments remains elusive .", "miRNA targets not only serve as substrates for RISC activity , but also influence RISC function and miRNA stability .", "A pioneering study in plants shows that target mimicry can act as an endogenous decoy for miRNAs , resulting in unproductive RISC and miRNA destabilization ( Franco-Zorrilla et al . , 2007 ) .", "Similar phenomena including miRNA sponges and competing endogenous mRNA ( ceRNAs ) that contain multiple miRNA-binding sites can modulate RISC activity and effectively inhibit miRNA function in animal systems ( Ebert and Sharp , 2010; Salmena et al . , 2011; Rubio-Somoza et al . , 2011 ) .", "In these organisms , miRNAs recognize target mRNAs through seed pairing ( Bartel , 2009 ) .", "Extensive pairing of 3’ miRNAs to target RNAs triggers miRNA trimming and tailing and an accompanying loss of mature miRNAs ( Ameres et al . , 2010; Xie et al . , 2012 ) .", "In human cells , highly complementary target RNAs destabilize the RISC and accelerate release of the guide RNA from AGO2 whereas partially complementary targets attenuate unloading of sRNAs and increase their stability ( De et al . , 2013 ) .", "Due to the presence of prevalent mismatches between the 3’ end of a guide RNA and its target in mammals , the majority of identified miRNA targets do not destabilize the interaction ( Bartel , 2009 ) .", "In contrast , plant miRNAs are nearly perfectly complementary to their target RNAs; and miRNA-RISC canonically functions to cleave target RNAs despite coherent presence of translation repression ( Li et al . , 2013 ) .", "However , whether RISC cleavage products regulate RISC function and miRNA abundance is unknown .", "Arabidopsis encodes nine functional AGOs , among which , AGO1 is a principal contributor to RNA silencing as it recruits most miRNAs , and a variety of siRNAs ( Mi et al . , 2008; Wang et al . , 2011 ) .", "AGO10 is the closest genetic paralog of AGO1 , but functions to specifically sequester a group of miRNAs , miR165/166 , to antagonize their silencing activity through AGO1 ( Zhu et al . , 2011; Zhou et al . , 2015; Yu et al . , 2017 ) .", "Here , we successfully used proteomics analysis to identify a novel AGO10-bound partner , RICE1 .", "We showed that RICE1 and its genetic paralog , RICE2 , interacted with both AGO10 and AGO1 , suggesting a common role in regulation of miRNA-RISC activity .", "RICE1 and RICE2 function as 3’-to-5’ exoribonucleases that specifically degraded ss RNAs .", "We found that overexpression of RICEs increased miRNA levels , whereas downregulation of RICE1 and RICE2 through artificial miRNA technology decreased miRNA accumulation .", "We also determined the crystal structure of RICE1 and observed that it formed a homohexameric ring with the active sites uniquely embedded at the interface between monomers .", "We further pinpointed the critical residues required for RICE1 catalytic activity and oligomerization .", "Whereas oligomerization-defective RICE1 variants were unstable in vivo , introduction of catalytically-inactive RICE1 significantly reduced miRNA levels in vivo , reminiscent of loss-of-function mutants of rice1 rice2 .", "Importantly , catalytically inactive RICE1 also caused the accumulation of longer uridylated 5’ cleavage RNA fragments from RISC activity .", "We propose that RICE1 and RICE2 act to clear 5’ uridylated fragments from RISC cleavage and to facilitate RISC recycling .", "Moreover , our study also suggests that the turnover of miRNA targets and miRNA accumulation might be related in vivo ." ], [ "To identify AGO10 cofactors , we generated stable A . thaliana transgenic suspension cell lines overexpressing Flag-4Myc-AGO10 to mimic plant stem cells where AGO10 is specifically expressed ( Figure 1A , B ) .", "Then , we purified the AGO10 complex through two-step immunoprecipitation and resolved the complexes on SDS-PAGE gradient gels ( Figure 1C ) .", "Distinct bands that were absent from the control IP with non-transgenic suspension cells were analyzed by mass spectrometry ( MS ) ( Figure 1C ) .", "We identified numerous cofactors , including HSP90 and cyclophilins , which are known to be required for RISC assembly ( Iki et al . , 2010 , 2012 ) , as well as novel factors ( Figure 1C; Figure 1—figure supplement 1A ) .", "The results of the AGO10 complex isolation and MS analysis were reproducible in three independent experiments ( Figure 1C ) .", "Here , we focus on RICE1 ( At3g11770 ) , which is annotated as a 23 kDa polynucleotidyl transferase and ribonuclease H-like superfamily protein in the TAIR database .", "RICE1 has a close paralog ( At5g06450 , RICE2 ) that was previously known as AtDECP , Arabidopsis DnaQ-like 3’-to-5’ exonuclease domain-containing protein ( Perry et al . , 2006; Smith et al . , 2013 ) .", "RICE1 and RICE2 share 67% sequence identity , but their functions have not been characterized . 10 . 7554/eLife . 24466 . 003Figure 1 . RICE1 is a novel RISC-bound cofactor in Arabidopsis .", "( A and B )", "Generation of transgenic calli and cell lines expressing 35S-Flag-4Myc-AGO10 .", "( C ) Gel-code blue stained SDS-PAGE of purified AGO10 complexes for proteomics analysis .", "( D–F )", "Specific RICE1-AGO interaction was confirmed in N . benthamiana ( N . bentha . ) by LCI ( D ) , co-localization ( E ) , and co-IP ( F ) assays .", "In ( D ) , the schemes of leaves show different combinations of infiltrated constructs that were fused either to N-terminal ( NLuc ) and C-terminal ( CLuc ) regions of luciferase .", "The LCI photographs on right showed the signals resulting from the protein-protein interaction .", "The red arrows indicate the infiltration positions .", "A combination of AGO1 and CMV 2b ( Zhang et al . , 2006b ) serves as a positive control .", "In ( E ) , RICE1-CFP and YFP-AGO proteins , when expressed alone in the N . bentha , were shown in top panels .", "The RICE1-CFP and YFP-AGOs , when co-expressed and detected in CFP and YFP channels separately , were shown in middle and bottom panels , respectively .", "Note: co-localization of RICE1-CFP and YFP-AGOs was observed predominantly in cytoplasm .", "In ( F ) , IP was done with an anti-Flag antibody .", "Western blot analyses were conducted using anti-Flag , or -HA antibodies .", "SE is a negative control .", "( G–I )", "Specific interaction between endogenous RICE1 and AGO1 in Arabidopsis was confirmed by SEC ( G ) and co-IP ( H and I ) assays .", "IP was performed with anti-AGO1 ( H ) or anti-Flag ( I ) and western blot analyses were detected with anti-AGO1 ( or -Flag ) , -RICE1 and -actin antibodies .", "Actin is a negative control .", "In ( I ) , the crude extract was applied with ( + ) or without not ( − ) 50 μg/ml RNase A before co-IP . DOI: http://dx . doi . org/10 . 7554/eLife . 24466 . 00310 . 7554/eLife . 24466 . 004Figure 1—figure supplement 1 . Identification and experimental confirmation of RICEs as novel Arabidopsis RISC-bound cofactors .", "( A ) Western blot analysis of transgenic Arabidopsis calli expressing Flag-4Myc-tagged AGO10 using an anti-Myc antibody .", "( B ) Peptides recovered from proteomics analysis of AGO10 complex .", "( C ) Cytoplasmic distribution of RICE1 and RICE2 in various tissues of Arabidopsis .", "Microscopic images were taken of stably transgenic Arabidopsis plants expressing 35S-RICE1 ( or −2 ) -CFP at the different growth stages .", "( D ) Schematic diagram of full-length and truncated forms of AGO1 and AGO10 .", "The numbers refer to the amino acid residues in AGO proteins .", "Locations of the N-terminal ( including the conserved PFAM domain ArgoN and DUF1785 ) , PAZ , MID , and PIWI domains are shown .", "( E and F )", "In vitro pull-down assays of MBP-tagged target proteins with the indicated His-tagged bait proteins using nickel bound nitrilotriacetate ( Ni-NTA ) resins .", "( E ) Input of bait proteins ( His-SUMO and His-SUMO-RICEs ) and target proteins ( MBP-tagged full-length or truncated AGOs ) .", "Coomassie brilliant blue R250 staining of the proteins shows their mobility .", "The major bands representing the target proteins are indicated with arrowheads .", "( F ) Output of in vitro pull-down assays of MBP-tagged target proteins .", "Western blots were analyzed with a monoclonal anti-MBP antibody .", "All His-SUMO-tagged bait proteins and MBP-tagged target proteins were purified from E . coli using nitrilotriacetate ( Ni-NTA ) and amylose resins , respectively .", "In all assays , 2 μg of target proteins were pulled down with the indicated bait proteins ( 2 μg each ) using Ni-NTA resins . DOI: http://dx . doi . org/10 . 7554/eLife . 24466 . 004 To examine whether RICE1 is a bona fide partner of AGO10 , we first carried out a split luciferase complementation assay ( LCI ) ( Zhang et al . , 2011 ) .", "In our LCI assays , both AGO10 and AGO1 displayed LUC complementation with RICE1 ( Figure 1D ) , as did AGO1 with the positive control , Cucumber mosaic virus-encoded 2b protein ( CMV 2b ) ( Zhang et al . , 2006b ) , suggesting that RICE1 may be a common factor in various RISCs ( Figure 1D ) .", "Next , using confocal microscopy , we observed that both AGO1 and AGO10 predominantly co-localized with RICE1 in the cytoplasm , suggesting a potential function of RICE1 in processing nucleic acids in the cytoplasm ( Figure 1E; Figure 1—figure supplement 1B ) .", "To further investigate RICE-AGO interactions , we performed co-immunoprecipitation ( co-IP ) experiments .", "To this end , we transiently expressed RICE1-3HA with either Flag-AGOs or -Serrate ( SE ) , a control protein involved in the miRNA pathway ( Manavella et al . , 2012 ) , by agroinfiltration in Nicotiana benthamiana ( N . bentha ) .", "We conducted an IP with anti-Flag antibody and detected co-immunoprecipitates with anti-HA antibody .", "RICE1-3HA was indeed co-immunoprecipitated with both AGO1 and AGO10 , but not with the control protein ( Figure 1F ) .", "We next conducted size exclusion chromatography ( SEC ) assays of Arabidopsis extracts in both wild type and 35S-RICE1 transgenic plants .", "Western blot with antibodies against endogenous AGO1 and RICE1 , respectively , indicated that a fraction of RICE1 protein was co-eluted with AGO1 ( Figure 1G ) .", "Additional IP experiments with the same extracts demonstrated the consistent coexistence of the two proteins in vivo ( Figure 1H ) .", "Similar co-IP results were also obtained in PAGO1-Flag-AGO1 complementation line .", "Furthermore , RNase A treatment in the co-IP process did not abolish the interaction between RICE1 and AGO1 , indicating that their interactions were RNA-independent ( Figure 1I ) .", "Finally , in vitro pull-down assays showed that 6His-SUMO-RICE1 and -RICE2 , but not 6His-SUMO , specifically and directly interacted with maltose-binding protein ( MBP ) -AGO1 and -AGO10 , but not control protein MBP , ( Figure 1—figure supplement 1D–F , lanes #1 , 9 and 10 ) .", "Moreover , RICEs interacted with several fragments of AGO10 proteins – AGO10 NC ( 127-336 ) , AGO10 PAZ ( 337-474 ) , and AGO10 PIWI ( 625-988 ) – with an apparently stronger affinity to the PAZ domain ( Figure 1—figure supplement 1F , lanes # 3–8 ) .", "In contrast , neither of the variable parts of AGO10 or AGO1 N-terminal domains could be pulled down by RICE proteins ( Figure 1—figure supplement 1F , lanes #2 and 11 ) .", "Collectively , these experiments demonstrated that RICEs are indeed novel AGO1/10-interacting proteins in plants .", "Computational analysis predicted that RICEs might function as RNase H proteins ( from TAIR website ) or DnaQ-like nucleases ( Smith et al . , 2013 ) .", "To characterize the biochemical function of RICEs , we prepared RICE proteins from E . coli through Ni-NTA affinity purification followed by SEC ( Figure 2A; Figure 2—figure supplement 1 ) .", "The major peaks in the SEC profiles of RICE1 and RICE2 corresponded to molecular weights of 130 and 180kD , respectively ( Figure 2—figure supplement 1B , C ) ; that are approximately six times the monomer size , indicating that RICE1 and RICE2 most likely form oligomers in solution .", "To identify bona fide substrates for RICE1 , we tested RICE1 enzymatic activity with 5’ 32P-labelled ss 21-nucleotide ( nt ) RNA or DNA using various reaction conditions , including the one described for its human structural homolog , Werner Syndrome Helicase exonuclease ( WRN-exo ) ( Perry et al . , 2006 ) .", "Our results show that RICE1 , in contrast to the control protein , human Sting protein ( hSting ) ( Shu et al . , 2012 ) , which underwent the same purification process , degraded the ss RNA , but not ss DNA , into shorter fragments , indicating that RICE1 principally acts as an RNase and not a DNase ( Figure 2B ) .", "Next , we performed cleavage assays of RICE1 on ds RNA substrates .", "To make the ds RNA substrate , miR166 was 5’ labeled and annealed to cold miR166* .", "Enzymatic assays showed that RICE1 readily degraded miR166 but not the miR166/* duplex , indicating that RICE1 , unlike the worm exonuclease Eri-1 that degrades siRNA duplexes ( Kennedy et al . , 2004 ) , specifically degrades ss RNAs ( Figure 2C ) . 10 . 7554/eLife . 24466 . 005Figure 2 . RICEs are 3’-to-5’ exoribonucleases preferring ss RNA .", "( A ) Coomassie brilliant blue staining of purified RICEs .", "hSting protein serves as a control throughout experiments .", "( B ) RICE1 favored ss RNA over ss DNA as a substrate .", "( C ) RICE1 did not degrade ds RNA .", "miR166/* , the 32P-labeled miR166 was annealed with unlabeled miR166* .", "( D ) RICE1 did not degrade nicked but not released miRNA/* .", "Top panel shows positions of labeled nucleotides ( * ) and schematic diagram of nicked but still annealed miRNA/* .", "The nucleotides in red are the 5’ 9-nt fragment of miR166* and the nucleotides in blue are the 3’ 12-nt fragment of miR166* .", "( E ) RICE1 was a 3’-to-5’ ribonuclease .", "Released single nucleotide is shown with an arrowhead .", "( F ) RICE1 was an exoribonuclease .", "The circular substrates were generated by the self-ligation of 5’ labeled linear RNAs with T4 RNA ligase .", "( G ) RICE1 efficiently degraded ss sRNA but not long RNA with secondary structures .", "Partial mRNAs of PHV and At4g29770 ( 165 nt and 162 nt in length ) serves as long ss RNA substrates .", "( H ) RICE1 efficiently degraded both sRNA and long RNA homopolymer ( poly-As in 21- , 50- , 70- , and 100-nt length ) .", "( I ) RICE2 , similar to RICE1 , is a 3’-to-5’ exoribonuclease .", "All the substrates in the enzymatic assay were 5’ 32P-labeled except the ones in ( E ) as indicated . DOI: http://dx . doi . org/10 . 7554/eLife . 24466 . 00510 . 7554/eLife . 24466 . 006Figure 2—figure supplement 1 . Purification of RICE1 and RICE2 proteins .", "( A ) Gel filtration of protein standards using Hiload 16/600 Superdex 75 .", "( B ) Size exclusion chromatogram shows that RICE1 forms hexamer in vitro .", "( C ) Size exclusion chromatogram of RICE2 shows that RICE2 oligomerizes in vitro . DOI: http://dx . doi . org/10 . 7554/eLife . 24466 . 006 In humans and fungi , following the loading of siRNA duplexes , the passenger strand is nicked by catalytically-active AGO proteins and is subsequently cleared via nucleases such as C3PO and QIP ( Liu et al . , 2009; Maiti et al . , 2007; Ye et al . , 2011 ) .", "To investigate whether RICE1 function resembles C3PO or QIP and removes the nicked passenger strand from sRNA duplexes , a 5’ 32P labeled 12-nt RNA fragment and cold 9-nt RNA fragment fully complementary to miR166 were annealed to cold miR166a ( Figure 2D ) to generate a nicked sRNA duplex .", "Similarly , an analogous nicked duplex was prepared by annealing 5’ 32P labeled 9-nt RNA fragment with cold 12-nt fragment and miR166a ( Figure 2D ) .", "While RICE1 cleaved the 5’ labeled 9-nt and 12-nt ss sRNAs effectively , the 9-nt and 12-nt RNA fragments in the duplex forms were resistant to RICE1 degradation ( Figure 2D ) .", "Thus , RICE1 , unlike previously characterized C3PO and QIP , did not degrade unreleased nicked ss RNAs .", "To distinguish whether RICE1 is a 5’-to-3’ or 3’-to-5’ ribonuclease , we repeated the RNase assays with the RNA substrates labeled on different ends .", "When a 5’ labeled substrate was used for the assay , truncated fragments of various sizes were observed; whereas when a 3' labeled ss RNA substrate was used , only a single one-nucleotide cleavage product was observed .", "This RNA substrate was more stable than the 5' labeled RNA and we infer that the 3' labeled phosphate inhibited the target degradation ( Figure 2E ) .", "These results indicate that RICE1 is a 3’-to-5’ ribonuclease that cleaves nucleotides off the RNA substrates progressively from the 3’ terminus .", "To further determine whether RICE1 is an exo- or endo-ribonuclease , a 5’-labeled 21nt ss RNA was circularized by T4 RNA ligase .", "While the circularized ss RNA was effectively degraded by RNase A , this RNA was resistant to RICE1 activity , suggesting that RICE1 is an exoribonuclease ( Figure 2F ) .", "We next tested the length requirement of RNA substrates for RICE1 .", "ss RNAs , with lengths ranging from 9 to 165-nt , were 5’ 32P labeled and subjected to RICE1 treatment .", "We observed that ss RNAs with lengths less than 40-nt were readily degraded by RICE1 while the RNAs that were 50-nt or longer were less vulnerable to the exonucleolytic activity of RICE1 ( Figure 2G ) .", "However , considering that double-strand secondary structures are more likely to occur in the long ss RNAs than short ones , we tested RICE1 enzyme activity against RNA homopolymers of different lengths .", "Consequently , we noticed that RICE1 enzyme activities on these 21-nt to 100-nt substrates were comparable ( Figure 2H ) , suggesting that RICE1 favored both small ss RNAs and long ss RNAs without secondary structure .", "We repeated the nuclease activity assay with RICE2 and observed that RICE2 functioned essentially identical to RICE1 ( Figure 2I; data not shown ) .", "Taken together , both RICE1 and RICE2 are exoribonucleases specifically targeting ss RNAs .", "To study the in vivo function of RICE1 and RICE2 genes , we examined their expression profiles using transgenic plants expressing a GUS reporter gene under the native RICE promoters .", "RICE1 promoter was ubiquitously active in various tissues and organs throughout development including germinating seeds , cotyledons , leaves and roots of young seedlings and adult plants , stems and inflorescence ( Figure 3—figure supplement 1 ) .", "In contrast , RICE2 promoter activity was more restricted to specific niches including shoot and root apical meristems , trichomes , and vascular veins , among other tissues ( Figure 3—figure supplement 1 ) .", "The partially overlapping expression domains of these two genes suggest their functional redundancy but also possible specificity for different biological processes .", "What are the functions for RICE1 and RICE2 in vivo ?", "Given that RICEs are housed in AGO1 and AGO10-centered complexes and that the proteins are 3’-to-5’ exoribonucleases specifically degrading ss RNAs , our initial hypothesis was that RICEs might function to degrade AGO-bound miRNAs in a manner similar to SDN1 ( Ramachandran and Chen , 2008 ) .", "To test this , we examined miRNA accumulation in transgenic plants with altered levels of RICE1 and RICE2 in vivo .", "First , we generated transgenic plants expressing artificial miRNA constructs specifically targeting RICE1 ( amiR-RICE1 ) , RICE2 ( amiR-RICE2 ) , or both ( amiR-RICEs ) ( Figure 3A; Figure 3—figure supplement 2A ) .", "Consistent with GUS-reporter line results , RICE1 transcripts were much more abundant than RICE2 transcripts ( Figure 3B; Figure 3—figure supplement 2B ) .", "Analyses of sRNA and RNA blots showed that artificial miRNAs targeting individual or both genes were efficiently processed and correspondingly , levels of RICE1 and/or RICE2 transcripts decreased approximately ~70–90% compared to wild type plants ( Figure 3B; Figure 3—figure supplement 2A–B ) .", "Knockdown of just RICE1 or RICE2 in the amiR-RICE1 and amiR-RICE2 transgenic lines seemed to have little effect on the levels of tested miRNAs ( Figure 3C–D; Figure 3—figure supplement 2C ) .", "However , concurrent knockdown of both RICE1 and RICE2 consistently decreased the abundance of tested miRNAs relative to the control plants or the sibling lines with barely detectable artificial miRNAs ( Figure 3C–D; Figure 3—figure supplement 2C ) .", "Correspondingly , mRNA transcripts targeted by the assayed miRNAs were upregulated ( Figure 3E ) . 10 . 7554/eLife . 24466 . 007Figure 3 . RICE1 and RICE2 are positive regulators for miRNA accumulation in vivo .", "( A ) Sequence alignment of artificial miRNAs and their targets .", "The nucleotides in red are perfect matched region between RICE1 and RICE2 target sites , while the nucleotides in blue are mismatched region between RICE1 and RICE2 target sites .", "( B ) Generation of artificial miRNA transgenic lines targeting specific RICE1 , RICE2 , or both transcripts .", "( C–E )", "Concurrent knockdown of RICE1 and RICE2 consistently reduced miRNA abundance ( C and D ) and upregulated the targeted transcripts ( E ) .", "( F ) Ectopic expression of RICE1-3HA in wild-type Ler significantly enhanced accumulation of tested miRNAs .", "sRNA and RNA gel blot analyses were done with the indicated 32P-labelled probes .", "U6 and total RNAs stained with methylene blue serves as loading controls .", "For ( C ) , additional two biological replicates were shown in Figure 3—figure supplement 2C .", "Quantification of three biological replicates was statistically analyzed and the significant difference ( Student t-test , p<0 . 05 ) shown by the asterisk ( * ) in ( D ) .", "Also see in Supplementary file 1A .", "Western blot analyses were performed with anti-HA antibody and Rubisco stained with Ponceau S serves a loading control .", "The amount of miRNAs or mRNAs in wild-type plants or transgenic plants harboring empty vectors was arbitrarily designated as 1 . 0 , and the relative amount in the other lines was normalized to that of control plants . DOI: http://dx . doi . org/10 . 7554/eLife . 24466 . 00710 . 7554/eLife . 24466 . 008Figure 3—figure supplement 1 . RICE1 is ubiquitously expressed in various organs and tissues throughout different developmental stages whereas RICE2 expression is more restricted and less abundant . Note: the expression patterns of RICE1 and RICE2 are inferred from GUS gene regulated through their native promoters .", "Expression of RICE1 and RICE2 in germinating seeds ( A ) , 3 day seedlings ( B ) , 10 day seedlings ( C and D ) , mature leaves ( E ) , stem ( F ) , inflorescence ( G ) , flowers ( H ) , and matured siliques ( I ) .", "Black scale bars represent 0 . 5 cm in length . DOI: http://dx . doi . org/10 . 7554/eLife . 24466 . 00810 . 7554/eLife . 24466 . 009Figure 3—figure supplement 2 . RICEs positively regulate miRNA accumulation in vivo .", "( A ) miR166 abundance in additional transgenic lines expressing artificial miRNAs specifically targeting RICE1 , RICE2 or both .", "sRNA blot analyses were done with the indicated 32P-labelled probe .", "U6 serves as a loading control .", "( B ) RNA gel blot analyses of RICE1 and RICE2 transcripts in the transgenic lines expressing various artificial miRNAs .", "Methylene blue staining of total RNAs in blots shows loading controls .", "( C ) Additional biological replicates for the miRNA abundance in artificial miRNA knockdown lines targeting RICE1 , RICE2 or both .", "( D ) Size exclusion chromatogram of purified RICE1-3HA/-CFP proteins show that the proteins form oligomers in vitro .", "( E ) Coomassie brilliant blue R250 staining of SDS-PAGE shows purity and mobility of RICE1-3HA/-CFP .", "( F ) Both RICE1-3HA and RICE1-CFP are catalytically-active exoribonucleases in vitro .", "( G and H )", "Ectopic expression of RICE1- or RICE2-CFP in wild-type Ler ( G ) and ago10pnh-2 background ( H ) significantly enhanced accumulation of tested miRNAs .", "U6 serves as a loading control .", "Western blot analyses were performed with anti-GFP antibody and Rubisco stained with Ponceau S shows the loading control .", "The numbers listed on the bottom of panels represented the relative ratios of miRNAs in each line compared with that of wild-type plants or transgenic plants harboring empty vectors where the ratio was arbitrarily assigned a value of 1 . 0 after normalization with the loading control . DOI: http://dx . doi . org/10 . 7554/eLife . 24466 . 009 In parallel , we produced transgenic plants constitutively expressing RICE1 and RICE2 tagged with 3 HA at the C-termini .", "SEC and in vitro enzymatic assays showed that purified HA tagged RICE1 proteins had biochemical features identical to wild type RICE1 protein , suggesting that these tagged proteins are able to recapitulate functions of RICE1 in vivo ( Figure 3—figure supplement 2D–F ) .", "RNA blot analysis showed that overexpression of RICE1 or RICE2 increased the levels of all tested miRNAs compared to the control plants transformed with empty vectors or sibling plants with undetectable accumulation of tagged RICE1 and RICE2 ( Figure 3F ) .", "We repeated these experiments with transgenic plants expressing CFP tagged RICEs in the wild type or ago10pnh-2 mutant background and obtained similar results ( Figure 3—figure supplement 2G , H ) .", "Given that the accumulation of free miRNAs outside RISC in Arabidopsis is negligible , the levels of miRNAs detected here are proposed to represent the accumulation of miRNAs in the mature RISC in vivo ( Wang et al . , 2011 ) .", "Thus , RICEs , different from SDN1 , function as positive regulators for miRNA accumulation in RISC .", "To elucidate the catalytic mechanism of RICE1 , we have determined the crystal structure of RICE1 at 2 . 9 Å resolution ( Supplementary file 2 ) .", "RICE1 crystallizes in space group I4122 with three molecules in the asymmetric unit .", "RICE1 molecules form two donut-shaped hexamers that interact with each other in a face-to-face manner in the crystal lattice ( Figure 4—figure supplement 1A ) .", "A similar hexamer was also observed in the crystal structure of RICE2 crystallized in a different unit cell ( Smith et al . , 2013 ) .", "RICE1 exhibits an α/β fold with a central seven-stranded β-sheet sandwiched between eight α-helices ( Figure 4A , B ) .", "Structural analysis of RICE1 using the DALI server ( Holm and Rosenström , 2010 ) revealed that RICE1 exhibits structural homology to the DnaQ family of 3’-to-5’ exonucleases including WRN-exo ( r . m . s . d 2 . 4 Å to 2E61 ) ( Perry et al . , 2006 ) and recently reported RICE2 ( r . m . s . d 0 . 7 ~ 1 . 2 Å ) ( Smith et al . , 2013 ) ( Figure 4—figure supplement 1B , C ) .", "RICE1 also shows considerable structural similarity to ribonuclease D ( r . m . s . d 2 . 7 Å to 1YT3 ) ( Zuo et al . , 2005 ) . 10 . 7554/eLife . 24466 . 010Figure 4 . Crystal structure of RICE1 . ( A ) The amino acid sequence and secondary structure of RICE1 .", "( B–D )", "Structures of RICE1 monomer and hexamer .", "In ( B ) α-helices , β-strands , and loops are colored in cyan , green , and pink , respectively .", "In ( A and B ) , residues ( D52 , Y54 , D114 and E187 ) in the active site are highlighted in blue .", "In ( C ) , each monomer is shown in different colors .", "Active site residues are shown by the sphere representations , with active site residues D52 of each monomer labeled .", "In ( D ) , the active sites of RICE1 are located at the interface between RICE monomers .", "Key residues ( D52 , Y54 , D114 and E187 ) in the active site are shown as ball-and-stick models .", "Salt bridges are denoted by blue dotted lines .", "( E ) Overlay of active sites residues of RICE1 and WRN exonuclease .", "( F ) Enzyme assays showed that EDTA did not affect the catalytic activity of RICE1 . DOI: http://dx . doi . org/10 . 7554/eLife . 24466 . 01010 . 7554/eLife . 24466 . 011Figure 4—figure supplement 1 . Structural features of RICE1 . ( A ) Double donut assembly of RICE1 hexamers in the crystal .", "( B ) Sequence alignment with RICE1 and its Arabidopsis paralog , RICE2 , as well as structural homolog , WRN-exo .", "( C ) Overlaid structures of RICE1 and WRN-exo . DOI: http://dx . doi . org/10 . 7554/eLife . 24466 . 011 The DnaQ-like exonuclease superfamily members have four conserved acidic residues ( DEDD ) in their active sites ( Perry et al . , 2006 ) .", "The well-conserved WRN-exo active site is located within a large cavity on one face of the molecule .", "A comparison of RICE1 with WRN-exo and other members of the DnaQ family revealed that the DEDD motif is not conserved in the active site of RICE1 ( Figure 4—figure supplement 1B ) .", "Instead , the glutamate and the last aspartate residues in the DEDD motif are replaced by Tyr54 and Glu187 ( Figure 4A , C–E ) .", "In addition , the highly conserved tyrosine residue ( Tyr212 ) near the active site of WRN-exo is replaced by Ala183 in RICE1 .", "To confirm that these residues form the active site of RICE1 , we expressed three RICE1 mutants , D52A , Y54S and E187A , and analyzed their oligomerization and catalytic activity .", "The SEC profiles of these mutants were identical to that of the wild type enzyme , indicating that these mutations do not affect the oligomerization of RICE1 ( Figure 5A ) .", "However , enzymatic activity assays showed that these mutations eliminated or severely reduced the exoribonuclease activity of RICE1 ( Figure 5B–C ) , indicating that these residues indeed form the active site of RICE1 and are indispensable for its exonuclease activity . 10 . 7554/eLife . 24466 . 012Figure 5 . Mutations in catalytic sites impair RICE1 function in vivo .", "( A ) Chromatograph of representative RICE1 variants with mutations in catalytic sites .", "( B ) SDS-PAGE of purified RICE1 mutants and control protein hSting .", "( C ) Representative RICE1 mutants were compromised exoribonucleases compared to wild type ( WT ) .", "( D ) Phenotypic anomaly of transgenic plants ectopically expressing catalytically-inactive RICE1 variants in different stages .", "Note: the severe sterility of 35S-3HA-RICE D52A transgenic plants .", "( E–G )", "Ectopic expression of RICE1 variants caused dominant-negative effect on miRNA accumulation in vivo .", "In ( E ) and ( G ) , sRNA blot analyses were done with the indicated 32P-labelled probes .", "The amount of miRNAs in wild-type plants was arbitrarily designated as 1 . 0 , and the relative amount in the other lines was normalized to that of control plants .", "In ( F ) , quantification of three biological replicates was statistically analyzed and the significant difference ( Student t-test , p<0 . 05 ) shown by the asterisk ( * ) .", "Additional biological replicates were shown in Figure 5—figure supplement 1C and Supplementary file 1B .", "Western blot analyses were performed with anti-HA or anti-actin antibodies . DOI: http://dx . doi . org/10 . 7554/eLife . 24466 . 01210 . 7554/eLife . 24466 . 013Figure 5—figure supplement 1 . Catalytic inactive RICE1 impairs the accumulation of miRNAs in vivo .", "( A and B )", "Western blot analyses of transgenic plants expressing RICE1 D52A and Y54S using anti-HA antibody .", "( C and D )", "Ectopic expression of HA-tagged ( C ) and untagged ( D ) RICE1 D52A caused dominant-negative effect on miRNA accumulation in vivo .", "sRNA blot analyses were done with the indicated 32P-labelled probes .", "The amount of miRNAs in wild-type plants was arbitrarily designated as 1 . 0 , and the relative amount in the other lines was normalized to that of control plants .", "The right column chart showed the quantification of sRNA blots and asterisks ( * ) indicated statistically significant differences between wild type and transgenic plants ( Student t-test , p<0 . 05 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 24466 . 013 Another structural feature of the DnaQ-like exonuclease is the involvement of two metal ions at the active sites ( Perry et al . , 2006; Zuo and Deutscher , 2001 ) .", "Notably , no obvious electron density corresponding to the active site Mg2+ ion of the DnaQ-like nucleases was observed near the active site of RICE1 ( Figure 4E ) , despite the inclusion of 5 mM of MgSO4 in the protein sample for crystallization .", "Asp52 and Asp114 , two residues that otherwise bind to divalent cations , interact with Lys102 and Arg127 of a neighboring RICE1 molecule .", "In line with this observation , EDTA did not affect the enzymatic activity of RICE1 ( Figure 4F ) .", "To ask whether RICE1 catalytic activity is critical for the miRNA accumulation in vivo , we generated transgenic plants constitutively expressing catalytically-inactive RICE1 variants ( D52A and Y54S ) .", "Although RICE1 D52A and Y54S mutations abolished the catalytic activity , they did not interfere with the RICE1-AGO10 interaction in the tobacco transient assay ( data not shown ) .", "We obtained numerous positive transgenic plants expressing 35S-3HA-RICE1 D52A and 35S-3HA-RICE1 Y54S as determined by western blot analysis ( Figure 5—figure supplement 1A , B ) .", "Overexpression of 35S-HA3-RICE1 D52A and 35S-HA3-RICE1 Y54S caused pleiotropic developmental defects including spoon-shaped cotyledons , twisted true leaves and infertile flowers ( Figure 5D ) , phenocopying hypomorphic ago1 mutants and transgenic plants expressing viral suppressors that disable the miRNA pathway in plants ( Zhang et al . , 2006b ) .", "Western blot analyses showed that the phenotypic severity was proportional to the expression levels of RICE1 mutants ( Figure 5E , G ) .", "Together , all these results indicated that RICEs play important roles in plant growth and development .", "Next , we measured miRNA expression in the transgenic plants .", "In a sharp contrast with RICE1 overexpression plants , over-accumulation of the catalytically-inactive forms of RICE1 D52A and RICE1 Y54S significantly decreased the levels of all tested miRNAs and trans-acting siRNA compared to Ler wild type and sibling transgenic plants with barely detectable transgene expression ( Figure 5E–G; Figure 5—figure supplement 1C , D; Figure 7B ) .", "These results were reminiscent of the observation in rice1 rice2 knockdown lines ( Figure 3C , D; Figure 5—figure supplement 1C; Figure 7—figure supplement 1D ) , indicating that catalytically-inactive mutations act in a dominant negative manner on miRNA accumulation in vivo .", "Wild type RICE1 elutes predominantly as a hexamer in the SEC assay .", "Moreover , the active site of RICE1 , unlike other DnaQ family members ( Perry et al . , 2006 ) , is located at the interface between RICE1 subunits within the hexameric ring ( Figures 4C and 6A ) .", "Thus , the oligomerization of RICE1 seems critical for its catalytic activity .", "Structural analysis revealed extensive intermolecular interactions between RICE1 molecules within the hexameric ring .", "RICE1 molecules interact with each other mainly through electrostatic interactions ( Figure 6A–C ) .", "Five salt bridges , between residues Arg47 and Asp166 , Lys102 and Asp52 , Lys102 and Asp114 , Lys119 and Glu113 , Arg127 and Asp114 , are observed at the interface between RICE1 subunits ( Figure 6B ) .", "Hydrogen bonds and van der Waals interactions make additional contributions to the interactions between RICE1 molecules .", "The total buried surface area between two neighboring RICE1 molecules is ~2800 Å2 .", "The two binding surfaces show both charge and shape complementarities to each other ( Figure 6B ) .", "In summary , the structural analysis revealed that RICE1 exhibits a DnaQ family exonuclease fold with an altered active site that is located at the interface between RICE1 molecules . 10 . 7554/eLife . 24466 . 014Figure 6 . Mutations at the dimer interface impair RICE1 function and protein stability in vivo .", "( A ) Interface between RICE1 monomers .", "Amino acid residues involved in the interaction are shown in ball-and-stick models .", "( B and C )", "The electrostatic potential at the surfaces of RICE1 monomers and hexamer .", "Positively and negatively charged surfaces are colored blue and red , respectively .", "In ( C ) , the active site of RICE1 ( indicated by the yellow asterisk ) is located at the bottom of a deep groove between RICE1 monomers .", "( D and E )", "SEC assays of RICE1 R47E and D114A mutants defective in oligomerization .", "Note: SEC assay was conducted using Hiload 16/600 Superdex 200 in ( E ) instead of routinely used Superdex 75 .", "( F ) SDS-PAGE of purified wild type , mutated RICE1 , and control protein hSting .", "( G ) Oligomerization is essential for RICE1 catalytic activity .", "( H ) RICE1 R47E mutant proteins failing in oligomerization are unstable in vivo .", "( J ) RICE1 D114A , unlike D52A , Y54S , and K119E mutants , was unstable and barely detectable in vivo .", "( K ) Ectopic expression of 3HA-RICE1 D114A had no significant effect on miRNA accumulation in vivo .", "Western blot analyses were performed with anti-HA antibody .", "sRNA blot analyses were done with the indicated 32P-labelled probes .", "The amount of miRNAs in wild-type plants was arbitrarily designated as 1 . 0 , and the relative amount in the other lines was normalized to that of control plants . DOI: http://dx . doi . org/10 . 7554/eLife . 24466 . 014 To examine how oligomerization affects the properties of RICE1 , we studied three RICE1 mutants in key residues at the RICE1 interface: R47E , K119E , and D114A .", "SEC showed that the K119E mutation did not affect RICE1 oligomerization ( data not shown ) .", "By contrast , the R47E mutant displayed a distinct chromatographic profile compared to wild-type RICE1 with the major peak corresponding to the monomeric RICE1 ( Figure 6D ) .", "Notably , the D114A mutant eluted as several peaks that correspond to the monomer , dimer and hexamer of RICE1 ( Figure 6E ) .", "These results indicated that residues Arg47 and Asp114 are critical , for the oligomerization of RICE1 .", "Importantly , nuclease assays showed that the enzymatic activity of the R47E mutant , but not the K119E mutant was abolished ( Figure 6F , G; data not shown ) , indicating the oligomerization of RICE1 is required for the nuclease activity , in line with the observation that the active site of RICE1 is located at the interface between RICE1 subunits .", "To examine how the oligomerization-compromised mutations affected the miRNA pathway , we also generated transgenic plants expressing 35S-3HA-RICE1 R47E and 35S-3HA-RICE1 D114A .", "Surprisingly , we seldom recovered positive transgenic plants by western blot screening of dozens of T1 transformants , although the transcript levels of these RICE1 variants were comparable with the ones extracted from the plants expressing 35S-3HA-RICE1 D52A/Y54S ( Figure 6H–J; data not shown ) .", "In line with this result , no obvious defects of development and miRNA accumulation were visible in any of RICE1 R47E and D114A transgenic lines ( Figure 6K; data not shown ) .", "Together , these results indicated that disruption of protein oligomerization led to protein instability .", "Because RICEs function as positive regulators for miRNA accumulation in vivo , miRNAs themselves are unlikely the direct targets of RICEs .", "An alternative hypothesis is that RICEs might clear the miRNA* strand and facilitate incorporation of miRNA strands into AGO proteins .", "Because how miRNA/miRNA* is exactly loaded into AGO protein remains unclear in plants , we hypothesized that if RICEs removed miRNA* after the incorporation of miRNA/* into AGO proteins , then a defect in RICE function might lead to enrichment of miRNA* in AGO proteins .", "To test this , we examined the miRNA accumulation in AGO1 protein in the lines overexpressing the dominant negative mutant RICE1 D52A .", "The accumulation of AGO1 was slightly increased in the mutant , likely due to its feedback regulation by miR168 ( Figure 7A; Figure 7—figure supplement 1A , B ) .", "However , AGO1 protein retained less miRNA in the dominant negative mutant , indicating that RICEs promote integrity of functional RISC .", "These results were reproducible and a similar pattern could also be observed in rice1 rice2 knockdown lines , although more modest relative to the dominant negative transgenic plants ( Figure 7—figure supplement 1A–C ) .", "Interestingly , no obvious enrichment of miRNA*s was detected in AGO1 immunoprecipitates ( Figure 7A; Figure 7—figure supplement 1A–C ) , suggesting that RICE1 might not clear miRNA*s that are embedded into RISC .", "On the other hand , we observed a coordinated decrease in the amount of miRNA and miRNA* in the transgenic plants ectopically expressing the dominant negative RICE1 mutants and the rice1 rice2 knockdown mutants by qRT-PCR ( Figure 7B; Figure 7—figure supplement 1D ) .", "To further examine the relative levels of miRNA and miRNA* on a global level , sRNA-seq was performed with wild type plants and the dominant negative RICE1 D52A mutants .", "The expression levels of ~40% of miRNAs in the dominant negative mutants were 1 . 5 fold lower than observed in the wild type , while only less than 5% were 1 . 5 fold higher than in the wild type ( Figure 7C; Supplementary file 1D ) .", "Concurrently , the significantly downregulated and upregulated miRNA*s in the dominant negative mutants were ~35% and 9% , respectively ( Figure 7D; Supplementary file 1E ) .", "Consistent with qRT-PCR results , there were also no significant changes in most of the miRNA to miRNA* ratios ( Figure 7E; Supplementary file 1F ) . 10 . 7554/eLife . 24466 . 015Figure 7 . Catalytically-inactive RICE1 extends uridylated 5’ cleavage fragments from RISC activity whereas decreases the abundance of RISC-harbored miRNA in vivo .", "( A ) Ectopic expression of catalytically-inactive RICE1 mutants impaired the accumulation of miRNAs in RISC .", "Western blot analyses were done with anti-AGO1 and anti-Actin antibodies .", "Actin serves as a loading control .", "sRNA blot analyses of sRNA recovered from AGO1 IP were conducted with 32P-labeled probe against miR166 .", "The ratio of miR166/AGO1 in wild type was arbitrarily designated as 1 . 0 , and the relative amount in each sample was normalized to that of control plants .", "Additional biological replicates were shown in Figure 7—figure supplement 1A , B .", "( B ) Concurrent reduction of miRNA and miRNA* abundance in the transgenic plants expressing dominant-negative RICE1 mutant .", "The relative level of miRNA or miRNA* was normalized to that in Ler plants where the amount was arbitrarily designated as 1 . 0 with ±SD from at least three experiments , asterisks ( * ) indicate statistically significant differences ( Student t-test , p<0 . 05 ) .", "See also in Supplementary file 1C .", "( C and D ) sRNA-seq analyses of miRNA or miRNA* expression in wild type and transgenic plants expressing dominant-negative RICE1 mutant .", "X and Y axes indicate the logarithm base two for the miRNA or miRNA* expression level in wild type and 35S-HA-RICE1 D52A transgenic plants , respectively .", "The labels in the bottom right of the chart indicated the categories of miRNA or miRNA*s: D52A/WT ≥1 . 5 , upregulated in 35S-HA-RICE1 D52A transgenic plants for more than 1 . 5 fold; WT/D52A ≥1 . 5 , downregulated in transgenic plants for more than 1 . 5 fold; Ratio <1 . 5 , the rest with fold changes less than 1 . 5 fold .", "The pie in the top left of the chart indicated the numbers of different categories of miRNA or miRNA*s .", "See also in Supplementary file 1D and 1E .", "( E ) Comparison of the ratio of miRNA vs miRNA* in wild type and transgenic plants expressing dominant-negative RICE1 mutant .", "The empty triangle indicates the logarithm base two for the ratios of individual pairs of RatiomiRNA/miRNA* in wild type relative to the ones in 35S-HA-RICE1 D52A transgenic plants .", "RatiomiRNA/miRNA* = miRNA expression level/miRNA* expression level .", "* , the median value of the logarithm values mentioned above in the whole populations of tested miRNA/miRNA* pairs .", "See also in Supplementary file 1F .", "( F ) Schematic diagram of 3’ al-RACE to detect the uridine addition ( red rectangle ) in the 3’ end of RISC-cleaved 5’ fragment .", "The miR159- and miR160-cleavage sites were shown with black triangle .", "( G–I )", "Uridylation of 5’ fragments in Ler and Ler; 35S-HA-RICE1 D52A .", "The representative length distribution patterns of uridylated MYB33-5’ ( G ) and ARF10-5’ ( H ) , and the fitting curves in dotted lines indicate the tendency of the length distribution , respectively; the mean length of the 3’ tails and the mean percentage of uridylated clones in total clones were shown in ( I ) and ( J ) , respectively; significant difference ( Student t-test , p<0 . 05 ) was indicated with the asterisk ( * ) .", "See the details for three biological replicates in Figure 7—figure supplement 2A–D and Supplementary file 3A and 3B .", "( K ) The abundance of MYB33-5’ in Ler and Ler; 35S-HA-RICE1 D52A .", "MYB33-5’ RNAs were detected by Northern blot in three biological replicates , using probes for MYB33-5’ from miR159-AGO1-mediated cleavage .", "FL , full-length MYB33 transcripts; 5’F , 5’ fragment .", "Methylene blue staining of total RNAs shows the loading controls .", "( L ) A proposed model for RICE functions in miRNA-RISC .", "RICEs facilitate the clearance of the uridylated tail of 5’ RISC-cleaved fragments and promote RISC recycling and miRNA stability . DOI: http://dx . doi . org/10 . 7554/eLife . 24466 . 01510 . 7554/eLife . 24466 . 016Figure 7—figure supplement 1 . RICEs affect the accumulation of miRNAs and miRNA* .", "( A–C )", "Ectopic expression of catalytically-inactive RICE1 mutants ( A and B ) and downregulation of RICEs ( C ) decreased miRNA accumulation in RISC .", "Western blot analyses were done with the crude extract ( Input ) and the IP products using anti-AGO1 antibody ( top panels ) .", "Actin serves as a loading control .", "sRNA blot analyses of sRNA recovered from AGO1 IP were conducted with 32P-labelled probe against miR166 .", "The ratio of miR166/AGO1 in wild-type Ler was arbitrarily designated as 1 . 0 , and the relative amount in each sample was normalized to that of control plants .", "( D ) Quantitative PCR showed the concurrent reduction of miRNA and miRNA* abundance in the transgenic plants expressing artificial miRNAs specifically targeting RICE1 , RICE2 or both .", "The relative level of miRNA and miRNA* was normalized to that in Ler plants where the amount was arbitrarily assigned a value of 1 with ±SD from at least three experiments , asterisks indicate statistically significant differences between wild type and transgenic plants .", "( E ) sRNA blot analysis of highly-enriched low-molecular-weight RNAs prepared from wild-type and RICE-compromised transgenic lines .", "Numerous 21-nt and truncated oligo probes complementary to miR165 ( a–b ) * , miR166 ( a–g ) * , miR159b* , miR160c* , miR162b* , miR164b* , miR167b* , miR168a* , and miR171a* were pooled for sRNA blot .", "Note: The region marked in red box is the place where 9-nt and 12-nt miRNA* fragments would have been detected , if present .", "( F ) In vitro RISC assembly assays to test the effect of the D52A mutation in RICE1 on removal of miRNA*s and nicked miRNA* in wheat germ .", "Flag-4Myc-AGO1 , RICE1 wild type and D52A mutant protein were synthesized in wheat germ extract from TNT T7 Quick Coupled transcription/translation system before application of miR166/166* duplexes containing either 32P-labelled miR166 or miR166* ( indicated by RED ) .", "The reaction mixtures were finally extracted with equal volumes of TE-saturated phenol .", "The RNA in the aqueous phase was recovered , analyzed with native 15–18% PAGE .", "The 32P signals were detected after exposure to a phosphorImager plate ( Molecular Dynamics ) .", "Negative control reaction using mock-translated wheat germ was performed in parallel .", "Note: 9-nt and 12-nt miRNA* fragments were not detected in the red box region . DOI: http://dx . doi . org/10 . 7554/eLife . 24466 . 01610 . 7554/eLife . 24466 . 017Figure 7—figure supplement 2 . RICEs affect the accumulation of the uridylated 5’ RISC cleavage fragments .", "( A–D )", "The length distribution pattern of uridylated MYB33-5’ ( A and B ) and ARF10-5’ ( C and D ) , and the fitting curves in dotted lines indicate the tendency of the length distribution pattern , respectively .", "( E ) semi RT-qPCR analysis of selected pri-miRNAs show that RICEs do not affect miRNA biogenesis in Arabidopsis . DOI: http://dx . doi . org/10 . 7554/eLife . 24466 . 01710 . 7554/eLife . 24466 . 018Figure 7—figure supplement 3 . Phylogenetic analysis of RICE proteins and their orthologs in eukaryotes . Alignment of RICE proteins and their orthologs in eukaryotes was done with Clustal Omega .", "The evolutionary relationships were inferred . DOI: http://dx . doi . org/10 . 7554/eLife . 24466 . 018 Studies on siRNA-RISC assembly in Drosophila show that once the siRNA duplex is incorporated into AGO proteins , the passenger strand serves as the first target of RISC and is cleaved into 1–9 nt and 10–21 nt fragments by the catalytically-active AGO proteins before degradation ( Matranga et al . , 2005; Rand et al . , 2005 ) .", "If the miRNA-RISC assembly were similar to that of siRNA-RISC , RICE proteins might have some impact on accumulation of 9-nt and 12-nt miRNA* .", "To test this , we enriched a large amount of low-molecular weight RNA and repeated sRNA blotting using pooled oligo probes targeting numerous families of miRNA*s .", "Whereas we appeared to detect residual full-length miRNA*s , we were unable to observe any nicked miRNA* strands on the blots ( Figure 7—figure supplement 1E ) .", "Next , we adopted the wheat germ system ( Tang et al . , 2003 ) to study in vitro reconstituted miRNA-RISC .", "Again , we failed to detect 9-nt and 12-nt miRNA* fragments in any of the reactions ( Figure 7—figure supplement 1F ) .", "These observations are consistent with the prevailing model of miRNA-RISC assembly in different organisms .", "During the loading of miRNA/miRNA* into AGO proteins , it is believed that the unwinding of the miRNA/* duplex and the removal of miRNA* are conducted through a slicer-independent mechanism due to the presence of mismatches in miRNA/* duplexes ( Kawamata and Tomari , 2010; Yoda et al . , 2010 ) .", "Under this scenario , the central mismatches facilitate the loading of miRNA/* duplexes into AGOs , while mismatches located elsewhere promote separation of the miRNA/* duplex and the subsequent release and degradation of miRNA* ( Kawamata and Tomari , 2010; Yoda et al . , 2010 ) .", "Similarly in plants , it is also proposed that unwinding of miRNA/* and subsequent clearance of miRNA* do not entail the cleavage activity of AGO1 ( Iki et al . , 2010; Carbonell et al . , 2012; Iki et al . , 2012 ) .", "As such , the production of nicked 9-nt and 12-nt miRNA* would not take place; and thus it would be very unlikely that RICE proteins target the truncated miRNA* .", "All together , these results further validated that RICE proteins positively regulate miRNA accumulation in vivo , while suggesting that RICEs are very unlikely to clear miRNA* to facilitate RISC assembly .", "RISC harbors target mRNAs besides miRNA and miRNA*; and RICE1 also could cleave long unstructured ss RNA substrates .", "Given that unproductive RISC can decrease miRNA amount in RISC in vivo and that overexpression of RICE D52A reduced miRNA abundance , we next hypothesized that RICE1 might act on 5’ fragments from RISC cleavage to maintain productive RISC .", "To test this , we conducted 3’ adapter ligation-mediated rapid amplification of cDNA ends ( al-RACE ) assays using total RNA prepared from Ler wild-type and 35S-HA-RICE1 D52A lines .", "We tested MYB domain protein 33 ( MYB33 ) and AUXIN RESPONSE FACTOR 10 ( ARF10 ) transcripts , which are the targets of miR159 and miR160 , respectively , and sequenced their al-RACE products of 5’ fragments from RISC activity ( MYB33-5’ and ARF10-5’ ) ( Figure 7F ) .", "Previous studies show that 5’ fragments generated by miRNA-RISC slicing undergo the 3’ uridylation in Arabidopsis ( Ren et al . , 2014 ) .", "Interestingly , here we found that 3’ uridylation tail length of 5’ fragments in 35S-HA-RICE1 D52A lines was significantly longer than the ones in wild type control ( 3 . 56 ± 0 . 14 vs 1 . 97 ± 0 . 13 for MYB33-5’ and 2 . 35 ± 0 . 55 vs 1 . 53 ± 0 . 23 for ARF10-5’ , Figure 7G–I; Supplementary file 3A and 3B ) , although the percentages of uridylated MYB33-5’ and ARF10-5’ in Ler; 35S-HA-RICE1 D52A is just slightly higher than the ones in Ler Wild type ( 91 . 30 ± 7 . 78% vs 77 . 61±10 . 48% for MYB33-5’ and 64 . 03 ± 6 . 17% vs 45 . 98 ± 9 . 62% for ARF10-5’ , Figure 7J; Supplementary file 3A and 3B ) .", "This pattern was reproducible in the independent biological replicates ( Figure 7—figure supplement 2A–D; Supplementary file 3A and 3B ) .", "Together with the in vitro enzyme activity assay , these results suggested that RICEs are involved in the degradation of 5’ fragments after uridylation modification .", "In Arabidopsis , HESO1 uridylates 5' cleavage fragments and triggers their degradation .", "As such , 5’ cleavage products exemplified by MYB33-5’ is accumulated in a heso1 mutant , compared with the amount in wild type ( Ren et al . , 2014 ) .", "To examine how RICE1 altered the abundance of 5’ fragments , we performed RNA blot to detect MYB33-5’ , one of very few species of 5’ fragments that can be readily detected in wild-type plants .", "We observed that the abundance of full-length MYB33 transcript was significantly increased in 35S-HA-RICE1 D52A transgenic line; and the accumulation was clearly due to lower amount of miRNAs in the transgenic lines .", "Notably , the accumulation of MYB33-5’ which should otherwise be reduced due to less cleavage of full-length transcripts , was comparable or even slightly increased in 35S-HA-RICE1 D52A , relative to that in Ler wild type ( Figure 7K ) .", "Together , we propose that RICEs mediate the clearance of 5’ uridylated fragments resulting from RISC cleavage and promote RISC recycling in vivo ." ], [ "Bioinformatics analysis predicted that RICEs contain an RNase H-like domain in the TAIR database .", "Structural analyses revealed that RICEs belong to the DnaQ-like 3’ to 5’ exonuclease superfamily ( Perry et al . , 2006; Smith et al . , 2013 ) .", "RICE1 also exhibits considerable structural similarity to RNase D ( Zuo et al . , 2005 ) .", "Whereas RNase D is involved in processing of structural RNAs , DnaQ-like protein family , often represented by the proofreading domains of DNA polymerases , acts to check the fidelity of newly synthesized DNA during DNA replication and to excise the mismatched nucleotides in the 3’-to-5’ direction .", "It was previously proposed that RICE2 functions as its structural homolog , WRN-exo , in DNA metabolism ( Perry et al . , 2006 ) .", "However , its enzymatic activity and DNA binding capability were not previously detected ( Smith et al . , 2013 ) .", "Here we demonstrate that RICEs are mainly RNases , not DNases , and function in the degradation of 5’ uridylated fragments from RISC .", "Compared to other DEDD superfamily enzymes , RICEs exhibit some additional unique biochemical properties .", "First , RICEs harbor non-canonical residues in their active sites instead of the canonical DEDD motif observed in the superfamily .", "Moreover , RICEs lack other conserved histidine and tyrosine residues that are critical for the catalytic activity of the DEDD superfamily ( Zuo and Deutscher , 2001 ) .", "Second , the enzymatic activity of RICEs do not appear to involve metal ions , which are otherwise characteristic of DEDD superfamily proteins such as human WRN-exo ( Perry et al . , 2006 ) , bacterial RNaseT ( Zuo et al . , 2007 ) , yeast Ngl3p ( Feddersen et al . , 2012 ) , among others ( Zuo and Deutscher , 2001 ) .", "Third , the active sites of RICEs are located at the interface between RICE1 subunits in the hexameric assembly; therefore , each subunit contributes to the catalytic activity .", "Together , the lack of a canonical catalytic motif and metal ions in the active site as well as the unique location of its active site suggest a novel catalytic mechanism in RICEs .", "The multimeric assembly of RICE complex with the active sites at the interfaces between monomers in the hexameric ring may indicate physiological significance .", "First , the hexameric assembly of RICEs would allow them to trap nucleic acids more efficiently for processing .", "Examination of surface electrostatic potential of RICE1 and RICE2 reveals that the backside of the RICE hexamers is mostly negatively charged , whereas the front surface of the hexamer contains unique positively charged patches around the active site ( Figure 6C ) .", "Thus , these positively charged patches could sequester nucleic acids and facilitate interactions between the enzyme and substrate .", "On the other hand , the negatively charged surfaces might contribute to determining the orientation or polarity of 3’ tails of 5’ fragments that are dislodged from RISC .", "Second , the assembly and disassembly of RICE hexameric rings might be a dynamic process , and likely represent a new layer of homeostatic regulation of RISC maturation .", "A previous study showed the in vitro monomer-to-hexamer transition of RICE2 using recombinant proteins prepared from E . coli ( Smith et al . , 2013 ) .", "In our studies , although we barely detected the presence of RICE1 monomers in solution , we did frequently observe both hexameric and dimeric conformations .", "Consistent with this observation , SEC profiling of plant cell extracts revealed that RICE proteins also displayed an array of monomer/dimer , hexamer , and even higher-order oligomers ( Figure 1G ) .", "Given that the RICE monomer is very unstable , oligomerization likely allows for proper folding and protects it from protease degradation ( Figure 4; Figure 6H ) so the dynamic assembly and disassembly of RICE oligomers may serve as an extra regulatory layer , inferring a plastic capability of substrate processing corresponding to various functional needs .", "Based on our biochemical and genetic results , we wished to propose that RICEs act to clear 5’uridylated fragments to maintain productive RISC and facilitate its recycling .", "Several pieces of evidence supported the notion: ( 1 ) RICEs directly reside on AGO proteins in cytoplasm and thus should function coordinately with RISC to regulate post transcriptional silencing events ( Figure 1; Figure 1—figure supplement 1 ) ; ( 2 ) RICEs are 3’-to-5’ exoribonucleases specifically targeting ss RNAs ( Figure 2 ) ; ( 3 ) overexpression of RICEs elevated miRNA levels while downregulation of RICEs and dominant negative variants of RICE1 decreased abundance of miRNAs and their retaining in AGO proteins , indicating that RICEs are very unlikely to degrade miRNAs in vivo ( Figure 3; Figure 5; Figure 7 ) ; ( 4 ) , miRNA* was not enriched in RISC from rice1 rice2 mutants or transgenic plants ectopically expressing dominant-negative RICEs ( Figure 7A–E ) .", "Thus , RICEs functionally differ from some other ribonucleases that are reported to be engaged in promoting RISC assembly by clearing miRNA* ( Maiti et al . , 2007; Xiao et al . , 2010; Xue et al . , 2012 ) ; ( 5 ) last but importantly , overexpression of catalytically-inactive forms of RICE1 not only caused extended 5’ uridylated fragments from RISC activity in vivo , but also increased their abundance ( Figure 7F–K; Figure 7—figure supplement 2A–D ) .", "All the evidence implicates RICE proteins in degradation of the uridylated fragments from RISC cleavage to maintain proper function of RISC .", "How do RICEs coordinate with RISC to clear the cleavage fragments ?", "Recent single-molecule studies show that AGO proteins themselves can generate a favorable environment that actively promotes the release of the cleavage products in vitro ( Jo et al . , 2015; Salomon et al . , 2015; Yao et al . , 2015 ) .", "In this setting , the released cleavage products might be channeled to AGO-bound RICEs for further degradation .", "Interestingly , we observed that hexameric assembly of RICE subunits creates a central pore with a diameter approximately 21–22 Å that is rich in positively charged residues ( Figure 6C ) .", "It has been shown that proteins with similar ring-structures such as PCNA interact with ds DNA via their central cavity , while other ring-structured proteins translocate processively and unidirectionally along ssDNA to separate the strands of double-stranded ( ds ) DNA aiding both in the initiation and fork progression during DNA replication , or translocate ss RNA in transcriptional termination ( Patel et al . , 2011; Thomsen and Berger , 2009 ) .", "Given that human AGO2 releases its cleaved products by destabilizing their interaction with the guide RNA ( Salomon et al . , 2015 ) , RICE proteins might be coupled with a yet unidentified helicase or even AGO proteins themselves to separate double-stranded miRNA/5’-cleavage targets .", "The separation would allow unidirectional translocation of 5’ cleavage products from RISC to RICEs for degradation , whereas retaining miRNAs inside AGO proteins ( Figure 6C ) .", "Further study on the physiological significance of hexameric assembly of RICE proteins will provide new insight into cooperation between RICEs and RISC in RNA silencing events .", "Besides clearance of uridylated 5’ cleavage products from RISC , RICE proteins also promote miRNA accumulation .", "How could RICEs enhance miRNA abundance in vivo ?", "First , we have clarified that RICEs , different from QIP , did not impact miRNA biogenesis because the abundance of pri-miRNAs was not affected in overexpression lines of wild-type RICEs nor dominant-negative RICEs ( Figure 7—figure supplement 2E ) .", "Second , RICE promotion of miRNA abundance is not through changing the level of the housing protein AGO1 because AGO1 amount remained stable or even slightly increased whereas miRNAs were down-regulated in the RICE-compromised lines ( Figure 7—figure supplement 1A–C ) .", "Third , RICE proteins appeared not to target miRNA* and it is not likely that RICE proteins promote miRNA accumulation through degrading miRNA* to facilitate RISC assembly ( Figure 7A–E; Figure 7—figure supplement 1D–F ) .", "We speculate that the two functions of RICE proteins might be related .", "RISC performs multiple rounds of target cleavage ( Wee et al . , 2012 ) .", "Prompt removal of the cleaved products promotes RISC recycling and maintains productive AGO-miRNA complexes .", "On the other hand , 5’ cleavage fragments without prompt processing may be retained in RISC; this would result in a non-productive state that would be coupled to miRNA turnover .", "In this model , RICE function increases miRNA stability by clearing RISC products and promoting active RISC complexes .", "In the absence of RICEs , an increase in the proportion of non-productive RISC complexes would lead to miRNA turnover and thereby reduced miRNA levels .", "This scenario would be reminiscent of target mimicry in Arabidopsis in which , inactive interaction of targets with RISC causes miRNA decoy and repression ( Franco-Zorrilla et al . , 2007 ) .", "In other organisms , complementary targets could often switch their roles from regulated substrates to regulatory RNAs to inhibit the activity of miRNAs ( Ameres et al . , 2010; De et al . , 2013; Xie et al . , 2012 ) or additional species of small RNAs ( Figueroa-Bossi et al . , 2009 ) .", "Although the functions of RICE proteins in the promotion of miRNA accumulation and clearance of uridylated 5’ RISC products could be interconnected , it is also possible that these two roles for RICE proteins might be independent processes .", "That said , the mechanism of how RICE proteins enhance miRNA expression awaits future clarification .", "In conclusion , we have identified the enzymes that are specifically involved in RNA decay of uridylated 5’ cleavage products from RISC .", "By clearing the RISC products , RICE proteins can facilitate RISC recycling and maintain an active RISC pool ( Figure 7L ) .", "This study also raised several new outstanding questions: First , how could RICEs specifically select 5’ cleavage targets for degradation rather than promiscuously targeting miRNAs and miRNA* in RISC ?", "Second , does RICEs only chop out the single-stranded part of 5’ uridylated fragments , or degrade the entire 5’ cleavage fragments ?", "Considering that RICEs disfavor structured long ss RNAs , RICEs might only clear the linear ss RNA harbored in or near RISC .", "Until now , several candidates have been implicated in degrading the 5’ cleavage products ( Branscheid et al . , 2015; Ren et al . , 2014 ) .", "In this scenario , it might be necessary for RICEs to coordinate with additional 3’-to-5’ exosome nucleases or even with 5’-to-3’ exonuclease and accomplish the complete clearance of RISC cleavage products .", "Furthermore , it has been reported that HESO1 , the enzyme that accounts for uridylation of the cleavage targets , and the uridylated products are associated with AGO proteins ( Ren et al . , 2014 ) .", "Thus , it is tempting to hypothesize that uridylation , release and decay of 5’ cleavage fragments could be coordinated in vivo .", "Whether HESO1 and RICEs interacts or synergistically promote the functions for each other would be an exciting question to be addressed in the future .", "Equally interesting is whether there is interplay between RICEs and decapping machinery or deadenylation complexes in RNA decay .", "Last but not least , there are between one and four orthologs in most other angiosperm genomes ( Figure 7—figure supplement 3 ) ; and the RICE orthologs are also conserved through animals , implying their functional significance in these organisms .", "Further characterization of the RICE orthologs would certainly provide new mechanistic insights into RISC functionality in the eukaryotes ." ], [ "A . thaliana suspension MM1 cell lines expressing Flag-4Myc-AGO10 under the control of Cauliflower Mosaic Virus 35S promoter were generated following the standard transformation procedure ( Forreiter et al . , 1997; Menges and Murray , 2002 ) .", "Transgenic MM1 cells were collected and ground in liquid nitrogen and AGO10 protein complexes were extracted using four volumes of extraction buffer ( 20 mM Tris- HCl , pH 7 . 5 , 150 mM NaCl , 2 mM MgCl2 , 1 mM DTT , 1 mM EDTA , 10 µM MG132 , and EDTA-free protease inhibitor cocktail [Roche] ) .", "After removal of insoluble materials by centrifugation twice at 16 , 000 g for 10 min at 4°C , extracts were incubated with anti-Flag M2 magnetic beads ( Cat# M8823 , Sigma . AB_2637089 ) for 2 hr at 4°C .", "The beads were washed five times with the extraction buffer ( 5 min/each time ) before elution with 1 . 2 ml of elution buffer ( extraction buffer containing 100 µg/ml 3XFlag peptide ) .", "The eluate was then incubated with anti-Myc agarose beads ( Cat# A7470 , Sigma . RRID:AB_10109522 ) for 2 hr at 4°C .", "The beads were washed three times with the extraction buffer , and the dual-tagged AGO complexes were eluted by incubation with 400 µl of basic buffer ( 0 . 5 mM EDTA and 30 mM NH4OH ) for 20 min at room temperature .", "The eluted immunoprecipitates were frozen in liquid N2 and vacuum dried before being resolved in 4–20% gradient SDS-PAGE and proteomic analysis as described ( Castillo-González et al . , 2015 ) .", "Three independent protein samples were sequenced at three different mass spectrometry facilities ( Texas A and M; Cornell and Harvard Universities ) .", "Binary vectors including pBA-RICE1 , pBA-RICE1-3HA , pBA-RICE2-3HA , and pBA-3HA-RICE1 were transformed into two ecotypes ( Ler and Col-0 ) of A . thaliana as well as ago10pnh-2 mutant background ( Lynn et al . , 1999 ) by the floral dip transformation method ( Zhang et al . , 2006a ) .", "The tagged or untagged RICEs were detected in seven-day-old T2 transgenic seedlings by western blot analysis .", "Similarly , The binary vectors harboring 35S-driven artificial miRNAs targeting specifically to RICE1 , or RICE2 or both were introduced into Ler background .", "Transgenic plants were screened for the presence of artificial miRNAs and absence of target transcripts in seven-day-old T2 transgenic plants using sRNA or northern blot analyses .", "Constructs of PRICE1-GUS and PRICE2-GUS were transformed into Col-0 background for examination of the expression profiles throughout plant growth and development .", "Seeds from infiltrated plants were selected on standard MS medium containing the appropriate selective agents: 10 mg/L glufosinate ammonium ( Sigma , St Louis , MO ) together with 100 mg/L carbenicillin ( Sigma , St Louis , MO ) .", "Homozygous progeny of T2 transgenic plants that displayed 3:1 ratio on selection plates were further identified by the lack of segregation in the presence of selective agents on MS plates .", "Plants were grown in LP5 Metromix ( SunGro , Canada ) in a walk-in growth chamber at 22°C with relative humidity of 50% under long-day conditions ( 16 hr light/8 hr dark ) unless otherwise noted .", "Light illumination ( 150 mmol photons/m2s ) was provided by a Spec-tralux T5 high-output lamp ( Hydro warehouse ) .", "The majority of constructs were generated through a gateway system ( Zhang et al . , 2005 ) .", "Numerous destination vectors ( DC in our nomenclature ) used for transient expression in N . benthamiana and stable Arabidopsis transformation as well as protein expression in E . coli were described ( Zhang et al . , 2006b; Zhu et al . , 2011 ) .", "These destination vectors include the binary gateway vectors pBA-DC , pBA-Flag-4Myc-DC , pBA-3HA-DC , pBA-DC-3HA , pBA-3Flag-DC , pHyg-DC-CFP and pHyg-YFP-DC as well as E . coli-compatible pMAL-DC and pET28-SUMO-DC .", "Most cDNA and artificial miRNA genes were cloned into pENTR/D vectors using primers listed in Supplementary file 1G , confirmed by sequencing , and transferred to the appropriate vectors by recombination using the LR Clonase ( Invitrogen ) .", "Promoters for RICE1 and RICE2 members were cloned from Arabidopsis ( Col-0 ) genomic DNA into TOP-PCR ( Invitrogen ) and confirmed by sequencing .", "The promoter sequences covered 1184 and 1538 bp from the start codon of RICE1 and RICE2 , respectively .", "The confirmed promoter fragments were further introduced into appropriately digested binary vectors of pBA002a-GUS ( Zhang et al . , 2005 ) .", "pET28-SUMO-RICE1 and -RICE2 , which were used for protein purification , were cloned as follows: full-length cDNAs of RICE1 and RICE2 were cloned by PCR using two pairs of primers listed in Supplementary file 1G .", "The resultant PCR products were double digested with BamHI and SacI and ligated into BamHI /SacI-treated pET28a-SUMO ( Lu et al . , 2010 ) to produce pET28-SUMO-RICE1 and -RICE2 .", "The constructs were then confirmed by DNA sequencing .", "For analyses of residues potentially involved in catalytic activity and dimerization , numerous point mutations were introduced into RICE1 using pET28a-SUMO-RICE1 as a template and PCR-amplified using primers summarized in Supplementary file 1G .", "The constructs were sequencing confirmed before protein preparation .", "MBP-tagged full-length or truncated AGO proteins were purified as described ( Zhang et al . , 2006b ) .", "For purification of His-tagged recombinant proteins in E . coli , pET28a-SUMO-RICE1 , and -RICE2 as well as pET28a-SUMO-RICE1 variants were transformed into BL21 competent cells ( DE3 ) .", "The BL21 cells were grown at 37°C in LB medium in the presence of antibiotics ( 100 μg/ml kanamycin ) to an OD600 of 0 . 6–0 . 8 .", "Protein expression was induced by addition of isopropyl-β-D-1-thiogalactopyranoside ( IPTG ) to a final concentration of 0 . 5 mM .", "The cells were further incubated in a shaker for overnight at 16°C before pellet collection by centrifugation at 6000 rpm for 10 min at 4°C .", "For protein purification , cell pellets were re-suspended in lysis buffer ( 20 mM Tris-HCl pH 7 . 5 , 150 mM NaCl , 2 mM PMSF ) and disrupted by sonication at 60% amplitude for 5 to 10 min , using a Sonic Dismembrator ( Model 500 , Fisher Scientific ) .", "Cell lysate was cleared by centrifugation at 8000 rpm for 15 min at 4°C .", "Supernatant was further centrifuged at 16000 rpm for 30 min at 4°C to completely remove the cell debris .", "The 6His-SUMO-tagged fusion proteins were first purified by Ni-NTA resin .", "After the loading of soluble extracts , the resin was washed with first washing buffer ( 20 mM Tris-HCl pH 7 . 5 , 500 mM NaCl , 2 mM PMSF , and 25 mM imidazole ) and second washing ( 500 mM NaCl , 20 mM Tris-HCl pH 7 . 5 , 2 mM PMSF , and 100 mM imidazole ) buffer to remove nonspecific-binding proteins , prior to elution with elution buffer ( 150 mM NaCl , 20 mM Tris-HCl pH 7 . 5 , and 250 mM imidazole ) .", "For enzymatic assay purposes , the affinity purified RICE proteins were cleaved with SUMO protease for one hour at room temperature .", "The cleaved RICE proteins were further purified by gel filtration chromatography on a Superdex75 ( 1 . 6 × 60 ) column ( GE Healthcare ) eluted with the running buffer ( 20 mM Tris-HCl pH 7 . 5 , 150 mM NaCl ) .", "The purity of the proteins was analyzed by a 15% sodium dodecyl sulfate ( SDS ) polyacrylamide gel electrophoresis ( PAGE ) gel , which was stained with Coomassie Brilliant blue R-250 .", "RICE1 and RICE2 open reading frames in the pENTR vectors were cloned to destination vector pET28a-SUMO-DC by LR reaction to generate His-SUMO fusion proteins .", "AGO10 and AGO10 truncations in the pENTR vectors were cloned to destination vector pMAL-DC by LR reaction to generate Maltose-binding protein ( MBP ) fusion proteins .", "All the plasmids were transformed into BL21 competent cells ( DE3 ) .", "One microgram MBP-fusion Prey proteins of AGO1 , AGO10 and AGO10 truncated proteins were preabsorbed for 1 hr at room temperature in 1 mL of binding buffer ( 50 μl Ni-NTA resin beads , 50 mM Tris at pH 7 . 5 , 150 mM NaCl , 0 . 2% glycerol , 0 . 6% Triton X-100 , 0 . 5 mM β-mercaptoethanol , 1 mM PMSF ) .", "The mixture was cleared by centrifugation at 12 , 000g for 2 min .", "The resulting supernatant was transferred to a new tube containing 1 μg of the bait protein His-SUMO/His-SUMO-RICE1/RICE2 .", "After incubation at room temperature for 1 hr , Ni-NTA resin beads were added , and the incubation continued for another one hour in the same conditions .", "Finally , six further vigorous washes were performed with the washing buffer ( 50 mM Tris at pH 7 . 5 , 300 mM NaCl , 0 . 6% Triton X-100 , 1 mM PMSF ) .", "Pulled-down proteins were resolved by 6% SDS-PAGE and detected by Western blotting using the α-MBP antibody ( NEB E8032S , RRID:AB_1559732 ) .", "Long RNA substrates of PHV and At4g29770 were synthesized as described previously ( Zhang et al . , 2006b ) .", "For RNA homopolymer of 21- , 50- , 70- , and 100-nt in length , T7 promoter-fusion reverse primers listed in Supplementary file 1G were annealed with T7 promoter forward primer with 95°C for 10 min and naturally cooling-down in the heat block in room temperature for 1 hr; RNA homopolymers were synthesized by T7 RNA polymerase in 37°C for at least 2 hr .", "The RNA transcripts were separated by 6–15% PAGE and gel-purified .", "The RNA transcripts were dissolved in DEPC-treated water and ready for further labeling experiments .", "The RNA substrates were commercially synthesized by IDT and 5’- labeled with γ-32P-ATP by T4 PNK .", "For 3’ end labeling , RNA was ligated to α-32pCp by T4 RNA ligase followed by CIP treatment .", "For circular RNA substrate preparation , 5’ 32P – labeled ss RNA was circularized by T4 RNA ligase I according to the manufacture’s instruction .", "After the reaction , RNAs were extracted with a standard phenol-chloroform extraction method .", "The resulting RNA fragments were separated by 20% denaturing polyacrylamide gels and recovered by ethanol precipitation for overnight at −20°C .", "For enzymatic assays , 0 . 5 µM of wild type RICEs or RICE1 variants and 5 nM of substrates were incubated for normally one hour unless specifically indicated time at 37°C in 20 µl reaction buffer containing 20 mM HEPES , pH 7 . 0 , 50 mM KCl , 5 mM MgCl2 , 1/10 V/V RNase inhibitor SUPERaseIn ( Ambion ) and 1 mM DTT .", "The reaction mixtures were then separated with phenol-chloroform .", "The RNA in the aqueous phase was further precipitated with three volumes of absolute ethanol overnight at −20°C before size-fractionation on a denaturing polyacrylamide gel .", "Purified RICE1 was concentrated to 20 mg/ml and crystallized by hanging drop vapor diffusion method at 16°C with ~30% pentaerylthritol proxylate ( 5/4 PO/OH ) plus 0 . 2 M KCl in 50 mM HEPES at pH 7 . 5 .", "The crystals were harvested in mother liquor containing 35% pentarythritol proxylate and flash frozen in liquid nitrogen .", "The diffraction data were collected at beamline 5 . 0 . 1 of the Advanced Light Source ( ALS ) .", "The diffraction data were processed with the HKL2000 package .", "The structure of RICE1 was determined by molecular replacement using MOLREP in the CCP4 package .", "The structural model of At5g06450 ( PDB 1VK0 ) , which has 68 . 5% sequence identity to RICE1 , was used as search model .", "The model was rebuilt with Coot .", "The crystallographic asymmetric unit ( ASU ) contains three RICE1 molecules .", "The solvent content of the RICE1 crystal is ~80% , which corresponds to a Mathews coefficient ( Vm ) of 7 . 4 Å3/Da .", "Although the crystals diffract weakly , the electron density map was clear and continuous throughout the molecules .", "The molecules of RICE1 in the crystal assembled into two hexameric rings , interacting with each other in a face-to-face manner .", "The structure was refined using the Phenix package .", "Statistics of data collection and structural refinement were shown in Supplementary file 2 .", "All the structural figures were prepared with PyMol .", "Coordinates and structural factors have been deposited in the database of wwPDB with accession code 5V5F .", "A gateway destination cassette was cloned into pCAMBIA-NLuc and pCAMBIA-CLuc ( Chen et al . , 2008 ) to generate the gateway destination vectors , respectively .", "All the test genes were cloned into the destination vectors by LR reaction .", "All the constructs were transformed into A . tumefaciens strain GV3101 .", "LCI Assays were performed as described ( Zhang et al . , 2011 ) .", "Images were acquired using an electron multiplying charge coupled device camera ( EMCCD , Cascade II , Roper Scientific ) and processed by WinView software ( Roper Scientific ) .", "Agrobacterium harboring pHyg-35S-AGO1/10-YFP and pHyg-35S-CFP-RICE1/2 , respectively , were infiltrated separately and/or co-infiltrated into the leaves of Nicotina benthamiana .", "The infiltrated leaves were harvested 2-days-post-infiltration and used for the confocal imaging of subcellular localization in tobacco epidermis cell by confocal microscopy ( NIKON ) .", "Fluorescent signals of CFP and YFP were measured by excitation with CFPHQ Shutter ( emission was detected at 485 nm ) and with YFPHQ Shutter ( emission was detected at 540 nm ) , respectively .", "Similarly , transgenic Col-0 seedlings expressing pHyg-35S-CFP-RICE1/2 were collected to examine cellular localization of RICE1/2 by the confocal microscopy .", "Histochemical analysis of the GUS activity was conducted as described ( Zhou et al . , 2015 ) .", "Briefly , the germinating seeds , seedlings or other plant tissues were placed in a solution containing 0 . 2M Sodium Phosphate pH7 . 0 , 2 mM K3 [Fe ( CN ) 6] , 2 mM K4[Fe ( CN ) 6] , 2 mM 5-bromo-4-chloro-3-indolyl-D-glucuronide ( X-Gluc ) , and 10% methanol ) and vacuum infiltrated for about 30 min before incubation at 37°C for one or two days .", "The stained materials were rinsed with a series of ethanol dilutions and subsequently incubated in 70% ethanol solution overnight to remove chlorophylls .", "The plants were then mounted on microscope slides and examined using an Olympus DSU microscope .", "Polyclonal anti-RICE1 antiserum was generated in rabbits immunized with the recombinant full-length RICE1 prepared from above-mentioned three-step purifications ( Ni-NTA , SUMO protease cleavage and size-exclusive chromatography ) .", "For western blot analyses , RICE1 antibody was further affinity-purified from sera using the same recombinant proteins used for rabbit immunizations as previously described ( Zhang et al . , 2005 ) .", "For transient experiments , four-week-old N . benthamiana leaves were infiltrated with A . tumefaciens ABI harboring variety of binary plasmids .", "The ODs of A . tumefaciens cultures were diluted to 0 . 4 .", "N . benthamiana leaves were collected two days after agroinfiltration .", "For A . thaliana plants , seven-day-old transgenic seedlings were collected from MS plates .", "Total protein was extracted with the immunoprecipitation ( IP ) buffer containing 50 mM Tris-HCl , pH 7 . 5 , 300 mM NaCl , 4 mM MgCl 2 , 5 mM DTT , 0 . 1% Triton-100 , and the complete protease inhibitor cocktail ( Roche ) .", "Cleared protein extracts were immunoprecipitated with agarose-conjugated antibodies against Flag ( Sigma M8823 ) or Myc ( Sigma A7470 ) as described ( Zhang et al . , 2006b ) .", "Total RNA was extracted using Trizol reagent from Agrobacterium - transfected N . benthamiana leaves or A . thaliana tissues , including 7- ( or 10 ) -day-old seedlings and three week-old adult plants depending on the desired assays or from immunoprecipitates of AGO protein complexes .", "RNA blot hybridizations of low-molecular-weight RNAs ( sRNA blot ) were performed as described ( Zhang et al . , 2006b ) .", "Each lane contained sRNAs , which were recovered from isolated AGO complexes prepared from 0 . 4 g tissues .", "For input , 10 µg of total RNA was routinely used for each sample .", "Blots were hybridized with 32P-radiolabeled oligonucleotide probes complementary to the sRNAs of interest .", "In some experiments , the same blots were stripped and re-probed with 32P-labeled oligos complementary to the indicated miRNAs .", "U6 served as loading controls .", "RNA blots were detected after exposure to a phosphor plate and quantified using the Quantity One Version 4 . 6 . 9 according to the manufacturer’s instructions ( Bio-Rad ) .", "RNA blot hybridizations of high-molecular-weight RNAs ( northern blot ) were performed as described ( Zhang et al . , 2006b ) using random-labeled probes ( labeled by Amersham Rediprime II DNA labeling system , GE healthcare RPN1633 ) specifically targeting specific transcripts indicated .", "Western blot analyses were performed as previously described using antibodies against Flag ( Sigma F3165 ) , Myc ( Sigma C3956 ) , HA ( Sigma H9658 ) , actin ( A0480 ) as well as polyclonal antibodies against AGO1 ( Agrisera ) and RICE1 ( generated in Rockland ) ( Zhang et al . , 2006b ) .", "Western blots were developed with ECL+ , detected with ChemiDoc XRS+ and quantified using the ImageLab Software ( Bio-Rad ) .", "Approximately 200 µg total RNA extracted through Trizol reagent was used each time for each sample .", "mRNA and rRNA were then precipitated on ice for 1 hr after addition of PEG 8000 to a final concentration of 10% and NaCl to a final concentration of 0 . 5M .", "The mixture was then centrifuged at 12 , 000 rpm for 10 min at 4°C .", "Supernatant containing sRNA was precipitated with 3 vol of ethanol at −20°C for overnight .", "sRNAs were pooled from several experiments before sRNA blot assays .", "We used wheat germ system ( Tang et al . , 2003 ) to study the in vitro RISC assembly .", "The experimental protocol for this assay was modified from previous studies ( Iki et al . , 2010 , 2012 ) .", "Flag-4Myc-AGO1 , RICE1 wild type and D52A mutant protein were synthesized at 25°C for 1 hr in wheat germ extract from TNT T7 Quick Coupled transcription/translation system ( Promega , L1170 ) , mixed together ( 1:1 , v:v ) with 1 vol fresh wheat germ extract ( Promega , L4380 ) and incubated for 30 min at 25°C in the presence of additional 0 . 75 mM ATP , 1 . 8 mM MgCl2 , 20 mM creatine phosphate ( CP ) , and 0 . 4 mg/ml creatine kinase ( CK ) .", "Then , the mixture was further mixed with 5 nM miR166/166* duplexes containing either 32P-labelled miR166 or miR166* .", "The reaction mixtures were collected after incubation and diluted 10-fold with TR buffer ( 30 mM HEPES [pH 7 . 4] , 80 mM KOAc , 1 . 8 mM MgCl2 , 2 mM DTT , one tablet/50 ml complete protease inhibitor [Roche] ) and extracted with equal volumes of TE-saturated phenol .", "The resulting aqueous phase was recovered , mixed with 0 . 1 vol of 3M NaAc ( pH 5 . 2 ) , 2 volumes of ethanol and 1 μL of glycogen-blue and precipitated at −20°C overnight .", "Spin down the pellets at 4°C in 21 , 000g and washed the pellets with 80% ethanol .", "The pellets were dried up for 5 min and dissolved in 10 μL ddH2O .", "The dissolved RNAs were mix with an equal volume of native dye solution ( 1 X TBE ( 100 mM Tris , 90 mM boric acid , 2 mM EDTA-Na2 ) , 0 , 2 mg/ml bromophenol blue , 0 . 2 mg/ml xylene cyanol , 10% ( v/v ) glycerol ) , and analyzed with native 15–18% PAGE using 0 . 5 X TBE as running buffer in 4°C .", "The 32P signals were detected after exposure to a phosphorImager plate ( Molecular Dynamics ) .", "Negative control reactions using mock-translated wheat germ were performed in parallel .", "sRNAs were extracted from total RNA of whole plants of Landsberg erecta wild type and transgenic plant expressing 35S-HA-PNT1 D52A using Trizol ( Invitrogen ) , separated by 15% denature PAGE and sRNAs of 19–24 nt were gel-purified .", "sRNAs were ligated to a 3’ adaptor with/without different barcodes and a 5’ adaptor sequentially , and then RT-PCR amplified as described ( Zhu et al . , 2011; Hafner et al . , 2012 ) .", "The cDNA products were re-amplified using a pair of cloning primers and the products were gel-purified in low-melting agarose gel and subject to Illumina sequencing .", "After trimming adaptor sequences with fastx_toolkit-0 . 0 . 14 , sRNAs with lengths between 19- to 28-nt were selected and mapped to the Arabidopsis genomic sequences ( TAIR10 version ) with bowtie 1 . 1 . 2 and bedtools-2 . 26 . 0 .", "Sequences from different samples were normalized by the number of total tRNA reads with perfect genomic matches .", "Expression levels of miRNAs and miRNA* were examined by quantitative real-time PCR following the protocol in previous works ( Varkonyi-Gasic et al . , 2007; Turner et al . , 2013 ) .", "Total RNAs were prepared from control plants or different transformants and treated with DNase before being subjected to cDNA synthesis using Superscript III reverse transcriptase ( Invitrogen ) primed by miRNA/miRNA*/U6-Specific primers ( Supplementary file 1G ) .", "Quantitative real-time RT-PCR was performed in 384-well plates with an ABI 7900HT real-time PCR system using the SYBR Green I master mix ( Applied Biosystems ) in a volume of 10 μl .", "PCR conditions included one cycle at 50°C for 2 min , one cycle at 95°C for 10 min and 50 cycles of 96°C for 10 s followed by 60°C for 1 min .", "The U6 gene ( Turner et al . , 2013 ) was included as an internal control for normalization .", "Three biological replicates were performed , and the reactions were performed in triplicate for each run .", "The comparative CT method was used to evaluate the relative quantities of each amplified product in the samples .", "The threshold cycle ( CT ) was automatically determined for each reaction by the system .", "Al-RACE was performed according to the reference ( Ren et al . , 2014 ) with some modifications .", "2 µg total RNA was first ligated to 100 pmol RNA adaptor by T4 RNA ligase .", "3’ RT primer was used for first-strand cDNA synthesis .", "The pairs of 3’RT/MYB33 F1 and 3’RT/MYB33 F2 primers were used for nested PCRs of MYB33-5’ , while the pairs of 3’RT/PHB F1 and 3’RT/PHB F2 primers were used for nested PCRs of PHB-5’ .", "The PCR products were cloned into pGEM-T Easy Vector ( Promega ) and sequenced .", "Three biological replicates of total RNA samples were used for Al-RACE .", "The mean percentages of 5’ RISC fragments with 3’ U tails and the mean lengths of the 3’ tails were statistically calculated .", "Primer sequences are listed in Supplementary file 1G ." ] ]

[ "RNA-induced silencing complex ( RISC ) is composed of miRNAs and AGO proteins .", "AGOs use miRNAs as guides to slice target mRNAs to produce truncated 5' and 3' RNA fragments .", "The 5' cleaved RNA fragments are marked with uridylation for degradation .", "Here , we identified novel cofactors of Arabidopsis AGOs , named RICE1 and RICE2 .", "RICE proteins specifically degraded single-strand ( ss ) RNAs in vitro; but neither miRNAs nor miRNA*s in vivo .", "RICE1 exhibited a DnaQ-like exonuclease fold and formed a homohexamer with the active sites located at the interfaces between RICE1 subunits .", "Notably , ectopic expression of catalytically-inactive RICE1 not only significantly reduced miRNA levels; but also increased 5' cleavage RISC fragments with extended uridine tails .", "We conclude that RICEs act to degrade uridylated 5’ products of AGO cleavage to maintain functional RISC .", "Our study also suggests a possible link between decay of cleaved target mRNAs and miRNA stability in RISC ." ]

[ "DNA contains all the information needed to build a body , yet molecules of RNA carry these instructions to the sites in the cell where they can be used .", "Cells must control how much RNA they produce in order to ensure that they develop properly and can respond well to their environment .", "RNA silencing refers to a collection of mechanisms that use smaller RNA molecules called microRNAs to incapacitate certain RNA molecules and selectively switch off the genes that encode them to stop more from being made .", "One key player in RNA silencing is the multi-protein complex called RISC , which contains microRNA and a group of proteins called AGOs .", "Once the microRNA has identified its RNA target , the AGOs cut the RNA into two pieces , known as the 5’ cleavage fragment and 3’ cleavage fragment .", "The two resulting fragments need to be cleared away swiftly , so that the RISC can move on to the next target .", "While it was known how the 3’ cleavage fragment was removed , it was less clear how the 5’ cleavage fragment was dealt with .", "Previous studies had shown that the 5’ cleavage fragment was marked with a chemical called uridine , which somehow signals to the RISC that this fragment needs to be destroyed .", "Now , using biochemical techniques , Zhang et al . have identified two new proteins in the model plant Arabidopsis that attach to the AGO proteins and degrade the 5’ cleavage fragments that are marked with uridine .", "The two proteins are named RICE1 and RICE2 .", "Zhang et al . then analyzed the three-dimensional shape of RICE1 and identified the ‘active’ region that is responsible for degrading the RNA fragments .", "When these active regions were blocked , the microRNA levels were low , but the uridine-marked 5’ cleavage fragments were high .", "Also , the RISC complex could not work properly , which lead to problems during the development of the plant .", "These results suggest that RICE proteins degrade 5’ cleavage fragments modified with uridine to activate RISC .", "RICE proteins are conserved between plants and animals , and it is likely that their counterparts in humans will have a similar role to the plant proteins .", "The next challenge will be to explore how RICE proteins work in more details , which may lead to new ways to manipulate the levels of microRNAs to change the architecture of the plant and to improve their tolerance to various stress conditions ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "structural biology and molecular biophysics", "tools and resources" ]

[ [ "Electron microscopy of isolated macromolecules and their complexes ( single particles ) embedded in a thin layer of vitreous ice ( cryo-EM ) has recently led to a number of structures determined at near-atomic resolution ( Liao et al . , 2013; Allegretti et al . , 2014; Bartesaghi et al . , 2014; Wong et al . , 2014 ) , a level of detail that had previously been restricted to X-ray crystallography and NMR ( for a recent review of the technique , see Cheng et al . , 2015 ) .", "A limiting factor in the resolution of EM images of biological specimens is radiation damage because the imaging of such specimens ultimately relies on the interaction of electrons with the sample .", "Some of these interactions will result in energy being deposited in the specimen , and these will cause radiation damage ( Glaeser , 1971; Henderson , 1995 ) .", "The radiation damage fundamentally limits the information present in the images because the useful signal added per incident electron decreases with increasing electron exposure , while the added noise ( image contrast originating from other parts of the sample as well as inelastic scattering ) remains approximately constant .", "If the signal gain per unit exposure is known at a given resolution , an optimal exposure can be chosen that will maximize the signal-to-noise ratio ( SNR ) at that resolution ( Hayward and Glaeser , 1979; Baker et al . , 2010 ) .", "The rate of exposure-dependent signal decay has been measured by following the intensities of the fading diffraction spots in exposure series obtained from 2D and thin 3D crystals ( Unwin and Henderson , 1975; Hayward and Glaeser , 1979; Stark et al . , 1996; Baker et al . , 2010 ) .", "These studies demonstrated that higher resolution intensities tend to fade faster than lower resolution intensities , and that the rate of fading for all resolutions is slowed under liquid nitrogen conditions relative to room temperature conditions .", "The fading of the spots can be described by an exponential decay that is characterized by Ne , the resolution-dependent critical exposure after which spot intensities are reduced to 1/e of their starting values .", "To perform similar experiments with non-crystalline material , that is single particles , the resolution-dependent fading of the signal present in an image ( or average of images ) has to be determined .", "Unlike diffraction spots obtained from crystals , the signal in an image will give rise to signal amplitudes in the image Fourier transform .", "The squared Fourier amplitudes are equivalent to crystal diffraction intensities but will be affected by a number of additional factors , for example , the contrast transfer function ( CTF ) of the microscope and movement of the specimen , both attenuating the amplitudes .", "However , assuming a linear imaging system and constant amplitude-attenuating factors throughout an exposure series , the rate of fading of the signal in an image can be determined from the fading of the signal amplitudes in the image Fourier transform .", "While the fading of spots can be measured directly from diffraction patterns , the signal amplitudes calculated from an image are superimposed on the noise generated by the stochastic detection of electrons forming the image , as well as background due to the embedding ice and support film if present .", "Therefore , instead of measuring signal amplitudes directly , they have to be estimated from measurements of the SNR .", "Since the exposure , and therefore the noise , in each image within a series remains approximately constant , the SNR is directly proportional to the signal in each image .", "Analogous to the fading of diffraction spots ( Hayward and Glaeser , 1979 ) , the SNR of an image recorded after a prior exposure of N electrons/Å2 is therefore ( 1 ) SNR ( k , N ) =SNR ( k , 0 ) e−NNe ( k ) , where k is the spatial frequency , N is the accumulated exposure , and Ne is the resolution-dependent critical exposure defined above .", "We can see from Equation 1 that a plot of ln ( SNR ( k ) ) vs N should have a slope of −1/Ne ( k ) , and Baker et al . ( 2010 ) demonstrated that this relationship is true not just in the case of measurements of instantaneous signal , but also in the case of a detector that integrates measured intensities over time .", "The SNR of an image will continue to increase with electron exposures larger than the critical exposure .", "Hayward and Glaeser ( 1979 ) showed that the signal present in crystal diffraction spots is maximized at an optimal exposure of ∼2 . 5 times the critical exposure .", "The SNR increase per unit exposure is larger below the optimal exposure than the corresponding rate of SNR decay beyond the optimal exposure; thus , it is typically better to slightly overexpose a specimen than slightly underexpose .", "An exposure of 2 . 5 Ne is also expected to optimize the signal in images at a given resolution due to the analogy between image and crystal data as explained above .", "Despite studies of the general effects of radiation damage on single-particle specimens ( Conway et al . , 1993 ) , direct measurement of the critical exposure on non-crystalline specimens has proven difficult in the past for a number of reasons .", "Firstly , recording a series of images with increasing exposure would have led to images with SNRs so low that they would be difficult to analyze .", "Secondly , until recently the resolutions commonly obtained with the single-particle method were of insufficient quality to follow radiation damage at near-atomic resolution ( better than 4 Å ) .", "Finally , signal loss due to beam-induced specimen movement ( Brilot et al . , 2012; Campbell et al . , 2012; Li et al . , 2013; Scheres , 2014 ) was hard to quantify , and thus , the effects of radiation damage and movement could not be separated .", "The recent availability of direct electron detectors ( Milazzo et al . , 2005; Faruqi and Henderson , 2007; Li et al . , 2013 ) has largely alleviated these problems .", "The ability to record movies instead of single images allows for easy collection of a continuous exposure series , while allowing for measurement and correction of specimen movement ( Brilot et al . , 2012; Campbell et al . , 2012; Li et al . , 2013; Scheres , 2014 ) .", "Furthermore , the improved detective quantum efficiency of the detectors ( Ruskin et al . , 2013; McMullan et al . , 2014 ) has enhanced the signal present in the images , and this enhancement combined with motion correction has enabled single-particle reconstructions to reach higher resolutions .", "In this study , we used a K2 Summit direct detector ( Gatan , Inc . , Pleasanton , CA ) to collect a single particle data set of rotavirus double-layered particle ( DLP ) and estimate the critical exposure of a non-crystalline specimen .", "Rotavirus DLP is a spherical particle with a diameter of about 700 Å and a molecular mass of about 70 MDa ( Grigorieff and Harrison , 2011 ) .", "The capsid has icosahedral symmetry and consists predominantly of viral proteins VP2 and VP6 , forming an inner and outer layer , respectively .", "They have a combined mass of about 44 MDa and package 11 or 12 copies of viral proteins VP1 and VP3 , as well as 11 dsRNA segments that make up the viral genome ( Estrozi et al . , 2013 ) .", "VP6 assembles into 260 trimers that arrange in a T = 13 surface lattice containing a total of 780 VP6 monomers .", "The T = 13 lattice enables 13-fold averaging of density in addition to the 60-fold icosahedral symmetry .", "The large size of DLP and its high symmetry make it an ideal specimen for this study , yielding high-contrast particles with very low-alignment errors ( Henderson et al . , 2011 ) and almost a thousand averaged subunits per particle .", "The DLP data presented here are of sufficient quality to obtain sub-3-Å resolution reconstructions using just a few movie frames , enabling us to accurately measure the critical exposure over a large range of resolutions .", "To test the new filtering scheme , we also show its application to a recently published reconstruction of the Thermoplasma acidophilum 20S proteasome at 2 . 8 Å resolution ( Campbell et al . , 2015 ) , demonstrating optimization of SNR in the final reconstruction and improved alignment when the SNR in the images is very low ." ], [ "Examples of an aligned movie and particle images with different effective exposures are shown in Figure 1 .", "Processing exposure-filtered images ( see below ) of ∼4000 rotavirus particles and exploiting both their icosahedral and the 13-fold non-icosahedral symmetry of the outer layer led to a reconstruction of the VP6 trimer with clear density for side chains as well as the central Zn and Cl ions and a number of water molecules ( Figure 2 , Video 1 and 2 ) .", "At the estimated resolution of 2 . 6 Å ( Figure 3A ) , atomic model building is possible ( Grigorieff and Harrison , 2011 ) and an atomic model obtained by refinement of a previously published structure ( Mathieu et al . , 2001 ) agrees well with the starting model as indicated by an RMSD of 0 . 4 Å .", "Despite good overall agreement , there are a number of significant differences , localized mainly to areas on the periphery of the molecule , which may indicate effects of crystal packing and/or the presence of additional protein interactions in the full capsid as opposed to the isolated VP6 subunit . 10 . 7554/eLife . 06980 . 003Figure 1 . ( A ) Example of an aligned movie sum of the rotavirus double-layered particle ( DLP ) sample imaged with a total exposure of 100 e−/Å2 . ( B ) Particle sum created using all frames and ( C ) the first 3 frames of the movie , demonstrating the kind of data used in the analysis .", "In all cases , the scale bar represents 500 Å .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06980 . 00310 . 7554/eLife . 06980 . 004Figure 2 . ( A ) Density of an isolated VP6 subunit is shown as a mesh along with the docked atomic model . The model is colored using a rainbow spectrum , starting with the N-terminus in blue and ending with the C-terminus in red .", "( B ) Zoomed region of the VP6 subunit .", "( C ) At higher thresholds , small density features become visible that we interpret as water molecules because their locations are very close to water molecules found in the VP6 crystal structure ( Mathieu et al . , 2001 ) .", "A B-factor of −175 Å2 was applied to the DLP reconstruction before 13-fold non-icosahedral averaging to sharpen the VP6 map .", "In all cases , the scale bar represents 10 Å .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06980 . 00410 . 7554/eLife . 06980 . 010Video 1 . Density of an isolated VP6 subunit is shown as a mesh along with the docked atomic model ( see also Figure 2A ) .", "The model is colored using a rainbow spectrum , starting with the N-terminus in blue and ending with the C-terminus in red . DOI: http://dx . doi . org/10 . 7554/eLife . 06980 . 01010 . 7554/eLife . 06980 . 011Video 2 . Zoomed region of the VP6 subunit showing two β-sheet strands ( see also Figure 2B ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06980 . 01110 . 7554/eLife . 06980 . 005Figure 3 . ( A ) Fourier Shell Correlation ( FSC ) curves estimating the resolution of the final VP6 reconstruction calculated using exposure-filtered data . Two FSC curves were obtained , one from maps calculated from two halves of the data set , another using the modeled atomic coordinates .", "The dashed green line represents 15 Å , the upper resolution limit used during parameter refinement .", "( B ) FSC curves between half data set reconstructions of the VP6 subunit when using an exposure-filtered sum of frames 4–130 , an unfiltered sum of frames 4–130 , and an unfiltered sum of frames 4–21 , which were determined to be the best set of unfiltered frames by trial and error .", "Exposure filtering was applied only to the final reconstruction , and not during refinement ( C ) FSC curve for the reconstruction using only frames 25–27 , indicating a resolution of ∼3 . 4 Å after a pre-exposure of ∼19 e−/Å2 . DOI: http://dx . doi . org/10 . 7554/eLife . 06980 . 005 Calculating the Fourier Shell Correlation ( FSC ) ( van Heel and Harauz , 1986 ) between the VP6 map and the refined atomic model demonstrated a resolution of ∼2 . 7 Å ( Figure 3A ) .", "One possibility for the difference between this and the ∼2 . 6 Å obtained with the half map is the absence of full density for the most radiation-sensitive side chains in the map , while they are present in the model .", "Assigning higher atomic temperature factors to side chains with weak density may accommodate these discrepancies , but we did not explore this in our model refinement .", "A complication of using images for the calculation of critical exposure as opposed to diffraction intensities is the beam-induced movement of the specimen .", "Previous studies have demonstrated that the magnitude of this movement is not constant across a single exposure , typically being greater at the beginning of the exposure than at the end ( Brilot et al . , 2012; Campbell et al . , 2012; Li et al . , 2013 ) .", "Movement of a particle over the course of the exposure will lead to signal degradation , and if the movement is not constant across the entire exposure , it will lead to an exposure-dependent signal degradation unrelated to radiation damage .", "Non-constant movement across an exposure will therefore interfere with the critical exposure measurement and must be taken into account .", "In order to do this , we estimated per-frame shifts for each particle using a new movie alignment program called Unblur ( see ‘Materials and methods’ ) .", "During subsequent refinement ( see below ) , we also estimated particle rotations by aligning 3-frame sums ( see below ) .", "On average , particles rotate by ∼0 . 9° over the course of a movie , or an average of 0 . 02° per three frames .", "A rotation of 0 . 02° causes a translation of subunits on the surface of DLP of about 0 . 12 Å , too small to be relevant for our analysis of the fading signal .", "Movies were recorded such that the specimen was exposed to a total exposure of 100 electrons/Å2 , split over 130 movie frames ( 0 . 77 e−/Å2 per frame ) .", "By the 28th frame ( an accumulated exposure of 21 e−/Å2 ) , the average particle shift per frame had fallen to 0 . 2 Å , with 95% of particles shifting by less than 0 . 54 Å per frame .", "Between the 28th frame and the 130th frame , the average particle shift per frame remained constant at 0 . 2 Å , and 95% of the measured particle shift stayed between 0 . 4 Å and 0 . 54 Å per frame .", "Because the movement was almost constant between frames 28–130 , we do not expect differences in their signal to be significantly affected by translational movement , and for this reason , only frames 28–130 were used during the analysis .", "Movie frames for each particle were aligned individually , but in order to provide more signal , reconstructions were calculated from sums of every 3 movie frames ( 2 . 31 e−/Å2 per sum ) .", "Each of these reconstructions of the VP6 trimer was used to calculate an FSC curve ( e . g . , Figure 3C ) for the corresponding accumulated exposure .", "These FSC curves were used to estimate the SNR of a resolution shell ( k ) ( Grigorieff , 2000 ) : ( 2 ) SNR ( k ) =2FSC ( k ) 1−FSC ( k ) .", "Thus , using the measured FSC curves and Equation 2 , we were able to plot ln ( SNR ) for each resolution shell as a function of exposure .", "For each plot , we performed linear regression using the points from frame 28 up to the point at which ln ( SNR ) fell below the 3σ value expected from pure noise .", "Figure 4 shows the plots and regressions found for a number of different resolutions . 10 . 7554/eLife . 06980 . 006Figure 4 . Example plots of ln ( SNR ) vs accumulated exposure with associated linear fits at a number of different resolutions . Data used in this study are shown in darker color , while data for early frames excluded from the analysis due to specimen movement are shown in lighter color .", "The slopes of the lines become steeper as the resolution increases , corresponding to faster fading of the signal .", "The linear plots fit the data well , suggesting that in the analyzed regions a single-exponential process is dominant in the decay . DOI: http://dx . doi . org/10 . 7554/eLife . 06980 . 006 Good quality regressions were found for data from ∼22 Å to ∼3 . 8 Å , and the critical exposure values derived from these regressions are plotted in Figure 5A .", "At resolutions higher than 3 . 8 Å , there were too few points ( estimated SNR values ) for reliable fitting .", "At resolutions below 22 Å , the curves became much noisier resulting in poor quality fits .", "This may be due to the fact that at resolutions below 22 Å , errors due to inelastic scattering become more significant , and the FSC values at these spatial frequencies are so high ( typically greater than 0 . 999 ) that small errors lead to large changes in the estimated SNR .", "Additionally , our VP6 reconstruction has only 4 resolution shells at resolutions lower than 22 Å ( nominally at ∼27 Å , ∼37 Å , 55 Å , and 110 Å ) .", "These low-resolution shells thus effectively contain information from a wide range of frequencies , which may also cause errors in the estimation . 10 . 7554/eLife . 06980 . 007Figure 5 . ( A ) The measured critical exposure curve plotting the critical exposure at each measured resolution and the fit function for comparison .", "( B ) Curve plotting the optimal exposure obtained in this study alongside that obtained in a previous study on crystalline specimens , scaled by a factor of 1 . 25 to compensate for the fact that the previous study was conducted at 200 kV ( Baker et al . , 2010 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06980 . 007 We could fit a function to the recorded data , which takes the form ( 3 ) y=axb+c .", "The best fit to the data , with a R2 value of 0 . 997 , was a = 0 . 245 , b = −1 . 665 , and c = 2 . 81 , plotted in Figure 5A together with the experimental data .", "This function can be used to estimate the critical exposure at 300 kV; the critical exposure at 200 kV can be expected to be about 25% lower ( Yalcin et al . , 2006 ) .", "The optimal exposure , plotted in Figure 5B , is ∼2 . 5 times the critical exposure ( Hayward and Glaeser , 1979 ) .", "At higher spatial frequencies ( ∼4 Å ) , our values agree well with values previously measured on crystalline specimens ( Figure 5B , Baker et al . , 2010 ) , but they deviate toward lower spatial frequencies , suggesting that the signal at these frequencies in single-particle images is less sensitive to electron exposure than in images of crystalline specimens .", "A possible explanation is that when imaging crystalline specimens , there are two modes of damage , a short-range component caused by damage to the molecules themselves and a long-range component caused by loss of crystalline order .", "We would expect single particles to be affected only by the former and so appear to be less radiation sensitive .", "Rotavirus DLP may also be affected by lattice distortions because the surface lattice formed by the VP6 trimers may be considered analogous to the lattice of a small crystal .", "However , the extent to which deformations will attenuate signal will depend on the size of the lattice: small deformations can add up to larger unit cell displacements across larger distances compared to displacements across smaller distances .", "The crystals used for electron diffraction typically measure 1 μm or more in diameter ( Unwin and Henderson , 1975 ) , at least 5 times larger than the circumference of DLP .", "Furthermore , due to their geometry , 2D and thin 3D crystals are prone to in-plane and out-of-plane distortions , both of which can severely attenuate diffraction intensities .", "It is therefore essential for crystal diffraction studies to use a flat support film , usually amorphous carbon .", "Studies of beam-induced motion have shown that the observed motion in cryo-EM specimens involves both the ice and the carbon support ( Glaser et al . , 2011; Brilot et al . , 2012 ) .", "Some of the beam-induced motion may lead to distortions of the carbon support , causing long-range distortions in the crystals .", "Beam-induced distortions of the ice layer embedding the DLP particles may also perturb the particle structure .", "However , the tight packing of the capsids around the RNA genome is likely to yield a particle that is more resistant to distortions than the crystals .", "We therefore expect that , at least in terms of the structural properties relevant for this study , DLP is much closer to a single particle than a thin crystal .", "If the icosahedral surface lattice of DLP is also affected by lattice distortions due to radiation damage , we would expect the critical exposure for smaller , less symmetric samples to be higher still .", "A further difference to the crystal studies is that they used the fastest fading intensities in each resolution bin for their analysis ( Unwin and Henderson , 1975; Hayward and Glaeser , 1979; Stark et al . , 1996; Baker et al . , 2010 ) .", "With a single-particle specimen , we are limited to measuring the average loss of SNR in each resolution bin , which by definition is lower than the fastest fading components .", "However , the difference between the average and fastest fading components does not appear to be enough to explain the observed differences .", "Other factors may include the chemical makeup of the specimen , such as the presence of nucleic acids in DLP , as well as buffer and solvent components .", "However , the effect of these factors on radiation damage is not well understood .", "The first 27 frames of the movie were excluded from the analysis so as to prevent beam-induced movement from interfering with the results .", "Our results are therefore based upon data , which have effectively been pre-exposed by ∼20 e−/Å2 .", "If radiation damage indeed occurs as a single-exponential process , pre-exposure should have no effect on the result presented here .", "We cannot exclude the possibility that the damage may occur in two ( or more ) phases and that we are only observing the final phase , but there is no evidence for a multi-phased process , and previous studies on crystalline specimens also demonstrate a single-exponential decay .", "Moreover , for lower resolutions at which beam-induced movement is expected to have less of an effect , a plot of ln ( SNR ) vs accumulated exposure across all frames shows a single-exponential process ( see Figure 4 ) .", "We also note that a reconstruction calculated using only frames 25–27 ( ∼19 e−/Å2–∼21 e−/Å2 ) still yields a 3 . 35 Å resolution reconstruction with clear side chain density ( see Figure 6 ) , suggesting that the decay we have measured is relevant to the kinds of structural information we are interested in .", "As expected , the density of side chains fades with increasing exposure and dose deposited on the sample .", "The density of carboxyl groups ( e . g . , Asp29 , Figure 6 ) fades most rapidly and is already partially gone after an exposure of about 3 e−/Å2 , while those of aromatic groups ( e . g . , Tyr24 , Figure 6 ) persists even after an exposure of about 35–40 e−/Å2 .", "This general pattern agrees with that observed before in a number of cryo-EM ( Allegretti et al . , 2014; Bartesaghi et al . , 2014; Fromm et al . , 2015 ) and X-ray crystallography studies ( Fioravanti et al . , 2007 ) .", "We note also that the density of the α-helix backbone remains visible even after an exposure of ∼50 e−/Å2 .", "This high exposure exceeds the optimal exposure of about 30 e−/Å2 at 8 Å resolution where α-helical features become clearly visible .", "Such high tolerance of secondary structural features to radiation damage may help in the alignment of single-particle images recorded using high exposures ( see below ) . 10 . 7554/eLife . 06980 . 008Figure 6 . Surface rendering of an isolated small helix from different 3-frame reconstructions shown with the docked model . Each reconstruction is shown with its exposure range and resolution as calculated from the FSC using the 0 . 143 cut-off . DOI: http://dx . doi . org/10 . 7554/eLife . 06980 . 008 When using a detector , which outputs single images , the optimal exposure curve ( Figure 5B ) can be used to select the optimal exposure based on a targeted resolution , although this exposure will not be optimal for other resolutions .", "However , as has been suggested before ( Baker et al . , 2010; Campbell et al . , 2012; Scheres , 2014 ) , given a detector capable of recording movies , the optimal exposure curve can be used to filter frames based on their individual exposures .", "Filtering the frames in this way will result in a sum with an increased SNR relative to the unfiltered sum .", "Equation 1 describes the attenuation of the image SNR caused by radiation damage with increasing exposure .", "The attenuation of the SNR is the result of fading image Fourier amplitudes ( A ) , which will follow the square root of the exponential decay of the SNR: ( 4 ) A ( k , N ) =A ( k , 0 ) e−N2Ne ( k ) .", "The initial value of A at N = 0 is usually not known but is not needed for optimal exposure filtering; only relative differences are significant ( see below ) .", "Thus , the exposure-dependent amplitude attenuation is given by ( 5 ) q ( k , N ) =e−N2Ne ( k ) .", "Previous work has described the optimal 3D reconstruction from images , which have had their amplitudes attenuated by the CTF ( Sindelar and Grigorieff , 2012 ) .", "We can easily extend this formalism to take into account both the CTF and the amplitude attenuation due to radiation damage: ( 6 ) F3D ( k ) =∑i=1nCTFi∗ ( k ) q ( k , Ni ) Fi ( k ) ∑i=1n ( |CTFi ( k ) |q ( k , Ni ) ) 2+1/SNR ( k ) .", "Here , the sum over i includes all frames of all movies contributing to Fourier coordinate k , F3D is the 3D reconstruction Fourier transform , while Fi represents the measured image Fourier terms .", "Ni is the accumulated exposure of the frame , and SNR ( k ) is the average unattenuated SNR in the area of the particle within a single frame ( the ‘particle spectral SNR’ , or PSSNR in [Sindelar and Grigorieff , 2012] ) .", "While Equation 6 describes an optimal correction when 3D reconstructions are calculated from individual movie frames , it is often more practical to calculate a filtered sum of the frames for each aligned movie or individual particle .", "This sum can then be used for further processing without further consideration of the movie frames .", "In this case , for simplicity , we assume that the defocus does not change and thus disregard the CTF term , leaving it to be taken into account in later processing steps .", "The filtered 2D image FW is then given by ( 7 ) FW ( k ) =∑i=1nq ( k , Ni ) Fi ( k ) ∑i=1nq2 ( k , Ni ) +1/SNR ( k ) .", "SNR ( k ) is not known , but we can assume it to be very small for a single frame of a movie due to the small exposure .", "Its reciprocal value is therefore large compared to the first term in the denominator , which can be neglected , and thus ( 8 ) FW ( k ) ≈SNR ( k ) ∑i=1nq ( k , Ni ) Fi ( k ) .", "SNR ( k ) is an average across a resolution shell , and thus , the term serves to weight resolution shells with respect to each other to maximize the SNR of the final image .", "This will generally result in a low-pass filtering of the sums and may affect later processing steps .", "Furthermore , an estimate of the SNR is usually not available until later stages of processing .", "Thus , to minimize the alteration of features in the original images by the exposure filter , we formulate the filter as a weighted sum that maximizes the SNR in each resolution shell: ( 9 ) F˜W ( k ) =∑i=1nq ( k , Ni ) Fi ( k ) ∑i=1nq ( k , Ni ) 2 .", "Exposure filtering using the above scheme , combined with the measured critical exposure curve , has been integrated into the new Unblur program ( see ‘Materials and methods’ ) allowing the production of aligned and exposure-filtered movies .", "Increasing the SNR of the movie sum should increase the accuracy of particle alignments leading to improved reconstructions , particularly in the most difficult cases such as very small proteins .", "Furthermore , correct filtering of movie frames allows the use of all later frames in the reconstruction with no need to test the inclusion of different numbers of frames in order to find an optimum .", "In order to assess the effectiveness of applying the exposure filter , we first used trial and error to manually determine which range of frames , when summed , yielded the best VP6 reconstruction .", "The best reconstruction was found when using frames 4–21 .", "Separately , we calculated a reconstruction using frames 4–130 ( i . e . , to the final frame of the movie ) , weighted with the exposure filter .", "The two reconstructions are nearly identical as judged by the FSC and are both of better quality than a reconstruction using the unfiltered sum of frames 4–130 , suggesting that the exposure filter is weighting correctly ( see Figure 3B ) .", "While the reconstructions calculated using exposure filtering and by selecting frames 4–21 have the same resolution , the exposure-filtered reconstruction visually appears slightly less sharp .", "This higher B-factor is to be expected due to the inclusion of comparatively stronger lower resolution signal in the exposure-filtered sums when compared to the simple sum of frames 4–21 .", "After scaling the amplitudes to be the same for both reconstructions using diffmap ( http://grigoriefflab . janelia . org/diffmap ) , the two reconstructions are indistinguishable .", "In the case of DLPs , which exhibit extremely low-alignment error using traditional processing ( Henderson et al . , 2011 ) , increasing the SNR at low and intermediate resolutions would not be expected to lead to improved alignment .", "However , additional resolution gains can be expected for smaller particles with a lower molecular mass , when using exposure-filtered particle sums ( see below ) .", "Using recently published data to calculate a 2 . 8 Å reconstruction of the 20S proteasome , a 700 kDa complex with D7 symmetry ( Campbell et al . , 2015 ) , we applied the exposure filter described above to test if it also optimizes the reconstruction of a smaller , less symmetrical particle .", "Movies of 20S proteasome consisted of 38 frames collected on a Gatan K2 detector using a total exposure of 53 e−/Å2 ( ∼1 . 4 e−/Å2 per frame ) .", "Using the frame alignment obtained by Campbell et al . , we calculated four different frame sums: using the exposure filter , unfiltered sums of the first 9 frames ( corresponding to an effective exposure of 12 . 6 e−/Å2 ) , the first 14 frames ( corresponding to an effective exposure of 19 . 6 e−/Å2 ) , and all frames .", "An exposure of 12 . 6 e−/Å2 approximates the optimal exposure at ∼3 Å resolution measured using rotavirus DLP , while an exposure of ∼20 e−/Å2 is close to exposures used in previous high-resolution cryo-EM studies ( Zhang et al . , 2008; Wolf et al . , 2010 ) that were published prior to the use of direct detectors and movies .", "The same particles selected and used by Campbell et al . ( 49 , 954 particles ) were extracted from these four sets of micrographs , aligned against an initial model obtained from class averages ( see ‘Materials and methods’ ) and refined using Frealign v9 ( Lyumkis et al . , 2013 ) .", "A comparison between the previously published structure and our structure ( Figure 7A ) suggests that the exposure filter described here and the B-factor weighting implemented in Relion ( Scheres , 2014; see below ) and used by Campbell et al . perform equally well .", "Figure 7B shows FSC curves for all four test cases , indicating a resolution of 2 . 8 Å for all reconstructions except the one calculated from unfiltered frame sums that included all 38 frames .", "The lower resolution of this last reconstruction ( about 3 Å ) is expected due to the effective total exposure of 53 e−/Å2 , which exceeds the optimal exposure at 3 Å by a factor of 4 .", "The optimal exposure is also exceeded in the case of 19 . 6 e−/Å2 , albeit only by a factor of 1 . 5 , which does not appear to affect the resolution of the reconstruction significantly .", "To better follow the effectiveness of the exposure filter , we also plotted the average spectral SNR of the filtered particle images ( PSSNR , see above ) .", "Figure 7C shows that the SNR of the exposure-filtered sums of all frames equals or exceeds that of the other frame sums at all resolutions .", "Without exposure filtering , summing all 38 frames produces SNR values that are similar to the filtered sums at low resolution but are significantly lower as the resolution increases .", "At 3 Å resolution , the average SNR in the unfiltered sums is only about 30% of that of the filtered sums .", "The PSSNR curves estimated for the unfiltered sums , calculated from the first 9 and 14 movie frames , start out lower at low resolution than the other two curves as expected since they are missing the additional exposure to boosts the low-resolution signal .", "At 3 Å resolution , both curves converge with the curve calculated form the filtered sums using all 38 frames .", "Based on the effective radiation damage reflected in the data , we would expect the PSSNR at high resolution to be highest for the exposure-filtered sums , followed by the unfiltered sums corresponding to 12 . 6 e−/Å2 , 19 . 6 e−/Å2 , and 53 e−/Å2 .", "However , in practice , the estimates are subject to some error due to the weak signal at high resolution and the fact that all data sets were independently refined from low resolution ( see ‘Materials and methods’ ) .", "Therefore , only the data set with the most significant signal attenuation ( sums of 38 unfiltered frames ) deviates significantly from the rest , and the remaining three curves indicate very similar SNR values . 10 . 7554/eLife . 06980 . 009Figure 7 . ( A ) Comparison of an isolated helix from the previously published reconstruction ( Campbell , 2015 ) on the left , and the reconstruction using exposure-filtered data on the right . The two maps appear almost identical after scaling the amplitudes using diffmap ( http://grigoriefflab . janelia . org/diffmap ) , suggesting that in this case exposure filtering performs as well as the weighting based on B-factors implemented in Relion ( Scheres , 2014 ) .", "( B ) Plot of FSC curves for the various proteasome reconstructions .", "The exposure-filtered reconstruction has a resolution of ∼2 . 8 Å , matching the resolution previously obtained .", "( C ) Plot of the average particle signal-to-noise ratio ( SNR ) as a function of resolution .", "The exposure-filtered particles have equal or higher SNR than the other data sets at all resolutions .", "( D ) Plot of FSC curves from the signal-limited data set .", "In this case , the exposure-filtered reconstruction is of better quality than those derived from the other data sets .", "Recalculating the non-filtered reconstructions with particle alignment parameters obtained for the exposure-filtered data set increases the resolution to that of the filtered data set ( curves labeled ‘EF Alignment’ ) , demonstrating that the loss in resolution was due to particle misalignments . DOI: http://dx . doi . org/10 . 7554/eLife . 06980 . 009 The higher SNR present in exposure-filtered frame sums , compared with unfiltered sums containing a lower total exposure , is expected to help in the alignment of smaller particles where the alignment accuracy is significantly limited by the signal generated by the particle .", "The benefit of a higher total exposure is not apparent in the alignment of rotavirus DLP because the alignment errors of all tested frame sums is extremely small .", "Even the resolution of the reconstruction of the 20S proteasome with a total mass of 700 kDa does not appear to be limited by alignment errors at exposures of 12 . 6 e−/Å2 and higher ( see previous section ) .", "To demonstrate improved alignment at higher total exposures when the signal is weak , we generated additional data sets by adding images of areas of empty ice to the proteasome particle images .", "The ice images were taken from the same micrographs as the particles they were added to , and they contained the same number of frames ( total exposure ) and filtering .", "The ice images add approximately the same amount of noise and background that is also present in the particle images , thus , reducing the effective SNR in the resulting images approximately by 50% .", "At low resolution ( special frequencies larger than 1/10 Å−1 ) , the new data sets should therefore emulate data that would have been obtained by a particle of half the mass , that is , 350 kDa , while at higher resolution ( special frequencies smaller than 1/10 Å−1 ) the data should approximate the signal expected from a 175 kDa particle , that is , with a quarter of the mass ( Rosenthal and Henderson , 2003 ) .", "Figure 7D shows the results of the processing of three data sets with added ice background , derived from exposure-filtered sums of 38 frames , as well as unfiltered sums of the first 9 and 14 frames ( total exposures of 12 . 6 e−/Å2 , 19 . 6 e−/Å2 , respectively ) .", "Unlike the reconstruction calculated from the exposure-filtered original data ( see above ) , the reconstruction calculated form the exposure-filtered images with added noise displays a significantly improved FSC curve compared with the other two noise-added data sets .", "To test if the improved resolution with exposure filtering is due to the improved alignment of the images , we also calculated reconstructions from the noise-added data sets with total exposures of 12 . 6 e−/Å2 , 19 . 6 e−/Å2 using the alignment parameters obtained from the exposure-filtered data .", "As shown in Figure 7D , this parameter replacement increases the resolution for the two unfiltered reconstructions to that of the filtered reconstruction , similar to the results obtained for the original data sets in Figure 7B .", "The superior resolution of the exposure-filtered reconstruction is therefore due to more accurate particle alignments .", "High-quality data sets of smaller particles ( about 300 kDa or less ) collected using 50 to 100 e−/Å2 exposures are currently not available to demonstrate the improved particle alignment more directly with experimental data .", "However , the alignment accuracy of these particles is expected to be limited by weaker contrast ( Henderson , 1995 ) , and we expect improvements similar to those seen in our simulation when using exposures that significantly exceed the optimal exposure and applying the exposure filter described here ." ], [ "The optimal exposure curve we determined should correct well for radiation damage to the specimen , which has a substantial effect on the relative signal content of individual movie frames , in particular the later frames .", "As discussed earlier , the other major source of frame-dependent signal change is motion of the specimen , which tends to degrade the signal of earlier frames .", "For optimal filtering of the data , movement should also be included in the filtering , as should any other effects that change the relative signal in different frames .", "Currently , the exposure filter does not take movement into account and so will not filter the initial frames optimally .", "We plan to incorporate movement in a future version of the algorithm .", "An alternative method for frame filtering has recently been described ( Scheres , 2014 ) .", "This methodology uses the data set itself to estimate per frame weights based on fitting relative B-factors to reconstructions from individual frames .", "Although this is an elegant solution , which in the best case handles weighting of both radiation damage and movement , filtering with the exposure curve described in this paper should offer a number of advantages , at least in terms of filtering radiation damage .", "First , our filter can be applied at the beginning of processing without assessing the data , and thus , can help in the initial steps of picking particles and finding initial alignment and orientation parameters .", "Indeed , one could use the two methods in combination , by using the exposure filter described here for the initial refinement , then when a good quality reconstruction has been obtained , using frame filtering based directly on the data .", "Second , for some data , the estimate for the B-factor to be applied to each frame may be quite noisy and may lead to error , a problem , which is likely to be exacerbated with smaller data sets .", "Furthermore , accurate estimation of the B-factor relies on high-frequency signal and will thus fail on intermediate- and low-resolution structures .", "Third , even in situations in which a B-factor can be estimated , it is unclear how well a B-factor estimated using a specific resolution range will describe signal degradation due to radiation damage outside that range .", "In contrast , our calculated optimal exposure curve describes experimentally determined values at a wide range of resolutions .", "The tests carried out with the previously published 20S proteasome data ( see above ) show that the filtering is indeed close to optimal , not only for rotavirus DLP but also for smaller and less symmetrical particles , and that its performance matches that of B-factor weighting .", "Finally , exposure filtering can also be applied when B-factors are difficult to determine , such as in electron tomography .", "Using the filter described here , images collected at different specimen tilts can be filtered to optimize the final SNR and resolution in a tomogram .", "While a similar filtering could also be achieved using B-factors , the relatively low resolution of a tomogram and the presence of structural heterogeneity would likely prevent the determination of appropriate B-factors .", "The results we present here indicate that the electron exposures commonly used in cryo-EM experiments ( ∼10–20 e−/Å2 ) should be increased to obtain optimal image contrast .", "By using a considerably larger total exposure , the SNR of the images at intermediate and low resolutions will be increased , leading to greater accuracy in particle alignment and orientation determination in cases where the accuracy is limited by the signal , which will in turn lead to better reconstructions .", "This should especially be true for smaller particles where alignment errors can severely limit the attainable resolution ( Henderson et al . , 2011 ) ." ], [ "Rotavirus DLPs were prepared as previously described ( Street et al . , 1982 ) .", "Three microliters of sample with a concentration of 2 . 5–4 mg/ml was applied to C-flat 1 . 2/1 . 3 Cu 400 mesh grids ( Protochips , Raleigh , NC ) , which had previously been subjected to glow discharge for 45 s at 20 mA , and plunge-frozen using an FEI Vitrobot Mark 2 ( FEI Company , Hillsboro , OR ) with a 4 or 6 s blot time and at relative humidity between 65 and 80% .", "The data were collected on an FEI Krios microscope ( FEI Company , Hillsboro , OR ) operating at 300 kV .", "Movies were collected on a Gatan K2 Summit direct electron detector ( Gatan , Inc . , Pleasanton , CA ) in super resolution mode with a calibrated pixel size of 0 . 512 Å per super resolution pixel .", "The pixel size was calibrated by maximizing the cross correlation between the whole DLP reconstruction and a 3 . 8 Å crystal structure of the entire DLP ( McClain et al . , 2010 ) .", "Each exposure was 13 s long and recorded as a movie of 130 frames .", "The exposure per frame as reported by Digital Micrograph ( Gatan , Inc . , Pleasanton , CA ) was 0 . 769 e−/Å2 , which corresponds to an exposure of 8 electrons/pixel/s on the camera .", "Movies were collected at a range of underfocus between ∼0 . 4 μm and ∼2 . 0 μm .", "Throughout the data collection , the exposure per movie was checked regularly to make sure it hadn't deviated from a total exposure of 100 e−/Å2 .", "Super-resolution movie frames were initially corrected for a magnification distortion present on our FEI Titan Krios microscope by real space stretching using bilinear interpolation .", "The frames were then downsampled by a factor of 2 using Fourier cropping to a pixel size of 1 . 023 Å .", "The downsampled frames were motion-corrected using newly written software called Unblur ( Supplementary file 1 ) .", "Unblur is stand-alone software available for download from the Grigorieff lab web page ( http://grigoriefflab . janelia . org/unblur ) and is based on iterative alignments of each raw frame to the current best total sum of all other frames , leaving the frame which is currently being aligned out of the total sum to avoid the frame ‘finding itself’ in the sum .", "After each iteration , a spline is fit to both the X and Y shifts to reduce sensitivity to noise .", "Frame sums can be filtered according to the described exposure filter , or the filtering step can be skipped .", "Frame sums of already aligned frames can be recalculated using the program Summovie ( Supplementary file 2 ) , which is also available for download from the Grigorieff lab web page ( http://grigoriefflab . janelia . org/unblur ) .", "The filter constants ( Equation 3 ) are not user accessible but can easily be changed in the source code if needed .", "4178 DLP particles were picked manually from the aligned movie sums , and extracted into 1024 × 1024 boxes .", "Filtered amplitude spectra were calculated for each particle and were used to estimate the defocus and astigmatism values on a per-particle basis using the FindCTF program of the TIGRIS package ( http://tigris . sourceforge . net/ ) .", "TIGRIS consists of a set of programs for single-particle image analysis , including algorithms for CTF determination and correction , image alignment and classification , 3D reconstruction and a fully-featured display .", "FindCTF attempts to determine the optimal defocus parameters by maximizing the correlation between an amplitude spectra and a theoretical CTF , first performing a brute-force parameter search followed by downhill simplex optimization .", "Particles were then individually motion corrected using the Unblur algorithm on particles boxed from individual frames .", "An initial model was calculated from a single DLP image using Angular-Reconstitution in the IMAGIC package ( van Heel et al . , 1996 ) .", "Initial parameters were obtained on images resampled to 8 Å per pixel via Fourier cropping , which were aligned to the initial model with the brute force alignment program of the TIGRIS package .", "This alignment finds the highest cross-correlation peak across a specified set of in-plane rotations ( in this case every 1° ) of reference projections ( in this case projections of the model sampled at every 5° ) .", "These parameters were further refined using Frealign v9 ( Lyumkis et al . , 2013 ) using data sampled at 1 . 023 Å per pixel , and using information between 200 Å and 15 Å .", "In order to increase the signal present in the raw frames , 3-frame sums of the motion corrected particles were calculated , and these sub-sums were individually refined using Frealign again using data between 200 Å and 15 Å .", "Individual reconstructions were calculated from each set of 3 frame sums , providing 43 individual DLP reconstructions at increasing exposure .", "Frealign outputs two half map reconstructions used for calculating an FSC curve .", "For the two half maps of each of the 43 reconstructions , additional 13-fold averaging of the VP6 trimer was performed using the AVE program ( Kleywegt and Read , 1997 ) and matrices derived from the asymmetric unit of the previously published 3 . 8 Å crystal structure of the DLP ( McClain et al . , 2010 ) .", "The resulting VP6 half maps were masked with a soft-shaped mask and used to calculate FSC curves , resulting in 43 curves ( e . g . , Figure 3C ) each corresponding to a different exposure , which were used for the critical exposure estimation .", "In order to obtain the highest resolution reconstruction , subsets of frames were manually selected and a trial and error approach resulted in the determination that refinement using the sum of frames 4–21 and again the resolution range of 200 Å to 15 Å resulted in the highest resolution reconstruction as determined by the FSC between two half maps masked with a soft-shaped mask .", "The resulting resolution as determined by the 0 . 143 cut-off ( Rosenthal and Henderson , 2003 ) was 2 . 59 Å ( Figure 2A ) .", "Maps were rendered using UCSF Chimera ( Pettersen et al . , 2004 ) .", "To render specific sections of a map , a zone including specified amino acid residues within the fitted atomic model was defined , and the only densities within a radius of 3 Å of the zone were displayed ( command ‘zone’ in Chimera ) .", "Furthermore , disconnected density was removed from the display ( command ‘hide dust’ in Chimera ) in maps shown in Figures 2A , 6 ( except for the first panel ) , and Figure 7A .", "An initial fit of a previously determined crystal structure ( Mathieu et al . , 2001 ) fit well for the majority of the structure; however , it was clear that some regions needed adjustment , and therefore , real-space refinement was carried out in Coot ( Emsley et al . , 2010 ) exploiting all restraints .", "As a further validation , the resulting model was converted into a density map using the UCSF Chimera package ( Pettersen et al . , 2004 ) specifying 2 . 5 Å resolution , and an FSC curve between this density map and the optimum reconstruction was calculated ( Figure 2A ) .", "The resolution as determined by the 0 . 5 cut-off ( Rosenthal and Henderson , 2003 ) was 2 . 72 Å .", "Aligned micrographs were downloaded from the EMPIAR database ( EMPIAR-10023 ) , particle locations , and estimated defocus parameters corresponding to the best published reconstruction ( Campbell et al . , 2015 ) were kindly provided by the authors .", "Particle sums corresponding to 12 . 6 e−/Å2 , 19 . 6 e−/Å2 , and the total sum of 53 e−/Å2 were calculated , along with particle sums for the whole 53 e−/Å2 weighted with the exposure filter .", "Signal-limited particle sums were created as follows .", "First an area devoid of particles was selected from each movie sum .", "These were then extracted from movie sums corresponding to 12 . 6 e−/Å2 , 19 . 6 e−/Å2 , and 53 e−/Å2 weighted with the exposure filter .", "Particle sums corresponding to each of these total exposures were then added to the empty area corresponding to the movie sum they were extracted from .", "Class averages were generated using MSA within IMAGIC and the exposure-filtered particles .", "∼10 of these class averages were manually selected and given as input to e2initialmodel . py from the EMAN2 package ( Tang et al . , 2007 ) to generate an initial model .", "This initial model was used as a starting model for all subsequent refinements .", "Processing for all data sets followed the following procedure .", "Particles sampled to a 5 . 3 Å pixel size were aligned to the initial model using the TIGRIS brute-force alignment program .", "These initial parameters were then refined using Frealign , starting with a resolution cut-off of 10 Å and gradually increasing to a resolution cut-off of 5 Å for the final rounds .", "For all reconstructions , FSCs were calculated between the Frealign output half maps after first masking with a soft-shaped mask ." ] ]

[ "Biological specimens suffer radiation damage when imaged in an electron microscope , ultimately limiting the attainable resolution .", "At a given resolution , an optimal exposure can be defined that maximizes the signal-to-noise ratio in the image .", "Using a 2 . 6 Å resolution single particle cryo-EM reconstruction of rotavirus VP6 , determined from movies recorded with a total exposure of 100 electrons/Å2 , we obtained accurate measurements of optimal exposure values over a wide range of resolutions .", "At low and intermediate resolutions , our measured values are considerably higher than obtained previously for crystalline specimens , indicating that both images and movies should be collected with higher exposures than are generally used .", "We demonstrate a method of using our optimal exposure values to filter movie frames , yielding images with improved contrast that lead to higher resolution reconstructions .", "This ‘high-exposure’ technique should benefit cryo-EM work on all types of samples , especially those of relatively low-molecular mass ." ]

[ "Microscopes allow us to visualize objects that are invisible to the naked eye .", "One type of microscope—called the electron microscope—produces images using beams of particles known as electrons , which enables them to produce more detailed images than microscopes that use light .", "There are several ways to prepare samples for electron microscopy .", "For example , in ‘electron cryo-microscopy’—or cryo-EM for short—a sample is rapidly frozen to preserve its features before it is examined under the microscope .", "This technique generates images that can be analyzed by computers to produce three-dimensional models of individual viruses , proteins , and other tiny objects .", "Unfortunately , the samples need to be exposed to high-energy beams of electrons that will damage the sample while the images are gathered , which results in sample movement and blurry images that lack the finer details .", "The contrast between the sample and its background is one of the factors that determine the final quality of an image .", "The higher the contrast , the greater the level of structural information that can be obtained , but this requires the use of longer exposures to the electron beam .", "To overcome this issue , researchers found that instead of recording a single image , it is possible to record movies in which the movement of the sample under the electron beam can be tracked .", "After the movies are gathered , the movie frames are aligned using computer software to reduce the blurring caused by the sample moving and can then be used to make three-dimensional models .", "Grant and Grigorieff improved this method further by studying how quickly a large virus-like particle called ‘rotavirus double-layered particle’ is damaged under the electron beam .", "These experiments identified an optimum range of exposure to electrons that provides the highest image contrast at any given level of detail .", "These findings were used to design an exposure filter that can be applied to the movie frames , allowing Grant and Grigorieff to visualize features of the virus that had not previously been observed by cryo-EM .", "This method was also used to study an assembly of proteins known as the proteasome , which is responsible for destroying old proteins .", "Grant and Grigorieff's findings should be useful for cryo-EM studies on many kinds of samples ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "neuroscience" ]

[ [ "Hebbian remodeling of neural circuits , encapsulated by the phrase ‘neurons that fire together wire together’ , is the leading candidate mechanism for learning in the brain ( Shatz , 1992; Cooper , 2005 ) .", "Thus , the finding that synaptic plasticity often has properties compatible with Hebbian learning has been a powerful driver of experimental and theoretical studies of changes in synaptic weights .", "The simple idea is that coincident presynaptic and postsynaptic activity results in changes in synaptic strength such as long-term potentiation ( LTP ) .", "Many in vitro studies support a model in which modifiable synapses detect presynaptic activity through glutamate binding to postsynaptic receptors and detect postsynaptic activity through depolarization , which relieves magnesium block of N-methyl-D-aspartate ( NMDA ) receptors ( NMDARs ) and activates voltage-gated calcium ( Cav ) channels .", "Both of these effects mediate postsynaptic calcium entry , which is believed to be a critical activator of the biochemical steps necessary for increases in synaptic strength ( Madison et al . , 1991; Bliss and Collingridge , 1993; Blackstone and Sheng , 2002; Cavazzini et al . , 2005 ) .", "Four decades of research have led to this general picture , but important questions about the events leading to the critical calcium entry remain unanswered .", "Three distinct ideas have been considered for the type of postsynaptic depolarization required to produce the calcium entry leading to Hebbian LTP ( Williams et al . , 2007 ) :Postsynaptic axo-somatic action potential firing is necessary , and this signal reaches synapses in the form of backpropagating action potentials ( bAPs ) , Postsynaptic axo-somatic action potential firing is not necessary; rather , localized passive synaptic depolarization is sufficient , Postsynaptic axo-somatic action potential firing is not necessary and passive synaptic depolarization is not sufficient; rather , localized synaptic depolarization must activate dendritic nonlinearities , such as locally initiated dendritic spikes ( dSpikes ) .", "At any given synapse at any moment in time , these three mechanisms of postsynaptic activity detection are mutually exclusive .", "However , all three mechanisms may exist in the brain , for example , in different cell types or even within the same cell at different synapses , different stages of development , or possibly in response to different activation patterns at the same synapse .", "Such heterogeneity would be consistent with the idea that LTP is not a single phenomenon , but rather a collection of mechanisms that can produce a long-lasting increase in synaptic strength ( Malenka and Bear , 2004 ) .", "In each of the three cases above , the concerted action of multiple synapses is required—the so-called ‘cooperativity’ requirement for LTP ( Feldman , 2012 ) —but the patterns of synaptic activation that result in LTP are quite different .", "In the first case , the synapses that drive action potential firing could be located anywhere in the dendritic tree and plasticity would occur at all synapses that experience significant depolarization as a result of the bAP .", "This is the most conventional interpretation of Hebbian LTP .", "In the second and third conditions , synapses have to be co-localized in the dendrites in order to depolarize each other sufficiently to produce Hebbian LTP .", "In these conditions , the postsynaptic axon does not have to ‘fire’ at all; the third condition additionally requires that the co-localized synapses produce enough depolarization to trigger a dSpike , which may occur with or without axonal firing .", "Although these conditions may still be classified as ‘Hebbian LTP’ , because of the requirement for co-incident presynaptic and postsynaptic activation , the existence of LTP under these conditions would imply that Hebbian-like LTP occurs at finer spatial scales than the more cell-wide form that most current models employ .", "Thus , during behavior , neurons that fire may undergo LTP , but even a neuron that is synaptically activated but axonally silent during a behavioral event can be recruited to participate in the neural engram .", "Interestingly , hippocampal place cells behave in a manner suggestive of a form of Hebbian LTP that may not require postsynaptic action potential firing: many cells are silent when the animal first goes to a new place , and then they are recruited to participate in the network representation of the spatial map ( Frank et al . , 2004 ) .", "This suggests that if synaptic plasticity contributes to reshaping the network upon initial exposure to a new environment , it need not be conventional Hebbian plasticity , but rather a modified form of Hebbian plasticity that does not require axonal firing , such as the second and third conditions described above .", "Consistent with this idea , hippocampal neurons receive spatially tuned synaptic inputs even when they are silent ( Lee et al . , 2012 ) .", "Understanding whether and how such inputs can drive synaptic plasticity will be an important step toward understanding how spatial memories are formed in the hippocampus .", "If axonal action potential firing is required for synaptic plasticity , memories can only be stored in active neurons .", "On the other hand , if it is not required , memories can also be formed in silent neurons .", "Furthermore , plasticity that is induced by dSpikes that remain localized to individual dendritic branches has been proposed to enhance the memory-storing capacity of individual neurons ( Poirazi and Mel , 2001; Mehta , 2004; Wu and Mel , 2009 ) .", "Collectively , these considerations underscore the importance of understanding the dendritic events leading to the postsynaptic calcium entry necessary for the induction of LTP .", "At synapses from the perforant path ( PP; which carries predominantly spatial information from the entorhinal cortex ) to the distal apical tuft of hippocampal CA1 pyramidal neurons , LTP requires strong synaptic activation , and LTP induction can have a significant impact on the output of CA1 neurons ( Colbert and Levy , 1993; Remondes and Schuman , 2002; Ahmed and Siegelbaum , 2009; Takahashi and Magee , 2009 ) .", "In previous work , we showed that LTP at these synapses does not require bAPs; rather , LTP is correlated with the initiation of dSpikes , which often do not trigger action potential firing and bAPs ( Golding and Spruston , 1998; Golding et al . , 2002 ) .", "This pathway therefore offers an ideal opportunity to study the potential role of dSpikes in the induction of LTP .", "The hypothesis that dSpikes are a causal step in the induction of LTP has not been directly tested , owing to the difficulty of blocking them selectively .", "Three types of dSpikes have been described , which have been named according to the primary channel type supporting the regenerative event: dendritic sodium spikes ( Na-dSpikes ) and dendritic calcium spikes ( Ca-dSpikes ) are mediated primarily by voltage-gated sodium ( Nav ) channels and Cav channels , respectively , while dendritic NMDA spikes ( NMDA-dSpikes ) are mediated primarily by NMDAR channels ( Schwartzkroin and Slawsky , 1977; Andreasen and Lambert , 1995; Schiller et al . , 1997; Golding et al . , 1999; Larkum et al . , 1999; Spruston , 2008; Larkum et al . , 2009; Major et al . , 2013 ) .", "NMDAR and Cav channels are known to contribute to the induction of LTP at PP-CA1 synapses ( Golding et al . , 2002; Takahashi and Magee , 2009 ) , but it is difficult to disentangle the importance of the calcium permeability of these channels from their roles in mediating regenerative dendritic voltage changes .", "Furthermore , the importance of dendritic Nav channels and Na-dSpikes has not been addressed , mostly because these channels are essential for action potential firing in presynaptic axons and terminals , thus making it difficult to block them without inhibiting synaptic transmission .", "One strategy to block postsynaptic Nav channels without affecting presynaptic Nav channels is to use the intracellular blocker QX-314; however , this drug also blocks Cav channels , voltage-gated potassium ( Kv ) channels and has effects on other channels and receptors , making the overall consequences difficult to interpret ( Nathan et al . , 1990; Andrade , 1991; Oda et al . , 1992; Lambert and Wilson , 1993; Martin et al . , 1993; Otis et al . , 1993; Perkins and Wong , 1995; Talbot and Sayer , 1996 ) .", "As an alternative strategy , we used a relatively low concentration of bath-applied tetrodotoxin ( TTX; 20 nM ) to achieve partial block of the Nav channels ( Kaneda et al . , 1989; Madeja , 2000 ) , which we demonstrate is able to inhibit postsynaptic dSpikes mediated by Nav channels without blocking presynaptic action potential firing or synaptic transmission .", "This strategy works because the density of Nav channels is much lower in dendrites than in axons .", "As a result , the safety factor for spike initiation is lower in dendrites , so partial block of Nav channels affects postsynaptic ( dendritic ) dSpikes much more than presynaptic ( axonal ) action potentials ( Mackenzie and Murphy , 1998 ) .", "Using this strategy , we demonstrate that Na-dSpikes are necessary for the induction of LTP in response to theta-burst stimulation ( TBS ) of the PP synapses in the apical tuft dendrites of CA1 pyramidal neurons , and we offer an explanation for why these spikes are an essential mechanism ." ], [ "To determine whether a low concentration of TTX ( 20 nM ) could be used without interfering with synaptic transmission over a range of stimulus intensities , we performed whole-cell recordings from CA1 pyramidal neurons in rat hippocampal slices while stimulating the PP ( ‘Materials and methods’ ) to activate synapses in the distal apical tuft dendrites ( henceforth ‘PP → CA1tuft’ synapses ) .", "We adjusted stimulus intensity to yield single PP → CA1tuft excitatory postsynaptic potentials ( EPSPs ) of 3–10 mV at the soma , which likely corresponds to activation of several tens of synapses onto the apical tuft dendrites of the recorded neuron ( Golding et al . , 2005; Nicholson et al . , 2006 ) .", "We chose 20 nM TTX ( henceforth ‘low TTX’ ) because it was the highest concentration we could use without obviously reducing the size of EPSPs .", "The IC50 of TTX on Nav channels has been estimated as 6–10 nM in dissociated hippocampal neurons ( Kaneda et al . , 1989; Madeja , 2000 ) , but it may be higher for slice experiments due to limited drug penetration in brain slices .", "Single EPSPs in response to low-frequency ( single shock ) stimulation were not affected by bath application of low TTX ( control: 4 . 57 ± 0 . 36 mV , low TTX: 5 . 02 ± 0 . 49 mV , n = 7; p > 0 . 05 by Student's t-test; data not shown ) .", "We also tested the effects of low TTX on the responses to low-frequency or high-frequency ( 5 stimuli at 100 Hz ) activation of the PP , while using localized application of a higher concentration of TTX ( 10 µM in the application pipette ) to the vicinity of the soma and proximal axons ( ‘Materials and methods’; Figure 1A ) in order to prevent somatic or axonal action potential initiation .", "Bath application of low TTX had no significant effect on PP → CA1tuft EPSPs in response to either type of stimulation , across a wide range of stimulus intensities resulting in EPSPs from ∼3 mV ( single shock ) up to ∼27 mV ( high-frequency burst; Figure 1B–G; Figure 1—source data 1 ) .", "These results suggest that excitatory synaptic transmission is not affected by bath application of low TTX within the range of stimulus intensities and frequencies tested here . 10 . 7554/eLife . 06414 . 003Figure 1 . Reducing Nav channel availability with 20 nM TTX does not affect synaptic transmission at PP → CA1tuft synapses .", "( A ) Experimental configuration showing somatic whole-cell recording with presynaptic stimulation of the perforant pathway ( PP ) , 10 µM TTX locally applied to the soma , and bath application of 20 nM TTX .", "( B , C )", "Representative traces of somatically recorded voltage in response to single-shock stimulation ( B ) or high-frequency burst stimulation ( 5 stimuli at 100 Hz; C ) in control and 20 nM TTX .", "Traces are from three different cells .", "( D–G )", "Summary of effects of 20 nM TTX on EPSP amplitude and integral ( D , E , single-shock , n = 9; F , G , burst , n = 8 ) .", "Top .", "Scatter plots of the amplitude or integral of responses in 20 nM TTX vs control .", "Each point represents data from one cell .", "Solid lines represent a linear fit to data points , with shaded areas representing the 95% confidence band of the fit .", "Dashed lines are the unity line .", "Bottom .", "Bar graphs of the amplitude or integral of responses in control and 20 nM TTX . DOI: http://dx . doi . org/10 . 7554/eLife . 06414 . 00310 . 7554/eLife . 06414 . 004Figure 1—source data 1 . Source data for Figure 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 06414 . 00410 . 7554/eLife . 06414 . 005Figure 1—source data 2 . Source data for Figure 1—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 06414 . 00510 . 7554/eLife . 06414 . 006Figure 1—figure supplement 1 . Effects of reducing Nav channel availability with 20 nM TTX on somatic action potentials .", "( A ) Left , experimental configuration showing somatic whole-cell recording .", "Right , representative traces of somatically recorded voltage in response to a current step ( 100 ms ) at the level of action potential ( AP ) threshold ( rheobase ) injected at the soma in control and 20 nM TTX .", "Dashed lines indicate the apparent voltage threshold of the APs .", "( B , C )", "Summary of effects of 20 nM TTX on somatic action potentials ( n = 8 ) .", "***p < 0 . 001 by Student's paired t-test .", "( D ) Left , experimental configuration showing somatic whole-cell recording with antidromic stimulation of CA1 axons .", "Right , representative traces of somatically recorded voltage in response to antidromic TBS ( the same temporal pattern as used for LTP induction; see ‘Materials and methods’ ) at different stimulus intensities in control and 20 nM TTX .", "( E ) Summary of effects of 20 nM TTX on antidromic action potentials ( n = 7 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06414 . 006 Consistent with this result and with the notion of a high safety factor for action potential initiation in the axon ( Coombs et al . , 1957; Mainen et al . , 1995; Raastad and Shepherd , 2003; Clark et al . , 2005; Khaliq and Raman , 2006; Kole and Stuart , 2008; Hu and Jonas , 2014 ) , low TTX did not block action potential firing in response to somatic current injection , though it did raise the voltage threshold and reduce action potential amplitude slightly , as expected from a reduction of Nav channel availability ( Figure 1—figure supplement 1A–C; Figure 1—source data 2; see also Mackenzie and Murphy , 1998; Magee and Carruth , 1999; Fan et al . , 2005 ) .", "In addition , we tested the effects of low TTX on action potentials evoked by repeated bursts of antidromic stimulation of CA1 axons and found that low TTX only affected antidromic action potential firing at low stimulus intensities .", "At high stimulus intensities , antidromic spikes were not blocked by low TTX ( Figure 1—figure supplement 1D , E; Figure 1—source data 2 ) .", "Together with the lack of effect of low TTX on synaptic responses , these data suggest that the effects of low TTX on presynaptic action potential firing and glutamate release are minimal .", "To analyze the effects of low TTX on dSpike initiation , we performed recordings from the primary apical dendrite ( n = 9; 200–320 µm from the soma , mean distance = 250 µm; about 50–80% of the distance from the soma to the end of the apical tuft ) .", "Dendritic recording is necessary to increase the probability of detecting dSpikes originating in the apical tuft , because their steep attenuation makes them impossible to detect hundreds of microns away in somatic recordings .", "We used a stimulus pattern known to cause dSpikes ( Golding et al . , 2002 ) , which consisted of PP stimulation in high-frequency bursts ( 5 stimuli at 100 Hz ) , repeated five times at theta frequency ( 5 Hz; a ‘theta-burst stimulation’ , TBS , consisting of 25 stimuli in total ) .", "Stimulus intensity was set to evoke single PP → CA1tuft EPSPs of 4–10 mV recorded in the dendrites , approximately the same as that required to produce single PP → CA1tuft EPSPs of 2–5 mV recorded at the soma ( Golding et al . , 2005 ) .", "This stimulus pattern is intended to mimic the theta rhythm observed both in the hippocampus and in the entorhinal cortical neurons forming the PP ( Buzsáki and Moser , 2013 ) , and is known to be an effective stimulus for induction of LTP in the hippocampus ( Larson et al . , 1986; Phillips et al . , 2008 ) .", "A TBS is normally repeated multiple times to induce LTP , but in this first series of experiments , we used a single TBS in the presence of low TTX followed by another single TBS after washout of TTX .", "We used this order of drug application and avoided repeating TBS in each condition in order to prevent the induction of LTP , which would confound the comparison of responses in the presence and absence of TTX .", "Consistent with previous results ( Golding and Spruston , 1998; Golding et al . , 2002; Losonczy and Magee , 2006 ) , we observed three types of regenerative dendritic responses: ( 1 ) bAPs , ( 2 ) large dSpikes , and ( 3 ) small dSpikes ( also known as ‘spikelets’ ) .", "These three kinds of events were distinguishable in most cases by their voltage amplitude , first temporal derivative of voltage ( dV/dt ) and onset kinetics ( Figure 2A , B; Figure 2—source data 1; ‘Materials and methods’ ) .", "Large dSpikes reflect dSpikes that have propagated actively from their initiation site to the recording site .", "However , dSpikes can fall below threshold for active propagation ( often because of large impedance drops at branch points ) and then propagate passively to the recording electrode , where they appear as small dSpikes ( spikelets ) ( Golding and Spruston , 1998; Golding et al . , 2002; Jarsky et al . , 2005; Losonczy and Magee , 2006; Katz et al . , 2009 ) .", "dSpikes that are generated far from the recording site may not be visible at all .", "Observations of events with amplitudes in between those of large dSpikes and small dSpikes ( spikelets ) are rare , because the transition from a large dSpike to a small dSpike occurs over very short distances due to the large attenuation at branch points ( Katz et al . , 2009 ) .", "Accordingly , we observed a large gap in the distribution of event amplitudes , with no events having amplitudes in the 30–40 mV range ( Figure 2—figure supplement 1A; Figure 2—source data 2 ) .", "Small dSpikes ( spikelets ) were distinguishable from the cases without any regenerative events ( i . e . , EPSPs only ) because they had larger dV/dt values ( Figure 2B and Figure 2—figure supplement 1B; Figure 2—source data 2; see below and ‘Materials and methods’ ) . 10 . 7554/eLife . 06414 . 007Figure 2 . Reducing Nav channel availability with 20 nM TTX inhibits postsynaptic dSpikes evoked by presynaptic TBS of PP → CA1tuft synapses .", "( A ) Left , experimental configuration showing dendritic whole-cell recording with presynaptic stimulation of the PP .", "Right , traces of dendritically recorded voltage in response to a single burst of presynaptic theta-burst stimulation ( TBS ) from two different neurons with large dSpikes in control ( arrows; see Figure 2—figure supplement", "2 ) and the corresponding responses in 20 nM TTX .", "In the top traces , the large-amplitude dendritic events following the fourth and fifth stimulus in the burst are likely to be bAPs ( ‘Materials and methods’ ) .", "Recordings were performed at 280 and 320 µm from the soma for the top and the bottom traces , respectively .", "( B ) Representative traces of dendritically recorded voltage ( V; top ) and the first temporal derivative of voltage ( dV/dt; bottom ) in response to a single burst of presynaptic TBS from a neuron with a small dSpike ( spikelet; asterisk ) in control and the corresponding responses in 20 nM TTX .", "Experimental configuration is as shown in the inset in A . Recording was performed at 290 µm from the soma ( note: a large-amplitude dendritic event following the fifth stimulus in the burst is truncated . It is likely to be a bAP; see ‘Materials and methods’ ) .", "( C ) Summary of the dendritic recordings , showing peak first temporal derivative of dendritically recorded voltage ( dV/dt ) , normalized to the median value for Stim .", "# 1 of the five bursts ( in control ) from the given cell , plotted as a function of Stim .", "# in each burst in control and 20 nM TTX ( n = 9 cells; positions with a large-amplitude dendritic event in the control condition were excluded; see ‘Materials and methods’ ) .", "The solid line represents an exponential fit to the whole data set ( control and 20 nM TTX ) , and the dashed lines represent the prediction band at the 85% confidence level and the upper bound at the 99% confidence level .", "( D ) Summary of effects of 20 nM TTX on small dSpikes ( spikelets ) , showing the number of events above the upper bound of prediction band at different confidence levels ( i . e . , small dSpikes; see ‘Materials and methods’ ) in control and 20 nM TTX .", "**p < 0 . 01 , *p < 0 . 05 by binomial test . DOI: http://dx . doi . org/10 . 7554/eLife . 06414 . 00710 . 7554/eLife . 06414 . 008Figure 2—source data 1 . Source data of Figure 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 06414 . 00810 . 7554/eLife . 06414 . 009Figure 2—source data 2 . Source data of Figure 2—figure supplement 1–4 . DOI: http://dx . doi . org/10 . 7554/eLife . 06414 . 00910 . 7554/eLife . 06414 . 010Figure 2—figure supplement 1 . Peak amplitude and the first temporal derivative of dendritically recorded voltage in response to TBS plotted as a function of stimulus position in TBS .", "( A ) Additional analysis of the experiments in Figure 2A , showing peak amplitude of dendritically recorded voltage ( V ) in response to presynaptic TBS plotted as a function of stimulus position ( Stim . # ) in TBS .", "Gray area illustrates a gap in the distribution , dividing it into groups with ( w/ ) and without ( w/o ) large-amplitude dendritic events ( presumed to consist of both bAPs and large dSpikes; see ‘Materials and methods’ ) .", "( B ) Additional analysis of the experiments in Figure 2B , showing peak first temporal derivative of dendritically recorded voltage ( dV/dt ) in response to presynaptic TBS plotted as a function of Stim .", "# in TBS ( positions with a large-amplitude dendritic event in the control condition were excluded ) .", "Solid lines connect the data points from the same burst .", "Unconnected data points correspond to the events above the upper bound of the 99% prediction band in Figure 2C ( ‘Materials and methods’ ) ; these traces are shown in Figure 2—figure supplement 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 06414 . 01010 . 7554/eLife . 06414 . 011Figure 2—figure supplement 2 . Distinguishing between bAPs and large dSpikes in dendritic recordings .", "( A ) Analysis of the onset kinetics of large-amplitude dendritic events .", "A1 .", "Left , experimental configuration showing dendritic whole-cell recording with presynaptic stimulation of the PP .", "Right , traces of dendritically recorded voltage in response to a single burst of presynaptic TBS from two different neurons with large dSpikes in control ( same as the traces in Figure 2A , black ) .", "Purple arrows indicate a large dSpike in the early phase of each burst response ( purple ) , which had a more gradual onset ( insets , purple trace ) and a lower apparent voltage threshold .", "In contrast , a bAP in the late phase of the burst response ( red ) had a sharp kink at its onset ( inset , red arrow and trace ) and a higher apparent voltage threshold .", "A2 .", "Phase plots ( dV/dt vs V ) of the events color-coded in A1 .", "Insets show a linear fit ( dotted line ) to the initial phase of the large dSpikes ( purple ) and the bAP ( red ) ; ‘initial phase slope’ was measured as the slope of the fit .", "This measure represents the apparent voltage sensitivity of membrane current at the onset of the spike ( see ‘Materials and methods’ ) .", "( B ) Distinguishing between bAPs and large dSpikes on the basis of apparent voltage threshold and initial phase slope .", "B1 .", "Scatter plot of initial phase slope vs apparent voltage threshold of large-amplitude dendritic events .", "Gray areas illustrate gaps in the distribution of both measures .", "The events in the bottom-left quadrant ( the red circle and the black square , same as the ones indicated by the purple arrows in the top and the bottom traces in A1 , respectively; see also Figure 2A ) were identified as large dSpikes .", "The events in the top-right quadrant ( such as the one indicated by ‘c’ ) were identified as bAPs .", "The events in the other two quadrants ( indicated by ‘a’ , ‘b’ , ‘d’ , ‘e’ , and ‘f’ ) are ambiguous cases: those in the top-left quadrant ( ‘a’ and ‘b’ ) are most likely bAPs , because they have a large initial phase slope and are initiated in the falling phase of the dendritically recorded EPSPs; those in the bottom-right quadrant ( ‘d’ , ‘e’ , and ‘f’ ) are likely large dSpikes , because they have a small initial phase slope , and their broad width suggests a larger contribution from Cav and/or NMDAR channels .", "B2 .", "Traces of the events indicated by letters in B1 , shown together with the corresponding responses in 20 nM TTX . DOI: http://dx . doi . org/10 . 7554/eLife . 06414 . 01110 . 7554/eLife . 06414 . 012Figure 2—figure supplement 3 . Voltage and the first temporal derivative of voltage of the clearest small dSpikes ( spikelets ) .", "Traces of dendritically recorded voltage ( V ) and the first temporal derivative of voltage ( dV/dt ) in response to a single presynaptic burst of the PP for the events above the upper bound of the 99% prediction band shown in Figure 2C ( asterisk; i . e . , the small dSpikes identified by the most stringent criterion; see ‘Materials and methods’ ) .", "These are the same events as the data points disconnected from the solid lines in Figure 2—figure supplement 1B ( except for the green traces ) .", "Dotted boxes group data from the same cell , with color codes consistent to that used in Figure 2—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 06414 . 01210 . 7554/eLife . 06414 . 013Figure 2—figure supplement 4 . Small dSpikes ( spikelets ) identified by a second method .", "( A ) Alternative analysis of the experiments in Figure 2B , showing notched box plots of peak first temporal derivative of dendritically recorded voltage ( dV/dt ) , normalized to the median value for Stim .", "# 1 of the five bursts ( in control ) from the given cell , plotted as a function of Stim .", "# in each burst in control and 20 nM TTX ( n = 9 cells; positions with a large-amplitude dendritic event in the control condition were excluded ) .", "The edges of notch were defined by the 95% confidence intervals of the median , the end points of whiskers as the largest and smallest data values that are within the range of ( the 75th percentile + 1 . 5 × interquartile range ) and ( the 25th percentile − 1 . 5 × interquartile range ) , and individual data points are those outside this range .", "The upper and lower dashed lines represent normalized peak dV/dt of 1 . 8 and 1 . 4 , respectively .", "( B ) Summary of effects of 20 nM TTX on small dSpikes ( spikelets ) , showing the number of events above different thresholds ( i . e . , small dSpikes; see ‘Materials and methods’ ) in control and 20 nM TTX .", "***p < 0 . 001 , *p < 0 . 05 by binomial test . DOI: http://dx . doi . org/10 . 7554/eLife . 06414 . 013 In the presence of low TTX , both the number and the amplitude of large-amplitude dendritic events ( i . e . , bAPs and large dSpikes , all with amplitudes >40 mV; see ‘Materials and methods’ ) were reduced ( control: amplitude = 56 . 2 ± 1 . 8 mV , n = 39 events; low TTX: amplitude = 44 . 8 ± 1 . 7 mV , n = 17 events; p < 10−4 by Student's t-test ) .", "Although distinguishing between bAPs and large dSpikes was only possible in some cases ( Figure 2—figure supplement 2; Figure 2—source data 2; see ‘Materials and methods’ ) , the two events that were the most clearly large dSpikes were reduced to spikelets in response to the same stimulus in the presence of low TTX ( Figure 2A ) .", "Of the smaller events ( i . e . , <30 mV ) , some small dSpikes were relatively easy to identify in dendritic recordings ( e . g . , Figure 2B ) , because they were the most extreme outliers in the distribution of event dV/dt values .", "Other small dSpikes were more difficult to resolve because their attenuation from the site of origin makes them barely distinguishable from the cases without regenerative events ( i . e . , EPSPs only ) .", "Thus , rather than using a single definition for small dSpikes , we employed a range of definitions; then for each definition we compared the control and low TTX conditions .", "In this way , we could determine whether conclusions about the effects of low TTX on dSpikes were dependent on the definition used .", "We varied the dV/dt criterion for small dSpikes from a relatively inclusive one ( outside the 85% prediction band of a fit to the distribution of all events normalized dV/dt in control and low TTX; Figure 2C; see ‘Materials and methods’ ) to a more restrictive one ( outside the 99% prediction band ) .", "We found that application of low TTX dramatically reduced the number of small dSpikes defined by all confidence levels in this range ( Figure 2C , D; Figure 2—source data 1; see Figure 2—figure supplement 3 for all small dSpikes identified by the 99% confidence level; Figure 2—source data 2 ) .", "Similar results were obtained using other methods for identifying small dSpikes ( Figure 2—figure supplement 4; Figure 2—source data 2; see ‘Materials and methods’ ) .", "As both large dSpikes and small dSpikes ( spikelets ) reflect the presence of dSpikes initiated in the tuft dendrites , and both were inhibited by low TTX , we conclude that low TTX is an effective inhibitor of dSpikes generated in response to TBS of the PP .", "Given the ability of low TTX to inhibit dendritically initiated spikes , we tested its effects on the induction of LTP using TBS at PP → CA1tuft synapses ( using stimulus intensities yielding single EPSPs of 2–5 mV in somatic recordings ) .", "TBS was repeated three times ( TBSx3 ) and was either paired with brief somatic current injections ( TBSx3+Current ) to evoke action potential firing during each burst of presynaptic stimuli ( Figure 3A; Figure 3—source data", "1 ) or delivered with somatic voltage clamp ( TBSx3+SomaticVC ) to prevent somatic action potential firing ( holding potential −68 to −70 mV; see ‘Materials and methods’ ) .", "The magnitude of PP → CA1tuft LTP was similar in these two conditions ( Figure 3B–E; TBSx3+Current , potentiation ratio = 1 . 63 ± 0 . 16 , n = 9; TBSx3+SomaticVC , potentiation ratio = 1 . 53 ± 0 . 07 , n = 9; p = 1 . 00 by one-way ANOVA; Figure 3—source data 1 ) , suggesting that action potential firing did not contribute to LTP induction at these synapses ( see also Golding et al . , 2002 ) .", "Under both conditions , PP → CA1tuft LTP was blocked completely in the presence of low TTX ( Figure 3C–E; Figure 3—source data 1 ) . 10 . 7554/eLife . 06414 . 014Figure 3 . TBS-induced PP → CA1tuft LTP is blocked by reducing Nav channel availability with 20 nM TTX .", "( A ) Left , experimental configuration showing somatic whole-cell recording with presynaptic stimulation of the PP .", "Right , representative trace of somatically recorded voltage in response to presynaptic TBS paired with somatic current injections at 50 Hz .", "A TBS consisted of five high-frequency bursts repeated at 5 Hz , with each consisting of 5 stimuli at 100 Hz .", "Each TBS was delivered three times , at 30-s intervals .", "( B , C )", "Representative time course of EPSP amplitude before and after TBS was delivered three times , paired with somatic current injections ( TBSx3+Current; arrows ) in control ( B ) and 20 nM TTX ( C; 20 nM TTX was applied via the bath during the entire experiment ) .", "Top , representative traces ( single trials ) of EPSP before ( 1 ) and 25 min after ( 2 ) TBSx3+Current were delivered .", "The scale bar in B applies to all panels .", "( D ) Summary of the LTP experiments in control and 20 nM TTX .", "EPSP amplitude is normalized to the average EPSP amplitude before LTP induction .", "Solid lines and shaded areas represent mean and S . E . M . , respectively .", "( E ) Potentiation ratio in different experimental conditions: TBSx3+Current , TBS paired with somatic current injections; TBSx3+SomaticVC , TBS delivered with the soma voltage-clamped ( VC ) at ∼−70 mV to prevent action potential firing ( TBSx3+Current , n = 9; TBSx3+SomaticVC , n = 9; TBSx3+Current in 20 nM TTX , n = 8; TBSx3+SomaticVC in 20 nM TTX , n = 8 ) .", "##p < 0 . 01 for the effect of time on EPSP amplitude by one-way repeated measures ANOVA .", "**p < 0 . 01 ( TBSx3+Current , control vs 20 nM TTX ) , *p < 0 . 05 ( TBSx3+SomaticVC , control vs 20 nM TTX ) by one-way ANOVA with post-hoc means comparison using Tukey's test . DOI: http://dx . doi . org/10 . 7554/eLife . 06414 . 01410 . 7554/eLife . 06414 . 015Figure 3—source data 1 . Source data of Figure 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 06414 . 015 Because low TTX blocks both dSpikes and bAPs , it is conceivable that TTX inhibits LTP by blocking either or both of these sources of postsynaptic depolarization .", "However , consistent with our previous study ( Golding et al . , 2002 ) , we showed here again that PP → CA1tuft LTP is not affected by blocking bAPs .", "Thus , we attribute the effect of low TTX on this particular form of LTP to its effect on Na-dSpikes .", "This is a surprising result , because of the brief duration of Na-dSpikes , compared to Ca- or NMDA-dSpikes .", "One possible explanation for the requirement of Na-dSpikes during PP → CA1tuft LTP induction is that they are required to activate channels mediating synaptic calcium influx .", "We therefore sought to determine which calcium-permeable channels are responsible for the underlying sources of calcium influx .", "We previously showed that PP → CA1tuft LTP was reduced ∼50% by blocking NMDARs , ∼50% by blocking L- , T- , and R-type Cav channels , and almost completely by blocking NMDARs and Cav channels together ( Golding et al . , 2002 ) .", "We confirmed these results here and showed further that the effects of Cav channel blockers were attributable to blocking L-type Cav channels ( with 10 µM nimodipine ) , but not blocking T- , R- , or other types of Cav channels ( with 50 µM Ni2+; Figure 4; Figure 4—source data 1; see also Remy and Spruston , 2007 ) .", "Furthermore , 10 µM nimodipine had no effect on synaptic responses , either before or after the induction of PP → CA1tuft LTP ( Figure 4—figure supplement 1; Figure 4—source data 2 ) , suggesting that L-type Cav channels are involved in the induction rather than the expression of LTP . 10 . 7554/eLife . 06414 . 016Figure 4 . NMDAR and L-Cav channels mediate TBS-induced LTP at PP → CA1tuft synapses .", "( A–D )", "Representative time course of EPSP amplitude before and after TBSx3+Current was delivered ( arrows ) in 50 µM AP5 ( A ) , 10 µM nimodipine ( B ) , 50 µM AP5 and 10 µM nimodipine ( C ) , or 50 µM Ni2+ ( D ) .", "Drugs were applied via the bath during the entire experiment .", "Top , representative traces ( single trials ) of EPSP before ( 1 ) and 25 min after ( 2 ) TBSx3+Current was delivered .", "The scale bar in A applies to all panels .", "( E ) Summary of the LTP experiments in AP5 , nimodipine ( Nimo ) , AP5 and nimodipine ( AP5+Nimo ) , and Ni2+ , shown along with the data in control and 20 nM TTX ( Figure", "3 ) for comparison .", "EPSP amplitude is normalized to the average EPSP amplitude before LTP induction .", "Solid lines and shaded areas represent mean and S . E . M . , respectively .", "( F ) Potentiation ratio in different experimental conditions ( Control , n = 9; AP5 , n = 11; Nimo , n = 9; AP5+Nimo , n = 11; Ni2+ , n = 9; 20 nM TTX , n = 8 ) .", "##p < 0 . 01 , #p < 0 . 05 for the effect of time on EPSP amplitude by one-way repeated measures ANOVA .", "**p < 0 . 01 , *p < 0 . 05 ( all compared to control ) by one-way ANOVA with post-hoc means comparison using Tukey's test . DOI: http://dx . doi . org/10 . 7554/eLife . 06414 . 01610 . 7554/eLife . 06414 . 017Figure 4—source data 1 . Source data of Figure 4 . DOI: http://dx . doi . org/10 . 7554/eLife . 06414 . 01710 . 7554/eLife . 06414 . 018Figure 4—source data 2 . Source data of Figure 4—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 06414 . 01810 . 7554/eLife . 06414 . 019Figure 4—figure supplement 1 . L-Cav channels are not required for synaptic transmission before or after the induction of PP → CA1tuft LTP . Summary of effects of 10 µM nimodipine ( Nimo ) on somatically recorded voltage or current in response to single-shock stimulation of the PP before ( A , B ) or after ( C , D ) the induction of PP → CA1tuft LTP .", "( A , B )", "Scatter plots ( nimodipine vs control ) and bar graphs ( normalized to control ) of the amplitude or integral of voltage before LTP induction .", "( C , D )", "Scatter plots ( nimodipine vs control ) and bar graphs ( normalized to control ) of the amplitude or integral of current after LTP induction .", "Each point represents data from one cell .", "Solid lines represent a linear fit to data points , with shaded areas representing the 95% confidence band of the fit .", "Dashed lines are the unity line . DOI: http://dx . doi . org/10 . 7554/eLife . 06414 . 019 These results suggest that NMDAR and L-type Cav ( L-Cav ) channels are the two main sources of calcium entry during LTP induction by TBS of PP → CA1tuft synapses .", "The observation that partial block of Nav channels by low TTX blocked PP → CA1tuft LTP almost completely ( Figure 3B–E ) indicates that Nav channel-mediated events are required to activate these two types of calcium-permeable channels , and these events are unlikely to be bAPs , as we showed here and in our previous study ( see above ) .", "We therefore performed additional analyses on the dendritic recordings ( described above ) to determine how low TTX affects membrane potential changes that can activate these channels .", "As described above , low TTX blocked large and small dSpikes ( Figure 2 and Figure 2—figure supplement 4 ) .", "In one of the two dendritic recordings where we observed inhibition of a clear large dSpike by low TTX , we subsequently washed out TTX and applied 50 µM AP5 , which did not block the large dSpike ( Figure 5A; Figure 5—source data 1 ) .", "By contrast , low TTX had almost no effect on the slow depolarization produced by each burst of PP → CA1tuft EPSPs in response to TBS , whereas 50 µM AP5 had a much greater effect on the slow depolarization associated with each burst .", "This differential effect of low TTX and 50 µM AP5 on the slow depolarization was confirmed in additional dendritic recordings ( n = 9; for four cases , data in TTX and AP5 were obtained from the same cell; Figure 5B; Figure 5—source data 1 ) .", "Because low TTX was a more potent inhibitor of PP → CA1tuft LTP than 50 µM AP5 ( Figure 4F ) , these results suggest that the fast depolarization associated with Na-dSpikes is more important for the induction of PP → CA1tuft LTP than the slow synaptic depolarization mediated solely by EPSPs . 10 . 7554/eLife . 06414 . 020Figure 5 . Reducing Nav channel availability reduces the slow synaptic depolarization in distal apical trunk in response to TBS significantly less than blocking NMDAR channels .", "( A ) Differential effects of 20 nM TTX and 50 µM AP5 on dendritically recorded voltage in response to presynaptic TBS .", "A1 , Left , experimental configuration showing dendritic whole-cell recording with presynaptic stimulation of the PP .", "Right , example traces of dendritically recorded voltage in response to presynaptic TBS from a neuron with a large dSpike in control ( the same cell as the bottom case shown in Figure 2A ) and the corresponding responses in 20 nM TTX or 50 µM AP5 ( TTX was washed out before subsequent application of AP5 ) .", "A2 .", "Traces corresponding to the box in A1 .", "Note that the large dSpike was blocked by 20 nM TTX , but not 50 µM AP5 .", "( B ) Summary of effects of TTX ( n = 9 ) and AP5 ( n = 4 ) on burst responses ( normalized to control ) .", "Measured integrals include contributions from large-amplitude dendritic events , which were observed in some cells .", "**p < 0 . 01 by Student's t-test . DOI: http://dx . doi . org/10 . 7554/eLife . 06414 . 02010 . 7554/eLife . 06414 . 021Figure 5—source data 1 . Source data of Figure 5 . DOI: http://dx . doi . org/10 . 7554/eLife . 06414 . 021 To test this conclusion further , we designed a stimulation protocol expected to have differential effects on Na-dSpikes and slow synaptic depolarization .", "A modified TBS pattern consisting of only 2 stimuli in each burst ( 2-stim TBSx3 , Figure 6A; Figure 6—source data", "1 ) is expected to greatly reduce the slow synaptic depolarization while preserving about 50% of Na-dSpikes ( based on Figure 2C ) .", "This 2-stim protocol induced LTP at PP → CA1tuft synapses of approximately the same magnitude as various TBSx3 protocols consisting of 5 stimuli ( 5-stim ) in each burst ( Figure 6B , C; Figure 6—source data 1 ) .", "The 2-stim TBSx3 protocol yielded PP → CA1tuft LTP of 82% of the average LTP induced by all variants of the 5-stim TBSx3 protocol , despite a greatly reduced integral of the slow depolarization ( 27% of that with 5-stim TBSx3; Figure 6D , E; Figure 6—source data 1 ) . 10 . 7554/eLife . 06414 . 022Figure 6 . Brief synaptic stimuli are sufficient for the induction of PP → CA1tuft LTP .", "( A ) Top left , experimental configuration showing somatic whole-cell recording with presynaptic stimulation of the PP .", "Bottom right , representative trace of somatically recorded voltage in response to a modified TBS pattern , consisting of only 2 ( 2-stim TBS ) instead of 5 ( 5-stim TBS ) synaptic stimuli in each burst .", "( B ) Summary of the time courses of normalized EPSP amplitude before and after 2-stim TBS or 5-stim TBS was delivered three times ( 2-stim TBSx3 or 5-stim TBSx3; arrows ) , shown along with the data with TBSx3+Current or TBSx3+SomaticVC ( Figure", "3 ) for comparison .", "EPSP amplitude is normalized to the average EPSP amplitude before LTP induction .", "Solid lines and shaded areas represent mean and S . E . M . , respectively .", "( C ) Potentiation ratio in different experimental conditions ( 5-stim TBSx3+Current , n = 9; 5-stim TBSx3+SomaticVC , n = 9; 5-stim TBSx3 , n = 5; 2-stim TBSx3 , n = 13 ) .", "###p < 0 . 001 , ##p < 0 . 01 for the effect of time on EPSP amplitude by one-way repeated measures ANOVA .", "No statistically significant differences by one-way ANOVA ( p = 0 . 45 ) .", "( D ) Representative traces of somatically recorded voltage in response to 5-stim or 2-stim TBS .", "The action potentials are truncated .", "( E ) Summary of the integral of responses to the first repeat of 5-stim ( n = 5 ) or 2-stim ( n = 13 ) TBS .", "**p < 0 . 01 by Student's t-test . DOI: http://dx . doi . org/10 . 7554/eLife . 06414 . 02210 . 7554/eLife . 06414 . 023Figure 6—source data 1 . Source data of Figure 6 . DOI: http://dx . doi . org/10 . 7554/eLife . 06414 . 023 Collectively , the results above indicate that low TTX blocks PP → CA1tuft LTP by inhibiting fast Na-dSpikes , with minimal effect on the slow synaptic depolarization induced by each high-frequency burst during TBS .", "We next turned our attention to investigating the calcium entry that links Na-dSpikes with the induction of PP → CA1tuft LTP .", "Given that NMDAR and L-Cav channels are both voltage-dependent sources of calcium entry , and they both contribute to PP → CA1tuft LTP ( approximately equally , see Figure 4F ) , the most parsimonious hypothesis is that Na-dSpikes are effective activators of calcium entry via these pathways .", "Although LTP induction at these synapses may depend on the complex spatiotemporal properties of calcium entry , the simplest scenario is that Na-dSpikes produce particularly large calcium entry in synaptically activated portions of the tuft dendrites , which triggers LTP .", "To test this idea , CA1 pyramidal neurons were filled with 100 µM Oregon Green 488 BAPTA-1 ( OGB-1 ) and 50 µM Alexa Fluor 594 Hydrazide ( AF-594 ) and imaged on a two-photon laser-scanning microscope during stimulation of the PP with single burst of 5 stimuli at 100 Hz ( ‘Materials and methods’ ) .", "In these experiments , the soma was voltage-clamped at ∼ −70 mV in order to prevent action potential firing , thus limiting detected calcium influx to that induced by synaptic events and dendritically initiated spikes .", "For quantification of calcium signals , the calcium-induced change in OGB-1 fluorescence was measured as a fraction of the baseline fluorescence of the calcium-insensitive reference , AF-594 ( ΔG/R; see ‘Materials and methods’ ) .", "Although we could easily resolve dendritic spines , we could not determine which spines received direct synaptic input , so we restricted our analysis to the calcium signals in dendritic shafts ( Figure 7A ) , using the response integral as the best measure of dendritic calcium entry ( see ‘Materials and methods’; Figure 7—figure supplement 1; Figure 7—source data 2 ) . 10 . 7554/eLife . 06414 . 024Figure 7 . Reducing Nav channel availability reduces the calcium influx in distal apical tuft dendrites in response to high-frequency burst stimulation significantly less than blocking NMDAR channels .", "( A ) Bottom , experimental configuration showing somatic whole-cell recording with presynaptic stimulation of the PP , with a representative Z-stack image of a neuron ( filled with 50 µM AF-594 ) on which the imaging experiments were performed .", "The box indicates the field of view used during high-resolution line scan .", "Top , a single-scan image corresponding to the box in the image below .", "The red line represents a line profile used for line scan , which went through a short stretch of the dendritic shaft and crossed a neighboring spine ( not shown ) .", "Analysis was restricted to the data from dendritic shafts only .", "( B ) Representative traces of ∆G/R ( 100 µM OGB-1 ) from dendritic shaft and current ( ‘I’ ) from somatic voltage-clamp recording in response to a single burst stimulation in control and 20 nM TTX or 50 µM AP5 ( at least 15 min after drug application ) .", "Note that a spikelet was blocked by 20 nM TTX .", "( C ) Summary of effects of TTX ( n = 7 ) and AP5 ( n = 5 ) on ∆G/R ( normalized to control ) .", "*p < 0 . 05 by Student's t-test . DOI: http://dx . doi . org/10 . 7554/eLife . 06414 . 02410 . 7554/eLife . 06414 . 025Figure 7—source data 1 . Source data of Figure 7 . DOI: http://dx . doi . org/10 . 7554/eLife . 06414 . 02510 . 7554/eLife . 06414 . 026Figure 7—source data 2 . Source data of Figure 7—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 06414 . 02610 . 7554/eLife . 06414 . 027Figure 7—figure supplement 1 . Stability of two-photon calcium imaging over long recording durations .", "( A ) Experimental configuration showing somatic whole-cell recording with presynaptic stimulation of the PP , with a representative Z-stack image of a neuron ( filled with 50 µM AF-594 ) .", "The box indicates the field of view used during high-resolution line scan .", "Analysis was restricted to the data from dendritic shafts only .", "( B ) Time courses of basal fluorescence of AF-594 ( ‘R’ ) , ratio of the basal fluorescence of OGB-1 ( 100 µM ) to that of AF-594 ( ‘G0/R’ ) , and peak and integral of ∆G/R from dendritic shaft in response to a single high-frequency burst stimulation .", "Different symbols and colors denote data from different cells .", "For comparison , data were normalized with respect to the time point shared by all the cells .", "These control experiments were performed with somatic voltage-clamp or current-clamp recording , and control ACSF was ‘washed in’ and ‘washed out’ ( i . e . , solution switched with no drugs ) in the same manner as the pharmacological experiments ( Figure 7 and Figure 8–figure supplement 1 ) .", "As the AF-594 fluorescence ( R ) increased with dialysis of the indicators into the cells , the ratio G0/R ( as a readout of basal calcium concentration ) remained stable .", "Note that the integral of ∆G/R was more stable than peak ∆G/R .", "( C ) Summary of stability of the two-photon calcium imaging and brain slice conditions ( n = 3 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06414 . 027 Burst stimulation of the PP resulted in clear ΔG/R responses ( 79 ± 14% , n = 17 cells ) , which were significantly reduced by 50 µM AP5 ( Figure 7B; Figure 7—source data 1 ) , indicating that we were able to detect calcium entry dependent on synaptic activation—likely mediated by multiple channel types—when imaging from dendritic shafts .", "Importantly , however , low TTX was far less potent at inhibiting intracellular calcium elevations than 50 µM AP5 ( Figure 7B , C; Figure 7—source data 1 ) .", "This result was surprising , given that it is opposite to what one would expect from the result that low TTX was a far more potent inhibitor of PP → CA1tuft LTP than 50 µM AP5 .", "This suggests that the effectiveness of low TTX as a blocker of LTP cannot be explained by its effect on the measured dendritic calcium signals .", "To assist with the interpretation of our results , we used a compartmental model of a CA1 pyramidal neuron .", "The model was adapted from a previous model that contained Nav and Kv channels in the dendrites to reproduce experimental data on bAPs and dSpikes ( Golding et al . , 2001; Jarsky et al . , 2005; Katz et al . , 2009 ) ; it was extended to include NMDARs at all synapses and L-Cav channels ( high-voltage-activated Cav channels ) in the dendrites , as well as simple models of calcium buffers ( including endogenous buffer and OGB-1 ) , calcium diffusion , and calcium extrusion ( ‘Materials and methods’ ) .", "The model was able to reproduce the effects of low TTX ( simulated by 50% reduction of Nav channel conductance ) and 50 µM AP5 ( simulated by complete block of NMDARs ) on dSpikes and EPSPs recorded from the distal apical trunk ( Figure 8A–C; Figure 8—source data 1 ) .", "The simulations were also able to reproduce the effects of TTX and AP5 on dendritic calcium transients ( Figure 7 ) , as measured by the modeled increase in the concentration of simulated calcium-bound OGB-1 ( [Ca2+]OGB ) in response to burst activation of simulated synapses ( Figure 8B , C; Figure 8—source data 1 ) .", "The ability of the model to reproduce these experimental observations allowed us to use it to explore the possible mechanism by which Na-dSpikes mediate calcium influx that may be critical for the induction of PP → CA1tuft LTP . 10 . 7554/eLife . 06414 . 028Figure 8 . Computational modeling reveals a large , rapid , Na-dSpike-mediated calcium current through NMDAR and L-Cav channels .", "( A ) Left , Z-stack image of a neuron ( filled with 50 µM AF-594 ) .", "Right , morphology of the model neuron .", "Red dots indicate the locations of simulated synapses .", "Arrow indicates the imaged compartment with a simulated synapse ( blue dot ) for the example shown in B and D . ( B ) Representative traces of simulated voltage recorded from the distal apical trunk and concentration of calcium-bound OGB-1 ( [Ca2+]OGB ) recorded from a compartment with a simulated synapse on the distal apical tuft ( as indicated in A ) in response to a single burst stimulation in control and simulated 20 nM TTX or 50 µM AP5 .", "( C ) Summary of the simulations and a comparison with the experiments in Figures 5 , 7 .", "The bar graphs show mean ± S . D . ( normalized to control; S . D . was used to emphasize the distribution and variability of the drug effects across different imaging locations ) .", "For normalized peak amplitude , only the cell with a dSpike initiated presumably the closest to and actively propagating to the recording electrode in our dendritic recordings ( Figure 5A ) is illustrated for comparison .", "For normalized peak ∆[Ca2+]OGB and normalized integral of ∆[Ca2+]OGB , data were pooled from all the compartments on the distal apical tuft ( the number of compartments = 440 ) , to mimic the experimental condition in which the imaging locations were arbitrarily selected .", "( D ) Representative traces of simulated voltage and instantaneous fractional calcium current through NMDAR channels ( ICa , NMDA ) and L-Cav channels ( ICa , L-Cav ) .", "Modeled voltages and currents are from a compartment with a simulated synapse on the distal apical tuft ( as indicated in A ) in response to a single burst stimulation in control and simulated 20 nM TTX or 50 µM AP5 .", "Note the presence of a spikelet ( asterisk ) , which was due to a Na-dSpike initiated in a neighboring dendritic branch and failing to propagate reliably to the recorded branch . DOI: http://dx . doi . org/10 . 7554/eLife . 06414 . 02810 . 7554/eLife . 06414 . 029Figure 8—source data 1 . Source data of Figure 8 . DOI: http://dx . doi . org/10 . 7554/eLife . 06414 . 02910 . 7554/eLife . 06414 . 030Figure 8—source data 2 . Source data of Figure 8—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 06414 . 03010 . 7554/eLife . 06414 . 031Figure 8—figure supplement 1 . Minimal effect of blocking L-Cav channels on the calcium influx in distal apical tuft dendrites is consistent with the prediction of the model .", "( A ) Left , experimental configuration showing somatic whole-cell recording with presynaptic stimulation of the PP , with a representative Z-stack image of a neuron ( filled with 50 µM AF-594 ) on which the imaging experiments were performed .", "The box indicates the field of view used during high-resolution line scan .", "Right , a single-scan image corresponding to the box in the image on the left .", "The red line represents a line profile used for line scan .", "Analysis was restricted to the data from dendritic shafts only .", "( B ) Representative traces of ∆G/R ( 100 µM OGB-1 ) from dendritic shaft and current ( ‘I’ ) from somatic voltage-clamp recording in response to a single high-frequency burst stimulation in control and 10 µM nimodipine ( at least 15 min after drug application ) .", "Note the presence of a spikelet both in control and in 10 µM nimodipine .", "( C ) Summary of effects of nimodipine ( Nimo , n = 5 ) on ∆G/R ( normalized to control ) .", "( D ) Morphology of the model neuron .", "Red dots indicate the locations of simulated synapses .", "Arrow indicates the imaged compartment with a simulated synapse ( blue dot ) for the example shown in E and G . ( E ) Representative traces of simulated concentration of calcium-bound OGB-1 ( [Ca2+]OGB ) recorded from a compartment with a simulated synapse on the distal apical tuft ( as indicated in D ) in response to a single burst stimulation in control and simulated 10 µM nimodipine .", "( F ) Summary of the simulations and a comparison with the experiments in A–C .", "The bar graphs show mean ± S . D . ( normalized to control; S . D . was used to emphasize the distribution and variability of the drug effects across different imaging locations ) .", "For normalized peak ∆[Ca2+]OGB and normalized integral of ∆[Ca2+]OGB , data were pooled from all the compartments on the distal apical tuft ( the number of compartments = 440 ) , to mimic the experimental condition in which the imaging locations were arbitrarily selected .", "( G ) Representative traces of simulated voltage and instantaneous fractional calcium current through NMDAR channels ( ICa , NMDA ) and L-Cav channels ( ICa , L-Cav ) .", "Modeled voltages and currents are from a compartment with a simulated synapse on the distal apical tuft ( as indicated in D ) in response to a single burst stimulation in control and simulated 10 µM nimodipine .", "Note the presence of a spikelet ( asterisk ) , which was due to a Na-dSpike initiated in a neighboring dendritic branch and failing to propagate reliably to the recorded branch . DOI: http://dx . doi . org/10 . 7554/eLife . 06414 . 031 In response to burst activation of simulated synapses a single large dSpike was observed in the apical trunk .", "However , in small apical tuft branches , where direct patch-clamp recording cannot be performed , simulations revealed that a dSpike was initiated on several branches in response to burst activation of spatially distributed synaptic inputs , appearing as multiple dSpikes on the apical tuft branch ( Figure 8D; Figure 8—source data 1 ) .", "This is consistent with the observations in the experiments that multiple spikelets occurred in response to burst stimulation ( see Figure 2—figure supplement 3 ) .", "The simulations also offered insight into how Na-dSpikes could provide the calcium influx necessary for PP → CA1tuft LTP induction .", "By comparing the dendritic calcium signals ( reported by simulated OGB-1 ) and the calcium influx through the synaptic channels in the model , we could clearly distinguish the difference between dendritic ‘bulk’ calcium signals and the calcium signals near the mouth of channels .", "Simulations at the synapses revealed that the largest instantaneous calcium currents ( ICa ) through NMDAR and L-Cav channels were mediated by locally generated Na-dSpikes , owing to efficient relief of magnesium block of NMDAR channels and strong activation of L-Cav channels , respectively ( Figure 8D; Figure 8—source data 1 ) .", "These simulations , combined with the experimental results , suggest that a large , rapid , Na-dSpike-mediated component of calcium entry may produce transient , localized increases in intracellular calcium concentration that are essential for the induction of TBS-induced PP → CA1tuft LTP .", "To further test this model , we experimentally tested two predictions .", "First , we posited that blocking L-Cav channels , which inhibits LTP by ∼50% , would have minimal effect on measurements of bulk calcium entry in the distal apical dendrites , as indicated in our model ( Figure 8—figure supplement 1D–F; Figure 8—source data 2; see also Tsay et al . , 2007 ) .", "Indeed , we found that 10 µM nimodipine did not have a significant effect on dendritic calcium responses during burst stimulation of the PP ( Figure 8—figure supplement 1A–C; Figure 8—source data 2 ) .", "This result is consistent with our hypothesis that L-Cav channels contribute to the induction of PP → CA1tuft LTP by mediating increases in localized calcium near the channel pores rather than in dendritic bulk calcium .", "Second , we posited that LTP would be inhibited by chelating intracellular calcium , but only when the buffering kinetics were fast enough to disrupt localized calcium signaling close to the channel pores ( see ‘Discussion’ ) .", "1 , 2-Bis ( 2-aminophenoxy ) ethane-N , N , N' , N'-tetraacetic acid ( BAPTA ) has a much faster calcium binding kinetics than ethylene glycol-bis ( 2-aminoethylether ) -N , N , N' , N'-tetraacetic acid ( EGTA ) ( Tsien , 1980 ) , and we therefore tested this hypothesis by buffering cytoplasmic calcium elevations with either a relatively low or high concentration of EGTA or BAPTA ( Maravall et al . , 2000; Henneberger et al . , 2010 ) , while maintaining the basal calcium concentration at 50 nM by adding an appropriate amount of CaCl2 ( ‘Materials and methods’ ) .", "We predicted that only a high concentration of BAPTA should buffer intracellular calcium increases rapidly and effectively enough to block PP → CA1tuft LTP , and this was indeed found to be the case ( Figure 9; Figure 9—source data 1 ) .", "These results suggest that NMDAR and L-Cav channels contribute to the induction of TBS-induced PP → CA1tuft LTP by mediating calcium influx , and are consistent with the hypothesis that it is the large , transient , localized increases in intracellular calcium concentration mediated by Na-dSpikes that are critical for this form of LTP . 10 . 7554/eLife . 06414 . 032Figure 9 . TBS-induced PP → CA1tuft LTP is blocked by intracellular calcium buffering with a high concentration of BAPTA , but not a low concentration of BAPTA or low or high EGTA .", "( A–D )", "Representative time course of EPSP amplitude before and after TBSx3+Current was delivered ( arrows ) from cells buffered with 0 . 5 mM EGTA ( A ) , 0 . 5 mM BAPTA ( B ) , 10 mM EGTA ( C ) , or 10 mM BAPTA ( D ) .", "CaCl2 was added to maintain basal calcium level ( ∼50 nM; see ‘Materials and methods’ ) .", "Calcium buffer was included in the intracellular solution .", "Top , representative traces ( single trials ) of EPSP before ( 1 ) and 25 min after ( 2 ) TBSx3+Current was delivered .", "The scale bar in A applies to all panels .", "( E ) Summary of the LTP experiments with different calcium buffering .", "EPSP amplitude is normalized to the average EPSP amplitude before LTP induction .", "Solid lines and shaded areas represent mean and S . E . M . , respectively .", "( F ) Potentiation ratio in different experimental conditions ( 0 . 5 mM EGTA , n = 8; 0 . 5 mM BAPTA , n = 5; 10 mM EGTA , n = 5; 10 mM BAPTA , n = 8 ) .", "##p < 0 . 01 , #p < 0 . 05 for the effect of time on EPSP amplitude by one-way repeated measures ANOVA .", "*p < 0 . 05 ( compared to 10 mM EGTA ) by one-way ANOVA with post hoc means comparison using Tukey's test . DOI: http://dx . doi . org/10 . 7554/eLife . 06414 . 03210 . 7554/eLife . 06414 . 033Figure 9—source data 1 . Source data of Figure 9 . DOI: http://dx . doi . org/10 . 7554/eLife . 06414 . 033" ], [ "Our results advance our understanding of the mechanisms underlying PP → CA1tuft LTP induction in three important ways .", "First , whereas previous evidence for the role of dSpikes in the induction of PP → CA1tuft LTP ( Golding et al . , 2002 ) was correlative ( stimuli that evoked dSpikes also induced LTP , while stimuli that failed to evoke dSpikes also failed to induce LTP ) , this work supports a more direct causal link between the occurrence of dSpikes and the induction of PP → CA1tuft LTP .", "Second , our results firmly establish a role for dendritic Nav channels in the induction of PP → CA1tuft LTP .", "Previously observed correlations between dSpikes and PP → CA1tuft LTP ( Golding et al . , 2002 ) could not discern whether the relevant dSpikes were mediated by Nav , Cav , or NMDAR channels .", "One possibility is that these channels could be activated in concert and support relevant dendritic plateau potentials via a ‘spike-chain mechanism’ ( Schiller et al . , 2000 ) to trigger PP → CA1tuft LTP , but our results suggest that Na-dSpikes are the major contributors , and slow NMDAR-dependent synaptic depolarization is neither sufficient nor necessary for LTP at these synapses ( Figures 5 , 6 ) .", "Our evidence for the necessity of Na-dSpikes in synaptic plasticity adds to the list of important functions of dendritic Nav channels , which also includes contributions to synaptic integration leading to action potential initiation and a central role in action potential backpropagation ( Stuart and Sakmann , 1994; Golding et al . , 2001; Larkum and Nevian , 2008; Spruston , 2008 ) .", "Third , our results provide insight into the mechanisms by which Na-dSpikes are coupled to the induction of LTP through calcium influx at PP → CA1tuft synapses , as discussed in detail below .", "The block of LTP only by the fast calcium chelator BAPTA strongly supports the hypothesis that the large , localized increases in intracellular calcium concentration are necessary for the induction of PP → CA1tuft LTP .", "This is in interesting contrast to the block of LTP also by the slow calcium chelator EGTA at the more proximal Schaffer collateral synapses of CA1 pyramidal neurons ( Lynch et al . , 1983 ) , which suggests a different underlying mechanism for the calcium dependence of LTP at these synapses .", "The most parsimonious explanation for our results is that the effective block of LTP by low TTX is through the inhibition of Na-dSpikes per se; however , a few alternative interpretations should be considered .", "For example , LTP induction could depend on a non-ionic mechanism directly coupled to the conformational changes of Nav channels or an ionic mechanism mediated by sodium flux through them ( but little has been reported regarding these speculative mechanisms ) .", "Alternatively , during synaptic activation , Nav channels may affect membrane potential and calcium entry in spines independently of dSpikes ( Araya et al . , 2007; Bloodgood and Sabatini , 2007; note that blocking Nav channels enhanced synaptic calcium influx in Bloodgood and Sabatini , 2007 ) .", "Finally , TTX may have a presynaptic effect , as presynaptic sodium has been shown to regulate synaptic strength constitutively at the Calyx of Held ( Huang and Trussell , 2014 ) .", "A few observations argue against these possibilities , such as the lack of effect of low TTX on synaptic responses at PP → CA1tuft synapses ( Figure 1 ) , the strong correlation between dSpikes and PP → CA1tuft LTP ( Golding et al . , 2002 ) and the reduction in Na-dSpikes that accompanies the block of PP → CA1tuft LTP by low TTX ( Figures 2 , 3 ) .", "However , additional experiments would be required to test these alternative possibilities directly .", "The finding that Na-dSpikes play such a critical role in the induction of PP → CA1tuft LTP is somewhat surprising , given the brief duration of these Na-dSpikes compared to slow depolarization mediated by EPSPs , Ca-dSpikes , or NMDA-dSpikes .", "The fact that simultaneously blocking NMDAR and L-Cav channels eliminated PP → CA1tuft LTP ( Golding et al . , 2002; Remondes and Schuman , 2003; Ahmed and Siegelbaum , 2009 ) suggests that Na-dSpikes contribute to the induction of PP → CA1tuft LTP by increasing calcium influx through these channels .", "But how does this work , given that Na-dSpikes have a brief duration and thus a limited contribution to the increase in ‘bulk’ calcium concentration ( i . e . , the calcium measurable with the spatiotemporal resolution provided by imaging with calcium-sensitive dyes ) compared to slow synaptic depolarization ?", "A simple explanation , supported by our modeling ( Figure", "8 ) and the subsequent experiments ( Figure 9 ) , is that the calcium influx during Na-dSpikes ( via NMDAR and L-Cav channels ) is vigorous and brief , whereas the calcium influx during slow synaptic depolarization ( via NMDAR channels ) is smaller , but longer lasting .", "Although the latter ultimately contributes more to the total amount of calcium entry during a burst and comprises the vast majority of the bulk calcium measured with dendritic calcium imaging , the vigorous and brief calcium entry during Na-dSpikes is more critical for the induction of PP → CA1tuft LTP .", "During TBS , Na-dSpikes produce the largest voltage excursions and maximize ICa through both NMDAR and L-Cav channels ( Figure 10 ) .", "Activation of NMDAR channels is strongly voltage dependent ( through voltage-dependent magnesium block ) as is activation of L-Cav channels ( through high-voltage-activated conformational changes ) .", "Thus , these calcium-permeable channels that we implicate in PP → CA1tuft LTP are activated more by the Na-dSpike than by the slow synaptic depolarization .", "The large ICa caused by the Na-dSpike produces a brief , high calcium concentration near the mouth of the channels , which will quickly diffuse away and contribute modestly to the bulk calcium concentration in the rest of the dendritic spine and shaft .", "On the other hand , most of the bulk calcium is generated by the smaller , but longer-lasting flux of calcium through the NMDAR channels activated during the slow synaptic depolarization .", "The calcium concentration near the mouth of the channels is proportional to the instantaneous amplitude of ICa , while the bulk calcium concentration is proportional to the time integral of ICa through the calcium-permeable channels ( Augustine et al . , 2003; Eggermann et al . , 2012; Tadross et al . , 2013 ) . 10 . 7554/eLife . 06414 . 034Figure 10 . Proposed model for the critical role of Na-dSpikes in the induction of PP → CA1tuft LTP .", "( A ) Synaptic membrane potential and calcium currents in response to a high-frequency burst activation of glutamatergic synapses ( re-plotted from Figure 8D ) .", "( 1 ) AMPA-/NMDA-EPSP , ( 2 ) Na-dSpike , ( 3 ) ICa through NMDAR channels , ( 4 ) ICa through L-Cav channels .", "Note also the presence of a spikelet ( asterisk ) .", "( B ) Schematic illustration of the events leading to the induction of PP → CA1tuft LTP .", "Strong activation of PP → CA1tuft synapses results in EPSPs ( 1 ) and , on some trials , subsequently leads to initiation of Na-dSpikes ( 2 ) .", "The locally generated Na-dSpike mediates the largest ICa through both NMDAR channels ( 3 ) and L-Cav channels ( 4 ) , thus resulting in a high , localized calcium concentration near the mouth of the channels ( dark red ) , which activates a series of biochemical events necessary for the induction of LTP .", "This intracellular calcium diffuses away , contributing only modestly to the ‘bulk’ calcium concentration throughout the dendritic spine and shaft ( pink ) , which is eventually removed from the cytoplasm by pumps in the plasma membrane and in organelles such as the endoplasmic reticulum .", "In contrast , the smaller , longer-lasting ICa through NMDAR channels generated during the slow synaptic depolarization produces a lower localized calcium concentration near the channel pore , but contributes more to the bulk calcium concentration . DOI: http://dx . doi . org/10 . 7554/eLife . 06414 . 034 The efficacy of low TTX as a blocker of LTP by inhibiting Na-dSpikes could be explained by the existence of a calcium sensor near the channels that triggers a series of LTP-inducing biochemical events ( Figure 10 ) .", "The affinity and co-operativity of the calcium sensor would have to be matched to the spatiotemporal features of the calcium concentration profiles near the mouths of NMDAR and L-Cav channels in response to TBS .", "The affinity of the sensor would need to be relatively low , in order to respond only to the high calcium concentration near the calcium-conducting pores during a large Na-dSpike .", "As our model predicts a modest increase in peak ICa ( proportional to local calcium concentration ) during a dSpike , relative to the slow synaptic depolarization , it is likely that the sensor would also need high cooperativity in order to respond selectively during the dSpike .", "Importantly , experimentally observed Hill coefficients are indeed high enough for this operation; for example , assuming a Hill coefficient of 4 , a modest dSpike-driven difference in peak ICa ( ∼twofold; Figure", "8 ) would increase calcium sensor activation from 20% to 80% .", "The identities of the calcium sensors and the downstream biochemistry leading to LTP induction are the subjects of ongoing research ( Amici et al . , 2009 ) , but calmodulin may have the properties necessary to serve this function , namely low affinity , rapid binding kinetics , and high cooperativity for calcium binding ( Faas et al . , 2011 ) .", "Calmodulin has been postulated to be the initial calcium sensor coupled to other key LTP-inducing molecules such as calcium/calmodulin-dependent protein kinase II ( Bradshaw et al . , 2003; Lisman et al . , 2012 ) .", "Alternatively , Na-dSpikes might couple activation of NMDAR or L-Cav channels to LTP via mechanisms that operate independently of postsynaptic calcium influx or in concert with it .", "For example , it is well known that the gating of L-Cav channels is coupled to muscle contraction independently of calcium flux through the channels and that similar mechanisms may exist in neurons ( Kaczmarek , 2006 ) .", "Indeed , activation of NMDARs in the absence of calcium influx through the channels has been suggested to be sufficient for induction of long-term depression ( LTD; Nabavi et al . , 2013; but see Babiec et al . , 2014 ) .", "In keeping with this possibility , we observed LTD in neurons buffered with 10 mM BAPTA .", "However , the block of LTP by intracellular calcium buffering ( Figure", "9 ) suggests that NMDAR and L-Cav channels contribute to the induction of PP → CA1tuft LTP by their ionotropic rather than metabotropic functions .", "Moreover , high-frequency stimulation of PP → CA1tuft synapses has been shown to result in LTP expression mediated by functional upregulation of presynaptic Cav channels ( Ahmed and Siegelbaum , 2009 ) , but the lack of effect of nimodipine on synaptic responses after LTP induction ( Figure 4—figure supplement", "1 ) indicates that the effect of nimodipine on TBS-induced PP → CA1tuft LTP cannot be explained by the block of a presynaptic expression mechanism mediated by L-Cav channels .", "Nevertheless , we cannot rule out a possible role of presynaptic NMDARs in LTP induction ( Kunz et al . , 2013 ) .", "The requirement for Na-dSpikes has interesting functional implications for PP → CA1tuft LTP .", "The activation of multiple synapses on a single dendritic branch leads to initiation of a dSpike ( Losonczy and Magee , 2006 ) , but dSpikes tend to fail at branch points ( Spruston , 2008; Katz et al . , 2009 ) .", "Thus , the spatial extent of a Na-dSpike-dependent LTP event may be restricted to a particular dendritic branch , or even a segment of a branch containing the synapses that contribute to initiation of a Na-dSpike .", "If true , such compartmentalization of plasticity ( Losonczy et al . , 2008; Makara et al . , 2009; Makino and Malinow , 2011 ) would have substantial implications for the granularity of associations learned at the dendritic level , and consequently , the information-carrying capacity of a pyramidal neuron , as information may be stored discretely in individual dendritic branches or segments , rather than in an entire neuron ( Poirazi and Mel , 2001; Mehta , 2004; Wu and Mel , 2009 ) .", "There are many forms of LTP , so it is not clear how general the requirement for Na-dSpikes will prove to be , even at the same synapses but with a different induction protocol .", "PP → CA1tuft LTP has also been reported to occur in response to combined activation of the PP and the more proximal Schaffer collaterals ( SCs; Takahashi and Magee , 2009 ) .", "In those experiments , dendritic Nav channels would be critical because bAPs were necessary to trigger calcium plateau potentials ( a form of Ca-/NMDA-dSpike ) that induced LTP .", "For this reason , it may prove difficult to disentangle the contributions of dendritic regenerative events mediated primarily by dendritic Nav channels ( such as Na-dSpikes and bAPs ) from dendritic regenerative events mediated by other channels ( such as Ca- and NMDA-dSpikes ) to the induction of PP → CA1tuft LTP with this paradigm .", "Moreover , although NMDARs and R-type Cav channels were shown to be important for this form of LTP ( Takahashi and Magee , 2009 ) , the role of dendritic L-Cav channels is unclear .", "L-Cav channels were not required for plateau potentials , but whether their activation by plateau potentials contributes to the induction of this form of PP → CA1tuft LTP has not been tested .", "Given the remarkable capacity of the brain to store information , it is no surprise that there are many different forms of LTP .", "Thus , we expect our findings generalize to some synapses but not others .", "For example , LTP has been induced at SC synapses on CA1 pyramidal neurons using many different protocols , such as those that produce synaptic depolarization alone , without bAPs or dSpikes ( Gustafsson et al . , 1987; Isaac et al . , 1995; Liao et al . , 1995; Han and Heinemann , 2013 ) .", "Another interesting form of LTP at SC synapses depends on an instructive signal from coincident PP activation ( Dudman et al . , 2007 ) .", "This form of LTP does not depend on bAPs or dSpikes either , but is mediated by endocannabinoid release from dendrites , induced by summation of EPSPs from both pathways , which ultimately downregulates feedforward inhibition ( Xu et al . , 2012; Basu et al . , 2013 ) .", "At these same SC synapses , other forms of LTP have been reported to depend on postsynaptic action potential firing , presumably via bAPs ( Magee and Johnston , 1997; Thomas et al . , 1998; Wittenberg and Wang , 2006; Buchanan and Mellor , 2007 ) .", "Still other forms of LTP at SC synapses , however , are correlated with the initiation of dSpikes ( Remy and Spruston , 2007; Hardie and Spruston , 2009 ) , but it is unclear what role Na-dSpikes play , relative to Ca-dSpikes or NMDA-dSpikes .", "At excitatory synapses on other cell types , LTP has been reported to result from a range of postsynaptic mechanisms ( Gordon et al . , 2006; Holthoff et al . , 2006; Kampa et al . , 2007; Froemke et al . , 2010; Hsu et al . , 2010 , 2012; Brandalise and Gerber , 2014 ) .", "Importantly , however , a recent study demonstrated that rhythmic whisker stimulation induced LTP in somatosensory cortex , even when the stimulus did not drive somatic action potential firing ( Gambino et al . , 2014 ) .", "Thus , LTP can occur in vivo , in response to naturalistic simulation , even in the absence of axo-somatic spiking .", "Nevertheless , action potentials and bAPs are likely to be important for other forms of synaptic plasticity .", "The diversity of LTP induction mechanisms is likely to contribute to the powerful learning capacity of the brain .", "In summary , the experiments presented here argue for a critical , causal role of dendritic Nav channels , by triggering Na-dSpikes , in the induction of PP → CA1tuft LTP .", "With this conclusion , we have now established possible functional roles for Na-dSpikes not only in the rapid , moment-to-moment processing of internal and external information ( by influencing synaptic integration; Golding and Spruston , 1998; Jarsky et al . , 2005; Losonczy and Magee , 2006; Katz et al . , 2009 ) , but also in the long-term storage of this information ( by a Na-dSpike-dependent form of LTP ) .", "Dendritic Nav channels were previously known to contribute to the cellular basis of memory by mediating forms of LTP that require action potential backpropagation .", "In this case , the Hebbian plasticity rule—‘neurons that fire together wire together’—is implemented by bAPs .", "Our results suggest that dendritic Nav channels can also contribute to memory formation via a critical role for Na-dSpikes in some forms of LTP .", "Our view is that this form of LTP is Hebbian on a finer spatial scale , for which the rule can be rephrased as ‘neurites that fire together wire together’ .", "Dendritic Nav channels play a central role in the cellular implementation of this rule , by determining which postsynaptic neurites fire ." ], [ "All animal procedures were approved by the Animal Care and Use Committees at the HHMI Janelia Research Campus and Northwestern University .", "3- to 7-week-old male Wistar rats were decapitated under deep isoflurane anesthesia , and the brain was transferred to an ice-cold dissection solution containing ( in mM ) : 110 Choline-Cl , 0 . 2 NaCl , 2 . 5 KCl , 1 . 25 NaH2PO4 , 25 NaHCO3 , 15 Dextrose , 2 . 4 Na-Pyruvate , 1 . 3 Na-Ascorbate , 0 . 5 CaCl2 , 3 MgCl2 ( pH 7 . 4 , oxygenated with 95% CO2 and 5% O2 ) .", "300- to 350-µm thick , near-horizontal slices were sectioned using a vibrating tissue slicer ( Vibratome 3000 , The Vibratome Company , St . Louis , MO; or Leica VT 1200S , Leica Microsystems , Wetzlar , Germany ) .", "CA3 and superficial layers of the entorhinal cortex were removed to limit polysynaptic activation .", "The slices were transferred to a suspended mesh within a chamber filled with artificial cerebrospinal fluid ( ACSF ) containing ( in mM ) : 119 NaCl , 2 . 5 KCl , 1 . 25 NaH2PO4 , 25 NaHCO3 , 25 Dextrose , 2 CaCl2 , 1 MgCl2 ( 3 Na-Pyruvate , 1 Na-Ascorbate were added when two-photon imaging experiments were performed; pH 7 . 4 , oxygenated with 95% CO2 and 5% O2 ) .", "After 30 min of incubation at 35°C , the chamber was maintained at room temperature .", "All recordings were performed using slices submerged in the recording chamber of an Axioskop 2 ( Carl Zeiss Microscopy , Jena , Germany ) or SliceScope ( Scientifica , East Sussex , UK ) upright microscope constantly perfused with oxygenated ACSF at 33–35°C .", "SR-95531 ( 2 µM ) and CGP 52432 ( 1 µM ) were added to block GABAA and GABAB receptors , respectively .", "Patch pipettes were pulled from thick-wall borosilicate glass and fire polished , resulting in resistance of 3–5 MΩ and 7–9 MΩ for somatic and dendritic recording , respectively , when filled with intracellular solution containing ( in mM ) : 115 K-Gluconate , 20 KCl , 10 Na2-phosphocreatin , 10 HEPES , 4 Mg-ATP , 0 . 3 Na-GTP , and 0 . 1% biocytin .", "When EGTA or BAPTA was included in the intracellular solution , the basal level of cytoplasmic calcium concentration ( ∼50 nM for CA1 pyramidal neurons; Maravall et al . , 2000; Henneberger et al . , 2010 ) was maintained by adding CaCl2 to the intracellular solution , calibrated based on the mass-action reactions between calcium ion and the particular calcium chelator used ( calculated with MaxChelator by Chris Patton , http://web . stanford . edu/∼cpatton/webmaxc/webmaxcS . htm ) .", "The dissociation constant ( Kd ) for Ca2+ at 33°C with the ionic strength 0 . 16 N , pH 7 . 3 is 91 . 7 and 229 nM for EGTA and BAPTA , respectively .", "The concentrations of calcium chelator and CaCl2 included are ( in mM ) 0 . 18 CaCl2 for 0 . 5 EGTA; 3 . 53 CaCl2 for 10 EGTA; 0 . 09 CaCl2 for 0 . 5 BAPTA; 1 . 79 CaCl2 for 10 BAPTA .", "Dendritic recordings were obtained 200–320 µm away from the soma .", "Patch pipette series resistance was always lower than 50 MΩ .", "Recordings were made using a Dagan BVC-700A amplifier ( Dagan Corporation , Minneapolis , MN ) .", "Data were low-pass filtered at 3 or 5 kHz and digitized at 50 kHz via an ITC18 digital-analog converter ( HEKA Instruments Inc . , Bellmore , NY ) under control of custom macros programmed in IGOR Pro ( Wavemetrics , Lake Oswego , OR ) .", "For synaptic stimulation , theta-glass or capillary micropipettes ( ∼40–50 µm in diameter ) filled with ACSF were used for bipolar or monopolar stimulation via a stimulus isolator ( BSI-950 , Dagan Corporation; or Model 4AD , Getting Instruments , San Diego , CA ) , placed in stratum lacunosum-moleculare ( SLM ) ∼200–300 µm away ( usually toward subiculum ) from the recorded neuron .", "For LTP induction , stimulus intensities were set to give EPSP amplitudes of 2–5 mV in somatic recordings and 4–10 mV in dendritic recordings , consistent with approximately 50% attenuation of EPSPs between the dendrite ( 200–300 µm from the soma ) and the soma ( Golding et al . , 2005 ) .", "For testing the effects of 20 nM TTX on synaptic transmission ( Figure 1 ) , a larger range of stimulus intensities ( giving EPSP amplitudes of 3–10 mV in somatic recordings ) was used .", "PP → CA1tuft EPSPs were monitored every 20 s , and interleaved test pulses were used to monitor the recording quality ( series resistance , bridge balance , pipette capacitance compensation ) and the input resistance of the cell throughout the experiment .", "To induce PP → CA1tuft LTP , we used TBS , which consisted of five burst stimuli grouped at 5 Hz , with each consisting of 5 synaptic stimuli at 100 Hz .", "This stimulus was repeated three times ( TBSx3 ) , at 30-s intervals .", "Each TBS was delivered under one of the following three conditions: ( 1 ) paired with brief ( 2 ms ) somatic current injections at 50 Hz to evoke 3 action potentials during each burst ( TBSx3+Current ) , ( 2 ) with the soma voltage-clamped at −68 to −70 mV ( TBSx3+SomaticVC ) , or ( 3 ) alone ( 5-stim TBSx3 ) .", "In most cases of somatic voltage clamp , the soma was clamped well enough to prevent action potential firing in response to TBS; however , in three cases , escape spikes were observed , so these experiments were rejected from the dataset .", "For pharmacological experiments , PP → CA1tuft EPSP amplitude was monitored before and 10 min after bath application of drugs to confirm that synaptic transmission was unaffected .", "To block NMDARs , the D-isomer of AP5 was used to maximize the effect ( Watkins and Olverman , 1987; Morris , 1989 ) .", "Notably , 50 µM AP5 alone inhibited LTP as much as the combination of AP5 and MK-801 used in our previous work ( Golding et al . , 2002; Remy and Spruston , 2007 ) , suggesting that the limited block of LTP by AP5 could not be attributed to glutamate competing away binding of AP5 to NMDARs .", "When EGTA or BAPTA was included in the intracellular solution , cells were dialyzed for at least 20–25 min before the LTP induction protocol was applied .", "For antidromic stimulation , bath application of CNQX ( 10 µM ) and AP5 ( 50 µM ) was used to block ionotropic glutamate receptors ( in addition to the GABA receptor blockers included in all experiments ) , and a stimulating electrode ( as described above ) was placed in stratum oriens , ∼20–50 µm away from the recorded neuron .", "Several different stimulus intensities were used to trigger action potentials antidromically .", "In some experiments ( Figure 1 ) , 10 µM TTX was applied manually via pressure through a patch pipette positioned near the soma .", "To limit TTX diffusion toward the apical dendrites , slices were positioned to have bath flow directed from the apical dendrites toward the soma .", "During perisomatic TTX application , EPSP amplitude was monitored and remained unchanged .", "In one experiment , action potentials were not completely eliminated in response to burst stimulation , so it was excluded from the analysis of burst responses ( but not from the analysis of single-shock EPSPs ) .", "Nickel chloride ( Ni2+ ) , SR-95531 , 6-cyano-7-nitroquinoxaline-2 , 3-dione disodium salt hydrate ( CNQX ) , biocytin , sodium pyruvate , ( + ) -sodium L-ascorbate , phosphocreatine disodium , adenosine 5'-triphosphate magnesium ( Mg-ATP ) , guanosine 5'-triphosphate sodium ( Na-GTP ) , 4- ( 2-hydroxyethyl ) piperazine-1-ethanesulfonic acid ( HEPES ) , potassium D-gluconate , dextrose , and choline chloride were from Sigma–Aldrich ( St . Louis , MO ) .", "Calcium chloride and magnesium chloride were from Fluka ( St . Louis , MO ) or Sigma–Aldrich .", "D- ( − ) -2-amino-5-phosphonopentanoic acid ( D-AP5 ) , tetrodotoxin citrate ( TTX ) , nimodipine , CGP 52432 , EGTA , and BAPTA were from Tocris Bioscience ( Minneapolis , MN ) .", "Sodium chloride , potassium chloride , sodium bicarbonate , and sodium phosphate monobasic were from Fisher Scientific ( Waltham , MA ) .", "Calcium imaging in distal tuft dendrites of CA1 pyramidal neurons was performed using a galvanometer-based two-photon laser scanning system ( Prairie Ultima; Prairie Technologies , Middleton , WI ) equipped with an epifluorescence microscope ( BX61WI; Olympus , Tokyo , Japan ) and a water-immersion objective lens ( 40X , 0 . 8 NA; Olympus ) .", "Neurons were filled with 100 µM Oregon Green 488 BAPTA-1 , hexapotassium salt ( OGB-1; Invitrogen , Waltham , MA ) and 50 µM Alexa Fluor 594 Hydrazide ( AF-594; Invitrogen , Waltham , MA ) .", "An ultrafast , Ti:Sapphire pulsed laser ( Chameleon Ultra; Coherent , Auburn , CA ) was tuned to 880 nm to acquire reference images and 920 nm to perform calcium imaging .", "Laser power was controlled with an electro-optical modulator ( Model 350-80; Conoptics , Danbury , CT ) .", "Line-scan imaging along dendritic shafts ( typically 5–10 µm in length ) and across spines was performed using the Ultima scanner at 250–500 Hz with a dwell time of 10 µs , and fluorescence was collected by multi-alkali photomultiplier tubes ( PMTs; Hamamatsu Photonics , Hamamatsu City , Japan ) .", "Laser power was adjusted to ensure a good signal-to-noise ratio of the fluorescence signal without photo-bleaching the dyes or photo-damaging the dendrites ( see below ) .", "In some experiments , multiple locations on several branches or longer stretches of the branch ( up to ∼35 µm ) were imaged to determine the spatial profile of the calcium signals .", "All the data acquisition and device controls were performed using BNC-2090 and BNC-2110 boards ( National Instruments , Austin , TX ) with Prairie View and TriggerSync software ( Prairie Technologies ) .", "To achieve an equilibrium concentration of the dyes sufficient for calcium imaging in the distal dendrites ( distance from the soma: 312–673 µm; average = 411 ± 18 µm ) , cells were dialyzed for at least 30 min before imaging commenced .", "Calcium signals were quantified as the increase in ( green ) OGB-1 fluorescence from the baseline before stimulation ( 50–100 ms ) divided by ( red ) AF-594 fluorescence ( ∆G/R ) .", "This quantification is insensitive to small variations in basal calcium concentration and independent of the volume of imaged structures ( Sabatini et al . , 2002 ) .", "For calcium imaging , single high-frequency burst stimulation ( instead of full TBS ) was applied to the PP with an interval of at least 30–120 s; PP → CA1tuft EPSP amplitude was constantly monitored to determine the stability of synaptic responses .", "Consistent with previous theoretical and experimental studies ( Helmchen et al . , 1996; Sabatini et al . , 2002 ) , control experiments showed that the integral of ∆G/R is a more stable measure of calcium entry than peak ∆G/R ( Figure 7—figure supplement 1; Figure 7—source data 2 ) .", "Control experiments suggested that dye saturation did not occur under our conditions ( data not shown ) .", "The largest peak ∆G/R achieved in the distal dendrites ( ∼250% , by pairing TBS with somatic current injection to evoke bAPs ) was well above the largest value observed in response to a single high-frequency burst stimulation .", "Furthermore , the dye was not saturated by a single burst in general because calcium responses were able to facilitate upon delivery of additional bursts in TBS with the ratio ΔG/R ( Max ) :ΔG/R ( first burst ) = 1 . 52 ± 0 . 31 ( n = 4 ) .", "Importantly , no correlation was observed between normalized drug effects and the ∆G/R measure in control ( data not shown ) , which would be expected if dye saturation had resulted in an underestimate of pharmacological inhibition .", "The following indications of phototoxicity were monitored carefully: basal fluorescence of both dyes and its ratio ( G0/R as a readout of basal calcium concentration; see Figure 7—figure supplement 1 ) , morphological changes of dendrites ( swelling or fragmentation ) , long-lasting depolarization following synaptic stimulation or current injection , and a sudden loss of calcium signals .", "Experiments were terminated if any signs of photo-damage were observed .", "At the end of each experiment , the path distance and the depth ( usually 25–55 µm from the surface ) of the imaging sites were measured , and high-resolution Z-stack images were collected .", "In initial experiments , we compared the effects of 20 nM TTX across different PP → CA1tuft LTP induction protocols ( see above ) , and found reduction of calcium signals in all conditions ( data not shown ) .", "However , during high-frequency burst stimulation of the PP , bAPs could contribute to calcium signals in distal dendrites ( data not shown ) .", "To focus on the calcium signaling mediated by dSpikes rather than bAPs ( which are not required for the induction of PP → CA1tuft LTP; Figure 3E; Golding et al . , 2002 ) , we conducted calcium imaging with the soma voltage-clamped at ∼ −70 to −75 mV .", "No escape spikes were observed under these conditions in this series of experiments .", "For all recordings , the peak amplitude of events was measured as the difference between the resting and peak membrane potentials .", "For somatic recordings , the apparent voltage threshold of action potentials was measured as the voltage at which dV/dt crossed 40 V/s .", "In dendritic recordings , the voltage responses after each synaptic stimulus position ( Stim . # ) could be divided into two populations with non-overlapping amplitude distributions ( Figure 2—figure supplement 1A ) .", "Large-amplitude dendritic events ( defined as those with the peak amplitude >40 mV ) were presumed to consist of both bAPs and large dSpikes .", "The apparent voltage threshold of large-amplitude dendritic events was measured as the voltage at which the second temporal derivative of voltage d2V/dt2 crossed 7 . 5 mV/ms2 .", "bAPs typically have a high apparent voltage threshold , because large dendritic EPSPs are required for the axonal EPSPs to be large enough to reach the threshold for action potential initiation after EPSP attenuation between the dendrites and the axon .", "In contrast , large dSpikes have a lower apparent threshold , because they are initiated closer to the dendritic recording electrode .", "bAPs and large dSpikes also differ in their onset kinetics , quantified as ‘initial phase slope’ , which was measured as the slope of a linear fit to the initial portion of the phase plot ( dV/dt vs V; Figure 2—figure supplement 2A ) .", "As dV/dt is proportional to membrane current , the initial phase slope can be conceptualized as the apparent voltage sensitivity of membrane current .", "It is an apparent voltage sensitivity because the source of the current ( e . g . , the Nav channels ) and the membrane potential responsible for the current are at a remote location , and therefore the observed voltage sensitivity does not necessarily reflect the voltage dependence of the activation of the channels per se .", "In dendritic recordings , actively propagating spikes initiated in the axon ( i . e . , bAPs ) typically have a sharp ‘kink’ at their onset , because they are initiated by axial current flowing from a remote location of spike initiation ( i . e . , the axon ) , which precedes local dendritic Nav channel-mediated currents .", "The ‘kink’ is reflected as a large initial phase slope ( >3 . 5 ms-1 ) , and thus a steep apparent voltage sensitivity of the membrane current , because the axial current is driven by the voltage difference between the axon and the dendrite , thus resulting in a current that is not driven by changes in the local dendritic depolarization and therefore does not require activation of local channels .", "As a result of this sudden current , the onset of these backpropagating spikes shows a kink in the time domain and a steep initial slope in the phase domain .", "In contrast , large dSpikes have a more gradual onset , because they are initiated by local Nav channel-mediated currents generated closer to the dendritic recording electrode , and therefore the voltage sensitivity of the membrane current ( as measured by initial phase slope ) more accurately reflects the voltage sensitivity of the Nav channels ( Shu et al . , 2007; Yu et al . , 2008; Brette , 2013; Smith et al . , 2013 ) .", "On the basis of these two criteria , some events could be identified as clear bAPs ( high threshold and large initial phase slope ) or clear large dSpikes ( low threshold and small initial phase slope ) , while the other events were ambiguous ( Figure 2—figure supplement 2; Figure 2—source data 2 ) , perhaps owing to complications such as coincident local dendritic depolarization ( which may reduce the initial phase slope of bAPs ) or very distal locations of dSpike initiation ( which may result in large initial phase slope of propagating dSpikes ) .", "Three large-amplitude dendritic events with relatively small initial phase slope but high threshold also had relatively broad halfwidth , suggesting a larger contribution from Cav and/or NMDAR channels ( Figure 2—figure supplement 2B; Figure 2—source data 2 ) .", "Small dSpikes ( spikelets ) were identified as outliers in the distribution of peak dV/dt values ( stimulus positions with a large-amplitude dendritic event in the control condition were excluded for this analysis ) for each of the five stimulus positions ( Stim . # ) in each burst ( Figure 2C ) .", "We observed a trend toward decay of peak dV/dt in each burst as a function of stimulus position ( Figure 2—figure supplement 1B ) .", "Therefore , we normalized peak dV/dt to the median value for Stim .", "#1 in control ( i . e . , of the five bursts in control from the given cell ) and plotted normalized dV/dt as a function of stimulus position in each burst in control and in the presence of 20 nM TTX .", "A fit of an exponential decay function to the whole population of data ( control and 20 nM TTX ) was then performed .", "Small dSpikes were identified as events that fell outside the prediction band of the fit; in this way , a single criterion was applied to both experimental conditions ( i . e . , control and 20 nM TTX ) .", "Because there is no perfectly objective way to determine which events should be called small dSpikes , we analyzed the data for prediction bands at confidence levels ranging from 85% ( least stringent; largest number of small dSpikes ) to 99% ( most stringent; smallest number of small dSpikes ) .", "Two additional statistical criteria ( based on the median or standard deviation of normalized peak dV/dt for each stimulus position; data not shown ) and another approach applying a single ‘hard’ threshold ( Figure 2—figure supplement", "4 ) for identifying small dSpikes were also used , and the qualitative results remained the same .", "For notched box plots ( Figure 2—figure supplement 4A ) , the edges of notch were defined by the 95% confidence intervals of the median , the end points of whiskers as the largest and smallest values that are within the range of ( the 75th percentile + 1 . 5 × interquartile range ) and ( the 25th percentile − 1 . 5 × interquartile range ) , and individual data points are those outside this range .", "The potentiation ratio was calculated as the average EPSP amplitude 26–30 min after LTP induction normalized by the average EPSP amplitude before induction .", "Calcium and current signals were averaged over 3–6 trials , with a slight Gaussian smoothing ( standard deviation of the kernel = 0 . 064 ms ) , and the peak amplitude and integral ( up to 800 ms after stimulation ) were measured .", "Special caution was taken to confirm that the smoothing reduced random noise without changing the kinetics and amplitude of the original signals .", "Tissue autofluorescence was determined to be negligible in the two-photon experiments , so no background subtraction was carried out .", "The two-photon Z-stack images of neurons were generated by two-dimensional projection of maximal AF-594 fluorescence intensity from three-dimensional Z-stacks .", "All analyses were performed using IGOR Pro ( Wavemetrics , Lake Oswego , OR ) , MATLAB ( The MathWorks , Natick , MA ) , ImageJ ( National Institutes of Health , Bethesda , MD ) , and Microsoft Office Excel ( Microsoft , Redmond , WA ) .", "In most cases ( unless noted otherwise ) , data were presented as mean ± S . E . M . Significance of the difference in normalized EPSP amplitude between experimental conditions was tested by one-way ANOVA with post hoc multiple comparisons by Tukey's method .", "Significance of LTP expression for each experimental condition was tested by one-way repeated measures ANOVA .", "Significance of the difference in number of events counted as small dSpikes ( spikelets ) between two experimental conditions was tested using a binomial test; rejection of the null hypothesis suggests an unequal likelihood of an event occurring above threshold between two conditions .", "All other comparisons between two experimental groups/conditions were done with Student's t-test .", "Statistical analyses were performed using Prism ( GraphPad Software , La Jolla , CA ) and OriginPro ( OriginLab Corporation , Northampton , MA ) .", "All data presented in the figures has been deposited to our laboratory website ( www . janelia . org/lab/spruston-lab/resources ) and figshare ( Spruston et al . , 2015 ) .", "Simulations were performed using a compartmental model based on the morphology reconstructed from a rat CA1 pyramidal neuron ( Golding et al . , 2001 ) .", "The passive and active properties of the model were used the same as those used previously , which reproduced experimental data on bAPs and dSpikes ( Golding et al . , 2001; Jarsky et al . , 2005; Katz et al . , 2009 ) with adaptations described below .", "All simulations were performed using the NEURON simulation software ( Hines and Carnevale , 1997 ) with a fixed time step ( dt = 0 . 025 ms ) .", "Code for the simulations has been deposited to our laboratory website ( www . janelia . org/lab/spruston-lab/resources ) and the ModelDB database ( Hsu et al . , 2015 ) .", "The model included active conductances simulating the following channels: Nav channels , A-type potassium ( KA ) channels , delayed-rectifier potassium ( KDR ) channels , and L-Cav channels .", "Properties and distributions of KA and KDR channels were the same as previously stated ( Golding et al . , 2001; Katz et al . , 2009 ) .", "The L-Cav channel model ( Poirazi et al . , 2003 ) was inserted into the apical tuft with a uniform density gCa = 1 . 25 mS/cm2 .", "The properties of the dendritic Nav channels , including slow inactivation , were implemented using a model from Migliore et al . , 1999 .", "Inclusion of slow inactivation improved the fit of the limited effect of 20 nM TTX on the dendritically recorded voltage in response to single high-frequency burst stimulation ( Figures 2A , B , 5 ) .", "Following a recent study ( Lorincz and Nusser , 2010 ) , in the apical dendritic tree a linearly decreasing gradient of Nav channel density was implemented using the equation:gNa ( x ) =gNa , soma+ΔgNa⋅xwhere x is the distance from the soma ( in μm ) , and gNa ( x ) is the Nav channel conductance at the distance x; gNa , soma = 42 mS/cm2 , ΔgNa=−0 . 025 ( mS/cm2 ) /μm , and for the remaining compartments , gNa=gNa , soma .", "Synaptic stimulation was simulated as 150 simultaneous events distributed randomly across all of the apical tuft branches .", "Spines were not explicitly modeled , but the associated surface area was accounted for by decreasing specific membrane resistivity ( Rm ) and increasing specific membrane capacitance ( Cm ) by a factor of 2 for compartments >100 μm from the soma .", "In additional simulations , synapses simulated on explicitly modeled spines yielded qualitatively similar results .", "Every synapse was taken to have α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ( AMPA ) receptor ( AMPAR ) and NMDAR conductances ( gAMPA=0 . 18 nS , gNMDA=0 . 18 nS ) , and both were modeled as a difference of two exponentially decaying functions with rise and decay time constants of 0 . 2 and 2 ms for AMPARs ( Katz et al . , 2009 ) and 1 and 50 ms for NMDARs ( Hestrin et al . , 1990; Spruston et al . , 1995 ) .", "The voltage-dependent magnesium ( Mg2+ ) block of NMDARs was modeled as gMg=[1+0 . 2801⋅Mgext2+⋅exp ( −0 . 062⋅ ( V−10 ) ) ]−1 ( Spruston et al . , 1995; Maex and De Schutter , 1998 ) with the Mg2+ concentration in bath Mgext2+=1 mM .", "The fractional calcium influx through NMDAR channels was taken to be 10% of the total current ( Schneggenburger et al . , 1993; Burnashev et al . , 1995; Garaschuk et al . , 1996 ) .", "The temperature used in the model was 35°C .", "A calcium handling mechanism was implemented ( Hines and Carnevale , 2000 ) which included buffers ( endogenous buffer and OGB-1 ) , diffusion ( radial and longitudinal ) , and extrusion ( a pump ) .", "The parameters of OGB-1 were as follows ( Schmidt et al . , 2003 ) : diffusional mobility 0 . 3 µm2/ms , calcium-binding forward rate constant 430 mM−1ms−1 , backward rate constant 0 . 14 ms-1 , and effective concentration 0 . 05 mM .", "The endogenous buffer was immobile , and the parameters were as follows ( Yamada et al . , 1989 ) : calcium-binding forward rate constant 100 mM−1ms−1 , backward rate constant 0 . 1 ms-1 , and concentration 0 . 003 mM .", "The calcium extrusion by a calcium pump was modeled as a two-step reaction with mass-action kinetics:Cain2++ pump ⇄ Ca2+×pumpCa2+×pump ⇄ pump+Caout2+ The forward and backward rate constants for the first step were 1 mM−1ms−1 and 0 . 005 ms-1 , and for the second step 1 ms-1 and 0 . 005 mM−1ms−1 , respectively .", "The calcium pump density was 10−11 mol/cm2 .", "With these calcium buffers and calcium pump , the free calcium concentration ( [Ca2+]free ) was in the linear operation range of OGB-1 ( i . e . , the dissociation constant ) , and the kinetics of [Ca2+]OGB signals matched the imaging data .", "To simulate bath application of 20 nM TTX , we reduced the conductance of Nav channels to 50% of control , consistent with the previous observations of TTX efficacy on hippocampal neurons ( Kaneda et al . , 1989; Madeja , 2000 ) ; to simulate bath application of 50 µM AP5 or 10 µM nimodipine , we reduced the NMDAR or L-Cav channel conductance to zero .", "For all simulations , either a somatic voltage clamp at −70 mV was simulated or gNa , soma and gNa , axon were set to be zero in order to mimic the experimental paradigm in which axo-somatic action potential firing was prevented .", "Although the model contains a large number of parameters , many of these values were constrained by a wealth of available experimental data on CA1 pyramidal neurons ( this study; Magee and Johnston , 1995; Spruston et al . , 1995; Helmchen et al . , 1996; Hoffman et al . , 1997; Golding et al . , 2001; Otmakhova et al . , 2002; Sabatini et al . , 2002; Katz at al . , 2009; Bittner et al . , 2012 ) .", "As in all modeling , some assumptions had to be made , so we were careful to test a range of parameter values .", "Our conclusions were robust using several combinations of parameters .", "As always , however , we make no claim that the model is ‘correct’ in an absolute sense .", "Its purpose is to demonstrate the plausibility of our interpretation and the proposed model with parameter values constrained by those available in the literature ." ] ]

[ "Dendritic integration of synaptic inputs mediates rapid neural computation as well as longer-lasting plasticity .", "Several channel types can mediate dendritically initiated spikes ( dSpikes ) , which may impact information processing and storage across multiple timescales; however , the roles of different channels in the rapid vs long-term effects of dSpikes are unknown .", "We show here that dSpikes mediated by Nav channels ( blocked by a low concentration of TTX ) are required for long-term potentiation ( LTP ) in the distal apical dendrites of hippocampal pyramidal neurons .", "Furthermore , imaging , simulations , and buffering experiments all support a model whereby fast Nav channel-mediated dSpikes ( Na-dSpikes ) contribute to LTP induction by promoting large , transient , localized increases in intracellular calcium concentration near the calcium-conducting pores of NMDAR and L-type Cav channels .", "Thus , in addition to contributing to rapid neural processing , Na-dSpikes are likely to contribute to memory formation via their role in long-lasting synaptic plasticity ." ]

[ "When we explore somewhere new , we activate a region of the brain that processes spatial information called the entorhinal cortex .", "This brain region stimulates the brain's memory-formation center , known as the hippocampus , which in turn forms a spatial memory of the new place .", "The process of forming these memories involves strengthening nerve connections , including those between the entorhinal cortex and the hippocampus .", "Groups of neurons that produce synchronized electrical activity will naturally strengthen the nerve connections between them .", "This led scientists to predict that synchronized electrical activity between neurons in the entorhinal cortex and the hippocampus may contribute to the formation of spatial memories .", "Previous research revealed that hippocampal neurons produced short bursts of electrical activity that are localized at specific sites along their branched nerve processes that extend out of the cell body and are where inputs from other neurons are received .", "These types of localized electrical activity have been associated with a strengthening of the nerve connections between the entorhinal cortex and the hippocampal neurons .", "Ion channels that allow calcium to flow through these neurons' cell membranes had been identified as a potential source of these local electrical activities , and calcium is responsible for the strengthening of nerve connections .", "But it remained unclear whether channels that allow only sodium ions to flow through might also be involved .", "Kim , Hsu et al . have now investigated this question by devising a way to selectively block the electrical activity produced by sodium ion channels on the branched nerve processes of hippocampal neurons .", "Slices of rat brain were collected and an inhibitor that specifically affected the sodium channels was delivered to the brain slices .", "Electrodes were used to stimulate the inputs from the entorhinal cortex , and to monitor the resulting electrical activity in the hippocampal neurons .", "Kim , Hsu et al . analyzed the results and reproduced them using computer simulations , which showed that sodium ion channels are essential for triggering brief electrical events within the individual branches of nerve processes .", "These local electrical events appeared to activate calcium channels to produce highly concentrated , short-lived calcium signals that are necessary for strengthening nerve connections .", "Future studies will determine whether local electrical activity mediated by sodium channels is also involved in strengthening nerve connections between other types of neurons , and how this mechanism affects the formation of memories ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "structural biology and molecular biophysics" ]

[ [ "The F1Fo ATP synthase is a membrane-embedded nano-machine and a member of the rotary ATPases ( F- , V- and A-ATPases ) , which are found in energy-converting membranes of all eukaryotes , bacteria , and archaea ( Muench et al . , 2011 ) .", "The enzyme catalyses the formation of ATP from ADP and inorganic phosphate ( Pi ) using the energy stored in a trans-membrane electrochemical gradient of protons or sodium ions ( Boyer , 1997; von Ballmoos et al . , 2009 ) .", "The bovine heart enzyme has a molecular mass of approximately 600 kDa and consists of 17 different subunits ( α3 , β3 , γ , δ , ε , a , b , c8 , d , e , f , g , A6L , F6 , oligomycin sensitivity conferral protein [OSCP] , DAPIT , and a 6 . 8 kDa protein ) ( Meyer et al . , 2007; Runswick et al . , 2013 ) .", "The enzyme can be subdivided into four essential functional parts: the catalytic part , ( αβ ) 3 , which binds and converts ADP and Pi to ATP; the membrane-embedded part ( a , b , c8 , e , f , g , A6L , DAPIT , and the 6 . 8 kDa protein ) through which protons move across the membrane; the central stalk , γδε , which transmits the rotation of the membrane-embedded rotor-ring ( c8 ) to the catalytic region; and the peripheral stalk ( b , d , F6 and OSCP ) which holds the catalytic part stationary relative to the membrane region .", "The catalytic and central stalk regions form the F1 subcomplex and the remainder , the Fo subcomplex .", "Structural studies of the intact F1Fo ATP synthase complex have been held back by the tendency of the enzyme to dissociate when extracted from the membrane .", "Nevertheless , a number of atomic models have been obtained by x-ray crystallography for various parts of the yeast and bovine mitochondrial F1Fo ATP synthase including the F1 subcomplex ( Abrahams et al . , 1994 ) , the F1/c-ring subcomplex ( Stock et al . , 1999; Watt et al . , 2010 ) , the peripheral stalk subcomplex ( Dickson et al . , 2006 ) , and the F1/peripheral stalk fragment ( Rees et al . , 2009 ) .", "All these structures except the c-ring belong to the soluble region of the F1Fo ATP synthase .", "The membrane-embedded part of the mitochondrial F1Fo ATP synthase has two important functions .", "First , it allows protons to cross the membrane , thereby driving ATP synthesis; and second , it causes the dimerisation and oligomerisation of the enzyme in the membrane .", "The first structure of the membrane-embedded region of an F1Fo ATP synthase dimer was recently determined for the colourless green algae , Polytomella sp .", ", by single-particle cryo-EM ( Allegretti et al . , 2015 ) .", "At 7 Å , the map shows that the conserved a-subunit forms a pair of horizontal helix haripins adjacent to the c-ring , whereas the peripheral stalk forms an intricate and unique dimerisation interface which is different from that of the bovine or yeast enzyme ( Davies et al . , 2011 ) .", "In mitochondria , F1Fo ATP synthases occur as rows of dimers on highly curved ridges in cristae membranes ( Strauss et al . , 2008; Davies et al . , 2011 , 2012 ) .", "Knock-out studies in yeast demonstrated the involvement of subunits e and g in dimer formation and cristae morphology , suggesting a crucial role of the F1Fo ATP synthase dimers in cristae formation ( Paumard et al . , 2002; Davies et al . , 2012 ) .", "Subtomogram averaging of the F1Fo ATP synthase dimers in situ revealed an angle of 86° between the monomers ( Davies et al . , 2012 ) .", "Coarse-grained molecular dynamic simulations have indicated that the shape of the F1Fo ATP synthase dimer alone is sufficient to deform the lipid bilayer and drive the self-assembly of the F1Fo ATP synthase dimers into rows ( Davies et al . , 2012 ) .", "However , a recent single-particle cryo-EM map of detergent solubilised , bovine F1Fo ATP synthase monomers found the detergent micelle around the Fo domain was bent leading to the suggestion that the monomer alone could deform lipid bilayers ( Baker et al . , 2012 ) .", "Electron cryo-crystallography and electron cryo-tomography allow the structure of membrane-embedded proteins to be investigated in a lipid environment ( Fujiyoshi and Unwin , 2008; Davies and Daum , 2013 ) .", "To determine the structure of membrane-embedded bovine heart F1Fo ATP synthase , we generated 2D crystals of intact and active bovine heart F1Fo ATP synthase using the synthetic lipid 1 , 2-dimyristoyl-sn-glycero-3-phosphocholine ( DMPC ) .", "By a combination of subtomogram averaging and electron crystallography of tomographic slices , we determined the in situ structure and packing of the enzyme complex in 2D crystals .", "The oblique orientation of individual complexes in the membrane results in a zigzag arrangement of the lipid bilayer , which perfectly matches the observed bend in the detergent micelle of the isolated complex ( Baker et al . , 2012 ) .", "Our results demonstrate that the transmembrane region of bovine mitochondrial F1Fo ATP synthase monomer is sufficient to bend the lipid bilayer .", "This membrane deformation is likely to be a prerequisite for the self-association of F1Fo ATP synthases into dimers and dimers into rows , as observed in mitochondrial cristae ." ], [ "Monomeric mitochondrial F1Fo ATP synthase was purified from bovine heart muscle tissue by sucrose density gradient centrifugation and ion-exchange chromatography .", "For 2D crystallisation , fractions exhibiting high oligomycin-sensitive ATPase activity ( >95% ) and a high content of native lipids ( >100 lipid molecules per F1Fo ATP synthase ) were mixed with synthetic DMPC .", "When the detergent was removed by dialysis , abundant crystalline vesicles formed that were stable for weeks ( Figure 1—figure supplement 1 ) .", "Analysis by SDS-PAGE and mass spectrometry confirmed that the crystalline vesicles contained all subunits of the bovine F1Fo ATP synthase , including the small Fo subunits e , f , g , A6L , DAPIT , and the 6 . 8-kDa protein ( Figure 1—figure supplement 2 ) .", "ATPase assays and blue-native PAGE performed on digitonin-solubilised crystalline vesicles demonstrated that the F1Fo ATP synthase complex in the 2D crystals was intact , active , and highly sensitive to oligomycin ( >95% ) ( Figure 1—figure supplement 3 ) .", "The 2D crystals were insufficiently ordered for electron crystallographic processing despite numerous attempts to improve their order and size .", "Thus , to gain insight into the structure of the membrane-embedded F1Fo ATP synthase , we performed electron cryo-tomography and sub-tomogram averaging .", "In the tomographic volumes , the F1 subcomplex appears as a 10-nm spherical density attached to the membrane by a thin stalk and is arranged in an alternating up-and-down orientation relative to the central lipid bilayer ( Figure 1A , B ) .", "Tomographic cross-sections of crystalline vesicles embedded in thick vitreous ice ( >200 nm ) showed a zigzag morphology of the membrane ( Figure 2 ) .", "Fourier transforms of flat areas of crystalline vesicles showed that the degree of crystalline order varied over short distances .", "The most highly ordered regions were found in vesicles with a rectangular appearance , in which two membranes were closely apposed ( Figure 1C , D ) .", "These ordered membrane regions were chosen for further analysis . 10 . 7554/eLife . 06119 . 003Figure 1 . 2D crystals of F1Fo ATP synthase in vitreous ice .", "( A ) Tomographic slice of a vesicle reconstituted with F1Fo ATP synthase .", "The ATP synthases appear as 10 nm spherical densities located 15 nm above the membrane .", "( B ) Enlarged view of red boxed area in A showing the zigzag membrane structure ( arrowhead ) .", "( See also Figure 2 . ) ( C ) Fourier transform of the blue-boxed area in ( A ) .", "( D ) Cross-section along the yellow dashed line in ( A ) .", "Scale bar: ( A ) 100 nm , ( B ) 20 nm , ( D ) 50 nm . DOI: http://dx . doi . org/10 . 7554/eLife . 06119 . 00310 . 7554/eLife . 06119 . 004Figure 1—figure supplement 1 . Crystalline vesicles of F1Fo ATP synthase in negative stain .", "( A ) Overview of an EM grid square with numerous rectangular crystalline vesicles ( red arrowheads ) .", "Scale bar 3 μm .", "( B ) Rectangular crystalline vesicle at higher magnification with boxed area enlarged .", "F1 heads ( white arrowheads ) .", "Scale bar 200 nm . DOI: http://dx . doi . org/10 . 7554/eLife . 06119 . 00410 . 7554/eLife . 06119 . 005Figure 1—figure supplement 2 . Subunit composition of F1Fo ATP synthase isolated from 2D crystals .", "( A ) SDS-polyacrylamide gradient gel ( 10–20% ) showing the subunit composition of purified F1Fo ATP synthases before ( lane", "2 ) and after ( lane", "3 ) 2D crystallisation .", "Lane 1 , molecular marker ( BenchMark ) .", "( B ) Mass spectrometry profile of small subunits ( 5000–12 , 000 Da ) of the F1Fo ATP synthase isolated from 2D crystals .", "All F1Fo ATP synthase subunits are present in the 2D crystals . DOI: http://dx . doi . org/10 . 7554/eLife . 06119 . 00510 . 7554/eLife . 06119 . 006Figure 1—figure supplement 3 . Blue-native polyacrylamide gel and activity assay of F1Fo ATP synthase isolated from 2D crystals .", "( A ) BN polyacrylamide gradient gel ( 4–12% ) of purified F1Fo ATP synthase solubilized in decylmaltoside ( lane 1 and 2 , 15 μg and 2 μg respectively ) and of digitonin-resolubilised 2D crystals ( lane 3 and 4 , 30 μg and 90 μg respectively ) .", "Bands of lower molecular weight subcomplexes , that is , Fo or c8 , are weak or absent .", "The dimer band in lane 1 and 2 presumably stems from mitochondrial F1Fo ATP synthase dimers preserved by mild purification conditions .", "The dimer band visible in lane 3 and 4 most likely represents non-physiological F1Fo ATP synthase dimers that form in the 2D crystals .", "( B ) Typical ATPase activity and oligomycin-sensitivity of digitonin-resolubilised F1Fo ATP synthase isolated from 2D crystals .", "The hydrolysis of ATP by the F1Fo ATP synthase was monitored using an enzyme couple assay by detecting NADH oxidation at 340 nm at 20°C in the absence ( blue ) or presence ( green ) of oligomycin .", "Oligomycin inhibits ATP hydrolysis through F1 by stopping rotation of the Fo motor , that is , oligomycin sensitivity gives a measure of the amount of coupled , intact F1Fo ATP synthase complexes in the preparation , in this case >95% .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06119 . 00610 . 7554/eLife . 06119 . 007Figure 2 . Zigzag membrane structure .", "( A ) Projection image of a vesicle reconstituted with F1Fo ATP synthase .", "( B ) and ( C ) tomographic slices of boxed area in ( A ) at different z-heights .", "( D ) Fourier transform of an oblique tomographic slice through boxed area in ( A ) showing weak diffraction .", "( E ) Tomographic slice of rectangular crystalline vesicle .", "( F ) Close-up of boxed area in ( E ) .", "White arrowhead , indicates membrane plane , yellow arrowhead , line of F1 head groups .", "Scale bar: ( A ) 50 nm , ( B , C , and F ) 10 nm , ( E ) 100 nm . DOI: http://dx . doi . org/10 . 7554/eLife . 06119 . 007 A total of 2100 F1Fo ATP synthase particles from the flattest regions of rectangular crystalline vesicles were manually selected and averaged by gold standard procedures ( Scheres and Chen , 2012 ) .", "According to the 0 . 5 FSC criterion , the resulting F1Fo ATP synthase average had a resolution of 24 Å ( Figure 3 and Figure 3—figure supplement 1 ) .", "Both the peripheral and central stalks as well as the individual α and β subunits are clearly visible .", "The α and β subunits form a non-symmetrical hexamer around the central stalk with alternating long and short sides , allowing an accurate assignment to their respective density regions ( Figure 3C , D , Figure 3—figure supplement 2 ) . 10 . 7554/eLife . 06119 . 008Figure 3 . Sub-tomogram average of bovine heart mitochondrial F1Fo ATP synthase calculated from 2D crystals .", "( A ) Surface view , ( B ) longitudinal section showing central stalk , ( C ) bottom view and ( D ) top view .", "Arrowheads: positions of the β subunits .", "d1 and d2: densities connecting peripheral stalk to F1 subcomplex .", "Threshold levels: light grey , 1 σ; mesh , 3 σ; dark grey , 5 σ .", "Scale bar: 20 Å .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06119 . 00810 . 7554/eLife . 06119 . 009Figure 3—figure supplement 1 . Resolution estimate of the ATP synthase monomer sub-tomogram average . Fourier shell correlation ( FSC ) curves were calculated by comparing averages generated from two independently processed data sets ( ‘Gold standard’; Scheres and Chen , 2012 , black line ) .", "To check for overfitting , phases beyond 40 Å were randomized ( Chen et al . , 2013 ) and the final alignment iteration repeated ( grey line ) .", "According to the FSC 0 . 5 criterion ( crosshairs ) , the ATP synthase monomer average has a resolution of 2 . 4 nm .", "Points on the FSC curves mark spatial frequency shells used in the FSC calculation . DOI: http://dx . doi . org/10 . 7554/eLife . 06119 . 00910 . 7554/eLife . 06119 . 010Figure 3—figure supplement 2 . The α/β hexamer .", "( A ) X-ray map of catalytic domain ( PDB code: 1BMF , Abrahams et al . , 1994 ) Fourier-filtered to 25 Å .", "( B ) Subtomogram average calculated from 2D crystals .", "Dashed lines , cross-section levels in i–iii .", "Arrowheads , beta subunits * , catalytic interface between α and β subunits .", "Contour levels drawn at intervals of 0 . 5 sigma above mean .", "Scale bar , 20 Å .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06119 . 010 The peripheral stalk is the strongest feature in the sub-tomogram average and is easily visible at contour levels >5σ above the mean .", "As in other cryo-EM maps ( Rubinstein et al . , 2003; Lau et al . , 2008; Baker et al . , 2012; Davies et al . , 2012 ) , the peripheral stalk of the F1Fo ATP synthase is arranged along the non-catalytic α/β interface and is offset towards the α-subunit , with which it makes contact midway along the stalk ( Figure 3 ) .", "The main contact of the peripheral stalk with the ( αβ ) 3 assembly occurs at the top of the complex ( Figure 3A , D ) .", "Two distinct densities are visible in this region: one above the α-subunit next to the peripheral stalk and the other above the remaining α-subunits ( Figure 3D , d1 and d2 , respectively ) .", "At lower contour levels , the two densities merge immediately above the β-subunit leaving the centre of the hexameric ring open .", "The first complex we docked into the subtomogram average was the x-ray structure of the F1/peripheral stalk fragment ( PDB: 2WSS Rees et al . , 2009 ) .", "All parts of this structure fitted well , with the N-terminal domain of the OSCP occupying the density immediately above the αβα subunits , and the C-terminal domain plus the peripheral stalk occupying the density above the remaining α-subunit .", "The peripheral stalk subcomplex ( PDB: 2CLY Dickson et al . , 2006 ) was then added to the fitted 2WSS structure but the resulting structure did not fit the map ( Figure 4—figure supplement 1 ) .", "The b-subunit residues 182–207 and the C-terminal domain of the OSCP subunit ( residues 115–188 ) from the 2WSS model were then added to the x-ray structure of the bovine peripheral stalk subcomplex ( PDB: 2CLY Dickson et al . , 2006 ) and fitted as a rigid body into the subtomogram average .", "The new model resulted in an excellent match with the EM density ( Figure 4—figure supplement 1 ) .", "Finally the c-ring from the F1/c8 x-ray structure ( PDB: 2XND Watt et al . , 2010 ) was fitted by superimposing the F1 subcomplex of this structure with that of 2WSS ( Figure 4 ) . 10 . 7554/eLife . 06119 . 011Figure 4 . Fitted atomic model of the bovine F1Fo ATP synthase .", "( A–C )", "Sub-tomogram average with fitted atomic models ( A ) side view , ( B ) top view , and ( C ) the peripheral stalk .", "Blue , catalytic domain; grey , central stalk; green , oligomycin sensitivity conferral protein ( OSCP ) from PDB:2WSS ( Rees et al . , 2009 ) .", "Purple , c-ring ( PDB:2XND ) ( Watt et al . , 2010 ) ; Yellow-red , peripheral stalk fragment ( PDB: 2CLY ) ( Dickson et al . , 2006 ) with additional residues from PDB:2WSS ( Rees et al . , 2009 ) .", "Pink , α-subunit helix thought to interact with the peripheral stalk .", "Dashed lines , position of membrane .", "Scale bar , 20 Å .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06119 . 01110 . 7554/eLife . 06119 . 012Figure 4—figure supplement 1 . Possible atomic models fitted to sub-tomogram average .", "( A ) Sub-tomogram average fitted with the F1/peripheral stalk complex ( PDB entry: 2WSS ) .", "( B ) as in ( A ) but peripheral stalk extended with the atomic model of the peripheral stalk fragment ( PDB:2CLY ) .", "( C ) as in ( B ) but the extended peripheral stalk sub-complex plus C-terminal domain of the OSCP subunit fitted to the average as a rigid-body subsequent to the fitting of the F1-subcomplex plus N-terminal domain of the OSCP subunit from 2WSS .", "( D ) Sequence of the peripheral stalk subunit b from bovine heart showing the position of the predicted trans-membrane helices ( blue ) and the known helices ( red and green ) determined by x-ray crystallography .", "The dashed purple box indicates the residues from PDB:2WSS by which the PDB:2CLY structure was extended for rigid-body fitting in ( C ) .", "Atomic model subunit colors: blue , α; dark blue , β; light blue , γ , δ , ε; yellow , b; red , F6; brown , d; green OSCP .", "Scale bar: 20 Å .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06119 . 012 Although the sub-tomogram average was calculated from protein reconstituted into membranes , the lipid bilayer itself was not resolved in the average .", "This is an effect of the missing wedge of information in the tomogram , which blurs out map features perpendicular to the electron beam , rendering the lipid bilayer in effect invisible ( Penczek and Frank , 2006 ) .", "An accurate estimate of the membrane position can however be obtained from the position of the c-ring in our fitted atomic model ( Figure 4 ) .", "To assess the packing of F1Fo ATP synthases in the 2D crystals , the sub-tomogram average was re-inserted into the original tomograms using the inverse of the parameters calculated during averaging ( Pruggnaller et al . , 2008; Video 1 ) .", "Defects in the 2D crystal lattice were apparent in the rotational orientation of individual complexes in a single layer ( Figure 5—figure supplement 1 ) .", "For a better understanding of the molecular packing , 400 particles were selected from a small region ( 235 × 285 nm ) of a single crystalline layer , which showed sharp diffraction spots to the third order .", "These particles were averaged and refined against a reference model calculated from the selected particles , which had been masked to contain a 3 × 3 array of F1Fo ATP synthase densities .", "The box size of the final average was enlarged to include particles of opposite orientation in the same membrane and the higher resolution subtomogram average ( shown in Figure", "3 ) was fitted multiple times into this volume ( Figure 5 and Video 2 ) . 10 . 7554/eLife . 06119 . 013Video 1 . Tomographic volume of a 2D crystal with re-inserted subtomogram average . The video moves through the z-stacks of the tomogram , revealing the location of the re-inserted sub-tomogram averages of the F1Fo ATP synthase .", "The four layers of F1Fo ATP synthase molecules are coloured in pink , blue , orange , and green .", "The predicted position of the vesicle membrane is shown in blue . DOI: http://dx . doi . org/10 . 7554/eLife . 06119 . 01310 . 7554/eLife . 06119 . 014Figure 5 . Packing of bovine F1Fo ATP synthase in the 2D crystal .", "( A ) Top view of the 2D crystal lattice of bovine F1Fo ATP synthase .", "Rectangles indicate the position of ATP synthase pairs .", "Arrows indicate cell axes .", "( B ) Side view of one pair .", "( C ) Cross-section of the crystal lattice indicated by the dashed box and arrowhead in ( A ) .", "F1Fo ATP synthases of opposite orientation in the membrane are connected via close interaction of their rotor-rings .", "The rotor-ring pairs are oriented 16° relative to the crystal plane ( single grey dashed line ) .", "( D ) Cross-section as in ( C ) but with the single-particle EM map ( Baker et al . , 2012 ) fitted into the subtomogram averages .", "The arrangement of the monomeric complexes on the 2D crystal lattice results in a locally kinked lipid bilayer ( bold dashed lines ) .", "( E ) Top view of the cross-section in ( D ) clipped to remove the catalytic domains of the upper F1Fo ATP synthase layer . DOI: http://dx . doi . org/10 . 7554/eLife . 06119 . 01410 . 7554/eLife . 06119 . 015Figure 5—figure supplement 1 . Lattice disorder .", "( A ) Side view of subtomogram averages repositioned into the lower face of a 2D crystal .", "( B ) Tomographic slice through a 2D crystal as indicated by line in ( A ) with a selection of repositioned sub-tomogram averages ( transparent ) .", "The tomographic slice is viewed from the membrane ( indicated by arrow in A ) .", "The repositioned subtomogram averages coincide with large and small circular densities , which correspond to the central and peripheral stalks , respectively ( orange arrowheads ) .", "Scale bar: 10 nm .", "( C ) Graphic representation of selected repositioned sub-tomogram averages in ( A ) highlighting the disorder in the crystal lattice .", "Small circles , peripheral stalk; double circle , F1-subcomplex + c-ring; rectangles indicate F1Fo ATP synthase pairs .", "Arrowheads correspond to those shown in ( B ) and are included for orientation purposes . DOI: http://dx . doi . org/10 . 7554/eLife . 06119 . 01510 . 7554/eLife . 06119 . 016Video 2 . Organization of bovine F1Fo ATP synthases in 2D crystals . The relative orientation of the F1Fo ATP synthases in a particularly well-ordered region of a 2D crystal is visualized by showing the re-inserted subtomogram averages , then by fitting the F1-c8 crystal structure and finally by fitting the single-particle map of Baker et al . , 2012 .", "F1Fo ATP synthases are orientated 16° to the crystal plane resulting in a zigzag arrangement of the lipid bilayer . DOI: http://dx . doi . org/10 . 7554/eLife . 06119 . 016 Figure 5A shows the typical packing of F1Fo ATP synthases in the most ordered regions of rectangular-shaped crystalline vesicles .", "The F1Fo ATP synthases form pairs of particles of twofold symmetry , which are in contact half-way up the peripheral stalks .", "The angle included by the long axes of the monomers in a pair is approximately 24° ( Figure 5B ) .", "Note that these ATP synthase pairs in the 2D crystals are structurally unrelated to the native dimers observed in mitochondrial membranes ( Strauss et al . , 2008; Davies et al . , 2011 ) .", "Pairs of F1Fo ATP synthase particles from opposite faces of the lipid bilayer interact via their c-rings and are related to each other by a ∼90° rotation ( Figure 5A ) .", "The interaction of c-rings in the membrane is best observed when viewing a cross-section of the map along the crystallographic a-axis ( Figure 5C ) .", "In this view , it becomes apparent that the pairs of opposing c-rings and the long axes of the F1Fo ATP synthases include an angle of 16° with the crystal plane .", "Therefore , in order for the c-rings to be fully embedded in the membrane , the lipid bilayer within the 2D crystals must adopt a zigzag topology , as observed in the vesicle cross-sections ( Figure 2 ) .", "To assess whether the Fo domain of the bovine F1Fo ATP synthase could in fact account for this curvature , we placed the segmented volume of the single-particle map of this enzyme ( Baker et al . , 2012 ) into our extended sub-tomogram average .", "To our surprise , the bend of the micelle-embedded membrane region of the single-particle map perfectly matched the kink in the membrane that is required to hold successive c-rings together ( Figure 5D , E ) .", "Thus the packing of F1Fo ATP synthases into the 2D crystal strongly supports the notion that the membrane domain of the monomeric bovine heart F1Fo ATP synthases is inherently bent and that this is sufficient to impose a local curvature on the lipid bilayer .", "The subtomogram average lacks well-defined density in the membrane-embedded part of the complex .", "Therefore , we validated our packing model by crystallographic image processing .", "Z-slices through the tomographic volume at the levels of the catalytic ( αβ ) 3 hexamer , the stalk region and the membrane-embedded domain , which all showed sharp diffraction spots to the third order , were transformed into projection images and processed by electron crystallographic routines without applying symmetry ( Figure 6 , Figure 6—figure supplement 1 and Figure 6—figure supplement", "2 ) ( Henderson et al . , 1986; Crowther et al . , 1996 ) . 10 . 7554/eLife . 06119 . 017Figure 6 . Projection maps of different z-slices in a 2D crystal of F1Fo ATP synthases .", "( A ) Side view of crystal packing from Figure 4 .", "( B ) Projection images ( left ) calculated from z-slices of the tomographic volume at z-height positions indicated by black bars in ( A ) , their Fourier transforms ( centre ) and projection maps ( right ) .", "( C ) z-slices through the crystal packing in ( A ) corresponding to the position of the projection maps shown in ( B ) .", "( A–C )", "Protein densities observed in the projection maps perfectly match the features shown in the corresponding z-slices of the crystal-packing model .", "Dashed orange outlines indicate a pair of F1Fo ATP synthases , red boxes indicate the unit cell of the crystal with dimensions of a = 179 . 1 Å , b = 171 . 4 Å , γ = 94 . 9° .", "Red arrowheads in the lower panel of ( B ) indicate lines of continuous protein density in the membrane plane . DOI: http://dx . doi . org/10 . 7554/eLife . 06119 . 01710 . 7554/eLife . 06119 . 018Figure 6—figure supplement 1 . Flow chart of electron crystallographic image processing of a tomographic volume . DOI: http://dx . doi . org/10 . 7554/eLife . 06119 . 01810 . 7554/eLife . 06119 . 019Figure 6—figure supplement 2 . Unit cell parameters and crystal symmetry .", "( A ) Projection images of tomographic z-slices containing the whole single-layered 2D crystal were processed to determine unit cell parameters and crystal symmetry .", "Image processing was performed using the MRC image processing programmes .", "( A ) Projection image ( left ) ; Fourier transform ( center ) ; Fourier synthesis map ( right ) .", "The red circle indicates the ( 0 , 1 ) reflection .", "( B ) The programme ALLSPACE did not indicate a clear plane-group symmetry for the 2D lattice . DOI: http://dx . doi . org/10 . 7554/eLife . 06119 . 019 The resulting projection maps were plotted at 30 Å resolution and showed good correlation with the crystal-packing model ( Figure 6 ) .", "The catalytic ( αβ ) 3 hexamer map featured twofold symmetric particles that were connected via a small protrusion due to the peripheral stalk .", "The stalk region showed four distinct densities related by a twofold rotation .", "The larger , more distal density corresponded to the central stalk and the thinner , more elliptical density to the peripheral stalk .", "For the membrane region , where no clear protein density is observed in the subtomogram average , continuous density is observed in the direction of the proposed c-ring/Fo interaction , but not in the perpendicular direction ( Figure 6 ) .", "Thus , the zigzag membrane geometry observed in Figure 5C was due to the membrane region of the F1Fo ATP synthase ." ], [ "Our structure of the bovine heart F1Fo ATP synthase reconstituted into a lipid bilayer is consistent with the monomeric detergent-solubilised complex determined by single-particle analysis ( Baker et al . , 2012 ) .", "Even though the resolution of our sub-tomogram average at 24 Å is nominally less good than that of the single-particle map at 18 Å , several features are more clearly resolved .", "This includes the densities of the central and peripheral stalks , the OSCP subunit and the α- and β-subunits , which are clearly separated in the sub-tomogram average ( Figure 4 ) .", "This may be a result of the more accurate particle alignment , which used 3D volumes rather than 2D projection images .", "In fact , Baker et al . ( 2012 ) recently reported that by reducing their data set by 82% to include only the best projection images , structural features of their cryo-EM map became clearer , although the nominal resolution , determined by the Fourier shell correlation , was unchanged .", "The resolution of our sub-tomogram average is about 1 . 5× better in the x–y plane than the z direction due to the single orientation of F1Fo ATP synthase particles in the crystal relative to the missing wedge of information ( Radermacher , 1988 ) .", "In our sub-tomogram average , the peripheral stalk is positioned along the non-catalytic αβ interface as previously reported ( Rees et al . , 2009; Baker et al . , 2012 ) but is more offset towards the α-subunit than in the x-ray structure ( Rees et al . , 2009; Figure 4 ) .", "A small connecting density visible between the midpoint of the peripheral stalk and α-subunit may account for this difference .", "A similar connection is seen in the single-particle cryo-EM map of the bovine complex ( Baker et al . , 2012 ) .", "Analysis of the fitted x-ray structure suggests possible ionic interactions between subunit d and residues 463–475 of the α-subunit , which is the point where the structures of the α and β-subunits diverge ( Walker et al . , 1982 ) .", "This additional contact may help the peripheral stalk in its role as a stator , but may also prevent the stalk from interfering with the catalytic cycle of the β-subunit by pulling it away from the α/β interface .", "Alternatively , the peripheral stalk may be pulled away from the α/β interface by its interactions in the membrane .", "The dominant density of the peripheral stalk in the sub-tomogram average suggests that this region is more rigid than other parts of the complex .", "In accordance with this , we were able to fit the bovine heart peripheral stalk fragment ( Dickson et al . , 2006 ) extended by several residues from the F1-peripheral stalk structure ( Rees et al . , 2009 ) into the density as a rigid body without the need to introduce hinges as previously suggested ( Baker et al . , 2012 ) ( Figure 4—figure supplement 1 ) .", "This fit therefore challenges the notion that the peripheral stalk acts as a flexible linker that stores torque or elastic energy during the catalytic cycle ( Sorgen et al . , 1998; Sorgen et al . , 1999 ) .", "This hypothesis was based on the bacterial enzyme , which has a peripheral stalk consisting of only two long alpha helices .", "The bovine peripheral stalk , in contrast , consists of one long alpha helix plus subunits d , F6 and the N-terminal domains of other Fo subunits , which would generate a more rigid structure ( Dickson et al . , 2006 ) .", "A rigid peripheral stalk would hold the ( αβ ) 3 hexamer in a stationary position relative to the membrane .", "This is probably more important in mitochondria than in bacteria , as the mitochondrial F1Fo ATP synthase has another important role in generating local membrane curvature and maintaining cristae morphology through the formation of dimer rows ( Davies et al . , 2012 ) .", "The dimer interface is located at the base of the peripheral stalk ( Davies et al . , 2011 , 2012 ) and thus any flexibility in the structure of the peripheral stalk may compromise the formation and stability of the dimer .", "Despite the prominent structural features visible in the membrane-extrinsic regions of the F1Fo ATP synthase , the membrane-embedded parts in our sub-tomogram average were not well-resolved ( Figure 3 ) .", "This was surprising as the c-ring , located directly beneath the central stalk , is easily visible in x-ray models filtered to 20 Å resolution .", "Therefore , if the sub-volumes are well enough aligned to give an overall resolution of 24 Å , we would expect to see the c-ring in the lipid bilayer .", "In our sub-tomogram average , we only see a weak density for the c-ring .", "The lack of detail in the membrane region is unlikely to be caused by the missing wedge as we have observed a similar phenomenon in sub-tomogram averages of other membrane proteins , which have no missing wedge of information , and in single-particle maps when the resolution does not extend beyond 15 Å .", "The extent of this problem appears to correlate with the composition of the surrounding lipid or detergent .", "Thus , the lack of protein density in the membrane region for our averages at this resolution seems to be caused mainly by contrast matching between the surrounding lipid and protein .", "The projection images generated from selected z-slices of tomographic volumes enabled us to use electron crystallographic image processing , even though the coherently packed areas of the 2D crystals were small .", "This improved the signal-to-noise ratio because noise originating from additional crystal layers or surrounding buffer was removed in silico by careful selection of the z-slices and Fourier filtering of the diffraction pattern .", "Thus 2D processing of tomographic 3D crystal volumes poses an attractive alternative for the electron crystallographic treatment of small 2D crystals of large membrane complexes that are not accessible to canonical image processing due to limited size and order .", "As exemplified in our 2D crystal of the F1Fo ATP synthase , the molecular packing of multi-subunit membrane complexes with large extramembranous domains can be complicated with multiple layers and multiple tilt angles relative to the crystal plane .", "These are circumstances under which projection maps alone are easily misinterpreted and our approach of combining electron tomography with electron crystallography enables a straightforward interpretation ( Gerle et al . , 2006; Tani et al . , 2013 ) .", "In mitochondria , the F1Fo ATP synthase forms rows of dimers along the highly curved ridges of cristae membranes ( Strauss et al . , 2008; Davies et al . , 2011 ) .", "Disruption of dimers prevents the formation of wild-type cristae and increases the generation time of the organism ( Paumard et al . , 2002; Davies et al . , 2012 ) .", "Molecular dynamics simulations have shown that the F1Fo ATP synthase dimer structure alone is sufficient to bend the lipid bilayer and drive row formation ( Davies et al . , 2012 ) .", "In addition , single-particle cryo-EM of mitochondrial ATP synthase complexes indicates that the bend of the dimer may originate from the structure of the Fo domain in a single monomer ( Baker et al . , 2012 ) .", "Through our analysis of the F1Fo ATP synthase packing in 2D crystals , we have shown that the Fo domain of monomeric bovine heart F1Fo ATP synthase indeed induces a substantial kink in the lipid bilayer ( Figures 5 ,", "6 ) explaining in molecular terms the zigzag appearance of the membrane in the 2D crystals ( Figure 2 ) .", "When two monomeric complexes join to form a dimer in the inner mitochondrial membrane , the resulting angle between the long axes of the monomers would be ∼86° ( Figure", "7 ) exactly as observed in the subtomogram averages of the bovine F1Fo ATP synthase dimer ( Davies et al . , 2012 ) .", "This dimer angle gives rise to the high local curvature of membranes required to shape the cristae ( Davies et al . , 2012 ) .", "We conclude that the F1Fo monomer is the basic building block of the ATP synthase dimer rows .", "Newly assembled monomeric complexes of the bovine heart F1Fo ATP synthase monomers would converge on pre-existing regions of high membrane curvature where they assemble into dimers , thus propagating the self-association of dimers into rows . 10 . 7554/eLife . 06119 . 020Figure 7 . Monomeric mitochondrial F1Fo ATP synthase as the factor determining cristae membrane curvature .", "( A ) Schematic diagram of bovine F1Fo ATP synthase in the 2D crystal lattice , with its transmembrane Fo stator domain imposing a local 43° kink and a 16° inclination of the c-ring relative to the crystal plane .", "Blue , stator; red , rotor .", "( B ) Schematic diagram of a single monomeric bovine F1Fo ATP synthase .", "CS: central stalk; PS: peripheral stalk; IMS: intermembrane space; TM Fo: transmembrane domain of Fo .", "( C ) Association of two monomeric F1Fo complexes into a dimer results in an angle of 86° , as observed in ATP synthase dimers in mitochondrial cristae ( Davies et al . , 2012 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06119 . 020 Using a combination of electron cryo-tomography , sub-tomogram averaging , and electron crystallography , we have determined the structure of an F1Fo ATP synthase in a lipid bilayer .", "This in vitro system is free from additional proteins , the enzyme is fully functional , and the proteoliposomes are in principle proton-tight .", "Therefore , this system may be suitable for studying the effect of physiological parameters such as ∆pH , ∆pmf , and substrate availability on the structure and function of F1Fo ATP synthase ( Walker , 2013 ) .", "In addition , with the recent advancements in electron detector development and image processing methods in cryo-EM , it may soon be possible to generate tomographic volumes with sub-nanometer resolution routinely ( Schur et al . , 2013 ) .", "This resolution should be sufficient to determine whether the structure of the enzyme changes under different physiological conditions .", "Only when these questions are answered will we really understand how the F1Fo ATP synthase works in the membrane ." ], [ "F1Fo ATP synthase was purified by a published procedure ( Maeda et al . , 2013 ) .", "Briefly , sub-mitochondrial particles in 40 mM HEPES pH 7 . 8 , 2 mM MgCl2 , 0 . 1 mM EDTA , and 0 . 1 mM DTT , isolated from fresh bovine hearts as described previously ( Shinzawa-Itoh et al . , 2010 ) , were solubilized on ice by the addition of deoxycholate and decylmaltoside to final concentrations of 0 . 7% ( wt/vol ) and 0 . 4% ( wt/vol ) , respectively .", "Subsequently , the suspension was centrifuged at 176 , 000×g for 50 min and the supernatant applied to a sucrose step gradient ( 40 mM HEPES pH 7 . 8 , 0 . 1 mM EDTA , 0 . 1 mM DTT , 0 . 2% [wt/vol] decylmaltoside and 2 . 0 M , 1 . 1 M , 1 . 0 M , or 0 . 9 M sucrose ) and centrifuged overnight at 176 , 000×g for 15 . 5 hr .", "Fractions with ATPase activity were loaded onto a Poros-20HQ ion-exchange column and eluted by a linear concentration gradient of 0–240 mM KCl in 40 mM HEPES pH 7 . 8 , 2 mM MgCl2 , 0 . 1 mM EDTA , 0 . 1 mM DTT and 0 . 2% ( wt/vol ) decylmaltoside .", "F1Fo ATP synthase fractions containing high amounts of native phospholipids were concentrated to 10 mg/ml ( Advantec Ultra filter , polysulfone , MWCO 200 kDa , Toyo Roshi , Tokyo , Japan ) .", "Synthetic , chemically pure 1 , 2-dimyristoyl-sn-glycero-3-phosphocholine ( Avanti Polar Lipids , Alabaster , AL ) was solubilised in decylmaltoside and mixed with freshly purified bovine F1Fo ATP synthase at a lipid to protein ratio of 0 . 2 .", "After overnight incubation on ice , the detergent was removed by dialysis using 20 μl Hampton dialysis buttons ( Hampton Research , Aliso Viejo , CA ) ( membrane cutoff 15 , 000 Da , SpectraPor#7 , Spectrum , Los Angeles , CA ) and 500 ml of dialysis buffer ( 40 mM Tris-HCl pH 8 . 2 , 100 mM NaCl , 0 . 02% [wt/vol] NaN3 , 0 . 5 mM ADP , 5 mM MgCl2 , 0 . 1 mM DTT , 0 . 1 mM EDTA ) .", "The dialysis buffer was exchanged daily and the sample was incubated at 27°C for 10 to 21 days .", "To determine the specific enzymatic activity and the proportion of coupled complexes of both the isolated F1Fo ATP synthases and the crystalline vesicles resolubilized with 4% ( wt/vol ) digitonin , an ATP-regenerating enzyme-coupled assay was used ( Pullman et al . , 1960 ) .", "The hydrolysis of ATP by the F1Fo ATP synthase was followed by NADH oxidation at 340 nm at 20°C in the absence or presence of oligomycin .", "To confirm the subunit composition and intactness of the bovine F1Fo ATP synthase , crystalline vesicles , resolubilized with sodium dodecyl sulfate were examined by denaturing SDS-PAGE and vesicles resolubilized with 4% ( wt/vol ) digitonin by non-denaturing blue-native PAGE ( Wittig et al . , 2006 ) .", "The presence of the lower molecular weight subunits ( 5000–12 , 000 Da ) of the F1Fo ATP synthase in the crystalline vesicles was confirmed by MALDI-TOF mass spectrometry ( Bruker Daltonics Inc . , MA , USA ) .", "2 . 5 µl of dialysed sample was applied to freshly glow-discharged , carbon-coated 400 mesh copper grids ( Veco , Nisshin , Tokyo , Japan ) , blotted and stained with 2% uranyl acetate solution .", "Grids were screened using a JEM1010 transmission electron microscope ( JEOL , Tokyo , Japan ) at 100 kV and images were acquired using a 2k × 2k slow-scan CCD camera ( Gatan , Pleasanton , CA ) .", "Images were recorded at a magnification of 40 , 000× , which corresponds to a pixel size of 6 Å using a 2 s exposure .", "Samples of 2D crystals were screened by negative stain EM immediately before freezing .", "Samples containing well-ordered arrays of F1 heads were selected and mixed 1:1 with fiducial gold markers ( 6 nm gold particles conjugated to protein A , Aurion ) .", "3 µl of the protein:gold sample were applied to glow-discharged quantifoil grids ( R2/2 , 300 copper mesh ) , blotted for 3 s ( #4 Whatman paper , Sigma-Aldrich , St . Louis , MI ) and plunge-frozen in liquid ethane using a home-built freezing device .", "Single-axis tilt series ( ±60° , step size 1 . 5° ) were collected on an FEI Krios microscope ( FEI , Hillsboro , OR ) operating at 300 kV and equipped with a post-column energy filter ( GIF Quantum , Gatan ) with a K2 summit direct detector in counting mode ( Gatan ) .", "Images were taken with a specimen pixel size of 0 . 33 nm and a defocus of 2 . 5 µm .", "Tilt series were aligned using the gold fiducials and back-projected to generate tomographic volumes using the IMOD package ( Kremer et al . , 1996 ) .", "For particle picking , tomograms were binned 4 × 4 and filtered by nonlinear anisotropic diffusion to increase contrast ( Frangakis and Hegerl , 2001 ) .", "F1 subcomplexes were picked manually in 3dmod with the F1 subcomplexes of opposite orientation assigned to different files .", "Particles were separated randomly into two data sets and processed independently .", "Particle alignment was performed in PEET , starting with the contrast-enhanced binned 2 × 2 tomograms , then the binned 2 × 2 tomograms without contrast enhancement and finally the unbinned ( 1 × 1 ) volume .", "An initial reference volume was calculated for each data set by averaging all selected subvolumes on one side of the membrane .", "Subvolumes containing F1 subcomplexes from the opposite side of the membrane were rotated 180° about the x-axis prior to alignment .", "Alignment parameters were then restricted to ±45° about the x and y-axis , and 12 pixels in xyz .", "No restriction was placed on the z-axis rotation .", "Initial alignment was performed with a resolution limit of 60 Å and gradually lowered to 30 Å in accordance with the FSC .", "To assess over-fitting , phases beyond 40 Å in the tomogram were randomized and the last alignment iteration was repeated ( Chen et al . , 2013 ) .", "The final average , calculated from 2500 subvolumes , was filtered to 18 Å using a Fermi filter with a temperature factor of 0 . 002 px−1 ( Frank , 1996 ) .", "Fourier shell correlation was performed in PEET using a box size of 96 voxels .", "Atomic models were docked into the EM density using the sequential fit routine of Chimera and superimposed using the matchmaker command ( Pettersen et al . , 2004 ) .", "Electron density maps of atomic models were calculated in CCP4 ( Winn et al . , 2011 ) .", "Two types of projection images were prepared from tomographic volumes of a single vesicular 2D crystal ( for a flow chart see Figure 6—figure supplement 1 ) .", "To determine the lattice parameters , a projection image of a single-layered 2D crystal was calculated from the corresponding z-slices of the tomographic volume by first extraction and conversion into a projection image using the IMOD package ( Kremer et al . , 1996 ) ( Figure 6—figure supplement 2A—left panel ) and then processing the projection images using the MRC image processing programmes ( Crowther et al . , 1996 ) ( Figure 6—figure supplement 2A , middle and right panel ) .", "The unit cell parameters of the crystal were: a = 179 . 1 Å , b = 171 . 4 Å , γ = 94 . 9° .", "As the programme ALLSPACE ( Valpuesta et al . , 1994 ) indicated only one plane of symmetry , data were merged in p1 ( Figure 6—figure supplement 2B ) .", "The single-layered 2D crystal in the tomographic volume was subdivided along the z-axis and a total of 64 projection images were generated at an interval spacing of 6 . 66 Å , corresponding to the sampling size of the tomographic volume .", "All images were processed with the MRC image processing programmes to correct for crystal lattice distortion ( Henderson et al . , 1986 ) .", "Only a few reflections had statistically significant amplitudes beyond 30 Å resolution and all projection maps were calculated within a 30 Å resolution limit ." ] ]

[ "We have used a combination of electron cryo-tomography , subtomogram averaging , and electron crystallographic image processing to analyse the structure of intact bovine F1Fo ATP synthase in 2D membrane crystals .", "ATPase assays and mass spectrometry analysis of the 2D crystals confirmed that the enzyme complex was complete and active .", "The structure of the matrix-exposed region was determined at 24 Å resolution by subtomogram averaging and repositioned into the tomographic volume to reveal the crystal packing .", "F1Fo ATP synthase complexes are inclined by 16° relative to the crystal plane , resulting in a zigzag topology of the membrane and indicating that monomeric bovine heart F1Fo ATP synthase by itself is sufficient to deform lipid bilayers .", "This local membrane curvature is likely to be instrumental in the formation of ATP synthase dimers and dimer rows , and thus for the shaping of mitochondrial cristae ." ]

[ "Cells use a molecule called adenosine triphosphate ( or ATP for short ) to power many processes that are vital for life .", "Animals , plants , and fungi primarily make their ATP in a specialised compartment called the mitochondrion , which is found inside their cells .", "The mitochondrion is often referred to as the powerhouse of cells as it captures and stores the energy that animals gain from eating food in the molecule ATP .", "Other enzymes in the cell break apart ATP to release the stored energy , which they use to power various cellular processes .", "The interior architecture of the mitochondrion includes a highly folded inner membrane where electrical energy is transformed into chemical energy .", "The tight folding of the inner membrane is thought to make this process more efficient .", "An enzyme named ATP synthase performs the final steps of the energy transformation process by producing ATP ( ATP synthase literally means ‘ATP maker’ ) .", "This enzyme sits in pairs along the edges of the inner membrane folds .", "This raises the question: does the ATP synthase cause the membrane to fold or does this enzyme just ‘prefer’ these folded edges ( which are instead caused by something else inside the mitochondrion ) ?", "To investigate this question , Jiko , Davies et al . extracted ATP synthase from the mitochondria of cow hearts and mixed them with modified fat molecules to form a ‘2D membrane crystal’: a membrane containing an ordered pattern of enzymes .", "An electron microscope was used to generate a three-dimensional volume of the 2D membrane crystal via a process similar to a MRI or CAT scan that one might have in hospital .", "In the three-dimensional volume of the membrane crystal , Jiko , Davies et al . discovered that instead of being flat as expected , the membrane of the 2D membrane crystal was rippled and that this ripple was caused by the membrane-embedded part of the ATP synthase .", "The geometry of the ripple exactly matched half of the bend at the edge of the membrane folds in the mitochondrion .", "Therefore , Jiko , Davies et al . concluded that a pair of ATP synthases , as found in mitochondria , was responsible for defining the tight folds of the inner mitochondrial membrane ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "structural biology and molecular biophysics", "immunology and inflammation" ]

[ [ "The HIV-1 envelope ( Env ) spike , a trimer of gp120-gp41 heterodimers , is the only target of neutralizing antibodies ( Abs ) .", "Rapid mutation combined with structural features of the Env trimer that hide conserved features of the HIV-1 spike result in induction of mainly strain-specific neutralizing Abs in most infected individuals .", "However , broadly neutralizing Abs ( bNAbs ) evolve in a minority of HIV-1–infected individuals after several years of infection .", "These Abs are of considerable interest for HIV-1 therapeutic efforts because they can prevent and treat infection in animal models ( reviewed in ( [West et al . , 2014] ) and exhibited efficacy against HIV-1 in a human clinical trial ( Caskey et al . , 2015 ) .", "Thus , it is believed that an immunogen that could elicit bNAbs would induce a protective immune response against infection by HIV-1 .", "Highly potent bNAbs against the conserved CD4-binding site ( CD4bs ) on gp120 that were derived from the VH1-2*02 variable heavy chain ( HC ) gene have been isolated from at least nine HIV-1–infected individuals ( Scheid et al . , 2011; Wu et al . , 2010; Zhou et al . , 2013; 2015; Wu et al . , 2011; Georgiev et al . , 2013 ) .", "These bNAbs exhibit unusually high levels of somatic hypermutation , with changes in both complementarity determining regions ( CDRs ) and framework regions ( FWRs ) ( Klein et al . , 2013 ) .", "As exemplified by VRC01 , the first such bNAb to be isolated ( Wu et al . , 2010 ) , VRC01-class bNAbs share a common mode of gp120 binding in which the VH1-2*02-derived variable heavy ( VH ) domain mimics CD4 ( Wu et al . , 2010; Zhou et al . , 2010; 2013; 2015; Diskin et al . , 2011 ) .", "Signature features of the HCs of VRC01-class bNAbs that explain their derivation from the VH1-02*02 gene segment include Trp50HC , Asn58HC , and Arg71HC; an additional signature residue , Trp100BHC within CDRH3 , is derived from the joining of the D and J gene segments to the VH gene segment during V ( D ) J recombination ( Diskin et al . , 2012 ) .", "The variable light ( VL ) domains of VRC01-class bNAbs , which can be derived from several different VL germline gene segments , require a short ( five residues ) CDRL3 loop to allow interactions with gp120 V5 and loop D ( Diskin et al . , 2012 ) and often include a deletion in CDRL1 to avoid clashes with an N-linked glycan attached to Asn276gp120 ( Zhou et al . , 2010; 2013; Scharf et al . , 2013 ) .", "The LC contact residues of VRC01-like bNAbs and the short CDRL3 are not germline encoded ( Diskin et al . , 2012 ) .", "Because VRC01-class bNAbs are highly potent , have demonstrated efficacy in animal and human trials , and evolved in multiple HIV-1–infected donors who were infected with different strains of HIV-1 , they are considered a promising target for elicitation by vaccination with designed immunogens .", "The first step in eliciting an Ab is binding to its precursor germline-encoded B cell receptor by an antigen or immunogen with adequate affinity to initiate B cell activation and affinity maturation ( Batista and Neuberger , 1998 ) .", "Although the unmutated VH1-2*02 gene segment includes the signature VH domain residues for interacting with the CD4bs , germline-reverted versions of VRC01-class bNAbs do not bind to Env trimers or to gp120 ( Scheid et al . , 2011; Zhou et al . , 2010; Diskin et al . , 2012; Scharf et al . , 2013; Hoot et al . , 2013; Ota et al . , 2012; McGuire et al . , 2013 ) .", "In previous studies , we investigated VRC01-class germline recognition of Env by solving a crystal structure of the clade A/E 93TH057 gp120 core bound to a half germline , half mature chimeric VRC01-class Ab ( NIH45-46CHIM ) in which the inferred germline HC was paired with the mature LC to permit binding to an unmodified gp120 ( Scharf et al . , 2013 ) .", "Two forms of designed gp120-based immunogens were subsequently engineered to bind and activate germline HC/LC VRC01-class B cell receptors: gp120 outer domain-only constructs ( eOD-GT6 and eOD-GT8 ) ( Jardine et al . , 2013; 2015 ) and gp120s modified from the clade C 426c Env ( McGuire et al . , 2013; 2014; 2016 ) .", "Crystal structures of the inferred germline Fab of VRC01 complexed with eOD-GT6 and NIH45-46CHIM complexed with gp120 revealed a similar angle of approach for binding as mature Fabs and conservation of the signature VRC01-class interactions with gp120 residues ( Scharf et al . , 2013; Jardine et al . , 2013 ) .", "However , questions concerning germline B-cell receptor recognition of gp120 remained because neither structure represented a complex between a fully germline VRC01-class Ab and a complete gp120 core .", "Here , we present crystal structures of inferred germline VRC01-like Abs alone and complexed with 426c-based gp120 immunogen cores and compare and contrast their gp120 recognition with structures of mature VRC01-class Ab/gp120 complexes .", "The analyses shed insight on the evolution pathway by which Abs derived from VH1-2*02 germline mature towards broad recognition of the CD4bs on gp120 and provide structural information that will facilitate design of immunogens to elicit VRC01-class bNAbs ." ], [ "The antigen-binding Fabs of two VRC01-class Abs isolated from different patients , NIH45-46 and 3BNC60 ( Scheid et al . , 2011 ) , were generated in their inferred germline forms ( NIH45-46GL and 3BNC60GL ) using sequences described in previous studies ( Scharf et al . , 2013; Hoot et al . , 2013; McGuire et al . , 2013; Dosenovic et al . , 2015 ) .", "Compared with mature NIH45-46 ( NIH45-46MAT ) , NIH45-46GL contained 40 HC and 23 LC substitutions in addition to a two-residue insertion in CDRL1 .", "3BNC60GL contained 38 HC and 25 LC substitutions in addition to a four-residue deletion in HC framework region 3 ( FWR3HC ) and a four-residue insertion in CDRL1 compared with 3BNC60MAT ( Figure 1 ) .", "Two versions of germline-binding gp120s were expressed as gp120 cores with N/C termini and V1-V2 and V3 loop truncations ( Zhou et al . , 2010 ) :", "( i ) 426c . NLGS . TM1△V1-3 ( hereafter referred to as 426c . TM1△V1-3 ) , a modified version of the clade C 426c Env in which the V1 , V2 and V3 loops were truncated and three potential N-linked glycosylation sites at gp120 residues Asn276gp120 , Asn460gp120 and Asn463gp120 were removed by mutation ( N276D , N460D , and N463D ) ( McGuire et al . , 2013; 2014 ) , and", "( ii ) 426c . TM4△V1-3 , a modification of 426c . TM1△V1-3 with additional substitutions ( D276N , S278R , G471S ) ( McGuire et al . , 2016; Dosenovic et al . , 2015 ) .", "426c . TM1△V1-3 binds to germline VRC01 and NIH45-46 ( McGuire et al . , 2013; 2014 ) , and 426c . TM4△V1-3 binds to germline versions of 12A21 , 3BNC60 , VRC-CH31 , VRC-PG19 and VRC-PG20 in addition to germline VRC01 and NIH45-46 ( McGuire et al . , 2016 ) . 10 . 7554/eLife . 13783 . 003Figure 1 . Sequence Alignments of Inferred Germline and Mature Forms of 3BNC60 and NIH45-46 . Alignments of ( A ) VH and ( B ) VL sequences of inferred germline progenitors ( 3BNC60GL and NIH45-46GL ) , mature 3BNC60 ( 3BNC60MAT ) , mature NIH45-46 ( NIH45-46MAT ) , and the predicted germline V gene segments from which they were derived .", "Antibody framework regions ( FWR ) and CDR loops ( CDR ) are marked and CDR loops are colored blue ( CDR1 ) , green ( CDR2 ) , and red ( CDR3 ) .", "CDR3 sequences for germline Fabs were taken from mature antibody sequences as done in previous studies ( Dosenovic et al . , 2015; Hoot et al . , 2013; McGuire et al . , 2013; Scharf et al . , 2013 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 13783 . 003 We used a surface plasmon resonance ( SPR ) -based assay to compare the binding of NIH45-46GL , 3BNC60GL , NIH45-46MAT , and 3BNC60MAT to gp120 cores including the 426c immunogens and a representative gp120 ( 93TH057 ) used for structural studies with mature bNAbs ( Figure 2 ) .", "As expected , the germline-reverted Abs did not bind detectably to 93TH057 gp120 , but NIH45-46GL bound to both 426c . TM1ΔV1-3 and 426c . TM4ΔV1-3 gp120s with equilibrium dissociation constants ( KDs ) of ~0 . 7 and 3 μM , respectively .", "As previously described ( McGuire et al . , 2013; 2014 ) , 3BNC60GL did not bind to 426c . TM1ΔV1-3 , but showed detectable binding to 426c . TM4ΔV1-3 gp120 .", "By contrast , NIH45-46MAT and 3BNC60MAT bound to all gp120s with nM or higher affinity ( Figure 2 ) . 10 . 7554/eLife . 13783 . 004Figure 2 . SPR binding assays . Representative sensograms ( red ) , fits ( black , where applicable ) , residuals , and KD , ka , and kd values ( mean ± standard deviation from 3 independent experiments ) for binding of germline and mature Abs to gp120 cores .", "NIH45-46GL , 3BNC60GL , NIH45-46MAT and 3BNC60MAT IgG were captured on a protein A biosensor chip , and 426c . TM1△V1-3 , 426c . TM4△V1-3 and 93TH057 gp120 cores were flowed over the chip as a 2-fold dilution series with top concentrations of 16 mM and 400 nM for germline and mature Abs , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 13783 . 004 Crystal structures of NIH45-46GL bound to 426c . TM1△V1-3 and 3BNC60GL bound to 426c . TM4△V1-3 were solved to 3 . 4 Å and 3 . 1 Å resolution , respectively ( Table 1 ) .", "We also solved structures of unbound 3BNC60GL and 426c . TM4△V1-3 ( at 1 . 9 Å and 2 . 0 Å resolution , respectively ) , and a complex structure of a 426c . TM4△V1-3 bound to 3BNC55MAT , a mature VRC01-class Ab ( Table 1 ) .", "We compared the new structures to previously-reported complex structures of NIH45-46CHIM/93TH057 gp120 and VRC01GL/eOD-GT6 ( PDB codes 4JDT and 4JPK ) , unbound structures of NIH45-46GL and VRC01GL ( PDB codes 4JDV and 4JPI ) ( Scharf et al . , 2013; Jardine et al . , 2013 ) , and representative mature VRC01-class Fab/gp120 complexes; e . g . , NIH45-46MAT/93TH057 gp120 and 3BNC117MAT/93TH057 gp120 ( PDB codes 3U7Y and 4JPV ) ( Zhou et al . , 2013; Diskin et al . , 2011 ) . 10 . 7554/eLife . 13783 . 005Table 1 . Data collection and refinement statistics , molecular replacement . DOI: http://dx . doi . org/10 . 7554/eLife . 13783 . 0053BNC60GL Fab ( 5F7E ) 426c . TM4ΔV1-3 ( 5FA2 ) 3BNC60GL Fab-426c . TM4ΔV1-3 complex ( 5FEC ) NIH45-46GL Fab-426c . TM1ΔV1-3 complex ( 5IGX ) 3BNC55MAT Fab-426c . TM4ΔV1-3 complex ( 5I9Q ) Resolution range34 . 13 - 1 . 9 ( 1 . 97 - 1 . 9 ) 35 . 8 - 2 . 0 ( 2 . 072 - 2 . 0 ) 37 . 44 - 3 . 1 ( 3 . 211 - 3 . 1 ) 39 . 37 - 3 . 4 ( 3 . 521 - 3 . 4 ) 37 . 54 - 2 . 8 ( 3 . 10 - 3 . 0 ) Space groupP212121C121P212121F222P3121Unit cell dimensionsa , b , c ( Å ) 74 . 9 , 74 . 9 , 83 . 1144 . 9 , 85 . 9 , 90 . 0103 . 1 , 134 . 1 , 195 . 0147 . 1 , 169 . 3 , 177 . 7122 . 9 , 122 . 9 , 265 . 0α , β , γ ( ° ) 90 . 0 , 90 . 0 , 90 . 090 . 0 , 104 . 8 , 90 . 090 . 0 , 90 . 0 , 90 . 090 . 0 , 90 . 0 , 90 . 090 . 0 , 90 . 0 , 120 . 0Total reflections243498 ( 23377 ) 416316 ( 24886 ) 335536 ( 34539 ) 207967 ( 20780 ) 710649 ( 51477 ) Unique reflections37256 ( 3651 ) 81715 ( 7961 ) 49653 ( 4885 ) 15410 ( 1508 ) 57299 ( 4353 ) Multiplicity6 . 5 ( 6 . 5 ) 4 . 6 ( 4 . 5 ) 6 . 1 ( 6 . 4 ) 13 . 5 ( 13 . 8 ) 12 . 4 ( 11 . 8 ) Completeness ( % ) 0 . 95 ( 0 . 98 ) 0 . 97 ( 0 . 95 ) 1 . 00 ( 1 . 00 ) 1 . 00 ( 1 . 00 ) 0 . 71 ( 95 . 7 ) Mean I/sigma ( I ) 16 . 56 ( 2 . 36 ) 13 . 35 ( 0 . 6 ) 9 . 75 ( 1 . 47 ) 21 . 76 ( 4 . 03 ) 8 . 65 ( 1 . 3 ) Wilson B-factor23 . 230 . 368 . 9897 . 4477 . 46R-merge0 . 09029 ( 0 . 8891 ) 0 . 06624 ( 1 . 36 ) 0 . 1729 ( 1 . 305 ) 0 . 1087 ( 0 . 6832 ) 0 . 327 ( 3 . 774 ) CC1/20 . 999 ( 0 . 831 ) 0 . 999 ( 0 . 437 ) 0 . 995 ( 0 . 483 ) 0 . 999 ( 0 . 909 ) 0 . 993 ( 0 . 186 ) CC*1 ( 0 . 953 ) 1 ( 0 . 78 ) 0 . 999 ( 0 . 807 ) 1 ( 0 . 976 ) 0 . 998 ( 0 . 56 ) Rwork0 . 1960 . 2070 . 2030 . 2790 . 240Rfree0 . 2100 . 2320 . 2670 . 2860 . 279Number of atoms3502586016611506111572macromolecules3226512416198495611258ligands320413105314Protein residues42967221826971537RMS ( bonds ) 0 . 010 . 0120 . 0140 . 0170 . 014RMS ( angles ) 1 . 281 . 5341 . 221 . 751 . 35Clashscore4 . 936 . 615 . 179 . 7728 . 65Average B-factor33 . 048 . 6684 . 9295 . 1486 . 29macromolecules31 . 1447 . 2783 . 9493 . 8186 . 98ligands70 . 79123 . 27165 . 3261 . 48solvent38 . 6748 . 77Statistics for the highest-resolution shell are shown in parentheses .", "To evaluate the structural plasticity of the inferred germline versions of the VRC01-class bNAbs , we superimposed available structures of inferred germline Fabs in their free and antigen-complexed forms .", "Superimpositions of bound and unbound forms of NIH45-46GL and 3BNC60GL Fabs ( this study ) and bound and unbound VRC01GL ( Jardine et al . , 2013 ) revealed no major structural changes ( Figure 3A , C , E ) .", "We previously noted slight differences between bound and unbound NIH45-46MAT in the conformations of CDRs L1 and H3 and positions of the Tyr89CDRL3 and Tyr74FWRH3 side chains ( Figure 3G ) ( Diskin et al . , 2011 ) , but the counterpart residues in NIH45-46GL were not affected by gp120 binding ( Figure 3F ) .", "In addition , superimpositions of bound and unbound 3BNC60GL Fab showed no major structural changes except in CDRL1 , which either flipped the backbone conformation of the Asp28LC-Asn31LC segment ( in the case of one Fab/gp120 complex in the crystallographic asymmetric unit; Figure 3H ) or was partially disordered ( in the case of the second Fab/gp120 complex in the asymmetric unit ) , thus avoiding clashes with loop D of gp120 despite a four-residue insertion in CDRL1 .", "In the mature Ab 3BNC60MAT ( and presumably its close relative 3BNC117MAT ) , a disrupted β-strand in the region of Pro61HC was reordered upon binding to gp120 ( Klein et al . , 2013 ) , but no notable differences in the bound and free forms of 3BNC60GL were seen in the counterpart region , which contained Ala61HC instead of Pro61HC ( Figure 3H , I ) .", "Root mean square deviations ( rmsds ) for superimposing all Cα atoms in the VHVL domains of bound and unbound Fabs of NIH45-46GL , 3BNC60GL , and VRC01GL were low ( 0 . 54 Å–0 . 73 Å ) ( Figure 3K ) .", "Thus the association of the VH and VL domains and the conformations of the CDR loops and surrounding FWRs found in the unbound forms were largely maintained in the germline-inferred Abs upon binding to their antigens . 10 . 7554/eLife . 13783 . 006Figure 3 . Overview of Bound and Unbound Structures of Germline and Mature Forms of NIH45-46 and 3BNC60 . Superposition of unbound ( grey ) and bound ( colored ) Fab structures of ( A ) NIH45-46GL ( blue ) , ( B ) NIH45-46MAT ( orange ) , ( C ) 3BNC60GL ( green ) , ( D ) 3BNC60MAT ( purple ) , and ( E ) VRC01GL ( teal ) .", "The crystal structures were superimposed on their VHVL domains and are shown as wire representations with CDR loops colored blue ( CDR1 ) , green ( CDR2 ) , and red ( CDR3 ) .", "Panels ( F–I ) show detailed areas of interest for the corresponding structure comparisons shown in panels ( A–D ) .", "Protein backbones are shown as wire diagrams , side chains are shown as stick representations ( red , oxygen; blue , nitrogen ) .", "( J ) Superposition of unbound ( grey ) and bound ( red ) structures of 426c . TM4△V1-3 shown as wire diagrams .", "( K ) Table summarizing Cα rmsds for the indicated Ab and gp120 pairs .", "Since no unbound crystal structure of 3BNC117MAT was available , the structure of the close clonal relative , 3BNC60MAT ( 93%HC/96% LC sequence identity ) was substituted .", "Similarly , no unbound structure of VRC01MAT was available , so that of NIH45-46MAT ( 88%HC/96% LC sequence identity ) was substituted , omitting its four-residue insertion in CDRH3 from the rmsd calculation . DOI: http://dx . doi . org/10 . 7554/eLife . 13783 . 006 Superpositions of bound and unbound forms of the mature counterparts of these bNAbs ( using a closely-related unbound Fab structure when necessary ) also revealed relatively small rmsd values with the exception of the 1 . 66 Å rmsd when comparing free 3BNC60MAT and complexed 3BNC117MAT due to the disrupted β-sheet structure near Pro61HC of free 3BNC60MAT ( Figure 3I ) ( Klein et al . , 2013 ) – when the disrupted β-strand was excluded , the rmsd dropped to 0 . 72 Å ( Figure 3K ) .", "Thus the mature bNAbs ( NIH45-46MAT , 3BNC60MAT , and VRC01MAT ) are overall largely preformed to recognize antigen , but exhibited more antigen binding-induced conformational flexibility within individual CDR loops and/or the FWRs than the germline-inferred Abs .", "We addressed potential changes in antigen structure resulting from germline and mature Ab binding by comparing structures of free and bound 426c . TM4△1–3 gp120 core .", "The 426c . TM4△1–3 structure was solved using crystals of the gp120 core alone and again as part of the analysis of the 3BNC60GL/426c . TM4△V1-3 crystals in which the crystallographic asymmetric unit included two copies of unbound 426c . TM4△V1-3 gp120 along with two copies of the 3BNC60GL/426c . TM4△V1-3 complex .", "We compared the unbound 426c . TM4△V1-3 structures to those of 426c . TM4△V1-3 complexed with Fabs from 3BNC60GL and from a mature VRC01-class Ab , 3BNC55MAT .", "We found no major structural rearrangements resulting from binding of either a germline or mature Ab ( Figure 3J , K ) , suggesting that the 426c gp120 core immunogens are preformed for binding to germline and mature forms of VRC01-class Abs .", "Since these immunogens lack the V1V2 and V3 loops , potential rearrangement of these regions in the context of the trimer upon binding remains to be studied .", "The new germline Fab/gp120 structures , NIH45-46GL/426c . TM1△V1-3 and 3BNC60GL/426c . TM4△V1-3 , showed interface interactions in which the germline Fab contacts with the CD4-binding loop and loops D and V5 of the gp120 outer domain were similar to those observed for mature VRC01-class Fab/gp120 complexes ( Zhou et al . , 2010; 2015; Diskin et al . , 2011 ) .", "This confirms the assumption of similar recognition modes derived from germline Fab-antigen complex structures that either did not contain a full gp120 core ( VRC01GL/eOD-GT6 ) ( Jardine et al . , 2013 ) or included a mature Ab LC ( NIH45-46CHIM/93TH057 ) ( Scharf et al . , 2013 ) .", "In particular , both NIH45-46GL and 3BNC60GL interacted with the CD4-binding loop on gp120 using conserved interactions that mimic CD4 binding , first described for the complex of VRC01MAT with 93TH057 gp120 ( Zhou et al . , 2010 ) , in which VRC01-class bNAbs mimic CD4 using backbone atoms in the VH domain C” strand to engage with the CD4-binding loop on gp120 ( Wu et al . , 2010; Zhou et al . , 2010; 2015; Diskin et al . , 2011 ) .", "As found in NIH45-46MAT and 3BNC117MAT complexes with 93TH057 gp120s , Gly54HC in NIH45-46GL and 3BNC60GL makes a main chain hydrogen bond with Asp368gp120 ( Figure 4A , B ) .", "The 3BNC60GL complex with 426c . TM4△V1-3 gp120 includes an additional main chain hydrogen bond between Gly57HC and Gly365gp120 that is not made in the 3BNC117MAT/gp120 complex .", "Similarly , NIH45-46GL Thr58HC makes an additional side chain hydrogen bond with Gly365gp120 .", "NIH45-46MAT further engages the CD4 binding loop of gp120 using water-mediated hydrogen bonds between Val57HC and Gly366gp120/Asp368gp120 ( Diskin et al . , 2011 ) .", "While the resolution of the germline Fab complex structures did not permit placement of water molecules , residues in the C′′ strand of both NIH45-46GL and 3BNC60GL are positioned to engage in water-mediated hydrogen bonds with additional CD4-binding loop residues in gp120 .", "Thus , our analyses showed that NIH45-46GL and 3BNC60GL , like their mature counterparts , use the C” strand in VH to mimic Leu44CD4 and Lys46CD4 interactions with the gp120 CD4 binding loop . 10 . 7554/eLife . 13783 . 007Figure 4 . Comparison of Signature VRC01-class and CD4-mimickry Contacts in Germline Ab-gp120 Immunogen Complexes . Top panels show ( A ) CD4 binding loop , ( C ) HC , ( E ) LC contacts in superimposed NIH45-46GL/426c . TM1△V1-3 and NIH45-46MAT/93TH057 ( PDB 3U7Y ) complexes .", "Bottom panels show ( B ) CD4-binding loop , ( D ) HC , ( F ) LC contacts in superimposed 3BNC60GL/426c . TM4△V1-3 and 3BNC117MAT/93TH057 ( PDB 4JPV ) complexes .", "Protein backbones are shown as wire diagrams , interacting residues are shown as stick representations ( red , oxygen; blue , nitrogen ) .", "Yellow dashed lines indicate putative hydrogen bonds ( distance < 3 . 5 Å , A-H–D angle > 90° ) .", "Ab coloring: blue , NIH45-46GL HC; light blue , NIH45-46GL LC; orange , NIH45-46MAT HC; yellow , NIH45-46MAT LC; green , 3BNC60GL HC; light green , 3BNC60GL LC; purple , 3BNC60MAT HC; light pink 3BNC60MAT LC .", "gp120 coloring: blue , CD4-binding loop; green , loop D; teal , loop V5 .", "( E , F )", "Interacting residues of the C” strand of Fab HCs and the CD4-binding loop of gp120 ( grey ) are shown as sticks .", "The Fab residue numbers are indicated since the amino acid sequence differs at some positions between germline and mature Abs . DOI: http://dx . doi . org/10 . 7554/eLife . 13783 . 007 We next examined the interactions of the VRC01-class signature residues ( Diskin et al . , 2012 ) .", "As found for mature VRC01-like Fab/gp120 complexes ( e . g . , PDB codes 3U7Y and 4JPV ) and the half-germline NIH45-46CHIM/93TH057 gp120 complex ( Scharf et al . , 2013 ) , the VH1-2*02 germline-encoded heavy chain signature residues ( Trp50HC , Asn58HC , Arg71HC ) engaged in the predicted interactions with 426c . TM1△V1-3 and 426c . TM4△V1-3 in their complexes with NIH45-46GL and 3BNC60GL: i . e . , Trp50HC and Asn58HC made hydrogen bonds with Asn280gp120 and Arg456gp120 , respectively , and Arg71HC formed a salt bridge with Asp368gp120 to mimic the Arg59CD4–Asp368gp120 interaction ( Kwong et al . , 1998 ) ( Figure 4C , D ) .", "The final signature interaction within the heavy chain , CDRH3 residue Trp100BHC , also made the predicted hydrogen bonding interaction with Asn279gp120 .", "In summary , NIH45-46GL and 3BNC60GL make all predicted HC VRC01-class signature contacts with the CD4-binding loop , the V5 loop , and loop D to bind to gp120 .", "Potent VRC01-class bNAbs pair with LCs that acquire signature residues , Trp67LC and Glu96LC , during somatic hypermutation and/or V-J joining , and are associated with short loops for two of the CDRs: CDRL1 ( resulting from a two- to four-residue deletion relative to the germline LC ) and a five-residue CDRL3 ( Diskin et al . , 2012 ) .", "Comparison of the NIH45-46GL/426c . TM1△V1-3 and 3BNC60GL/426c . TM4△V1-3 structures with their mature counterparts ( NIH45-46MAT/93TH057 and 3BNC117MAT/93TH057; PDB codes 3U7Y and 4JPV ) revealed no major structural differences between the LC contacts with gp120 cores ( Figure 4E , F ) .", "The greater number of residues in the CDRL1s in the germline Fab-containing complexes ( two and four additional residues in NIH45-46GL and 3BNC60GL , respectively ) result in wider , rather than longer , loops than their mature counterparts ( Figure 5A ) .", "The wider CDRL1s in the germline Fabs are not extended towards gp120 , thereby preventing clashes with the 426c . TM1△V1-3 and 426c . TM4△V1-3 gp120s ( Figure 5A ) .", "However , CDRL1 is near the position of the Asn276gp120N-linked glycan ( removed by mutagenesis in gp120-based immunogen candidates including the 426c . TM1△V1-3 and 426c . TM4△V1-3 gp120s and the eOD-GT gp120 outer domains ) ( McGuire et al . , 2013; 2014; 2016; Jardine et al . , 2013; 2015 ) .", "To address whether this glycan would clash with the larger CDRL1 loops of germline VRC01-class Abs , we superimposed the NIH45-46GL/426c . TM1△V1-3 and 3BNC60GL/426c . TM4△V1-3 complex structures with the structure of a mature Ab/gp120 complex that includes a partially ordered Asn276gp120 glycan ( the structure of 45-46m2 complexed with 93TH057 gp120 core; PDB code 4JKP ) ( Diskin et al . , 2013 ) .", "The superimposed germline Fabs showed no clashes between CDRL1 and ordered Asn276gp120 glycan residues ( Figure 5B–D ) .", "However , given the flexibility of N-linked glycans , some conformations of the Asn276gp120-linked glycan could interfere with binding germline CD4bs Abs .", "VRC01-class Abs VRC-CH31 and 12A21 do not have deletions in CDRL1 , but accommodate the Asn276gp120-linked glycan due to a pair of glycines that increase the conformational flexibility of CDRL1 ( Zhou et al . , 2013 ) .", "The CDRL1 of 3BNC60GL is of the same length as the VRC-CH31 and 12A21 CDRL1s and lacks glycines , yet has the conformational flexibility to avoid clashes with loop Dgp120 and the Asn276gp120 glycan ( Figure 5E ) .", "Therefore , CDRL1 deletions or enhanced loop flexibility due to somatically substituted glycine residues is not required for binding to gp120 although these adaptations likely lead to improved affinity in mature bNAbs adapted to bind to HIV-1 Envs that are not optimized to accommodate germline Ab binding . 10 . 7554/eLife . 13783 . 008Figure 5 . Accommodation of Asn276gp120 Glycan by Germline Ab Light Chains . Superposition of gp120 ( grey ) complexes with germline and mature Abs .", "( A ) CDRL1 from 45-46m2MAT ( yellow ) , NIH45-46GL ( blue ) and 3BNC60GL ( green ) is positioned near the Asn276gp120 glycan .", "Two- and four-residue insertions in NIH45-46GL and 3BNC60GL , respectively , result in a widening of the tip of CDRL1 rather than a more extended loop , which would clash with gp120 protein residues and/or the Asn276gp120 glycan .", "gp120 ( grey ) complexes with ( B ) 3BNC117MAT ( pink ) and 45-46m2MAT ( yellow ) ( C ) NIH45-46GL ( blue ) and 45-46m2MAT ( yellow ) , ( D ) 3BNC60GL ( green ) and 45–46 m2MAT ( yellow ) .", "Protein backbones are shown as wire diagrams and the Asn276gp120 glycan from the 45-46m2MAT/93TH057 gp120 complex ( PDB code 4JKP ) is shown as sticks ( yellow , carbon; red , oxygen; blue , nitrogen ) .", "The positions of CDRL1 and FWR3LC are indicated .", "( E ) CDRL1 from VRC-CH31MAT ( magenta ) , NIH45-46GL ( blue ) and 3BNC60GL ( green ) is positioned near the Asn276gp120 glycan .", "The CDRL1 loop of VRC-CH31MAT is of the same length as that of 3BNC60GL , and uses increased backbone conformational flexibility due to somatically mutated glycine residues to avoid clashes with gp120 protein residues and/or the Asn276gp120 glycan . DOI: http://dx . doi . org/10 . 7554/eLife . 13783 . 008 We previously noted that the buried surface area on both the Ab and the gp120 was larger in mature Fab/gp120 complexes than in the NIH45-46CHIM/93TH057 gp120 complex ( Scharf et al . , 2013 ) .", "Here , we extended this analysis to include comparisons with the HC/LC germline Fab complexes with 426c . TM1△V1-3 and 426c . TM4△V1-3 ( Figure 6A , B , Supplementary file 1 ) .", "The surface area buried on gp120 by both germline Fabs was only slightly smaller than that buried by the corresponding mature Abs ( 1 , 039 Å2 vs . 1 , 190 Å2 for NIH45-46GL vs . NIH45-46MAT and 939 Å2 vs . 1 , 033 Å2 for 3BNC60GL vs . 3BNC117MAT ) , with gains in buried surface area in loop D , inner domain and bridging sheet residues of gp120 ( Figure 6C ) .", "The surface area buried on the Fab HCs by gp120 was smaller than that buried by the corresponding mature Abs with no change in the area buried on the LCs ( 784 Å2 vs . 1 , 130 Å2 for NIH45-46GL HC vs . NIH45-46MAT HC and 731 Å2 vs . 802 Å2 for 3BNC60GL vs . 3BNC117MAT ) .", "The difference was most pronounced for the NIH45-46GL/NIH45-46MAT comparison , in which the mature Fab gained contacts in CDRH3 . 10 . 7554/eLife . 13783 . 009Figure 6 . Comparison of the Binding Interfaces in gp120 Complexes with Germline and Mature Abs .", "( A ) gp120 residues contacted by Fabs and ( B ) Fab residues contacted by gp120s are shown as surfaces over ribbon diagrams .", "gp120 domains are colored as in Figure 3 .", "Ab coloring: blue , NIH45-46GL HC; light blue , NIH45-46GL LC; purple , NIH45-46CHIM HC; light purple , NIH45-46CHIM LC; orange , NIH45-46MAT HC; yellow , NIH45-46MAT LC; green , 3BNC60GL HC; light green , 3BNC60GL LC; pink , 3BNC60MAT HC; light pink , 3BNC60MAT LC .", "( C ) Quantitation of buried surface areas ( Å2 ) depicted in ( A ) and ( B ) .", "The columns labeled total are the sums of areas for outer domain , bridging sheet and inner domain for gp120 , and of heavy chain and light chain for Abs .", "Surface areas buried due to complex formation were calculated using a 1 . 4 Å probe . DOI: http://dx . doi . org/10 . 7554/eLife . 13783 . 009 Mature VRC01-class Abs show similar angles of approach for binding to gp120 ( [Zhou et al . , undefined] and references therein ) .", "However , comparison of the orientations of the germline Fab interactions with gp120 in the NIH45-46GL/426c . TM1△V1-3 and 3BNC60GL/426c . TM4△V1-3 structures revealed differences compared with their mature Fab counterparts ( Figure 7A ) .", "To systematically analyze these differences , we calculated the rotation and translation for the VH domains of mature , chimeric , and germline Fabs bound to gp120s when compared with a reference Fab/gp120 structure , the VRC01MAT/93TH057 complex ( Zhou et al . , 2010 ) ( PDB code 3NGB ) ( Figure 7B ) .", "We found that the mature Fab/gp120 complex structures clustered in the recognition mode for VRC01-class Ab recognition of gp120 ( Zhou et al . , undefined ) .", "The germline Fab complexes with the 426c gp120 immunogens exhibited larger rotations and translations relative to the VRC01 reference structure , with the VRC01GL/eOD-GT6 complex showing an intermediate orientation .", "To verify that the orientations observed for the germline Fabs bound to the 426c gp120s are relevant for binding to Env trimer , we aligned the 3BNC60GL/426c . TM4△V1-3 structure onto the gp120 region of a native-like Env trimer structure ( BG505 SOSIP . 664; PDB code 5CEZ ) ( Garces et al . , 2015 ) , comparing this orientation with the alignment of a VRC01MAT/gp120 complex ( PDB code 3NGB ) ( Zhou et al . , 2010 ) onto the same Env trimer structure ( Figure 7C ) .", "This alignment suggests that the different orientation of the 3BNC60GL VH domain with respect to gp120 moves it away from the adjacent gp120 subunit , suggesting this orientation would be sterically compatible when binding to Env trimer . 10 . 7554/eLife . 13783 . 010Figure 7 . Comparisons of Binding Mode in Germline Ab-gp120 Immunogen Complexes .", "( A ) Superpositions of Fab-gp120 complexes depicted as wire diagrams .", "The following Fab/gp120 complexes were compared by alignment of their gp120s: 3BNC117MAT/93TH057 ( PDB code 4JPV ) , 3BNC117MAT/C1086 ( PDB code 4LSV ) , 3BNC117MAT/93TH057 ( PDB code 4JPV ) , 3BNC60GL/426c . TM4△V1-3 , NIH45-46MAT/93TH057 ( PDB code 3U7Y ) , VRC01MAT/KER . 2018 . 11 ( PDB code 4LSS ) , NIH45-46GL/426c . TM1△V1-3 .", "( B ) Rotation angle and translation distance of VH domains of mature , chimeric and germline Fabs in complex with gp120s relative to VRC01MAT in complex with 93TH057 gp120 ( PDB code 3NGB ) .", "Data points for complexes of mature Fabs bound to non-immunogen gp120s are shown as blue diamonds , complexes of germline Fabs bound to immunogen candidates are shown as red squares ( TM1 = 426c . TM1△V1-3 , TM4 = 426c . TM4△V1-3 , eOD = eOD-GT6 ) , the complex between the half mature , half germline NIH45-46CHIM and to a non-immunogen gp120 is shown as a purple diamond , and 3BNC55MAT bound to 426c . TM4△V1-3 is shown as a green square .", "When two complexes were found in the crystallographic asymmetric unit , rotation and translation parameters are shown for both complexes ( denoted as #1 and #2 ) .", "Standard deviations for the translation distance and rotation angle for mature VRC01-class bNAb–gp120 complexes shown as vertical and horizontal lines , respectively .", "( C ) Alignment of the 3BNC60GL/426c . TM4△V1-3 ( VHVL shown in red ) and VRC01MAT Fab/gp120 ( PDB code 3NGB ) ( VHVL shown in blue ) structures onto the gp120 region of a native-like Env trimer structure ( BG505 SOSIP . 664; PDB code 5CEZ ) ( gray ) .", "Modeled structures are shown looking down the trimer three-fold axis ( left panel ) and from the side ( right panel ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 13783 . 01010 . 7554/eLife . 13783 . 011Figure 7—source data 1 . Rotation angle and translation distance data of VH domains of mature , chimeric and germline Fabs in complex with gp120s relative to VRC01MAT in complex with 93TH057 gp120 . DOI: http://dx . doi . org/10 . 7554/eLife . 13783 . 011 The orientation differences for the NIH45-46GL/426c . TM1△V1-3 and 3BNC60GL/426c . TM4△V1-3 structures could result from structural characteristics of germline Fabs , immunogen characteristics allowing for germline Fab recognition , or a combination of these factors .", "Arguing in favor of the idea that the 426c immunogens could select for an altered binding mode , we note that a mature Ab ( 3BNC55MAT ) complex with 426c . TM4△V1-3 exhibited rotation and translation values closer to those of the germline Fab/426c immunogen complexes than those of mature Ab-gp120 complexes .", "In addition , the VRC01GL complex with another germline-binding immunogen , eOD-GT6 , exhibited a binding mode that differed from the VRC01 reference complex more than most of the mature Fab/gp120 complexes .", "We also note that the half germline NIH45-46CHIM Fab clustered with the mature Fab/gp120 complexes when bound to the 93TH057 gp120 ( Figure 7B ) .", "This is consistent with the idea that designed immunogens capable of binding germline VRC01-class bNAbs select for a slightly different antibody-binding mode than that adopted by mature VRC01-class Fabs bound to gp120s not capable of supporting germline antibody binding .", "To address potential global changes in VRC01-class bNAbs during maturation from germline to mature forms , we calculated electrostatic potentials of the binding surfaces of germline and mature Fabs .", "Comparisons of electrostatic surface potentials revealed a striking shift to more positively-charged antigen combining sites due to maturation ( Figure 8 ) .", "The more negatively-charged ( 3BNC60GL ) or neutral ( NIH45-46GL , VRC01GL ) properties of the antigen-binding sites of the germline Fabs may interfere with interactions with complex-type N-glycans on gp120 containing negatively-charged sialic acids; in particular , the Asn276gp120 carbohydrate , typically a complex-type N-glycan ( Binley et al . , 2010; Go et al . , 2011 ) , would likely make unfavorable interactions with the neutral or negatively charged surfaces of 3BNC60GL , NIH45-46GL , or VRC01GL Fabs .", "Such interactions were prevented in the 426c gp120- and eOD-based immunogens by mutation of the Asn276gp120N-linked glycosylation sequon ( McGuire et al . , 2013; 2014; Jardine et al . , 2013; 2015 ) .", "Similarly , potentially unfavorable electrostatic interactions between the germline Fabs and the Asn463gp120N-linked glycan , also usually a complex-type N-glycan ( Binley et al . , 2010; Go et al . , 2011 ) , were prevented by mutation of the Asn463gp120 glycosylation sequon in the 426c gp120- and eOD-based immunogens ( McGuire et al . , 2013; 2014; 2016; Jardine et al . , 2013; 2015 ) . 10 . 7554/eLife . 13783 . 012Figure 8 . Comparison of Electrostatic Surface Characteristics of Fab-gp120 Complexes . The binding surfaces of Fabs ( left panels ) and gp120s ( right panels ) are shown .", "Each binding partner is shown in an orientation looking into the binding interface; the corresponding complex would be obtained by rotating each binding partner by ~90˚ about the vertical axis .", "The top panel shows the locations of landmarks on surface representations of Fabs ( dark grey , VH; light grey , VL ) and gp120s ( yellow , outer domain; grey , inner domain; orange , bridging sheet; blue , CD4-binding loop; green , loop D; teal , loop V5; ordered residues of the Asn276gp120 glycan shown as yellow sticks ( normally a complex-type N-glycan , but a high mannose N-glycan in crystal structures ) ; approximate locations of Asn460gp120 and Asn463gp120 shown as light pink and magenta dots , respectively ) .", "The lower panels show electrostatic potentials on surface representations of Fabs ( left panels ) and gp120s ( right panels ) colored blue ( positive electrostatic potential ) to red ( negative electrostatic potential ) .", "The binding interfaces are outlined with a dotted black line .", "The approximate footprints of the complex-type Asn276gp120 glycan on Fab surfaces are indicated with a black triangle ( the Asn463gp120 glycan , also complex-type , is not resolved in any Env structures , thus its footprint on Fab surfaces cannot be shown ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 13783 . 01210 . 7554/eLife . 13783 . 013Figure 8—figure supplement 1 . The combining sites of germline ( left panels ) and mature Fabs ( right panels ) are shown as surface representations and electrostatic potentials are indicated using blue for positive electrostatic potential and red for negative electrostatic potential . Abs shown are CH58GL and CH58MAT ( PDB codes 4RIR and 4HPO ) , 4E10GL and 4E10MAT ( PDB codes 4ODX and 2FX7 ) , 10-1074GL and 10-1074MAT ( PDB codes 4FQQ and 4FQ2 ) ( shown in two orientations to better illustrate electrostatic changes ) , and CH103GL and CH103MAT ( PDB codes 4QHK and 4JAM ) .", "The antibody paratopes are outlined with a dotted black line .", "The approximate footprint of Asn137gp120 , Asn156gp120 and Asn332gp120 glycans on 10-1074GL and 10-1074MAT Fab surfaces are indicated with black triangles . DOI: http://dx . doi . org/10 . 7554/eLife . 13783 . 013" ], [ "The process by which B cells produce high-affinity Abs starts with antigen binding to an unmutated , germline B cell receptor produced by random joining of V , D , and J or V and J gene segments .", "Affinity maturation is the process by which Abs with higher antigen-binding affinity are created through somatic hypermutation of the germline B cell receptor ( Victora and Nussenzweig , 2012 ) .", "Many anti-HIV-1 bNAbs , including VRC01-class CD4bs bNAbs , are heavily somatically mutated , likely through successive rounds of hypermutation and selection of B cells in response to rapid mutation of HIV-1 Env ( West et al . , 2014; Klein et al . , 2013 ) .", "Mature VRC01-class bNAbs achieve nucleotide somatic mutation frequencies of 32% and 20% in HC and LC variable region genes , respectively , whereas the mutation frequencies of more typical affinity-matured human Abs rarely exceeds 10% ( Kwong and Mascola , 2012 ) .", "Although heavily somatically mutated , VRC01-class bNAbs are promising targets for vaccine design because they have evolved in multiple donors from a common human germline VH gene segment , VH1-2*02 , to recognize HIV-1 Env using conserved interactions ( Scheid et al . , 2011; Wu et al . , 2010; Zhou et al . , undefined; Zhou et al . , 2013; Diskin et al . , 2011; Zhou et al . , 2010 ) .", "The first step in targeted attempts to raise VRC01-class bNAbs by immunization requires identification of an immunogen that binds to the germline configuration of the B-cell receptor , but germline-reverted versions of VRC01-like bNAbs generally do not bind HIV-1 Env ( Scheid et al . , 2011; Zhou et al . , 2010; Diskin et al . , 2012; Scharf et al . , 2013; Hoot et al . , 2013; Ota et al . , 2012; McGuire et al . , 2013; Jardine et al . , 2013 ) .", "Although antigens designed to bind to predicted unmutated germline precursors of VRC01-class bNAbs have been constructed and evaluated ( McGuire et al . , 2013; 2014; 2016; Jardine et al . , 2013; 2015; Dosenovic et al . , 2015 ) , structural comparisons of germline and mature versions of VRC01-class bNAbs bound to gp120-based immunogens were not available .", "Here , we report the results of structural studies of immunogens that bind to the inferred germline precursors of VRC01-class bNAbs , discovering both general principles and details of interactions that can guide structure-based immunogen design .", "A surprising finding revealed by our structural comparisons of germline and mature VRC01-class Fabs was an increase in electropositive surface potential in the antigen-combining site for the mature Fab compared with the germline-inferred Fab , which we observed in the three VRC01-class Abs ( NIH45-46 , 3BNC60 , and VRC01 ) for which germline and mature Fab structures were available for calculating electrostatic potentials .", "This marked shift to a more electropositive antigen combining site may also occur in other VRC01-class bNAbs , particularly those whose LCs are derived from the LC germline KV1-33 gene segment .", "Mature VRC01-class bNAbs appear on average to have a higher net nominal charge of their VHVL domains ( +4 . 9 ) than the average human Ab ( +3 . 1; as calculated from sequenced human B-cell repertoires ) ( Table 2 ) ( Rubelt et al . , 2012 ) , and the VH1-2*02 gene segment yields a sequence with a nominal net charge of +5 , higher than the average human VH gene segment ( +2 to +3 ) .", "However , the KV1-33 LC germline gene segment , used by some VRC01-class Abs , yields a protein sequence with a nominal net charge of -4 , in contrast to the KV3-20 gene segment ( utilized by other VRC01-class Abs ) with a net charge of zero .", "Perhaps to compensate for these negative charges , after maturation of the KV1-33–derived VRC01-class bNAbs 3BNC117 , VRC-CH31 , and 12A12 , the portions of the VL domain encoded by the KV1-33 gene segment have net nominal charge changes of +5 , +3 , and +6 , respectively . 10 . 7554/eLife . 13783 . 014Table 2 . Nominal net charges of VRC01-class bNAbs and of the human Ab repertoire .", "Nominal net charge is the total charge of the sequence calculated with Asp/Glu as -1 and Arg/Lys as +1 .", "The human Ab repertoire average is calculated from 601 , 889 HC and 206 , 953 LC sequences reported in ( Rubelt et al . , 2012 ) .", "Light chain Vgene assignments are from ( Zhou et al . , 2015 ) .", "Standard deviations are indicated , except for human Ab repertoire VH + VL , which cannot be calculated with unpaired chains .", "*Two-tailed T test comparing the net charges of VRC01-class VHs with human repertoire VHs gives a p-value of 0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 13783 . 014Nominal Net ChargeAbVH + VLVHVLLC V-geneVRC01541K3-20NIH45-46981K3-203BNC117523K1-3312A12514K1-33VRC-PG04734K3-20VRC-CH31220K1-33VRC-PG2045-1L2-14VRC23541K3-15VRC18321K3-20VRC2746-2K1-33Average of VRC01-class bNAbs4 . 9 ± 1 . 93 . 7* ± 2 . 11 . 2 ± 1 . 9Human Ab repertoire average3 . 11 . 5 ± 2 . 41 . 6 ± 2 . 1 The trend towards increased electropositivity with Ab maturation does not apply to all HIV-1 Env-specific Abs: maturation of CH58 , a non-neutralizing Ab against the gp120 V2 loop that was raised by vaccination in the RV144 trial ( Liao et al . , 2013 ) , did not involve a shift to increased electropositive character ( Figure 8—figure supplement 1 ) , consistent with its epitope being centered on a positive residue , Lys169gp120 , within the gp120 V2 loop ( Nicely et al . , 2015 ) .", "However , the developmental pathway of potent V1V2-directed bNAbs involves increased positivity of the CDRH3 loop , which starts as highly electronegative in part due to sulfated tyrosines , but accumulates positive charges during maturation that partially compensate for the negatively-charged sulfates ( Doria-Rose et al . , 2014 ) .", "A trend towards electropositive combining sites in mature HIV-1 bNAbs could rationalize their tendencies towards polyreactivity ( binding more than one antigen ) , which has been observed at higher frequencies for broadly neutralizing , as compared with non-neutralizing , Abs against HIV-1 ( Liu et al . , 2015 ) .", "For example , increased non-specific binding to cardiolipin and nucleic acids , negatively-charged antigens commonly used in polyreactivity assays , correlates with polyreactive properties of Abs ( Mouquet et al . , 2010 ) .", "We suggest that evolution of increased electropositivity during maturation of VRC01-class bNAbs ( and perhaps other HIV-1 bNAbs; Figure 8—figure supplement 1 ) facilitates interactions with heavily glycosylated HIV-1 Env spikes that include complex N-linked glycans containing negatively charged sialic acids .", "Indeed , somatic hypermutation to increase electropositivity of an Ab combining site may be a strategy utilized by Abs against other viruses containing heavily glycosylated Env proteins , such as influenza and Ebola .", "In the case of VRC01-class bNAbs , this strategy may be part of a multi-pronged approach of the humoral immune response against the CD4 binding site of HIV-1 for accommodating and/or for avoiding the N-linked Env glycan attached to Asn276gp120 , a site that can include complex-type N-glycans ( Behrens et al . , 2016 ) , with other strategies including deletions to shorten CDRL1 and increasing CDRL1 flexibility by somatic mutations to glycine ( Zhou et al . , 2013 ) .", "In addition to these steric considerations for potentially unfavorable interactions between CDRL1 and the Asn276gp120-linked glycan , the neutral or negatively charged character of the germline Fab combining sites provides a structural justification for the necessity to eliminate the Asn276gp120- and Asn463gp120-linked N-glycans from eOD- and gp120-based immunogen candidates to achieve binding to inferred germline B-cell receptors ( McGuire et al . , 2013; Jardine et al . , 2013; 2015; McGuire et al . , 2014; 2016 ) .", "The structures presented here , taken together with immunogen requirements for achieving germline Ab binding and previous structural analyses of mature VRC01-class Abs ( Zhou et al . , undefined; Zhou et al . , 2010; 2013; McGuire et al . , 2013; 2014; 2016; Jardine et al . , 2013; 2015 ) , suggest that the initiating antigen ( s ) in a natural pathway to induce VRC01-class Abs in HIV-1–infected individuals involve viral variants that lack the Asn276gp120 ( 5% of HIV-1 strains in the Los Alamos Database ) and the Asn463gp120N-glycans ( ~80% of HIV-1 strains in the Los Alamos Database ) ( http://www . hiv . lanl . gov/content/index ) .", "Alternatively , a natural VRC01-class eliciting antigen could be the result of glycan heterogeneity within a single HIV-1 strain that produced variants containing high mannose , rather than complex-type , N-glycans at Asn276gp120 and/or Asn463gp120 , consistent with observations of glycan heterogeneity at individual N-linked glycosylation sites within single HIV-1 strains ( Go et al . , 2011; Behrens et al . , 2016 ) .", "Although antibody-antigen recognition modes represent a continuum of binding mechanisms , germline precursors to affinity-matured Abs have been suggested to display structural flexibility to allow expanded antigen recognition through induced fit mechanisms , whereas affinity maturation through somatic hypermutation was assumed to convert recognition to a lock-and-key model ( Foote and Milstein , 1994; Wedemayer et al . , 1997; Thorpe and Brooks , 2007 ) .", "Induced fit modes of Ab-antigen recognition involve changes in the conformations of backbone and sidechain atoms of both the Ab and the antigen ( Blackler et al . , 2011; Li et al . , 2007; Rosen et al . , 2005; Sinha and Smith-Gill , 2005; Verdaguer et al . , 1996 ) , with large changes in the Ab usually occurring in the CDR loops .", "By contrast , lock-and-key recognition involves a minimum of conformational changes between the bound and unbound states of the antigen and Ab .", "Here , we showed that for the VRC01 class of CD4bs bNAbs , the maturation pathway for antigen binding from germline Ab to affinity-matured Ab does not involve changes from induced fit to lock-and-key binding mechanisms .", "Instead , the 426c gp120 immunogens bind NIH45-46GL and 3BNC60GL without requiring notable conformational changes in either the Ab or the antigen; thus both are largely preformed for binding .", "The largely lock-and-key binding mechanism for germline VRC01-class recognition of antigen may be rationalized by the fact that germline VRC01-class CD4bs Abs face major steric constraints when binding to HIV-1 Env trimer due to the recessed location of the epitope and its shielding by glycans from the target and adjacent gp160 monomers ( West et al . , 2014; Zhou et al . , 2015 ) .", "VRC01-like CD4bs bNAbs bind Env at an acute angle using mostly CDRs and FWRs of VH in part because these steric constraints likely do not allow a Fab to bind perpendicular to the CD4bs on a closed Env trimer without clashes with the adjacent trimer subunit .", "These constraints may also not permit bNAbs or their germline precursors to undergo the major conformational changes characteristic of induced-fit binding and instead select for rare Ab sequences that are preformed to recognize their epitope in this highly sterically-constrained setting .", "The lock-and-key binding mechanism of VRC01-class recognition also may reflect that the binding interaction is dominated ( relative to other Abs ) by VH domain FWRs , and that these would be unlikely to undergo large induced-fit conformational changes .", "Structural analyses of germline and mature versions of the non-neutralizing HIV-1 Ab CH58 showed a more typical path for Ab-antigen recognition; the germline precursor used a combination of induced fit and lock-and-key binding modes to recognize a V2 peptide antigen: its CDRL2 loop was preformed for binding but CDRL3 changed conformation upon antigen binding ( Nicely et al . , 2015 ) .", "The conformation of CDRL3 became fixed in mature CH58 through limited somatic mutation compared with VRC01-class Abs ( 11 total substitutions in CH58 VHVL ) ( Nicely et al . , 2015 ) .", "Thus , the conversion from induced fit to lock-and-key binding may be applicable for HIV-1 Abs such as CH58 that include the relatively small numbers of somatic mutations typical for most Abs , but not for VRC01-class Abs , whose maturation process involves a much higher number ( >60 ) of mutations .", "The finding of what can be described as lock-and-key recognition for germline Fabs binding to the 426c gp120 immunogens contrasts with recognition of unmodified gp120s used for structural studies of complexes with mature VRC01-class bNAbs that do not bind or bind only poorly to germline Abs .", "For example , when bound to the unmodified 93TH057 gp120 , the CDRH3 of the germline HC in the NIH45-46CHIM-93TH057 complex structure was partially disordered , presumably to avoid clashes with a gp120 not optimized for binding to germline Abs ( Scharf et al . , 2013 ) .", "An example of structural rearrangements in a mature VRC01-class bNAb bound to an unmodified gp120 is illustrated by a study of the Ala61HCPro substitution in the C” β-strand of the β-sheet framework in the VH domains of the 3BNC60/3BNC117 HIV-1 bNAbs .", "This substitution is required for maximal neutralization potency of the mature bNAbs , yet it disrupts the C” β-strand of the VH domain in the unbound Fab and decreases its thermal stability ( Klein et al . , 2013 ) , with the β-sheet framework being restored through a large conformational change when the mature Fab binds gp120 ( Zhou et al . , 2013; Klein et al . , 2013 ) .", "Taken together , the findings of what appears to be lock-and-key style germline Fab recognition with increased flexibility upon maturation for the highly somatically-mutated VRC01-class bNAbs contradict the assumption of induced fit for germline Ab recognition and lock-and-key fit for mature Ab binding ( Foote and Milstein , 1994; Wedemayer et al . , 1997; Thorpe and Brooks , 2007 ) .", "This assumption was also challenged by studies of the maturation of other HIV-1 Abs: for example , CH103 , a non-VRC01-class CD4-binding site bNAb , exhibits changes in the relative orientation of its VH and VL domains during maturation ( Fera et al . , 2014 ) , and the HIV-1 gp41-directed bNAb 4E10 exhibits increased flexibility in its combining site during maturation ( Finton et al . , 2014 ) .", "Thus , the rare events that result in evolution of HIV-1 bNAbs can fall outside of typical Ab maturation pathways .", "We speculate that 426c-based immunogens are able to bind germline precursors of VRC01-class bNAbs as a complete gp120 core because they can engage the unbound conformation of these Abs .", "This may allow the gp120-based immunogens to overcome a loss of binding affinity due to slow on-rates in the context of the already weak binding affinities of immature B cell receptors .", "The improved binding of the 426c-based gp120 immunogens to some germline VRC01-class Abs upon removal of the V1V2 and V3 loops from the 426c . TM1△V1-3 and 426c . TM4△V1-3 gp120s could result from the removal of steric occlusion by the variable loops ( also not present in the eOD immunogens ( Jardine et al . , 2013; 2015 ) , and which may be flexible in the context of a gp120 or eOD ) , removal of glycans attached to these loops , or a combination of these factors .", "Thus , our results suggest implementation of a general strategy in which a germline-binding antigen is designed to be electrostatically compatible with the neutral or negatively charged antigen-binding surfaces of germline VRC01 Fabs and to fit in lock-and-key mode to the combining site of a germline Fab , with consideration paid to the slightly different angles of approach reported here for germline Fab binding to the gp120-based immunogens .", "The general principles established here for germline VRC01-class recognition of gp120 can be used to guide efforts to design and produce immunogens capable of eliciting broad and potent CD4bs Abs of the VRC01 class in uninfected people , facilitating the development of an efficacious vaccine to protect from HIV-1 infection ." ], [ "NIH45-46GL and 3BNC60GL were constructed as described previously by using the VH1-2*02 germline V gene segment , the appropriate germline VL gene segment , and mature sequences for CDRH3 and CDRL3 ( Scharf et al . , 2013; Hoot et al . , 2013; McGuire et al . , 2013; Dosenovic et al . , 2015 ) .", "The Abs were expressed and purified as described ( Scharf et al . , 2013 ) .", "Briefly , IgGs and 6xHis-tagged Fab fragments were produced by transient cotransfection of appropriate HC and LC plasmids into HEK293-6E cells followed by purification of the secreted proteins from cell supernatant using protein A ( GE Healthcare; Pittsburg , PA ) or Ni-NTA ( GE Healthcare ) affinity chromatography and Superdex 200 16/60 ( GE Healthcare ) size exclusion chromatography ( SEC ) .", "gp120 proteins were expressed as cores with N/C termini and V1-V2 and V3 loop truncations as described for previous structural studies ( Zhou et al . , 2010 ) by transient transfection of suspension-adapted HEK293-S cells .", "gp120s were purified using Ni-NTA affinity chromatography and Superdex 200 16/60 SEC .", "Proteins were stored in 20 mM Tris , pH 8 . 0 , and 150 mM sodium chloride ( TBS buffer ) supplemented with 0 . 02% ( wt/vol ) NaN3 .", "Crystals of 3BNC60GL Fab were obtained by combining 0 . 2 μL of a 15 mg/mL protein solution with 0 . 2 μL of 0 . 1 M bicine pH 9 . 1 and 10% ( w/v ) PEG 1 , 500 at 20°C and cryoprotected in mother liquor supplemented with 20% ( v/v ) glycerol .", "Crystals of 426c . TM4△V1-3 gp120 were obtained by combining 0 . 2 μL of a 10 mg/mL protein solution with 0 . 2 μL of 0 . 1 M sodium citrate tribasic dihydrate pH 5 . 0 , 10% ( w/v ) PEG 6000 and 0 . 2 M sodium thiocyanate at 20°C and cryoprotected in mother liquor supplemented with 30% ( v/v ) 2-propanol .", "Complexes of 3BNC60GL/426c . TM4△V1-3 , NIH45-46GL/426c . TM1△V1-3 and 3BNC55MAT/426c . TM4△V1-3 were produced by incubating Fabs and gp120s at a 3:1 molar ratio at 4°C for 16 hrs , followed by Endoglycosidase H ( New England BioLabs; Ipswich , MA ) treatment and SEC purification .", "Fractions containing complexes were combined and concentrated as indicated below .", "Crystals of 3BNC60GL/426c . TM4△V1-3 complex were obtained by combining 0 . 2 μL of a 20 mg/mL protein solution with 0 . 2 μL of 0 . 1 M imidazole pH 7 . 0 , 8% ( w/v ) PEG10000 , 10 mM calcium chloride dihydrate at 20°C and cryoprotected in mother liquor supplemented with 20% ( w/v ) ethylene glycol .", "Crystals of NIH45-46GL/426c . TM1△V1-3 complex were obtained by combining 0 . 2 μL of a 20 mg/mL protein solution with 0 . 2 μL of 0 . 1 M sodium citrate pH 5 . 5 , 22% ( w/v ) PEG 1000 , 3% ( w/v ) xylitol at 20°C and cryoprotected in mother liquor supplemented with 20% ( w/v ) ethylene glycol .", "Crystals of 3BNC55MAT/426c . TM4△V1-3 complex were obtained by combining 0 . 2 μL of a 15 mg/mL protein solution with 0 . 2 μL of 0 . 1 M sodium citrate pH 5 . 5 , 18% ( w/v ) PEG 1 , 000 , 3% ( w/v ) ethylene glycol at 20°C and cryoprotected in mother liquor supplemented with 20% ( w/v ) ethylene glycol .", "All crystals were flash cooled in liquid nitrogen .", "X-ray diffraction data were collected at the Stanford Synchrotron Radiation Lightsource beamline 12–2 outfitted with a Pilatus 6M pixel detector ( Dectris; Baden-Dättwil , Switzerland ) .", "XDS was used to index , integrate and scale the data ( Kabsch , 2010 ) .", "The structures were refined using an iterative approach of refinement with Phenix ( Adams et al . , 2010 ) and manual model building in Coot ( Emsley and Cowtan , 2004 ) .", "Crystals of 3BNC60GL Fab ( one molecule per asymmetric unit ) diffracted to 1 . 9 Å , and the structure was solved by molecular replacement using 3BNC117MAT Fab ( PDB code 4JPV ) VHVL with CDR loops removed and CH1CL as search models .", "The final model ( Rwork = 19 . 6% , Rfree = 21 . 0% ) has 99% , 1% , and 0% of residues in the favored , allowed and disallowed regions , respectively , of the Ramachandran plot .", "Crystals of NIH45-46GL/426c . TM1△V1-3 complex ( one Fab-gp120 complex per asymmetric unit ) diffracted to 3 . 4 Å , and the structure was solved by molecular replacement using 93TH057 gp120 core ( PDB code 4JDT ) and NIH45-46GL Fab VHVL with CDR loops removed and CH1CL ( from PDB code 4JDV ) as the search models .", "The final model ( Rwork = 27 . 9% , Rfree = 28 . 6% ) has 95 . 7% , 4 . 3% and 0% of residues in the favored , allowed , and disallowed regions , respectively , of the Ramachandran plot .", "Some parts of the CH1CL domain were not well ordered in the electron density , probably because this domain did not make crystal packing contacts .", "To account for regions of disorder , unresolved CH1CL residues were assigned occupancies of 0 .", "Crystals of 426c . TM4△V1-3 gp120 diffracted to 2 . 0 Å , contained two molecules in the asymmetric unit and the structure was solved by molecular replacement using 426c . TM1△V1-3 as the search model .", "The final model ( Rwork = 20 . 7% , Rfree = 23 . 2% ) has 97 . 7% , 2 . 3% and 0% of residues in the favored , allowed , and disallowed regions , respectively , of the Ramachandran plot .", "Crystals of 3BNC60GL/426c . TM4△V1-3 complex diffracted to 3 . 1 Å , contained two Fab-gp120 complexes and two unbound gp120 molcules in the asymmetric unit and the structure was solved by molecular replacement using 426c . TM4△V1-3 gp120 core and 3BNC60GL Fab VHVL and CH1CL with CDR loops removed as the search models .", "The final model ( Rwork = 20 . 3% , Rfree = 26 . 7% ) has 97% , 2 . 9% and 0 . 1% of residues in the favored , allowed and disallowed regions , respectively , of the Ramachandran plot .", "Crystals of 3BNC55MAT/ 426c . TM4△V1-3 complex diffracted to 3 . 0 Å , contained two Fab-gp120 complexes in the asymmetric unit and the structure was solved by molecular replacement using 426c . TM4△V1-3 gp120 core and 3BNC117MAT Fab ( from PDB code 4JPV ) VHVL and CH1CL with CDR loops removed as the search models .", "The final model ( Rwork = 24 . 0% , Rfree = 27 . 9% ) has 98% , 1 . 7% , and 0 . 3% of residues in the favored , allowed , and disallowed regions , respectively , of the Ramachandran plot .", "Buried surface areas were calculated using PDBePISA ( Krissinel and Henrick , 2007 ) and a 1 . 4 Å probe .", "Hydrogen bonds were assigned tentatively due to the low resolution of the complex structures using the following criteria: a distance of <3 . 5 Å , and an A-D-H angle of >90° .", "Structures were superimposed and molecular representations were generated with PyMOL ( Schrödinger LLC , 2011 ) and UCSF Chimera ( Pettersen et al . , 2004 ) .", "Rmsd calculations following pairwise Cα alignments were done in PyMOL without outlier rejection .", "All SPR measurements were performed on a Biacore T200 ( GE Healthcare ) at 20°C using HBS-EP+ ( GE Healthcare ) as the running buffer .", "A CM5 chip ( GE Healthcare ) containing 3000 resonance units ( RUs ) of primary amine-coupled protein A ( Pierce; Waltham , MA ) was used to capture HIV-1 IgGs ( 3BNC60GL , NIH45-46GL , VRC01GL , 3BNC60MAT , NIH45-46MAT ) and a non-gp120-binding control IgG ( mG053 ) by injecting 0 . 25 μg/mL or 0 . 1 μg/mL solutions of germline or mature IgG , respectively .", "Remaining protein A binding sites were blocked by injecting 1 μM Fc .", "gp120 cores ( 426c . TM1△V1-3 , 426c . TM4△V1-3 , 93TH057 ) were injected over the flow cells at increasing concentrations ( top concentration of 16 μM for germline Abs , 200 nM for mature Abs ) at a flow rate of 50 μL/min for 240 s and allowed to dissociate for 500 sec .", "Regeneration of flow cells was achieved by injecting one pulse each of 10 mM glycine pH 2 and 1 M guanidine-HCl at a flow rate of 90 μL/min .", "Kinetic analyses were used after subtraction of reference curves to derive on/off rates ( ka/kd ) and binding constants ( KDs ) with a 1:1 binding model with or without bulk refractive index change ( RI ) correction as appropriate ( Biacore T200 Evaluation software ) .", "The VRC01MAT/93TH057 gp120 complex ( PDB code 3NGB ) was used as the reference structure for comparisons of angles of approach of Fab recognition of gp120s ( Figure 7B ) .", "The center of mass of the VRC01 VH domain was placed at the origin and its principal axes of inertia were aligned with the Cartesian axes using AMORE from the CCP4 program suite ( CCP4 , 1994 ) .", "The 3NGB complex was then aligned with the centered VH domain .", "For comparisons with other complexes , each Fab/gp120 complex was aligned with the 3NGB gp120 chain using LSQMAN ( Kleywegt , 1996 ) .", "The transformation matrix between the aligned Fab/gp120 VH domain and the VRC01 VH domain was then calculated by LSQMAN ." ] ]

[ "Efforts to elicit broadly neutralizing antibodies ( bNAbs ) against HIV-1 require understanding germline bNAb recognition of HIV-1 envelope glycoprotein ( Env ) .", "The VRC01-class bNAb family derived from the VH1-2*02 germline allele arose in multiple HIV-1–infected donors , yet targets the CD4-binding site on Env with common interactions .", "Modified forms of the 426c Env that activate germline-reverted B cell receptors are candidate immunogens for eliciting VRC01-class bNAbs .", "We present structures of germline-reverted VRC01-class bNAbs alone and complexed with 426c-based gp120 immunogens .", "Germline bNAb–426c gp120 complexes showed preservation of VRC01-class signature residues and gp120 contacts , but detectably different binding modes compared to mature bNAb-gp120 complexes .", "Unlike typical antibody-antigen interactions , VRC01–class germline antibodies exhibited preformed antigen-binding conformations for recognizing immunogens .", "Affinity maturation introduced substitutions increasing induced-fit recognition and electropositivity , potentially to accommodate negatively-charged complex-type N-glycans on gp120 .", "These results provide general principles relevant to the unusual evolution of VRC01–class bNAbs and guidelines for structure-based immunogen design ." ]

[ "When human immunodeficiency virus-1 ( HIV-1 ) infects humans it can cause a serious disease that damages the immune system .", "Currently there is no cure for this disease and there are no vaccines available to halt the spread of the virus .", "Researchers are hoping to be able to develop a single vaccine that can protect individuals against every form ( or strain ) of HIV-1 , but this has proved difficult because many different versions of the virus exist .", "An effective vaccine triggers long-lasting immunity to a particular virus or microbe by activating the production of proteins called antibodies that identify and help to destroy the threat .", "Research has shown that most individuals infected with HIV-1 produce antibodies that can only recognize a few HIV strains .", "However , there are rare individuals who produce “broadly neutralizing antibodies”; that is , antibodies that can recognize and help to kill 90% or more of HIV-1 strains .", "Understanding how broadly neutralizing antibodies are produced in infected individuals may aid the development of a vaccine that can protect others from the many circulating strains of HIV .", "When an individual encounters a virus , immature antibodies are modified to generate mature antibodies that bind more effectively to specific virus proteins .", "Here , Scharf et al . investigated how a class of broadly neutralizing antibodies called VRC01-class antibodies , which bind to an HIV protein called gp120 , are produced .", "The experiments used a technique called X-ray crystallography to reveal the three-dimensional structures of immature versions of these antibodies when they are bound to gp120 .", "Scharf et al . discovered that , unlike most antibodies , the overall final structure of VRC01 antibodies is formed before the antibody matures .", "Instead of making large changes to the structure of these antibodies , the maturation process makes VRC01-class antibodies become more positively charged , which allows them to bind to gp120 proteins on a wider variety of HIV viruses .", "These findings suggest that it may be possible to use modified gp120 proteins in vaccines to trigger the production of broadly neutralizing antibodies against HIV ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "chromosomes and gene expression", "cell biology" ]

[ [ "Iron-deficiency anemia is estimated to affect one-third of the global population ( Camaschella , 2019 ) .", "Iron and heme are key components of hemoglobin , the primary oxygen transport molecule , and their cellular levels impact globin synthesis and red blood cell production .", "Specifically , globin is transcriptionally regulated by BACH1 ( BTB Domain and CNC homolog 1 ) ( Igarashi and Sun , 2006 ) and is regulated at the level of protein translation by HRI ( heme-regulated eIF2α kinase ) ( Chen , 2007 ) , both of which are heme-sensing proteins .", "During heme deficiency that is induced by dietary iron deficiency ( ID ) in mice , HRI is activated and phosphorylates the α subunit of eukaryotic initiation factor eIF2 ( eIF2α ) to inhibit translation of α- and β-globin mRNAs , thereby preventing proteotoxicity resulting from heme-free globin chains ( Han et al . , 2001 ) .", "Meanwhile , phosphorylated eIF2α ( eIF2αP ) selectively enhances the translation of activating transcription factor 4 ( Atf4 ) mRNA ( Suragani et al . , 2012; Chen , 2014 ) .", "This coordinated translational repression of general protein synthesis with the specific translational enhancement of Atf4 mRNA by eIF2αP is termed the integrated stress response ( ISR ) ( Harding et al . , 2003 ) .", "The ISR is a universal response to several types of cellular stress ( Chen , 2014; Pakos-Zebrucka et al . , 2016 ) and it is initiated by members of the eIF2α kinase family .", "Besides HRI , mammalian cells have three additional eIF2α kinases , which are expressed in distinct tissues and which combat specific physiological stress .", "PKR responds to viral infection ( Kaufman , 2000 ) , GCN2 senses nutrient starvations ( Hinnebusch , 1996 ) , and PERK is activated by endoplasmic reticulum ( ER ) stress ( Ron and Harding , 2000 ) .", "All four eIF2α kinases respond to oxidative and environmental stresses .", "In the erythroid lineage , HRI expression increases during differentiation , with higher expression occurring in hemoglobinized erythroblasts ( Liu et al . , 2008a ) .", "Starting at the basophilic erythroblast stage , HRI is the predominant eIF2α kinase and is expressed at levels that are two orders of magnitude greater than those of the other three eIF2α kinases ( Kingsley et al . , 2013 ) .", "At these stages , HRI is responsible for over 90% of eIF2α phosphorylation ( Liu et al . , 2008b ) .", "HRI-ISR is necessary for effective erythropoiesis during ID and acts by reducing oxidative stress and promoting erythroid differentiation ( Suragani et al . , 2012; Zhang et al . , 2018 ) .", "Furthermore , HRI-ISR represses the mTORC1 signaling that is activated by the elevated erythropoietin ( Epo ) levels occurring during ID specifically in the erythroid lineage ( Zhang et al . , 2018 ) .", "Thus , HRI coordinates two key translation-regulation pathways , eIF2αP and mTORC1 , during ID .", "However , the exact molecular mechanisms through which iron and heme regulate erythropoiesis are incompletely understood .", "Mitochondria not only are the energy powerhouses of the cell , but also are necessary for amino acid metabolism , nucleotide production , and the biosynthesis of heme and iron-sulfur clusters ( Shpilka and Haynes , 2018 ) .", "Translational regulation of mitochondrial biogenesis by mTORC1 is particularly important for erythropoiesis because of the high demand of heme for hemoglobin production and oxidative stress ( Liu et al . , 2017 ) .", "However , the roles of HRI and eIF2αP in mitochondrial biogenesis and function are still unknown .", "Transcriptional regulation during erythropoiesis has been studied extensively ( Kerenyi and Orkin , 2010; An et al . , 2014 ) , but much less is known about the translational control of this process ( Mills et al . , 2016; Khajuria et al . , 2018 ) .", "Ribosome profiling ( Ribo-seq ) has emerged as a powerful tool that can be used to interrogate translation genome-wide ( Ingolia et al . , 2009 ) .", "Here , we performed Ribo-seq and mRNA-seq in primary basophilic erythroblasts to investigate how in vivo translation is regulated by iron , heme , and HRI in order to gain a global understanding of the molecular mechanisms that govern erythropoiesis .", "We hypothesized that by globally surveying the landscape of translational and concomitant transcriptional changes that occur in the context of HRI deficiency ( comparing the changes seen in iron replete ( +Fe ) or iron deficiency ( –Fe ) conditions ) , we could gain important insights into the mechanisms through which iron and heme regulate the process of erythropoiesis .", "Our results demonstrate that heme and HRI mediate the translation of both cytosolic and mitochondrial ribosomal protein mRNAs .", "Furthermore , HRI–ATF4 mediated gene expression is essential during ID to prevent the accumulation of unfolded proteins in the cytoplasm , to maintain mitochondrial oxidative phosphorylation , and to enable erythroid differentiation in developing basophilic erythroblasts ." ], [ "Beginning at the basophilic erythroblast stage , erythropoiesis is finely regulated by iron and heme levels ( Chiabrando et al . , 2014; Muckenthaler et al . , 2017 ) .", "Thus , basophilic erythroblasts ( hereafter referred to as EBs for simplicity ) from Wt +Fe , Wt –Fe , Hri–/– +Fe , and Hri–/– –Fe fetal livers ( FLs ) were used as sources to generate Ribo-seq and mRNA-seq libraries for genome-wide analysis of transcriptional and translational changes ( Figure 1A ) .", "After applying standard protocols of quality control and preprocessing to remove rRNA and tRNA , we obtained 9 . 7–41 . 1 ( median 26 . 2 ) million reads of Ribo-seq and 29 . 4–66 . 6 ( median 42 . 5 ) million reads of mRNA-seq for subsequent mappings ( Supplementary file 1a ) .", "As expected , most of the reads from both Ribo-seq ( 84–88% ) and mRNA-seq ( 72–74% ) were mapped to protein coding sequence ( CDS ) , whereas some reads mapped to the 5′ and 3′ UTR or to other regions , mostly introns and the regions around transcription start sites ( Figure 1B ) .", "Our Ribo-seq data displayed excellent triplet periodicity , CDS enrichment , and limited 3′ UTR reads , validating the high quality of the ribosome-protected fragments ( RPFs ) that we obtained ( Figure 1B–C ) .", "The proportions of reads mapping to the 5′ UTRs of Ribo-seq data were similar to those from mRNA-seq ( Figure 1B ) , in agreement with reports of pervasive translation outside of annotated CDSs ( Ingolia , 2014 ) .", "Overall , 62 . 9% of the expressed genes in mRNA-seq were detected in Ribo-seq , and therefore appeared to be actively translated in EBs ( Figure 1D ) .", "As shown in Figure 1D , 99% of the mRNAs detected in Ribo-seq data ( with reads greater than 25 in at least one of the conditions ) were present in the mRNA-seq data , demonstrating the high quality of our Ribo-seq data and the correlation of RPFs and mRNAs ( Figure 1E ) .", "A total of 317 mRNAs were significantly differentially translated between Wt and Hri–/– EBs in the +Fe condition ( Figure 2A and Supplementary file 1b ) , supporting the role of HRI during normal fetal erythropoiesis .", "Twenty-five differentially translated mRNAs were common under both +Fe and –Fe conditions; these included the well-characterized ISR mRNAs , Atf4 , Ppp1r15a , and Ddit3 ( Figure 2A–B and Figure 2—figure supplement 1A ) as illustrated in Figure 8 .", "Atf4 mRNA was the most differentially translated mRNA between Wt and Hri–/– cells during ID ( 8 . 8-fold increase in translational efficiency ( TE ) ) , followed by the Ddit3 ( 8 . 1-fold ) and Ppp1r15a ( 3 . 7-fold ) mRNAs ( Figure 2B and Supplementary file 1b ) .", "Each of these mRNAs contains upstream open reading frames ( uORFs ) in their 5′ UTR , the use of which is upregulated by eIF2αP in cell lines under endoplasmic reticulum ( ER ) tostress or amino-acid starvation ( Pavitt and Ron , 2012 ) .", "We observed HRI-dependent regulation of the translation of these mRNAs involving ribosome occupancies in uORFs in vivo ( Figure 2C–D and Figure 2—figure supplement 1B–C ) .", "We also verified that changes in TE corresponded to concordant changes in ATF4 protein levels in EBs ( Figure 2E ) .", "As Eif2ak1 ( HRI ) and Atf4 are among the most highly expressed and efficiently translated mRNAs in Wt EBs ( top 3% , Supplementary file 1c ) , we investigated whether the translation of Atf4 mRNA is regulated by HRI in vivo .", "Atf4 mRNA has two well-characterized uORFs in its 5′ UTR .", "uORF1 encodes three amino acids and is translated regardless of stress and eIF2αP levels ( Pavitt and Ron , 2012 ) .", "We observed that uORF1 had exceptionally high ribosome occupancy ( 5 . 7-fold and 21 . 3-fold higher than eIF2s1 ( eIF2α ) and Rps6 initiating AUGs , respectively ( Supplementary file 1d ) ) .", "However , uORF2 and the canonical ORF of Atf4 were poorly translated in Hri–/-– compared to Wt EBs under both +Fe and –Fe conditions ( Figure 2C–D ) .", "Together , these data support the idea that Atf4 mRNA is primed for translation by HRI in developing EBs , and that HRI is a master translational regulator of key ISR mRNAs in vivo in primary EBs , especially during ID .", "Gene Ontology ( GO ) analysis of the differentially translated mRNAs revealed that the translation of components of ribosomes and mitochondria is most significantly upregulated in HRI- and iron-deficient states ( Figure 3A ) .", "Gene Sets Enrichment Analysis ( GSEA ) of differentially translated mRNAs further showed that translation of mRNAs that are involved in ribosome synthesis , mTORC1 signaling , and translation initiation and elongation were most highly elevated in Hri–/– –Fe EBs compared to Wt –Fe EBs ( Figure 3B–C ) .", "Increased 43S translation initiation complex formation in Hri–/– –Fe cells ( Figure 3B ) is consistent with the known function of HRI–eIF2αP in ternary and 43S complexes formation ( Chen , 2007 ) .", "Furthermore , we observed that Eef1a1 and Ccnd3 , which have been shown to be regulated by mTORC1 signaling ( Thoreen et al . , 2012 ) , were also more efficiently translated in Hri–/– –Fe EBs ( Figure 2B ) .", "Notably , 56 ribosomal protein ( RP ) mRNAs were more highly translated in Hri–/– –Fe EBs than in Wt-Fe EBs ( Figure 3D , Figure 3—figure supplement 1A–B and Supplementary file 2a ) .", "Whereas 38 of these RP mRNAs were known to be regulated by mTORC1 via 5′ terminal oligopyrimidine ( TOP ) motifs in their 5′ UTR ( Thoreen et al . , 2012 ) , translation of the other 18 RP mRNAs was regulated by HRI , but not mTORC1 .", "There was less overlap for non-RP targets between mTORC1- and HRI-mediated translation .", "Among the 20 non-RP mRNAs , translation of 12 mRNA was regulated by HRI but not by mTORC1 ( Figure 3D and Supplementary file 2a ) .", "The second set of most preferentially translated mRNAs were mitochondrial proteins ( Figure 3A–B ) .", "The translation of 163 mRNAs of nuclear-encoded mitochondrial proteins was upregulated in Hri–/– –Fe EBs compared to Wt –Fe EBs .", "These include mitochondrial ribosomal proteins , oxidative phosphorylation proteins ( comprising complexes I through V ) , and matrix proteins ( Figure 3—figure supplement 1C and Supplementary file 2b ) .", "Interestingly , this set of mRNAs encoding mitochondrial proteins that are differentially translated in HRI deficiency was distinct from those encoding mitochondrial proteins that are upregulated by mTORC1/eIF4EBPs-mediated translation ( Morita et al . , 2013 ) ( Figure 3D ) .", "Overall , these genome-wide results indicate roles for both HRI–eIF2αP and mTORC1 signaling in the global translation of ribosomal and mitochondrial proteins in developing EBs during ID .", "To examine the impact of increased translation of ribosomal proteins ( both cytosolic and mitochondrial ) , protein synthesis in the cytoplasm and mitochondria were measured in the primary erythroid cells isolated from bone marrows of both Wt –Fe and Hri–/– –Fe mice .", "We took advantage of the fact that cycloheximide ( CHX ) inhibits only cytoplasmic and not mitochondrial protein synthesis .", "As shown in Figure 3E , the rate of total protein synthesis in Hri–/– –Fe erythroid cells at 15–60 mins was greater than that in Wt –Fe cells .", "Interestingly , the rate of protein synthesis in cells under CHX treatment was also greater in Hri–/– –Fe erythroid cells than in Wt-Fe cells .", "Furthermore , protein synthesis under CHX treatment was inhibited by chloramphenicol ( Figure 3—figure supplement 2 ) , a selective inhibitor of mitochondrial translation ( Richter et al . , 2013 ) .", "These results demonstrate that HRI is necessary to inhibit both cytoplasmic and mitochondrial protein synthesis in iron-deficient erythroid cells .", "To discern the contributions of HRI–eIF2αP and mTORC1 activity in the regulation of mitochondrial protein synthesis , INK128 , a mTOR inhibitor , was employed in mitochondrial protein synthesis assays ( Figure 3—figure supplement 3 ) .", "INK128 treatment reduced mitochondrial protein synthesis by about 50% with a near complete inhibition of pS6 and p4EBP1 .", "These results show that the HRI–eIF2αP pathway contributes directly to the regulation of mitochondrial protein synthesis during ID , in addition to repressing mTORC1 activity .", "We then asked whether the increased protein synthesis in Hri–/– –Fe erythroid cells affects the activity and mass of mitochondria in these cells when compared with Wt –Fe cells .", "As iron and heme are cofactors for several enzymes in the respiratory electron transport chain , the oxygen consumption rates of primary erythroid cells from bone marrows of +Fe and –Fe mice were measured using the Seahorse assays ( Figure 4 ) .", "Wt erythroid cells were able to maintain their oxygen consumption rate in ID ( Figure 4A ) .", "There were no significant differences between Wt –Fe and Wt +Fe cells in either the basal or maximal respiration rates ( Figure 4B ) .", "By contrast , Hri–/– erythroid cells displayed decreased basal and maximal respiration under both +Fe and –Fe conditions , as compared to Wt erythroid cells ( Figure 4A–B ) .", "The similar extent to which respiration was reduced in Hri–/– +Fe cells and Hri–/– –Fe cells was somewhat unexpected because the phenotype of Hri–/–+Fe in vivo is milder than that of Hri–/– –Fe cells .", "This may be due in part to the stress experienced by Hri–/– +Fe cells during the Seahorse assays .", "Nonetheless , the significant difference between Wt -–Fe and Hri–/– –Fe cells demonstrates that HRI is necessary to maintain mitochondrial respiration under –Fe conditions .", "This impaired mitochondrial respiration in Hri–/– erythroid cells was not due to the reduced mitochondrial mass in these cells ( Figure 4C ) .", "The mitochondrial mass in primary erythroid cells was similar for Wt +Fe and Hri–/– +Fe mice , but was increased in Hri–/– –Fe cells as compared to Wt –Fe cells , especially during the later stages of terminal erythroid differentiation ( Figure 4C , populations II and III ) .", "There was no significant difference in mitochondrial DNA contents between Wt and Hri–/– erythroid cells in either +Fe or –Fe conditions ( Figure 4—figure supplement 1 ) .", "We next investigated the transcriptional impact of ID and the role of HRI in mediating the cellular response to the ID-induced stress response .", "Analysis of mRNA-seq data revealed that substantially more genes displayed significant differential expression between Wt –Fe and Wt +Fe EBs than between Hri–/– –Fe and Hri–/– +Fe EBs ( 232 vs 37 , Figure 5A and Supplementary file 1e ) , demonstrating the near-absolute requirement for HRI in regulating the transcriptional response to ID .", "GO analysis revealed that the most upregulated process in Hri–/– -Fe EBs when compared to Wt –Fe EBs is the process of protein folding and refolding , which involves many chaperone genes from the Hsp70 ( Hspa1a , Bag3 , Hspa1b , Hspa8 , HspA4L ) , Hsp90 ( Hsp90ab1 , Hsp90aa1 , Chordc1 , Ahsa1 ) , Hsp40 ( Dnaja4 , Dnajb1 ) , and Hsp110 ( Hsph1 ) families ( Figure 5A–B and Figure 6B ) .", "These are cytosolic chaperones that are upregulated specifically in response to cytoplasmic unfolded proteins .", "Chaperones involved in neither ER ( Hspa5 , Hsp90b1 ) nor mitochondrial ( Hspa9 , Hspd1 ) unfolded-protein responses ( UPR ) were not significantly increased .", "This comprehensive upregulation of cytoplasmic chaperones indicates the response to cytoplasmic unfolded proteins , such as heme-free globins and nuclear-encoded mitochondrial proteins , in Hri–/– –Fe EBs .", "Cytoplasmic chaperones were not induced in the presence of HRI during ID ( Figure 5A and Figure 6A ) , consistent with the essential role of HRI in inhibiting protein synthesis in iron/heme deficiency shown in Figure 3E .", "We have shown previously that HRI-ISR is activated in an ex vivo FL differentiation system and that erythroid differentiation of Hri–/– progenitors is impaired even under +Fe conditions ( Suragani et al . , 2012 ) .", "Here , we showed that there was no significant effect of HRI , eIF2αP , or ATF4 deficiencies on growth and viability during expansion and during up to 20 hr of erythroid differentiation ( Figure 5C–D ) .", "However , eIF2α Ala51/Ala51 ( AA ) erythroid precursors , which were devoid of eIF2αP , accumulated significant numbers of globin inclusions between 20 hr and 30 hr of erythroid differentiation , resulting in cell death and fragments of cell debris ( Figure 5C ) .", "A similar observation was found in Hri–/– erythroid precursors ( Figure 5C ) , but the effect was less severe in these cells because eIF2αP is completely absent from AA erythroid precursors , whereas Hri–/– cells still have a low level of eIF2αP .", "Together , the presence of aggregated protein inclusions in ex vivo differentiation and the induction of cytoplasmic UPR in Hri–/– EBs underscore the primary function of HRI–eIF2αP in inhibiting translation , and thereby coordinating protein homeostasis with available iron and heme concentrations to mitigate proteotoxicity during FL erythropoiesis .", "As shown in Figure 5D , Atf4–/– FL erythroid cells did not suffer from proteotoxicity due to the presence of functional HRI-eIF2αP in inhibiting globin mRNA translation ( Zhang et al . , 2018 ) .", "However , the differentiation of Atf4–/–erythroid precursors at 30 and 42 hr was impaired ( Figure 5D ) .", "These results demonstrate that the enhanced translation of Atf4 mRNA is also required for erythroid differentiation .", "The majority of highly induced genes in Wt –Fe EBs compared to Wt +Fe EBs were ATF4 target genes ( Figure 6A ) ( Pakos-Zebrucka et al . , 2016 ) .", "However , these genes were not upregulated in Hri–/– –Fe EBs ( Figure 6B , Figure 6—figure supplement 1A ) , indicating that HRI is required to activate ISR in ID ( Figure 2 ) .", "The GO terms for the biological processes that were most highly differentially expressed in Wt –Fe EBs as compared to Hri–/– –Fe EB were amino acid metabolism and biosynthesis ( Figure 6C ) .", "The corresponding upregulated ISR genes are involved in serine-glycine biosynthesis ( Phgdh , Psat1 , Psph , Shmt2 ) , one-carbon metabolism ( Shmt2 , Mthfd2 ) , proline-glutamine synthesis ( Pycr1 , Aldh18a1 , Asns ) and glutathione metabolism ( Chac1 ) ( Figure 6B , Figure 6—figure supplement 1B and Supplementary file 1e ) .", "Furthermore , the expression levels of these genes were lower in Hri–/– EBs than in Wt EBs under the +Fe condition ( Figure 6—figure supplement 1C ) , suggesting that HRI finetunes the ISR during iron replete erythropoiesis and thus has an important role even under normal conditions .", "Interestingly , some of these ATF4 target genes , most notably Atf5 , Trib3 and Chac1 , are also Epo-stimulated genes ( Figure 6—figure supplement 1D ) ( Singh et al . , 2012 ) , consistent with the interaction of the HRI-ISR pathway and Epo signaling ( Zhang et al . , 2018 ) .", "Having mapped the genome-wide translational and transcriptional responses to ID , we set out to characterize the function of one of the most highly induced ATF4 target genes , growth factor receptor-bound protein 10 ( Grb10 ) , in erythropoiesis .", "Grb10 has been reported to be part of a feedback mechanism that inhibits the mediation of mTORC1 signaling by growth factors such as insulin ( Plasschaert and Bartolomei , 2015 ) and stem cell factor ( SCF ) ( Yan et al . , 2016 ) .", "We employed ex vivo FL differentiation to interrogate the function of Grb10 in erythropoiesis .", "First , we validated that Grb10 and Atf5 , but not Atf4 , expression was increased in Wt –Fe EBs , but not in Hri–/– –Fe EBs ( Figure 7—figure supplement 1A ) .", "In addition , Grb10 expression in Wt EBs was increased during ex vivo erythroid differentiation from 20 to 30 hr , and was greatly reduced in Hri–/– , AA and Atf4–/– erythroblasts ( Figure 7—figure supplement 1B ) .", "We prepared eight shRNA recombinant retroviruses , all of which were able to knockdown Grb10 RNA expression by more than 80% during the expansion phase .", "Two of these retroviruses , shRNA_G3 and shRNA_G7 , were able to maintain persistent knockdown of Grb10 RNA during differentiation ( Figure 7A , Figure 7—figure supplements 2–3 ) .", "Reduction of Grb10 expression by shRNAs ( Figure 7D and Figure 7—figure supplement 3A ) increased the numbers of differentiating erythroblasts ( Figure 7B ) .", "This increase was mostly observed between 16 and 26 hr of differentiation ( Figure 7B ) , a period during which Epo concentrations were increased and SCF was withdrawn ( Figure 7A ) .", "We also observed increased expression of cyclin D3 , a known target of mTORC1 signaling ( Figure 7D ) .", "Importantly , terminal erythroid differentiation was inhibited in Grb10 knockdown cells , as indicated by an accumulation of polychromatic erythroblasts and a decrease in orthochromatic erythroblasts ( Figure 7C ) .", "Thus , the increased proliferation but decreased terminal differentiation upon reduction of Grb10 expression ( Figure 7 ) recapitulates the hallmarks of ineffective erythropoiesis observed in Hri–/– mice in ID ( Han et al . , 2001; Suragani et al . , 2012; Zhang et al . , 2018 ) .", "These results strengthen our global observation of an interaction between HRI-ISR and the Epo signaling pathway by showing that the induction of Grb10 by HRI-ISR serves as an important feedback mechanism for Epo signaling , resulting in reduced proliferation and thus promoting erythroid differentiation during stress erythropoiesis ( Figure 8 ) ." ], [ "Although the specific role of HRI in the translational regulation of globin and Atf4 mRNAs in erythroid cells has been appreciated , an understanding of the global impact of HRI-mediated translational regulation on erythropoiesis is lacking .", "Here , we report a global , unbiased assessment of iron , heme , and HRI-mediated translational and transcriptional alterations in primary EBs in vivo .", "We show that Eif2ak1 and Atf4 mRNAs are abundantly expressed in EBs and are poised to respond to ID during terminal erythroid differentiation .", "HRI is a major regulator of gene expression in erythropoiesis: only limited changes of mRNA expression are observed in Hri–/– EBs during ID .", "Our global analysis supports a distinct model of heme and HRI-regulated translational control involving several separate and interacting pathways that allow EBs to adapt to systemic ID ( Figure 8 ) .", "Activation of HRI in ID elicits three distinct pathways of translation .", "First and foremost , inhibition of protein synthesis prevents the accumulation of unfolded proteins and maintains protein homeostasis .", "Second , induction of Atf4 mRNA translation increases the expression of genes encoding mitochondrial UPR , redox homeostasis and metabolic reprogramming in order to maintain mitochondrial respiration and erythroid differentiation .", "Third , repression of Epo-mTORC1 signaling inhibits protein synthesis and enables erythroid differentiation .", "The present study identifies a previously unappreciated role of HRI in the translational regulation of cytosolic and mitochondrial ribosomal proteins during erythropoiesis .", "We observed , at a global level , additional evidence of mTORC1's involvement in translational regulation in Hri–/– –Fe EBs , further supporting the role of HRI-ISR in repressing Epo–mTORC1 signaling to mitigate ineffective erythropoiesis during ID ( Zhang et al . , 2018 ) .", "Importantly , we find that HRI–eIF2αP also controls the translation of 18 cytosolic RP mRNAs , which are not known targets of mTORC1 .", "Consistent with our results , mTORC1-independent translation of RP mRNAs requiring GCN2-eIF2αP has been reported recently in several cancer cell lines ( Li et al . , 2018 ) .", "Interestingly , the translation of many mitochondrial RPs and oxidative phosphorylation complexes is also increased in Hri–/– –Fe EBs .", "Furthermore , HRI directly inhibits mitochondrial protein synthesis in addition to repressing mTORC1 activity .", "Thus , both the HRI–eIF2αP and the mTORC1 pathways contribute to the increased mitochondrial protein synthesis in Hri–/– –Fe EBs .", "Increased protein synthesis in Hri–/– –Fe EBs requires additional protein folding capacity for heme-free globins and increased import into mitochondria of mitochondrial protein precursors synthesized in the cytoplasm .", "Indeed , Hri–/– –Fe EBs develop a prominent cytoplasmic UPR , as suggested here by the increased expression of cytoplasmic chaperones in these cells .", "These results are consistent with and help to identify the mechanisms underlying our previous reports of insoluble inclusions and protein precipitates accompanied by increased levels of Hsp70 and Hsp90 in erythroid cells of Hri–/– and eAA mice in ID ( Han et al . , 2001; Zhang et al . , 2018 ) .", "Nevertheless , activation of cytoplasmic UPR alone is not sufficient to compensate for the loss of HRI .", "Translational regulation of mitochondrial biogenesis by mTORC1 is particularly important for erythropoiesis because of the high demand for heme for hemoglobin production and the prevention of oxidative stress ( Liu et al . , 2017 ) .", "Most mitochondrial proteins are encoded by nuclear genes and synthesized by cytosolic ribosomes as precursors that are imported into mitochondria .", "Mitochondrial DNA encodes only 13 proteins , which are all components of the respiratory chain and oxidative phosphorylation ( Calvo and Mootha , 2010 ) .", "Therefore , the nuclear and mitochondrial synthesis of proteins that are involved in respiratory complexes needs to be coordinated to avoid excessive uncomplexed subunits ( Priesnitz and Becker , 2018 ) .", "In mitochondrial disorders , mitochondrial UPR ( UPRmt ) is activated to coordinate with nuclear transcription in order to enable restoration of mitochondrial function ( Shpilka and Haynes , 2018 ) .", "Interestingly , ISR is also activated in mitochondrial dysfunction and mediates UPRmt ( Fiorese et al . , 2016; Quirós et al . , 2017; Dogan et al . , 2014 ) .", "However , the eIF2α kinase responsible for UPRmt remains unknown .", "Our observations that Hri–/– erythroid cells have impaired mitochondrial function and no significant increase in the expression of mitochondrial chaperones suggest that HRI may be the eIF2α kinase responsible for the activation of UPRmt and might thereby contribute to the proper maintenance of the mitochondrial function of erythroid cells under ID .", "While inhibition of protein synthesis by the activation of HRI is the first line of defense in reducing proteotoxicity in ID , the second arm of the HRI-ISR , inducing ATF4 target gene expression as a consequence of enhanced translation of Atf4 mRNA , is the most highly activated transcriptome pathway in ID .", "These ATF4 target genes are largely unstudied factors with yet unknown functions in erythropoiesis .", "They are involved in redox and amino acid metabolism , particularly one-carbon metabolism .", "Interestingly , these ATF4-ISR genes are also upregulated under mitochondrial stress ( Quirós et al . , 2017; Bao et al . , 2016 ) in order to maintain mitochondrial redox homeostasis .", "The deficiency of induction of ATF4-ISR gene expression is likely to be responsible for elevated oxidative stress in erythroid cells from iron-deficient Hri–/– , eAA and Atf4–/– mice ( Suragani et al . , 2012; Zhang et al . , 2018 ) , as well as the mitochondrial dysfunction reported here .", "Hyporesponsiveness to Epo therapy is commonly encountered in ID ( Goodnough , 2007 ) .", "Our recent studies provide evidence that HRI-ISR constitutes one feedback mechanism of erythroid homeostasis in ID ( Zhang et al . , 2018 ) .", "We show here that Grb10 is probably one of the ATF4 target genes involved in inhibiting Epo–mTORC1 signaling in ID .", "The increased proliferation of differentiating erythroblasts upon knockdown of Grb10 is consistent with the growth suppressor function of Grb10 ( Plasschaert and Bartolomei , 2015 ) .", "In hematopoiesis , Grb10 regulates the self-renewal and regeneration of hematopoietic stem cells ( HSCs ) .", "Grb10-deficient HSCs exhibit increased proliferation that is dependent on the SCF-AKT/mTORC1 pathway ( Yan et al . , 2016 ) , consistent with our Grb10 knockdown results .", "Grb10 has also been shown to be a GATA2 target gene and to play a role in the transition from BFU-E to CFU-E ( Mehta et al . , 2017 ) .", "Our results support an additional role of ATF4-induced Grb10 expression in terminal erythropoiesis in which GATA2 expression is low .", "Notably , Atf4 mRNA is most highly expressed in Ter119+CD71+ erythroblasts among the 16 differentiation stages of murine bone marrow hematopoietic cells ( Figure 8—figure supplement 1 ) ( Lara-Astiaso et al . , 2014 ) .", "Altogether , our findings underscore the role of HRI-mediated translation of Atf4 mRNA in terminal erythroid differentiation .", "In summary , our genome-wide study reveals the prominent and coordinated contribution of heme- and HRI-mediated translational regulation in maintaining protein homeostasis , mitochondrial activity and erythroid differentiation during iron-restricted erythropoiesis ." ], [ "Mice were maintained at the Massachusetts Institute of Technology ( MIT ) animal facility , and all experiments were carried out using protocols approved by the Division of Comparative Medicine , MIT .", "Hri–/– , Atf4–/– and universal eIF2α Ala51/+ heterozygote knockin mice were as described previously ( Han et al . , 2001; Masuoka and Townes , 2002; Scheuner et al . , 2001 ) .", "Diet-induced iron deficiency in mice was achieved as previously described ( Han et al . , 2001; Zhang et al . , 2018 ) .", "Iron-sufficient and -deficient mice ( 8–12 weeks old ) were mated for E13 . 5 or E14 . 5 fetal livers as sources of erythroid precursors .", "Under these iron-deficient conditions , embryos were pale and anemic with decreased hematocrits in embryonic blood ( Liu et al . , 2008b ) .", "EBs from E14 . 5 FLs of Wt and Hri–/– mice maintained under +Fe or –Fe conditions were sorted using anti-Ter119 and anti-CD71 antibodies by flow cytometry using FACS Aria ( BD Biosciences , San Jose , CA ) ( Figure 1A ) .", "In order to have sufficient EBs for Ribo-seq library , FLs from embryos of the same mother were pooled then sorted for EBs as one biological replica .", "Two ( 5 million cells each ) and three biological replicas ( 1 million cells each ) for each condition from separate mothers were collected for preparation of Ribo-seq and mRNA-seq libraries , respectively .", "The third replica of Ribo-seq using 3 million cells was unsuccessful , probably because of lower cell numbers .", "All procedures for labeling and washing of cells for sorting were carried out at 4°C .", "Cells were sorted into tubes with 20% fetal bovine serum ( FBS , Atlanta Biologicals , Norcross , GA ) to help to preserve cell integrity .", "To preserve polyribosomes , sorted EBs were washed twice with cold phosphate-buffered saline ( PBS ) and then treated with cycloheximde ( 100 μg/mL ) at 37°C for 5 min , followed by isolation of RPFs as previously described ( Guo et al . , 2010; Ingolia et al . , 2011 ) using the ARTseq-Ribosome Profiling Kit ( Illumina , San Diego , CA ) .", "Briefly , cells were lysed in cold polysome buffer on ice for 10 min .", "To generate RPFs , lysates were digested with Rnase 1 .", "Monosomes were then purified by S-400 gel filtration spin columns ( GE Healthcare , Chicago , IL ) .", "RNAs in monosomes were extracted and purified .", "Size selection of 28–34 nucleotide RPFs was carried out using denaturing polyacrylamide gel electrophoresis .", "Ribosomal RNA contaminants in the RPF preparations were removed using an Ribo-Zero rRNA Removal Kit ( Illumina , San Diego , CA ) .", "RPFs were then purified by 15% urea-polyacrylamide gel electrophoresis .", "Libraries of RPFs were prepared as described previously ( Ingolia et al . , 2009; Ingolia et al . , 2011 ) for Illumina next generation DNA sequencing .", "RPFs were dephosphorylated and linkers were ligated using T4 RNA ligase .", "RNA samples were then reverse transcribed , circularized and PCR amplified for 12 cycles .", "PCR products were subjected to gel purification before sequencing .", "The results of Ribo-seq presented in Figure 2 were from the second set of experiments .", "For the first set of Ribo-seq experiments , EBs were treated with harringtonine ( 2 μg/ml ) at 37°C for 2 min followed by cycloheximide treatment as described above .", "However , harringtonine treatment at this condition did not work well for EBs and consequently ribosome occupancy was observed throughout many mRNAs .", "For the second set of Ribo-seq experiments , harringtonine was not used .", "Nonetheless , increased translation of Atf4 , Pppr115a and Ddit3 mRNAs was also observed in both sets of Ribo-seq .", "Total RNAs were extracted using an RNeasy Plus kit ( Qiagen , Germantown , MD ) and polyA+ mRNAs were isolated using an Oligotex mRNA kit ( Qiagen , Germantown , MD ) .", "mRNA-seq cDNA libraries were prepared by the MIT BioMicro Center .", "cDNA libraries of RPFs and mRNA were sequenced on an HiSeq 2000 platform ( Illumina , San Diego , CA ) at the MIT BioMicro Center .", "After standard preprocessing and quality control analysis , reads were mapped to mouse genome mm10 ( UCSC ) , beforedownstream analyses were performed .", "Raw data ( fastq files ) were trimmed by Cutadapt to remove adapters and reads with a base quality score of less than 10 .", "Reads with a length of less than 26 or 9 nucleotides were also discarded for Ribo-seq and mRNA-seq samples , respectively .", "For Ribo-seq samples , reads containing rRNA and tRNA sequences were further removed using Bowtie2 .", "After quality-control analysis using FastQC , reads were mapped to mouse genome mm10 ( UCSC ) using STAR aligner with maxima of two mismatches and eight multiple loci .", "The quality of the Ribo-seq data was examined by the triplet periodicity using RibORF ( Ji et al . , 2015 ) .", "For the visualization of ribosome occupancies , all mapped reads ( bam files ) that overlap each bin ( bin size = 1 ) were first calculated and normalized to effective mouse genome size to get a 1x depth of coverage ( RPGC ) using bamCoverage of deepTools ( Reimand et al . , 2016; Ji et al . , 2015 ) .", "Then , ribosome occupancies were visualized using the Integrative Genomics Viewer ( Broad Institute of MIT and Harvard ) .", "As there was no significant change in mRNA levels among four conditions , only mapped reads of Wt + Fe EBs are shown for mRNA-seq data in Figure 2C–D , Figure 2—figure supplement 1B–C and Figure 3—figure supplement 1B–C .", "The uniquely mapped reads were counted using HTseq for gene coverage analysis , transcript per million ( TPM ) calculation , translational efficiency ( TE ) calculation and analysis of differentially expressed mRNAs ( DEG ) .", "Genes with greater than 25 uniquely mapped reads in at least one of the conditions were used for gene coverage analysis and TPM calculation .", "Generally , RPFs are piled up around the start codons and stop codons because of the slower kinetics of translation initiation and termination .", "The use of cycloheximide to freeze polysomes during the preparation of RPFs also enhances the pile up of reads near start codons .", "We therefore removed the first 15 and the last 5 codons from reads counting for the TE calculation of Ribo-seq data , in which TE is defined as the ratio of RPF counts to mRNA counts .", "TE was calculated using the xtail package of R/Bioconductor , with parameter ‘bins = 10000’ , in order to identify genes that are differentially translated between different conditions ( Xiao et al . , 2016 ) .", "Genes with reads greater than 50 in at least one of the conditions were included in each comparison and were used for TE calculation ( Supplementary file 1b ) .", "Genes with adjusted p-value<0 . 05 together with log2 ( foldchange of TE ) >1 . 5 or <−1 . 5 were considered to be significantly differentially translated ( Figure 2A ) , whereas those with adjusted p-value<0 . 05 together with log2 ( foldchange of TE ) >1 or < −1 were considered to be significantly differentially translated ( Figure 2B , Figure 3A , Figure 2—figure supplement 1A and Figure 3—figure supplement 1A ) .", "For Gene Set Enrichment Analysis ( GSEA ) , pre-ranked gene lists were obtained using the formula –Log10 ( adjusted p-value ) × Log2 ( foldchange of TE ) .", "Mouse gene symbols were converted into human symbols using the biomaRt package of R/Bioconductor .", "GSEA was conducted using the GSEA tool from the Broad Institute with the c2 . all . v6 . 0 . symbols . gmt database , which includes 4738 gene sets .", "Gene sets shown in Figure 3B–C were REACTOME_FORMATION_OF_THE_TERNARY_COMPLEX_AND_SUBSEQUENTLY_THE_43S_COMPL , KEGG_RIBOSOME , WONG_MITOCHONDRIA_GENE_MODULE , KEGG_OXIDATIVE_PHOSPHORYLATION and BILANGES_SERUM_AND_RAPAMYCIN_SENSITIVE_GENES .", "Differentially expressed genes from mRNA-seq data were determined using the DESeq2 package of R/Bioconductor , and genes with the mean of reads for all conditions greater than 100 were used for further analysis ( Supplementary file 1e ) .", "Genes with adjusted p-value<0 . 05 were considered as significantly differentially ( Figures 5–6 ) .", "Lists of 5′ TOP/TOP-like genes were obtained from Thoreen et al . ( 2012 ) , whereas the integrated stress response ( ISR ) gene list was derived from Palam et al . ( 2015 ) .", "Gene ontology analysis was performed using g:Profiler for the TE and DEG datasets ( Reimand et al . , 2016 ) .", "The START App was employed to generate the volcano plots , in which red dots indicate significantly differentially translated or expressed genes , while green and gray dots indicate non-significantly changed genes ( Nelson et al . , 2017; Reimand et al . , 2016 ) .", "Heatmaps were plotted using GENE-E tool from the Broad Institute .", "All Ribo-seq data and mRNA-seq described in this paper are available at the Gene Expression Ominibus ( http://www . ncbi . nlm . nih . gov/geo/ ) under accession GSE119365 .", "Gene expression was performed by RT-qPCR and western blot analyses as previously described ( Zhang et al . , 2018 ) .", "Primers are listed in Supplementary file 3a .", "Antibodies used in western blots are described in Supplementary file 3b .", "Gapdh was used as the internal control for RT-qPCR .", "β-actin or eIF2α was used as a loading control for western blots .", "The incorporation of puromycin into nascent polypeptide chains was used as a measure of protein synthesis as described previously ( Schmidt et al . , 2009; Zhang et al . , 2018 ) .", "Briefly , erythroid cells from bone marrow ( BM ) samples were isolated as previously described ( Zhang et al . , 2018 ) .", "Isolated cells were further subjected to dead-cell removal using the MACS Dead Cell Removal Kit according to the manufacturer's protocol ( Miltenyi Biotech ) .", "Then , cells were first treated with DMSO or cycloheximide at 100 μg/mL ( Sigma-Aldrich , St . Louis , MO ) , with chloramphenicol at 100 μM ( Sigma-Aldrich ) or with INK128 at 0 . 2 μM ( LC Laboratories , Woburn , MA ) for 15 min at 37°C , followed by the addition of puromycin ( Sigma-Aldrich , St . Louis , MO ) at 5 μg/mL .", "Cells were collected after 15 , 30 , 45 or 60 min for western blot analysis using anti-puromycin antibody to measure protein synthesis .", "Oxygen consumption rate ( OCR ) was measured on a Seahorse XFe96 Analyzer with a Seahorse XFe96 FluxPak ( Agilent , Santa Clara , CA ) .", "Erythroid cells from BM of mice were isolated as previously described ( Zhang et al . , 2018 ) .", "After recovery in IMDM with 10% FBS and 3 U/mL Epo at 37°C for 1 hr , 300 , 000 cells/well were seeded onto an XF96 cell culture microplate coated with Cell-tak ( Corning , Corning , NY ) and subjected to seahorse assay according to the protocol of the manufacturer .", "Three OCR measurements — basal , ATP-linked and maximal respiration — were performed after sequential injections of 1 µM oligomycin , 1 µM FCCP , and 0 . 5 µM Antimycin A/Rotenone .", "Five technical replicas from the same sample were performed for OCR measurement .", "Mitochondrial mass was determined by Mitotracker through flow cytometry analysis .", "The mitochondrial DNA ( mtDNA ) content was measured by qPCR as previously described ( Liu et al . , 2017 ) .", "Briefly , after DNA isolation from the purified erythroid cells using a DNeasy Blood and Tissue Kit ( Qiagen , Germantown ) , qPCR with specific primers for mtDNA and genomic DNA ( gDNA ) ( Supplementary file 3a ) was performed to measure the ratio of mtDNA versus gDNA using the ΔΔCt method .", "Erythroid progenitors from E13 . 5 FLs of Wt + Fe embryos were enriched by magnetic sorting using EasySep Magnet ( StemCell Technologies , Vancouver , Canada ) as described ( Thom et al . , 2014 ) ( Figure 7—figure supplement 2 ) .", "In brief , total FL cells were mechanically dissociated by pipetting in PBS containing 2% FBS ( Atlanta Biologicals , Norcross , GA ) , 2 . 5 mM EDTA and 10 mM glucose .", "Cells were labeled with biotin-conjugated anti-B220 , anti-CD3 , anti-CD11b , anti-CD11c , anti-GR1 , anti-Ter119 ( all BioLegend , San Diego , CA ) and anti-CD41 ( eBioscience , San Diego , CA ) antibodies .", "Lineage negative ( Lin–Ter119– ) cells were subjected to a second purification step to obtain Lin–Ter119–CD71– erythroid progenitors using biotin-conjugated anti-CD71 antibodies ( BioLegend , San Diego , CA and BD Biosciences , San Jose , CA ) .", "Purified Lin–Ter119–CD71– erythroid progenitors were cultured in the expansion medi described by Thom et al . ( 2014 ) for three days at an initial cell density of 0 . 5 × 106 cells per mL .", "This expansion medium was StemPro-34 medium complemented with 10% supplement , 2 mM L-glutamine , 1% penicillin-streptomycin ( P-S ) , 10−4 M β-mercaptoethanol , 10−6 M dexamethasone , 0 . 5 U/mL Epo , and 100 ng/mL mouse stem cell factor ( mSCF ) .", "Then , cells were washed and cultured in differentiation medium at an initial cell density of 1 × 106 cells per mL .", "The differentiation medium was Iscove-modified Dulbecco medium ( IMDM ) containing 10% FCS ( fetal calf serum , Gemini Bio-Products , West Sacramento , CA ) , 10% plasma-derived serum ( Animal Technologies , Tyler , TX ) , 2 mM L-glutamine , 1% P-S , 10−4 M β-mercaptoethanol , and 5 U/mL Epo .", "Cells were collected for analysis at different time points as indicated in Figure 7A .", "The knockdown of Grb10 expression was performed by using recombinant retroviruses containing an shRNA-expressing murine stem cell retroviral vector , MSCV-pgkGFP-U3-U6P-Bbs , a kind gift from the laboratory of Dr . Harvey F Lodish ( MIT ) .", "DNA sequences of eight shRNA oligonucleotides ( Supplementary file 3c ) targeting different regions of Grb10 mRNA were either obtained from the Genetic Perturbation Platform of the Broad Institute or as previously reported ( Doiron et al . , 2012; Zacharek et al . , 2011; Park et al . , 2013 ) and were synthesized by Integrated DNA Technologies .", "Preparations of the plasmid constructs and recombinant retroviruses were performed as described previously ( Hu et al . , 2014 ) .", "Lin–Ter119–CD71– erythroid progenitors enriched from Wt + Fe E13 . 5 FLs were infected with retroviruses as described previously ( Thom et al . , 2014 ) .", "Cells were expanded for 72 hr after retroviral infections followed by differentiation for up to 42 hr as indicated .", "Erythroid differentiation was performed by flow cytometry using anti-Ter119 and anti-CD71 antibodies ( BioLegend , San Diego , CA ) as described previously ( Zhang et al . , 2018 ) on FACS LSR II ( BD Biosciences , San Jose , CA ) .", "4′ , 6-diamidino-2-phenylindole ( DAPI , Roche Diagnostics , Basel , Switzerland ) was used to exclude the dead cells .", "Data were analyzed with FlowJo ( Tree Star , Ashland , OR ) .", "Erythroid differentiation was also analyzed by cell morphology on cytospin slides stained with May-Grunwald/Giemsa staining ( Sigma-Aldrich , St . Louis , MO ) .", "Cell proliferation was determination by counting nucleated cells daily using crystal violet stain .", "The independent t test ( two-tailed ) was used to analyze the experimental data .", "Pearson correlation analysis was performed to determine the correlation coefficient .", "Data are presented as means ± SEs .", "*p<0 . 05 was considered statistically significant .", "**p<0 . 01; ***p<0 . 001 ." ] ]

[ "Iron and heme play central roles in the production of red blood cells , but the underlying mechanisms remain incompletely understood .", "Heme-regulated eIF2α kinase ( HRI ) controls translation by phosphorylating eIF2α .", "Here , we investigate the global impact of iron , heme , and HRI on protein translation in vivo in murine primary erythroblasts using ribosome profiling .", "We validate the known role of HRI-mediated translational stimulation of integratedstressresponse mRNAs during iron deficiency in vivo .", "Moreover , we find that the translation of mRNAs encoding cytosolic and mitochondrial ribosomal proteins is substantially repressed by HRI during iron deficiency , causing a decrease in cytosolic and mitochondrial protein synthesis .", "The absence of HRI during iron deficiency elicits a prominent cytoplasmic unfolded protein response and impairs mitochondrial respiration .", "Importantly , ATF4 target genes are activated during iron deficiency to maintain mitochondrial function and to enable erythroid differentiation .", "We further identify GRB10 as a previously unappreciated regulator of terminal erythropoiesis ." ]

[ "Red blood cells use a molecule called hemoglobin to transport oxygen around the body .", "To make hemoglobin , cells require iron to build a component called heme .", "If an individual does not get enough iron in their diet , the body cannot produce enough red blood cells , or the cells lack hemoglobin .", "This condition is known as iron deficiency anemia , and it affects around one-third of the world’s population .", "Researchers did not know exactly how iron levels control red blood cell production , though several proteins had been identified to play important roles .", "Heme forms in the cell's mitochondria: the compartments in the cell that supply it with energy .", "When heme levels in a developing red blood cell are low , a protein called HRI reduces the production of many proteins , most importantly the proteins that make up hemoglobin .", "HRI also boosts the production of a protein called ATF4 , which switches on a set of genes that help both the cell and its mitochondria to adapt to the lack of heme .", "In turn , HRI and ATF4 reduce the activity of a signaling pathway called mTORC1 , which controls the production of proteins that help cells to grow and divide .", "To understand in more detail how iron and heme regulate the production of new red blood cells , Zhang et al . looked at immature red blood cells from the livers of developing mice .", "Some of the mice lacked the gene that produces HRI , and some experienced iron deficiency .", "Comparing gene activity in the different mice revealed that in the developing blood cells of iron-deficient mice , HRI largely reduces the rate of protein production in both the mitochondria and the wider cell .", "At the same time , the increased activity of ATF4 allows the mitochondria to carry on releasing energy and the cells to continue developing .", "Zhang et al . also found that a protein that inhibits the mTORC1 signaling pathway needs to be active for the new red blood cells to mature .", "Overall , the results suggest that drugs that activate HRI or block the activity of the mTORC1 pathway could help to treat anemia .", "The next step is to test the effects that such drugs have in anemic mice and cells from anemic people ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "cell biology", "structural biology and molecular biophysics" ]

[ [ "Programmed cell death occurs during tissue development and homeostasis to remove superfluous cells or alternatively , during disease states to remove damaged or rapidly dividing cells ( Song and Stellar , 1999; Salvesen and Dixit , 1997; Green and Evan , 2002; Danial and Korsmeyer , 2004; Thompson , 1995 ) .", "In general , this process allows cellular components to be recycled without activating inflammatory pathways ( Green and Evan , 2002; Inohara and Nuñez , 2003 ) .", "In humans , intrinsic cell death signals converge on mitochondria to release pro-apoptotic factors through outer membrane pores formed by Bcl-2 family members ( Brunelle and Letai , 2009; Tait and Green , 2010 ) .", "These factors include cytochrome c , which up-regulates assembly of the Apoptotic protease activation factor-1 ( Apaf-1 ) to form the apoptosome ( Li et al . , 1997 ) .", "Other released factors , such as Smac/DIABLO and Omi/HtrA2 ( reviewed in Tait and Green , 2010 ) , are pro-apoptotic and interact with inhibitory proteins to unblock procaspases –9 , –3 and –7 .", "This multi-pronged pathway allows activation of procaspase-9 on the apoptosome and subsequent proteolytic activation of procaspase-3 and –7 , which results in selective proteolysis of target proteins and cell death ( Bratton and Salvesen , 2010 ) .", "Apaf-1 is a member of the AAA+ ATPase family , has seven domains and exists as an inactive monomer in the cytoplasm of healthy cells ( Danot et al . , 2009; Leipe et al . , 2004 ) .", "When released , cytochrome c binds to β-propellers in Apaf-1 and may trigger nucleotide exchange with ATP/dATP replacing bound ADP/dADP ( Liu et al . , 1996; Li et al . , 1997; Zou et al . , 1997 , 1999; Hu et al . , 1998 , 1999 ) .", "This leads to the assembly of a heptameric apoptosome ( Acehan et al . , 2002 ) .", "Caspase recognition domains ( CARDs ) are present as N-terminal effector domains in both Apaf-1 and procaspase-9 ( pc-9 ) .", "Thus , CARD-CARD interactions target inactive pc-9 monomers to the apoptosome ( Yuan et al . , 2011a; Hu et al . , 2014 ) .", "During this step , a CARD 'disk' is formed that sits above the central hub , and is attached by linkers to the nucleotide binding domain ( NBD ) ( Yuan et al . , 2010 , 2011a , 2013 ) .", "The nature of the disk is not known but a recent hypothetical model suggests that two CARD-CARD interfaces may be used to form a quasi-helical stack , akin to structures in Death Domain complexes ( Hu et al . , 2014 ) .", "Local proximity of procaspase-9 monomers on the apoptosome leads to protease activation .", "However , the precise mechanism of pc-9 activation after recruitment to the apoptosome is still being debated .", "Catalytic domains may associate in solution or they may interact with the apoptosome to form an active protease ( Yuan et al . , 2011a , Bratton and Salvesen , 2010; Boatright et al . , 2003; Hu et al . , 2014; Würstle and Rehm , 2014 ) .", "We now present a near atomic structure of the active human apoptosome determined with cryo-EM at a global resolution of ~4 . 1 Å .", "This work provides a model of Apaf-1 in an extended conformation with bound dATP and shows monomer interactions within the apoptosome .", "Improved clarity reveals interactions of cytochrome c with β-propellers in the V-shaped sensor domain .", "For the first time , molecular details are provided of a tilted , acentric disk formed by N-terminal CARDs from four Apaf-1 and three or four pc-9 molecules .", "In brief , the disk contains four pairs of Apaf-1/pc-9 CARDs in one turn of a spiral with the fourth pc-9 CARD being present at lower occupancy .", "This arrangement creates a mismatch between the CARD spiral in the disk and the c7 symmetry of the platform .", "We also verified that a pc-9 catalytic domain may be parked on the central hub ( Yuan et al . , 2011a ) , adjacent to a pc-9 CARD in the disk .", "In addition , three Apaf-1 CARDs are excluded from the disk but may retain a limited ability to bind pc-9 molecules , depending upon steric constraints .", "Stoichiometry measurements have suggested that 2 to 5 pc-9 molecules may be recruited by Apaf-1 CARDs on the apoptosome ( Malladi et al . , 2009; Yuan et al . , 2011a; Hu et al . , 2014 ) .", "Hence , at any instant one or two pc-9 dimers may be tethered to the apoptosome .", "Implications for Apaf-1 assembly and apoptosome function are discussed ." ], [ "We assembled an active human apoptosome from Apaf-1 and a two chain pc-9 with cytochrome c and dATP in Buffer A . Procaspase-9 apoptosomes were run on a linear glycerol gradient and visualized by SDS-PAGE to reveal the expected components ( Figure 1—figure supplement 1A ) .", "We also used a fluorescence assay to monitor the LEHDase activity of pc-9 apoptosomes used in this work ( Figure 1—figure supplement 1B ) .", "For single particle work , we froze freshly assembled samples on holey carbon grids and collected super-resolution movies on a Titan-Krios electron cryo-microscope equipped with an energy filter to remove inelastically scattered electrons and provide better contrast for alignments ( Materials and methods ) .", "After movie frame corrections for global movements during imaging ( Li et al . , 2013 ) , ~135 , 000 particles were extracted from the resulting micrographs and processed by 2D and 3D classification in RELION ( Scheres , 2012 ) to identify the best particles ( ~93 , 000 ) .", "These particles were 'polished' and refined with seven-fold symmetry ( c7 ) in RELION to produce a map with a global resolution of 4 . 1 Å , as determined with a gold standard FSC ( Figure 1—figure supplement 1C; Scheres , 2014 , 2012 ) .", "However , the resolution is not isotropic .", "As estimated by ResMap ( Kucukelbir et al . , 2014 ) the central hub is at a nominal resolution of 3–4 Å and this is supported by the visibility of larger side chains .", "The extended arm and peripheral sensor domains are in the range of 4 . 5–10 Å resolution ( Figure 1—figure supplement 1E ) .", "A sharpened and normalized 3D map from the global 3D refinement was rendered as an iso-surface to show the wheel-like geometry of the apoptosome , which has dimensions of 270 x 270 x ~75 Å .", "Many rod-like α-helices are visible in the map that dominate the central hub and seven arms , while a cylindrical CARD disk sits atop the platform and shows little structural detail ( Figure 1A and inset ) .", "A previous model of an extended Apaf-1 ( Yuan et al . , 2013; PDB 3J2T ) was docked into the map with some rebuilding ( Materials and methods ) and pertinent statistics are summarized in Figure 1—source data 1 . 10 . 7554/eLife . 17755 . 003Figure 1 . Overview of 3D density maps for the active apoptosome .", "( A ) A top view of the global 3D density map is shown with the platform in blue and the blurred CARD disk in magenta .", "Inset: The CARD disk was low pass filtered to ~14 Å resolution and is shown from the side .", "( B ) Top view of the composite 3D density map .", "The sensor domain ( with β-propellers ( β7 , β8 ) and cytochrome c ) and the CARD disk are from maps obtained with focused 3D classification at ~6 Å resolution .", "The strong tilt of the acentric disk is shown in the inset .", "( C , D )", "Bottom and top views of the platform .", "See also Figure 1 — table and figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 17755 . 00310 . 7554/eLife . 17755 . 004Figure 1—source data 1 . Data collection and refinement statistics . Model statistics in the table were derived from an analysis with Molprobity ( Davis et al . , 2007 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 17755 . 00410 . 7554/eLife . 17755 . 005Figure 1—figure supplement 1 . Sample and structure characterization for the active apoptosome .", "( A ) SDS PAGE of peak fractions ( 6–8 ) that contain the pc-9 apoptosome from a glycerol gradient .", "A two-chain pc-9 was used in the experiments .", "( B ) Histogram of LEHD protease activity for the pc-9 apoptosome ( green bar ) relative to a pc-9 control ( magenta bar ) .", "Measurements were done in triplicate .", "( C ) Gold standard FSC curves were calculated from half-data sets for the full apoptosome , the unblurred CARD disk and the sensor domain maps , as indicated .", "( D ) Model versus map FSC curves are shown for the central hub with HD2 arms , the unblurred CARD disk and the sensor region with cytochrome c .", "( E ) Local resolution was estimated with Resmap ( Kucukelbir et al . , 2014; see color scale ) and is displayed as a graduated color scale on the entire platform and thick slices from the global map , as viewed along the seven-fold axis .", "The thick slices are shown at a reduced scale . DOI: http://dx . doi . org/10 . 7554/eLife . 17755 . 005 While this work was being completed , a near atomic structure was published of the ground state human apoptosome ( Zhou et al . , 2015; PDB 3JBT ) , with improved resolution for the V-shaped sensor domain formed by tandem 7- and 8-blade β-propellers .", "This prompted us to re-analyze this region in our map of the active apoptosome with focused 3D classification in RELION , coupled with local remodeling ( Scheres , 2012; Zhou et al . , 2015; Materials and methods ) .", "To complete our analysis , we used focused 3D classification to reveal the architecture of the acentric CARD disk at ~5 . 8 Å ( Materials and methods ) .", "A model for the disk was constructed by rigid-body docking of individual Apaf-1 and pc-9 CARDs from a crystal structure ( Hu et al . , 2014; PDB 4RHW ) into the improved 3D map with Chimera ( Pettersen et al . , 2004 ) .", "A composite 3D map for the active apoptosome was constructed by zoning with appropriate domain models in global and local maps with Chimera , and these density maps were combined for display ( Figure 1B–D ) .", "Gold standard FSC curves were computed from independent half volumes using the entire global map , and for local maps containing the sensor domain with cytochrome c and the unblurred CARD disk .", "This provided estimated resolutions of ~4 . 1 , 6 . 1 and 5 . 8 Å , respectively .", "Model versus map FSC curves suggest that over-fitting is not a major issue .", "( Figure 1—figure supplement 1C , D ) .", "An over-view of the model for the heptameric platform is shown with color-coded domains that include the CARD , NBD , helix domain 1 ( HD1 ) , winged helix domain ( WHD ) , helix domain 2 ( HD2 ) and β-propellers ( Figure 2A , C; top views with and without the map ) .", "In addition , the disk and a single Apaf-1 molecule are highlighted in the active apoptosome without the map ( Figure 2B ) , and a close-up is shown of the disk within the map ( Figure 2D ) .", "Subunit interfaces , the quality of individual Apaf-1 domains and domain boundaries , cytochrome c and the pc-9 CARD are shown in Figure 2—figure supplements 1–3 , with color coding that is used throughout this work .", "We also created a sequence alignment in Chimera ( Pettersen et al . , 2004 ) for Apaf-1 , Dark and CED-4 apoptosomes ( Figure 2—figure supplement 4 ) .", "This alignment provides detailed information on secondary structure motifs and is a useful guide when discussing this large structure .", "The heptameric platform appears to be mostly unchanged during pc-9 activation relative to the ground state , with an rmsd of 1 . 7 Å for Cα's , when comparing Apaf-1 and cytochrome c in the two complexes ( Zhou et al . , 2015; this work ) .", "However , the active apoptosome contains an acentric disk formed by Apaf-1 and pc-9 CARDs , with a catalytic domain of pc-9 bound to the central hub in some particles ( as discussed later; Yuan et al . , 2010 , 2011a , 2013 ) .", "Importantly , structures are now available for apoptosomes from C . elegans ( CED-4; Qi et al . , 2010 ) , D . melanogaster ( Dark; Pang et al . , 2015; Yuan et al . , 2011b; Cheng et al . , unpublished ) and H . sapiens ( Apaf-1; Zhou et al . , 2015; this work ) , which together provide a wealth of structural data to interpret function . 10 . 7554/eLife . 17755 . 006Figure 2 . Model of the active apoptosome .", "( A , C )", "Top views are shown of the heptameric platform from the apoptosome model , with and without the composite 3D map .", "Apaf-1 domains are highlighted in color ( see color key ) .", "( B , D )", "A top view is shown of the complete model of the active apoptosome without the 3D map .", "In addition , a close-up of the acentric disk and part of the NBD ring is shown within the composite map ( panel D ) .", "Domains in a single Apaf-1 and the acentric CARD disk are color-coded , relative to the remaining Apaf-1 subunits , which are shown in grey .", "See also Figure 2—figure supplements 1–4 . DOI: http://dx . doi . org/10 . 7554/eLife . 17755 . 00610 . 7554/eLife . 17755 . 007Figure 2—figure supplement 1 . An Apaf-1 subunit with a bound pc-9 CARD within the active apoptosome .", "( A–C )", "Top , tilted and bottom views are shown of a color-coded Apaf-1 subunit along with the associated Apaf-1/pc-9 CARD pair , docked within the composite density map .", "The pc-9 CARD is shown in purple and the Apaf-1 CARD is at position 1a in the disk .", "The remaining CARDs are colored dark grey . DOI: http://dx . doi . org/10 . 7554/eLife . 17755 . 00710 . 7554/eLife . 17755 . 008Figure 2—figure supplement 2 . Segmented domain maps for the Apaf-1 subunit with a bound pc-9 CARD . Domains and their maps are color-coded and show a clear demarcation of α-helices and β-sheets .", "At this resolution all helices in cytochrome c are visible at a higher threshold ( not shown ) .", "The view direction for the β-propellers is indicated by colored arrow heads . DOI: http://dx . doi . org/10 . 7554/eLife . 17755 . 00810 . 7554/eLife . 17755 . 009Figure 2—figure supplement 3 . Representative electron density is shown for the four domains of the central hub and HD2 arm . At the nominal resolution of 3–4 Å , side chains are visible throughout the map for the platform region , although the quality falls off in the HD2 arm , due to local motions and effects of alignment errors at higher radius . DOI: http://dx . doi . org/10 . 7554/eLife . 17755 . 00910 . 7554/eLife . 17755 . 010Figure 2—figure supplement 4 . Sequence alignment for Apaf-1 , Dark and CED-4 subunits from their respective apoptosomes , prepared in Chimera ( Pettersen et al . , 2004 ) .", "The truncated HD2 in CED-4 has been omitted .", "Note that helix α7 is only present in one subunit of the AB pair in the CED-4 crystal structure and creates a more compact CARD-NBD linker . DOI: http://dx . doi . org/10 . 7554/eLife . 17755 . 010 The central hub of the apoptosome 'wheel' contains seven copies of the NBD , HD1 and WHD in two nested rings ( Figure 3A ) .", "In addition , HD2 forms an arm in each subunit that extends outwards from the hub and ends in a V-shaped sensor domain formed by 7- and 8-blade β-propellers with cytochrome c bound between them ( Figure 2 and Figure 2—figure supplements 1 , 2 ) .", "The central hub is formed by lateral interactions between adjacent NBDs including a ring of paired α-helices that surrounds the central pore ( Figure 3B , C ) .", "This cylindrical picket-fence is formed by helix α12 , the initiator specific motif ( ISM; Danot et al . , 2009 ) , which is connected to helix α13 by a long loop that lines the pore itself .", "The two α-helices in this motif are held together by a hydrophobic interface ( Figure 3D ) , while an extensive network of hydrogen bonds and salt bridges mediate interactions between adjacent α12-loop-α13 motifs ( Figure 3E ) .", "Possible interactions involve: Ser213 , Glu210’ and Gln214’; Arg215 , Glu222 and Thr204’ and Asn219 , Glu221 with Asn201' ( where prime denotes the adjacent subunit; not shown ) .", "The nucleotide dATP is bound in a pocket at the interface between the NBD and HD1 . 10 . 7554/eLife . 17755 . 011Figure 3 . Interactions within the central hub .", "( A ) The central NBD ring and the outer HD1-WHD ring are shown as ribbons in a segmented map of the hub .", "( B , C )", "The α12-α13 helices and linker between them interact with other copies of this motif to form a cylindrical picket fence that lines the central pore ( see pink dashed circle , ISM-ring ) .", "These features are shown with atomic interactions in the map .", "( D , E )", "The α12-loop-α13 motif is stabilized by an extended hydrophobic core and interacts with adjacent motifs in the ring through numerous hydrogen bonds and salt bridges .", "( F ) An overview is shown of the outer HD1-WHD ring .", "( G , H )", "The HD1-WHD loop forms part of the dATP binding pocket and interacts with an extended region that replaces helix α15 in the NBD of an adjacent subunit .", "Possible interactions are indicated by dashed lines . DOI: http://dx . doi . org/10 . 7554/eLife . 17755 . 011 An outer ring in the hub is formed by interactions between an HD1 and the WHD in adjacent subunits to compliment extensive interactions between HD1-WHD pairs in each subunit ( Figure 3F ) .", "There are also interactions between the NBD and the HD1-WHD pair within a single subunit .", "The HD1-WHD loop is involved in dATP binding and interacts with the NBD of an adjacent monomer .", "This surface is formed in part by possible interactions between Ser356 and Ser358 in the HD1-WHD loop to Asp271 and Ser272 in an extended region , which follows strand β4 in the NBD ( Figure 3G , H; Figure 2—figure supplement 4 ) .", "However , some of these interactions are in the 4 . 5–5 Å range and thus , could be mediated in part by water molecules that have not been resolved .", "Overall , the large number of hydrogen bonds and salt bridges between domains and subunits may be responsible for the tendency of the apoptosome to disassemble at higher salt concentration .", "Also note that the WHD-HD2 interface is similar to that observed in crystal structures of an inactive Apaf-1 ( Riedl et al . , 2005; Reubold et al . , 2011 ) .", "This interface stabilizes the extended HD2 arm and side chain densities are visible for many residues in the WHD and HD2 .", "Sensor domains in the apoptosome are formed by 15 WD40 repeats that form tandem 7- and 8-blade β-propellers in the C-terminal half of the molecule ( Yuan et al . , 2011a , 2013; Reubold et al . , 2011 ) .", "This region in the global density map is at lower resolution ( Figure 1 and Figure 1—figure supplement 1 ) .", "The radial fall-off in resolution may be due to rotational alignment errors in this thin and extended particle , coupled with local flexibility .", "However , a focused 3D classification ( Materials and methods; Zhou et al . , 2015 ) allowed us to obtain an improved density map with a resolution of ~6 . 1 Å for this region .", "Structures of human Apaf-1 β-propellers ( Reubold et al . , 2011; Yuan et al . , 2013; 3J2T ) were docked into the density map , along with bovine cytochrome c and a close-up view reveals their interactions ( Figure 4A , B ) . 10 . 7554/eLife . 17755 . 012Figure 4 . The sensor domain and interactions of β-propellers with cytochrome c .", "( A , B )", "An overview of β-propellers , cytochrome c and HD2 arm is shown with and without the density map .", "This view is from the central hub .", "( C ) A reverse view is shown of the sensor domain .", "The linker from HD2 to the 7-blade β-propeller is indicated with black dots .", "( D ) A calculated molecular surface is shown for the sensor domain super-imposed on color coded ribbon models .", "Interfaces between cytochrome c and the β-propellers are marked 1 and 2 .", "( E , F )", "The β-propellers are viewed along their pseudo-symmetry axes from within the V-shaped cleft .", "The β-propellers are overlayed with their counterparts ( in grey ) from the crystal structure of mouse Apaf-1 .", "See also Figure 4—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 17755 . 01210 . 7554/eLife . 17755 . 013Figure 4—figure supplement 1 . Connections between cytochrome c and the β-propellers .", "( A–C )", "Contact regions are apparent at a lower density threshold and are indicated by boxed areas labeled with '1' and '2' respectively , while a smaller contact is marked with an asterisk ( * ) .", "These regions contain lysine residues in cytochrome c that may extend outwards to help form these contacts .", "They include: Lys39 , Lys55 and Lys73 in box 1; Lys72 and Lys79 ( along with Trp884 ) in the smaller contact ( * ) and Lys25 and Lys27 in box 2 .", "( D ) An expanded view of box 2 shows four possible connections that can be modeled by hydrogen bonds and salt bridges between extended side chains , which cross the gap between cytochrome c and the 7-blade β-propeller .", "Higher resolution will be required to verify these interaction pairs . DOI: http://dx . doi . org/10 . 7554/eLife . 17755 . 013 Beta propellers are cylindrically symmetric with each blade formed by a 4-stranded anti-parallel β-sheet ( strands a-d ) .", "The a-strand of each β-sheet is near the central pore while the d-strand is located at the outer surface of the cylinder .", "Individual blades of 7- and 8-blade β-propellers are well resolved in the map ( Figure 2—figure supplement 2 ) .", "Starting at the distal end of the HD2 arm , a cleft is present at the base of the V-shaped sensor domain which accommodates four helices of HD2 ( α29-α32 ) to form an interface reminiscent of a ball and socket joint , as viewed from front and back ( Figure 4B , C and inset ) .", "The socket is formed by loops between blades 6 and 7 ( Ser1169-Gly1178 ) and between blades 7 and 8 in the 8-blade β-propeller , along with the outer surface of blades 7 and 1 in the 7-blade β-propeller .", "In the HD2 arm , helices α30 and α31 and the loop between them are inserted into this interface , while helix α29 makes contact with blade 1 of the 7-blade β-propeller .", "The HD2 arm is connected to the 7-blade β-propeller by an unusual linker ( Yuan et al . , 2010 ) that is present in an Apaf-1 crystal structure ( Reubold et al . , 2011 ) .", "The HD2-β7 linker starts at helix α32 of the HD2 arm ( Figure 4C , traced by black dots ) and runs along the C-terminal blade of the 8-blade β-propeller , where it replaces the outermost d-strand .", "The polypeptide chain then crosses over to the 7-blade β-propeller to form the outer d-strand of the last blade ( Asp589-Thr615 ) and then starts the innermost a-strand of blade 1 .", "The interaction between blades 7 and 1 closes the cylindrical fold of the 7-blade β-propeller .", "Clear density is present for both of the linkers between β-propellers , including a short turn of α-helix in the linker between the 7- and 8-blade β-propellers ( see asterisk , Figure 4C and Figure 2—figure supplement 2 ) .", "Cytochrome c is released from mitochondria to trigger apoptosome assembly from Apaf-1 monomers in the cytoplasm and is located between the two β-propellers ( Yuan et al . , 2010 , 2013; Zhou et al . , 2015 ) .", "We were able to accurately dock cytochrome c into the improved density map ( Figure 2—figure supplement 2 ) .", "All α-helices in cytochrome c are resolved when the map is viewed at a much higher threshold ( not shown ) and the overall fit is similar in ground state and active apoptosomes ( this work , Zhou et al . , 2015 ) .", "Importantly , a rotation of ~90 degrees was required to position cytochrome c , relative to our previous model , due to an ambiguity in the two top docking solutions when fitting with a map at a global resolution of 9 . 5–10 Å ( Yuan et al . , 2013 ) .", "The cytochrome c molecule appears to interact preferentially with the 8-blade β-propeller through an extensive contact region , which is apparent when the map is viewed at a lower threshold , which provides a better estimate of the all atom volume ( boxed region 1 , Figure 4—figure supplement 1A ) .", "Additional contacts with the 7-blade β-propeller are found at the bottom of the V-shaped cleft and on the surface opposite from the 8-blade β-propeller ( see asterisk and box 2 in Figure 4—figure supplement 1B–D ) .", "However , contacts to the 8-blade β-propeller are more extensive and involve numerous van der Waals interactions , while a gap of ~3–5 Å is present over much of the interface between cytochrome c and the 7-blade β-propeller ( Figure 4D , interfaces labeled 1 and 2 , respectively ) .", "Some possible hydrogen bond and salt-bridge interactions are shown in box 2 , which span the gap between cytochrome c and the 7-blade β-propeller ( Figure 4—figure supplement 1D ) .", "In addition , a strong density at the base of the V-shaped cleft may arise from a tryptophan ( Trp844 ) in the 7-blade β-propeller .", "Two lysine residues from cytochrome c ( Lys72 and Lys79 ) are also present in this region .", "In total , 7 lysines from cytochrome c are found in the major contact regions and include: lysines 25 , 27 , 39 , 55 , 72 , 73 and 79 ( also see Yu et al . , 2001 ) .", "However , higher resolution will be needed to pin down the precise nature of the interactions ( also see mutation studies in Zhou et al . , 2015 ) .", "Cytochrome c binding has a direct impact on the structure of the β-propellers , relative to their conformation in a crystal structure of unliganded Apaf-1 ( Reubold et al . , 2011 ) .", "An overlay of 7-blade β-propellers without and with bound cytochrome c shows a symmetric radial displacement with an rmsd of 1 . 9 Å ( Figure 4E ) .", "Extensive interactions of cytochrome c with the 8-blade β-propeller may be responsible in part , for a distortion of blades 6 , 7 , 8 and 1 , relative to the crystal structure , while blades 2–5 overlay quite well ( overall rmsd of 2 . 9 Å . Figure 4F ) .", "This distortion of the 8-blade β-propeller may also arise from the large displacement of the 7-blade β-propeller upon cytochrome c binding , which affects the interface between the two β-propellers , and could reflect altered interactions with helices α29-α32 in HD2 .", "The dATP binding site in Apaf-1 is located at the NBD-HD1 interface and is formed in part by the Walker A loop and the HD1-WHD loop ( Figure 5A ) .", "Side chain resolution in the central hub allowed us to identify molecular interactions as shown by a cross-section from the density map of the dATP binding site ( Figure 5B; for clarity only a few hydrogen bonds are shown ) .", "In particular , the adenine base is bound in a deep hydrophobic pocket lined by NBD residues Pro120 , Pro123 , Phe126 , Val127 , Val162 , Leu163 , Leu286 , Lys290 , Ileu294 along with Pro321 from HD1 ( Figure 5C ) .", "The adenine base is in an anti configuration relative to the deoxyribose ring and makes hydrogen bonds to the backbone carbonyl and amide of Val127 to help position the fused rings ( Figure 5D ) .", "A conserved arginine ( Arg129 ) is located above the adenine base at the 'top' of the pocket and plays a structural role .", "We note that the anti orientation of the adenine base is present in crystal structures of Apaf-1 with bound ADP ( Riedl et al . , 2005; Reubold et al . , 2011 ) and in near atomic structures of Dark and CED-4 apoptosomes ( Cheng et al . , unpublished; Qi et al . , 2010 ) .", "However , the adenine ring in the ground state apoptosome was modeled in a syn conformer with the base oriented above the deoxyribose ring ( Zhou et al . , 2015; PDB 3JBT ) .", "The syn orientation of the adenine base is a higher energy conformer .", "Therefore , we re-fit the Apaf-1 subunit ( PDB 3JBT ) into the published density map for the ground state apoptosome using the anti conformer of dATP without a Mg+2 ion .", "The resulting structure for dATP is in good agreement with the model of an active apoptosome ( rmsd 0 . 54 Å; Figure 5—figure supplement 1 ) . 10 . 7554/eLife . 17755 . 014Figure 5 . dATP binding by Apaf-1 in the apoptosome .", "( A ) The dATP binding pocket lies between the NBD and HD1 and is formed in part by the Walker A loop and HD1-WHD loop .", "dATP is shown in ball and stick representation .", "( B ) dATP is shown within the binding pocket overlayed with the density map .", "A few hydrogen bond and salt bridge indications are indicated by dashed lines .", "The HD1-WHD loop forms the bottom of the dATP binding pocket .", "( C ) The binding pocket for the adenine base is formed mainly by hydrophobic residues , whose side chains are shown in ball and stick representation .", "( D ) Interactions of dATP within the binding pocket are summarized in a schematic ( see text for details ) .", "See also Figure 5—figure supplements 1 , 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 17755 . 01410 . 7554/eLife . 17755 . 015Figure 5—figure supplement 1 . The anti conformer of dATP and the Apaf-1 subunit ( PDB 3JBT; Zhou et al . , 2015 ) were fit into the published apoptosome density map ( EMD-6480 ) without a Mg+2 ion using real-space refinement with Phenix . This produced a well fit structure ( in tan ) that overlays with our model for dATP .", "This suggests that the lower energy anti conformer is correct and supports a possible role for Ser325 as a modulator of nucleotide binding affinity . DOI: http://dx . doi . org/10 . 7554/eLife . 17755 . 01510 . 7554/eLife . 17755 . 016Figure 5—figure supplement 2 . A comparison of nucleotide binding pockets .", "( A ) An overlay is shown for the Apaf-1 monomer with bound ADP ( PDB 3SFZ; in light blue ) , aligned to Apaf-1 with dATP in the active apoptosome .", "An anti conformation is adopted by the adenine base in both structures .", "When ADP is bound Ser325 ( the modulator ) hydrogen bonds with the 2' OH on the ribose ring , whereas this residue interacts with the 3' OH when dATP is present .", "Intriguingly , the role of Tyr359 ( in the HD1-WHD loop ) in recognizing the γ-phosphate of dATP ( and its relative position ) are subsumed by His438 ( in the WHD ) , which interacts with the β-phosphate of ADP in the closed configuration of Apaf-1 .", "( B ) The A-subunit from the crystal structure of the octameric CED-4 apoptosome ( PDB 3LQQ ) is overlayed with the Apaf-1 subunit with bound dATP .", "For clarity only the ATP molecule for CED-4 is shown in the binding site .", "In CED-4 , a Mg+2 ion is bound and one ligand ( Lys191 ) is replaced by Ser186 in Apaf-1 to weaken and possibly eliminate the binding site for this ion in the human complex .", "In addition , Met334 is present on helix α18 in CED-4; hence , Thr367 functionally replaces Ser325 and makes hydrogen bonds to both the 2' and 3' OH's of the ribose ring .", "A small repositioning of the HD1-WHD loop in CED-4 does not prevent Tyr369 ( equivalent to Tyr359 in Apaf-1 ) from interacting with the γ-phosphate of ATP as an HD1 sensor . DOI: http://dx . doi . org/10 . 7554/eLife . 17755 . 016 In our model , the HD1-WHD loop forms the bottom of the nucleotide binding pocket .", "Both tyrosine side chains in the loop are in good density with Tyr359 forming a hydrogen bond to the γ-phosphate ( Figure 5B , D ) .", "Thus , Tyr359 in the HD1 may act as a sensor for dATP akin to sensor I ( Arg265 ) in the NBD .", "In addition , the oxygen in the deoxyribose ring may form a hydrogen bond to the amide of Leu322 .", "Importantly , the side chain of Ser325 in helix α18 is located within ~4 Å of the 2’ carbon of the deoxyribose ring , and is held in position by hydrogen bonds with the 3’ OH in the sugar ring and the backbone carbonyl of Leu322 ( Figure 5B , D ) .", "In addition , Ser325 may interact with the 2' OH of the ribose ring when ATP is bound instead of dATP , as found in the crystal structure of Apaf-1 with ADP ( Riedl et al . , 2005; Reubold et al . , 2011; Figure 5—figure supplement 2A ) .", "The close apposition of Ser325 to the 2’ carbon of the deoxyribose ring may be responsible for the higher affinity exhibited by Apaf-1 for dATP over ATP ( Jiang and Wang , 2000 ) .", "This effect is more obvious at lower nucleotide triphosphate concentrations ( Reubold et al . , 2009 ) .", "Remarkably , a similar interaction occurs in the Dark apoptosome where a close approach of the Ser325 side chain to the 2' carbon of the sugar ring may be responsible for the absolute preference for dATP during nucleotide-dependent assembly ( Cheng et al . , unpublished; Yu et al . , 2005 ) .", "Thus , Ser325 may act as a modulator that defines the relative affinity for dATP and ATP during assembly of Apaf-1 and Dark apoptosomes .", "Residues in the Walker A box ( Gly157-Val162; Figure 2—figure supplement 4 ) make numerous hydrogen bond interactions with the triphosphate moiety and are present in a loop that precedes helix α10 and also form the base of this helix ( Figure 5A , D ) .", "In particular , Lys160 , Ser161 and Val162 interact with α and β phosphates .", "The side chain of the absolutely conserved Lys160 interacts with the γ phosphate via three hydrogen bonds .", "In addition , the side chain of Arg265 ( sensor I ) makes three potential hydrogen bonds to the terminal phosphate and is stabilized in this position by interactions with Glu366 in the WHD .", "In the Walker B motif , carboxyl side chains of Asp243 and Asp244 are hydrogen bonded to Trp184 and Trp246 , respectively .", "In addition , Aspartate 243 makes a hydrogen bond to Ser161 in the Walker A motif , and the side chain of Ser161 then interacts with the β phosphate .", "Aspartate 244 does not interact directly with the γ-phosphate of dATP but is positioned within 5 Å .", "Hence , water molecules that are not detected at this resolution could mediate a possible interaction .", "To investigate the role of nucleotide exchange , we aligned models of closed and extended Apaf-1 on the NBD ( Figure 5—figure supplement 2A ) .", "In the closed state , ADP is bound by residues in Walker motifs in a manner similar to the extended state with bound dATP , except that Arg265 ( sensor I ) is too far removed from the β-phosphate of ADP to make a salt bridge interaction .", "However , a major rotation of the NBD-HD1 pair occurs when dATP is bound , such that the HD1-WHD loop rotates into position at the bottom of the nucleotide pocket .", "This is coupled with a large rotation of the NBD-HD1 pair about the WHD ( Yuan et al . , 2013; Figure 6 ) , which positions the NBD , HD1 and WHD to form lateral contacts in the inner and outer rings of the central hub ( Yuan et al . , 2010 , 2013 ) .", "This large rotation also breaks a hydrogen bond between His348 in helix α24 of the WHD and the β-phosphate , which may help stabilize the closed configuration ( Danot et al . , 2009; Riedl et al . , 2005; Reubold et al . , 2011; Figure 5—figure supplement 2A ) .", "Hence , the HD1-WHD loop effectively replaces helix α24 in the transition from a closed to an extended conformation .", "In addition , a carboxyl triad in the closed state comprises side chains from Asp244 in the Walker B motif , along with Asp392 and Asp439 in the WHD .", "This side chain grouping should be strongly repulsive but is partially neutralized by Arg265 ( sensor I; Reubold et al . , 2011 ) .", "As a result of conformational rearrangements during nucleotide exchange , Asp244 in the Walker B motif becomes hydrogen bonded to Trp246 , Asp439 in the WHD forms hydrogen bonds with Lys318 ( HD1 ) and Lys391 ( WHD ) , while Asp392 remains in close proximity to Asp439 .", "As predicted , Arg265 does not have to move much in order to interact with the γ-phosphate of dATP during nucleotide exchange and thus , is unlikely to play a direct role in triggering the conformational change ( Figure 5—figure supplement 2A; Danot et al . , 2009 ) . 10 . 7554/eLife . 17755 . 017Figure 6 . Apoptosome assembly .", "( A ) Band shift native gels: ( lanes 1–4 ) interactions of cytochrome c with Apaf-1 create a shifted ladder and higher order aggregation ( lane 3 ) in the absence of dATP; ( lanes 4–7 ) dATP and dADP addition to Apaf-1 do not induce band shifts .", "The apoptosome is assembled when cytochrome c and dATP are both present ( lane 8 ) .", "( B ) Ribbons cartoon showing the closed to extended transition for an Apaf-1 monomer triggered by cytochrome c and dATP binding during assembly .", "( C ) Apaf-1 domains are shown enclosed within calculated surfaces for closed and extended conformations .", "Reorganization of the NOD creates an HD1-NBD-WHD triad that promotes lateral dimer and ring formation , while the CARD is repositioned to bind pc-9 .", "( D ) Band shift native gels demonstrate the accessibility of the Apaf-1 CARD to pc-9 .", "Left panel ( lanes 1 , 2 ) : wild type pc-9 induces a band shift relative to Apaf-1 alone .", "Right panel ( lanes 1–3 ) : a pc-9 mutant with a thrombin site in the CARD-NBD linker also induces a band shift for Apaf-1 and thrombin cleavage releases the catalytic domain ( p20/p10 ) to shift the bands backwards .", "However , the pc-9 CARD/Apaf-1 bands are still shifted relative to Apaf-1 .", "See also Figure 6—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 17755 . 01710 . 7554/eLife . 17755 . 018Figure 6—figure supplement 1 . The V-shaped sensor domain undergoes a large conformational change upon cytochrome c binding .", "( A , B )", "The 8-bladeb-propeller rotates in the plane and 4 helices of HD2 at the base of the sensor domain rotate slightly about pivot point 1 towards the V-shaped cleft , while the 7-bladeb-propeller swings about in a clamping motion to bind cytochrome c .", "( C ) Concurrent with cytochrome c binding , theNBD , HD1 andCARDmust rotate about pivot point 2 near helixa20 to invert their orientations relative to the WHD .", "This large scale motion avoids a steric clash with the sensor domain and creates a more extended Apaf-1 molecule that may exchange boundADPmore readily .", "( D ) The open conformation of the HD1-NBD-WHD triad creates an extended Apaf-1 that can form lateral dimers and rings .", "In panels C and D , an asterisk marks the position of a loop that replaces helixa15 in the NBD .", "During the transition from a closed to an extended conformation this loop undergoes a large scale movement and forms contacts with the HD1-WHD loop in an adjacent subunit ( see also Figure 3G , H ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 17755 . 018 The Apaf-1 monomer has very limited ATPase activity ( ~4–5 ATPs/Apaf-1/hr; Reubold et al . , 2009 ) .", "This is in stark contrast to AAA+ ATPase family members that function as processive machines and catalyze multiple cycles of ATP hydrolysis .", "Instead , nucleotide exchange may trigger conformational changes in Apaf-1 that allow the platform to assemble .", "Experimentally , it has been shown that nucleotide hydrolysis does not occur in the apoptosome ( Reubold et al . , 2009; Kim et al . , 2005 ) .", "In support of this data , no arginine finger is contributed by an adjacent monomer in the apoptosome to promote catalysis .", "Furthermore , a bound Mg+2 ion also plays an important role in catalysis by AAA+ ATPases .", "By comparison with the crystal structure of the CED-4 apoptosome , Ser161 and Asp243 in the Walker A motif are predicted ligands for a possible Mg+2 ion in the nucleotide binding site of Apaf-1 but a third ligand , equivalent to Lys191 in CED-4 , is replaced by Ser186 in Apaf-1 , and this side chain is not able to interact with a cation ( Qi et al . , 2010; Figure 5—figure supplement 2B ) .", "Importantly , no clear density is present in our map of the human apoptosome for a Mg+2 ion and modeling with this ion in a previous structure appears to have distorted the model for dATP ( Zhou et al . , 2015 ) .", "Also note that Buffer A in the frozen pc-9 apoptosome sample contained 1 . 5 mM MgCl2 , 1 .", "0 mM EDTA and 1 . 0 mM EGTA .", "Hence , some free Mg+2 ion would have been available for binding to Apaf-1 .", "Intriguingly , a bound Mg+2 ion is present in chain A of CED-4 and causes a kink in the triphosphate of ATP .", "However , Tyr369 in the HD1-WHD loop of CED-4 still makes hydrogen bonds to the γ-phosphate and thus , may act as sensor for HD1 .", "In addition , Thr367 in this loop is hydrogen bonded to both the 3' and 2' OH groups on the ribose ring , thereby subsuming the role of Ser325 on helix α18 of Apaf-1 , as this serine has been replaced by a methionine in CED-4 ( Figure 5—figure supplement 2B ) .", "The transition from a closed to an extended conformation of Apaf-1 may allow lateral dimers to form .", "The sequential addition of further subunits would then overcome entropic barriers to favor apoptosome assembly ( Yuan et al . , 2010; reviewed in Yuan and Akey , 2013 ) .", "In theory , the order of cytochrome c binding and nucleotide exchange may not be fixed during the transition to an extended Apaf-1 monomer ( Reubold et al . , 2009 ) .", "However , ADP is deeply buried in the binding pocket of the compact monomer in crystal structures ( Riedl et al . , 2005; Reubold et al . , 2011 ) , which suggests that a major conformational change may be required to facilitate nucleotide exchange .", "To understand this process , we investigated Apaf-1 assembly with native gradient gels , which are sensitive to changes in mass , shape and charge .", "However , these experiments were complicated by the presence of a ladder with up to seven bands in purified and functional Apaf-1 preparations .", "This ladder may be due to lateral subunit interactions at the non-physiological protein concentration .", "Concerted band shifts were visible when Apaf-1 was incubated with increasing amounts of cytochrome c ( Figure 6A , lanes 2–3 ) , coupled with a depletion of the original Apaf-1 bands .", "In addition , higher order bands were formed relative to control lanes with Apaf-1 alone ( Figure 6A , lanes 1 , 4 ) .", "This suggests that cytochrome c binding may enhance the ability of Apaf-1 to sample more open conformations and in the absence of nucleotide exchange this may lead to aggregation ( Figure 6A , lane 3 ) .", "We next studied the effects of nucleotide triphosphate on Apaf-1 in the absence of cytochrome c .", "Thus , we added dATP at 1 mM to Apaf-1 and used dADP as a control .", "In this case , there was no apparent band shift with either nucleotide and no sign of higher order aggregation ( Figure 6A , lanes 4–7 ) .", "When dATP and cytochrome c are both added , the aggregation pathway is by-passed and a high molecular weight band is formed that corresponds to the apoptosome ( Figure 6A , lane 8 ) .", "Also note that excess cytochrome c in the assembly reaction also induced upward band shifts of unassembled Apaf-1 , while the ladder is greatly diminished in intensity .", "When taken together , these experiments support a sequential model in which cytochrome c binding is required before nucleotide exchange can occur .", "This idea is consistent with the nature of the conformational changes observed in the transition from a closed to an extended Apaf-1 conformation ( see below ) .", "The structure of the human apoptosome reveals subunit interactions required for correct assembly .", "To further investigate the first steps in assembly , we created a consensus model for full-length Apaf-1 from partial and overlapping models of human and mouse Apaf-1 ( Riedl et al . , 2005; Reubold et al . , 2011 ) .", "We then over-layed Apaf-1 structures with and without bound cytochrome c , by focusing the alignment on the WHD and HD2 arm , as these domains remain relatively unchanged during the transition from a closed to an extended conformation .", "During activation , cytochrome c most likely binds first to the 8-blade β-propeller since this region is accessible and the resulting interface is quite extensive .", "At this stage , the 8-blade β-propeller and helices α29 to α32 of the HD2 undergo a small upwards rotation relative to the core of the HD2 arm ( helices α25 to α28 ) , to bring this region into proximity with the 7-blade β-propeller ( Figure 6B , C; Figure 6—figure supplement 1A–C , Video 1 ) .", "At the same time , the 7-blade β-propeller undergoes a large local twist and rotation to clamp cytochrome c between the two propellers .", "This rotation breaks interactions between the 7-blade β-propeller , NBD and HD2 arm .", "However , the 7-blade β-propeller is not able to complete its rotation without clashing with the NBD ( Yuan et al . , 2013; Figure 6C ) .", "Thus , initial movements of the 7-blade β-propeller may increase domain flexibility in the NOD , as suggested by cytochrome c binding experiments ( Figure 6A ) .", "This flexibility may facilitate nucleotide exchange as the NBD-HD1 pair rotates into a more extended position , since the ADP molecule would become more accessible ( Riedl et al . , 2005; Reubold et al . , 2011 ) . 10 . 7554/eLife . 17755 . 019Video 1 . Conformational change of the Apaf-1 monomer during assembly . This video shows a morph between a model of the closed Apaf-1 molecule and the extended conformation that forms the apoptosome .", "During this process cytochrome c is bound between the β-propellers and dATP is then exchanged for bound ADP . DOI: http://dx . doi . org/10 . 7554/eLife . 17755 . 019 As described previously ( Yuan et al . , 2013 , 2010 ) , a large rotation of the NOD occurs about the HD1-WHD interface and the pivot point appears to lie roughly along the axis of helix α20 ( Figure 6—figure supplement 1C 'pivot 2'; Video 1 ) .", "In this scenario , nucleotide exchange would occur after cytochrome c binds to Apaf-1 .", "This two-step process would stabilize an extended conformation of Apaf-1 to drive apoptosome assembly .", "Side views of Apaf-1 molecules show the large extension that is achieved ( Figure 6B , C ) .", "This conformational change places domains of the NOD in a position to properly associate with additional Apaf-1 monomers that are in an extended or nearly extended conformation , to form lateral dimers , trimers and higher order oligomers that lead to ring formation ( Figure 6—figure supplement 1D; Video 2 ) . 10 . 7554/eLife . 17755 . 020Video 2 . Structure and assembly of the c7 platform and spiral CARD disk in the human apoptosome . This video documents a model for the stepwise assembly of a c7 platform from Apaf-1 subunits containing the NOD , HD2 arm and β-propellers .", "The presentation then focuses on the structure and molecular docking of Apaf-1 and pc-9 CARDs within the acentric , quasi-helical disk , which sits atop the platform . DOI: http://dx . doi . org/10 . 7554/eLife . 17755 . 020 The consensus model of a closed and inactive Apaf-1 also suggested that the N-terminal CARD may be accessible for interactions with pc-9 .", "This idea is supported by the observation that the Km of Apaf-1 for dATP is lower in the presence of pc-9 and cytochrome c ( 1 . 7 to 0 . 86 uM; Jiang and Wang , 2000 ) .", "To test this idea , we did band shift experiments with native gels in which the Apaf-1 ladder amplified the observed interactions .", "We added pc-9 or pc-9t ( with a thrombin site in the CARD-p20 linker; Yuan et al . , 2011a ) to Apaf-1 and observed a significant upwards shift of all bands in the ladder ( Figure 6D , left and right hand panels respectively ) .", "Thrombin cleavage of the pc-9t linker released the catalytic domains ( p20/p10 ) and the resulting Apaf-1/pc-9 CARD bands were shifted downward on the gel , but were still vertically displaced relative to the corresponding Apaf-1 bands ( see lane 1 in Figure 6D , right panel ) .", "Based on this data , it is clear that the Apaf-1 monomer is able to form a 1:1 complex with pc-9 that is mediated by CARD-CARD interactions .", "This has implications for the assembly mechanism ( see Discussion ) .", "Molecular details of the CARD disk have not been visualized in the active apoptosome , due to a possible symmetry mismatch between the disk and central hub ( Yuan et al . , 2010 , 2011a ) .", "However , 3D maps calculated with c1 symmetry in EMAN2 revealed additional features including a defined tilt and an acentric location of the disk , relative to the central seven-fold axis ( Yuan et al . , 2011a ) .", "To resolve the disk at ~5 . 8Å resolution we used focused 3D classification without additional refinements in RELION ( Materials and methods; Scheres , 2012; Zhou et al . , 2015 ) .", "In the resulting map , the acentric disk contains seven well-ordered CARDs arranged as a clockwise spiral when viewed from above .", "At this resolution , CARD-NBD linkers and distinctive features of the 6 helix bundles of Apaf-1 and pc-9 CARDs were resolved , including ~40 α-helices in the disk , which allowed an unambiguous rigid-body docking of the CARDs into the improved map .", "Moreover , a fourth pc-9 CARD is present at lower occupancy so there are four Apaf-1 CARDs and three or four pc-9 CARDs in the disk at any instant .", "Apaf-1 and pc-9 CARDs are shown as color coded pairs based on their interactions in the disk along with four CARD-NBD linkers colored in gold ( Figure 7A , B ) . 10 . 7554/eLife . 17755 . 021Figure 7 . Formation of a CARD disk on the active apoptosome .", "( A ) A composite map of the active apoptosome reveals density for eight CARDs in the acentric disk shown in top , tilted and side views .", "Density for the pc-9 CARD in position 8p has been included , although it is present at lower occupancy .", "Apaf-1/pc-9 CARD pairs that share a Type II interface have been color-coded and Apaf-1 subunits that contribute an ordered CARD to the disk are indicated by color-coded arrow heads .", "Apaf-1 CARD-NBD linkers are shown in gold and the linker to CARD 7a is marked with a black dot .", "( B ) Close-up views are shown of the CARD disk , viewed in three equi-spaced angular orientations about the center of the apoptosome .", "The α-helical nature of the CARDs is clearly visible along with the progressive tilt of the pairs as they spiral about the center of the disk .", "CARD pairs are rendered as surfaces and individual CARD positions are indicated ( 1a , 3a , 5a and 7a for Apaf-1; 2p , 4p , 6p and 8p for pc-9 ) .", "Individual linkers are shown as segmented density in gold for the four Apaf-1 CARDs .", "( C ) Similar views to panel B are shown , but as transparent surfaces with docked ribbon CARD models color-coded green for Apaf-1 and purple for pc-9 .", "Only two CARD pairs are shown with ribbons in each panel to highlight their interactions .", "Some Type I and II interfaces are indicated .", "CARD-NBD linkers have been omitted for clarity . DOI: http://dx . doi . org/10 . 7554/eLife . 17755 . 021 In the disk , four Apaf-1/pc-9 CARD pairs form a quasi-helical spiral and three Apaf-1 CARDs are in direct contact with the NBD ring ( Figure 7C ) .", "The rise of the spiral creates a tilted disk with a large gap between the disk and the NBD ring on one side ( Figure 7A , top right ) .", "An Apaf-1 CARD comes into close contact with the NBD ring and is adjacent to the gap in the clockwise direction , when viewed from above .", "We defined this Apaf-1 CARD as position 1a in the spiral ( Figure 7B , in red ) , while an adjacent pc-9 CARD in the clockwise direction is vertically and laterally offset from the first Apaf-1 CARD and occupies position 2p .", "Overall , Apaf-1 and pc-9 CARDs are present at odd and even positions around the spiral , respectively , ( Figure 8A , B ) .", "In this notation , Apaf-1/pc-9 CARD pairs are located in positions 1a-2p , 3a-4p , 5a-6p and 7a-8p , where they define a one-start , quasi-helical spiral .", "An Apaf-1 CARD in position 7a is located above the gap where it contacts the top of the Apaf-1 CARD in position 1a ( Figure 7B , left panel ) .", "Finally , weaker density is present at position 8p in the average map .", "However , this region could be visualized in a normalized map at a lower threshold .", "This density may correspond to a pc-9 CARD , which is present at lower occupancy and thus , would interact with the Apaf-1 CARD in position 7a to complete the fourth CARD pair .", "The pc-9 molecule at position 8p may be readily exchangeable , while the other pc-9 CARDs make more extensive contacts in the disk . 10 . 7554/eLife . 17755 . 022Figure 8 . CARD packing in the disk .", "( A ) A ribbons diagram of the disk is shown with color coded Apaf-1/pc-9 CARD pairs and individual positions labeled .", "( B ) Schematic of CARD positions in the disk with Type I and II interfaces indicated .", "( C , D )", "Alignment of pc-9 CARDs relative to their respective Apaf-1 CARD in Type II and Type I interface pairs .", "The crystal structure pair ( PDB 4RHW ) is in blue .", "( E ) Top view of the disk with a calculated surface .", "Individual CARD positions are indicated .", "( F ) A side view of CARD pairs is shown at the junction between the first and fourth pair , where a dislocation is present in the spiral disk .", "A weak and quite open interface is present between adjacent pc-9 CARDs ( 2p , 8p ) while adjacent Apaf-1 CARDs ( 1a , 7a ) form a novel interface .", "See also Figure 8—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 17755 . 02210 . 7554/eLife . 17755 . 023Figure 8—figure supplement 1 . Catalytic domains of pc-9 are parked on the central hub of the active apoptosome .", "( A ) A top view is shown of the active apoptosome after c1 refinement in EMAN2 .", "A blurred , yet acentric and tilted disk is resolved ( magenta ) , along with density for catalytic domains of a single pc-9 ( in blue ) .", "The final model for the platform ( grey ribbons ) is docked into the semi-transparent map .", "( B ) A view similar to panel B is shown with the unblurred 8-CARD disk superimposed onto the platform of the aligned c1 density map after aligning with the composite 3D map .", "Catalytic domains of pc-9 are docked on the NBD ring of the central hub , at a position with the most lateral room created by acentric positioning of the disk .", "( C ) A close-up is shown of the acentric and tilted 8-CARD disk and catalytic domains of a single pc-9 are docked into the density .", "The most favored position for the catalytic domains is adjacent to the pc-9 CARD at position 2p ( but see text ) .", "For this depiction , five pc-9 molecules would be bound to the apoptosome with four pc-9 and four Apaf-1 CARDs in the 8 CARD disk .", "Apaf-1 CARD-NBD linkers are not shown . DOI: http://dx . doi . org/10 . 7554/eLife . 17755 . 023 In total , four of the seven Apaf-1 CARDs in the apoptosome are used to construct the disk .", "In addition , CARD-NBD linker densities connect the C-terminal α-helix of each CARD to helix α8 of its respective NBD , when viewed at an appropriate threshold ( in gold , Figure 7B ) .", "The linkers in this figure are clearly defined after map segmentation with SEGGER ( Pintilie et al . , 2010 ) and are shown at a lower threshold .", "Molecular models of the linkers have not been built due to the limited resolution of these features .", "The four Apaf-1 subunits that contribute CARDs to the disk are marked by color-coded arrow heads ( Figure 7A ) .", "The remaining three Apaf-1 CARDs in the apoptosome are not added to the top of the disk to form a third layer , presumably due to restraints imposed by the length of their CARD-NBD linkers .", "In addition , steric clashes or increasing distortions in the CARD-CARD interfaces of the quasi-helical spiral ( see below ) may preclude the addition of a third layer to the disk .", "We surmise that the three excluded Apaf-1 CARDs are disordered since they are not visible , but they may contribute to the propensity of the particles to aggregate .", "In addition , band shift experiments with Apaf-1 monomers and pc-9 suggest that excluded Apaf-1 CARDs may , in principle , bind to pc-9 molecules in the absence of steric restraints .", "However , a measured stoichiometry of 2 to 5 pc-9 molecules per apoptosome under various conditions , places an upper limit on the number of Apaf-1 CARDs that may recruit pc-9 molecules ( Malladi et al . , 2009; Yuan et al . , 2011a; Hu et al . , 2014 ) .", "Thus , Apaf-1 CARDs in the disk and perhaps one excluded CARD may be able to recruit pc-9 molecules to the apoptosome .", "The CARD spiral is located acentrically relative to the seven-fold symmetry axis of the apoptosome and rests in a tilted position on the NBD ring ( Figure 7 ) .", "In all of the Apaf-1 CARDs , the N-terminal helix is positioned closer to the NBD ring , while the adjacent C-terminal helix is located above the N-terminal helix with both termini facing outwards from the disk .", "Thus , the C-terminal helix of each Apaf-1 CARD is positioned so that its linker to the NBD is located on the outside of the disk ( Figure 7A , B ) .", "Contacts made by Apaf-1 CARDs to NBDs in the central hub vary due to their position in the spiral .", "At position 1a , the first 10 residues in the N-terminal α-helix of the Apaf-1 CARD make contacts with helices α8 and α13 in two adjacent NBDs .", "At position 3a the N-terminal helix of the Apaf-1 CARD may interact with helices α8 and α11 in a single NBD .", "Due to the gradual rise of the spiral , the Apaf-1 CARD at position 5a makes weaker contacts with helix α11 in a single NBD , while the Apaf-1 CARD at position 7a does not contact the central hub , although it is attached to its NBD by a CARD-NBD linker ( Figure 7B ) .", "A gap is present between the CARD disk and the platform at this position ( marked '7a' , Figure 7B , left panel ) .", "Surprisingly , only three well ordered pc-9 CARDs and a single more weakly bound molecule are present in the disk .", "An oversized mask was used to compute the disk structure with focused 3D classification; hence no density has been omitted from the final map due to restrictions imposed by the mask .", "Thus , our data suggests that seven well ordered CARDs form the disk .", "In addition , no direct contacts are present between pc-9 CARDs and the central hub , and all pc-9 domains are located on the top surface of the disk-like spiral with their N- and C-termini pointing towards the outside .", "This would allow easy access of the long linker from pc-9 CARDs in the disk to catalytic domains bound to the apoptosome and in bulk solution .", "The overall architecture of the disk is distinct from a recent model , which suggested that the disk would contain three layers of alternating Apaf-1 , pc-9 and Apaf-1 CARDs ( Hu et al . , 2014 ) .", "This model was based on a crystal structure of a CARD heterotrimer in which a single pc-9 CARD is sandwiched between two Apaf-1 CARDs and used a crystal structure of Death Domain complexes as a template ( Hu et al . , 2014; reviewed in Ferrao and Wu , 2012 ) .", "However , coordinates for this model are not available .", "The unusual arrangement of Apaf-1 and pc-9 CARDs in the crystallized heterotrimer creates two interfaces , which were denoted Type I and II , with the Type I interface being similar to the contact surface in the structure of an Apaf-1/pc-9 CARD dimer determined previously ( Qin et al . , 1999 ) .", "The disposition of Type I and II interfaces in the disk is shown in Figures 7C and 8B–D with Type II interfaces occurring between CARDs in color-coded pairs ( Figure 8A , B ) .", "Two overlays are shown for the relevant CARD pairs centered on Type II and Type I interfaces with color coded pc-9 CARDs , after alignment to the appropriate Apaf-1 CARD in the crystal structure ( Figure 8A , C , D; Hu et al . , 2014 ) .", "In general , the interfaces become more distorted in moving clockwise around the disk from position 1a , due to the spiral geometry of the CARDs .", "The rmsd for Cα backbones of 'sequential' Apaf-1/pc-9 CARD pairs , aligned with the Apaf-1 CARD of the relevant crystal pair are 3 , 3 . 2 and 5 . 1 Å for the Type II interface and 2 . 1 , 1 . 5 and 3 . 2 Å for the Type I interface .", "The '7a-8p' CARD pair was not included in this analysis since the crystal structure was used to model this pair .", "The CARD spiral results in a rather open interface between two adjacent pc-9 CARDs at positions 8p-2p , and a novel interface is formed between Apaf-1 CARDs at positions 7a-1a ( Figure 8E , F ) .", "Mutations were made in Apaf-1 and pc-9 CARDs using the crystal structure of the heterotrimer as a guide ( Hu et al . , 2014; PDB 4RHW ) .", "The zig-zag packing of three consecutive CARDs in the disk at positions 1 to 3 , 3 to 5 and 5 to 7 , is due to the presence of alternating Type I and II interfaces , as described in the crystal .", "Hence , mutations that disrupt Type I and Type II interfaces are supported by our model of the disk .", "These mutations would disrupt CARD-CARD interactions and may decrease pc-9 binding and activity on the apoptosome .", "Two additional Apaf-1 CARD mutations ( K58E , K62E ) were found to greatly diminish pc-9 activation and occur outside of interfaces I and II ( Hu et al . , 2014 ) .", "These mutations are located on the bottom surface of the disk-like spiral and may interfere with Apaf-1 CARD interactions to the NBD ring .", "These twin point mutations are not positioned where wild type residues may interact with pc-9 catalytic domains and participate in activation .", "However , these two point mutations suggest that proper disk formation may be a pre-requisite for proper activation of pc-9 zymogens .", "Procaspase-9 catalytic domains consist of two subunits denoted p20 and p10 that are generated by zymogen cleavage .", "We showed previously that density for catalytic domains of a single pc-9 could be identified on the central hub , adjacent to the disk ( Yuan et al . , 2011a , 2010 ) .", "With this in mind , we re-processed our pc-9 apoptosome data with c1 symmetry in EMAN2 ( Tang et al . , 2007 ) and used e2refinemulti . py for 3D classification ( Materials and methods ) .", "A similar feature for pc-9 was found on the central hub with this large data set , and the density accommodates a single copy of the p20/p10 domains .", "However , the density was not modeled precisely due to local flexibility of this feature ( Figure 8—figure supplement 1A ) .", "Intriguingly , we could not detect the pc-9 catalytic domain with a focused 3D classification approach in RELION due in part to its small size and local heterogeneity .", "Refinement with c1 symmetry in EMAN2 uses a density based approach in real space and may reveal the pc-9 catalytic domain at the expense of some blurring of the disk .", "The success of this approach may also be due to the use of supervised 3D classification , which allowed the identification of particles that may contain bound pc-9 catalytic domains , prior to 3D refinements .", "We also note that c1 refinement in EMAN2 resolved the platform at α-helical resolution ( not shown ) .", "We suspect that pc-9 catalytic domains may also bind at sites adjacent to the one identified on the central hub in some particles , as this might explain , in part , why this small ( ~40 kDa ) feature was lost during focused 3D classification in RELION .", "We also surmise that pc-9 catalytic domains may be bound in a dynamic manner , since only ~50% of the particles contained the density in the aligned location .", "We next determined the most likely binding site ( s ) for the p20/p10 domains of pc-9 in relation to the unblurred disk .", "Thus , we aligned the composite 3D map with a well resolved disk relative to the map refined with c1 symmetry using Chimera .", "This alignment was guided by the acentric and tilted disk , since the 7 spokes are similar in both maps .", "Intriguingly , the catalytic domain can be located in two possible positions that are adjacent to a pc-9 CARD at position 2p within the CARD disk .", "For simplicity , we have shown the position with the highest cross-correlation , while the next most likely candidate with a similar probability , is rotated by one Apaf-1 subunit in the clockwise direction ( Figure 8—figure supplement 1B , C ) .", "This proximity would allow pc-9 catalytic domains parked on the hub to be connected to CARDs at all positions in the disk due to the long CARD-p20 linker ." ], [ "In the current assembly model , cytochrome c binding and nucleotide exchange produce a large conformational change in Apaf-1 ( Yuan et al . , 2013; Pang et al . , 2015; Reubold et al . , 2009 ) .", "We suggest that cytochrome c binding is a pre-requisite for nucleotide exchange during assembly .", "In particular , we observed a significant band shift and aggregation of Apaf-1 on native gels when cytochrome c was added , while similar changes could not be detected when dATP was added in a separate reaction .", "Conformational changes due to cytochrome c binding may involve a small rotation of the 8-blade β-propeller and the supporting four α-helices of HD2 , along with a much larger rotation of the 7-blade β-propeller about the base of the HD2 arm , to form the appropriate contact surface within the V-shaped sensor region for cytochrome c ( Yuan et al . , 2013 ) .", "During this reaction , the NBD-HD1 pair must begin to rotate away from the 7-blade β-propeller to avoid a clash , while allowing nucleotide exchange .", "Alternatively , a hinge-like rotation of the NBD-HD1 pair relative to the rest of Apaf-1 may be dynamic .", "Hence , the NBD-HD1 pair could fluctuate between ADP and ATP/dATP bound conformations until they are fixed in place by the binding of both ligands .", "Thus , cytochrome c and a bound nucleotide triphosphate would predispose Apaf-1 to adopt an extended conformation that promotes lateral assembly of subunits to form the ring .", "In the absence of nucleotide exchange , binding by cytochrome c leads to a local conformational change ( or alternatively to a change in the dynamics of the closed form ) and subsequent aggregation of Apaf-1 molecules .", "The low intrinsic ATPase activity of Apaf-1 ( ~4–5 ATP per Apaf-1/hr ) is probably not required for assembly per se ( Reubold et al . , 2009 ) , as exchange of bound ADP/dADP for a nucleotide triphosphate ( dATP , ATP or even GTP at 1 mM ) is all that is required to promote conformational changes that drive assembly .", "Near atomic structures of Apaf-1 monomers ( Riedl et al . , 2005; Reubold et al . , 2011 ) and the apoptosome ( Zhou et al . , 2015; this work ) suggest that the molecule has diverged from the canonical architecture of AAA+ ATPases .", "This includes the 'loss' of a stable binding site for a Mg+2 ion adjacent to the γ-phosphate of dATP/ATP , which greatly diminishes the intrinsic ATPase activity of the monomer .", "However , as Apaf-1 folds during translation or shortly thereafter , the protein may bind adenine nucleotide diphosphate or adenine nucleotide triphosphate , as both are present in the cytoplasm .", "If nucleotide diphosphate is bound , then Apaf-1 will be locked into a closed conformation , until cytochrome c is released from mitochondria in response to intrinsic cell death signals .", "Alternatively , if newly synthesized Apaf-1 binds a nucleotide triphosphate then a limited hydrolysis activity would ensure that the molecule adopts a closed , inactive conformation to prevent aggregation or further assembly into the cell death machine .", "Moreover , no detectable ATPase activity has been found in apoptosomes ( Reubold et al . , 2009; Kim et al . , 2005 ) .", "This may be correlated with the lack of an arginine finger and the absence of a stably-bound Mg+2 ion at the nucleotide binding site .", "We also find that pc-9 is able to interact with Apaf-1 to form a 1:1 complex and this step is mediated by CARD-CARD interactions .", "Hence , Apaf-1 and pc-9 may co-assemble to form an active apoptosome .", "In this case , preformed Apaf-1/pc-9 complexes would bind cytochrome c and undergo nucleotide exchange .", "Alternatively , Apaf-1 may adopt an extended conformation triggered by cytochrome c and dATP/ATP , and then bind pc-9 as it interacts with other copies of itself during assembly .", "Remarkably , the concentration of pc-9 and Apaf-1 in HeLa cervical cancer cells is estimated to be ~30 nM and ~340 nM , respectively ( Würstle and Rehm , 2014 ) .", "This translates to roughly one pc-9 molecule for every 10–11 Apaf-1 molecules and with an assembly efficiency of ~70% this would give a pc-9 to Apaf-1 ratio of roughly 1:7 or 1:8 in active complexes .", "However , in non-cancer cells the ratio of pc-9 to Apaf-1 may be higher .", "Even so , this raises the possibility that preformed Apaf-1/pc-9 heterodimers may undergo preferential recruitment to apoptosomes during their assembly , due to the additional cooperativity of disk formation .", "We have presented a structure for the central hub and extended arms of an active apoptosome at near atomic resolution ( ~3 . 5–4 Å ) .", "Improved structural clarity has revealed the sensor domain and for the first time , we have visualized the packing of CARDs within the acentric disk ( Figure 9 , side and close-up views; Video 2 ) .", "Members of the 6-helix Death Domain family ( CARDs and Death Domains ) have been tailored to assemble into quasi-helical arrays ( reviewed in Ferrao and Wu , 2012 ) , which form the heart of various activation machines .", "These include the human apoptosome ( Yuan et al . , 2011a; Hu et al . , 2014 ) , the NLRC4 inflammasome ( Zhang et al . , 2015; Hu et al . , 2015; Diebolder et al . , 2015 ) , the PIDDosome ( Park et al . , 2007 ) , the Myddosome ( Lin et al . , 2010 ) and the FAS-FADD death disc ( Wang et al . , 2010 ) .", "However , the disk in the human apoptosome uses 7–8 CARDs to form a spiral , instead of 10–14 domains found in other Death Domain complexes .", "Various attempts have been made to reconstitute a disk from individual CARDs of Apaf-1 and pc-9 .", "Recently , a meta-stable complex was observed by gel filtration and estimated to contain 7–8 CARDs .", "These studies led to the crystallization and structure determination of a unique CARD heterotrimer ( Hu et al . , 2014 ) .", "In our study , we find that the path of paired Apaf-1/pc-9 CARDs in the disk describes roughly one turn of a spiral .", "In addition , Type I and II interfaces in the crystal structure of the Apaf-1/pc-9/Apaf-1 CARD trimer ( Hu et al . , 2014; 4RHW ) are present in the disk , but with some distortions to accommodate the spiral geometry .", "Strikingly , only three well ordered pc-9 molecules are recruited to the disk , while a fourth pc-9 is present at lower occupancy . 10 . 7554/eLife . 17755 . 024Figure 9 . Models of the active apoptosome . In these models , procaspase-9 dimers ( PDB 1JXQ ) are flexibly-tethered to the CARD disk and may represent the activated enzyme .", "CARD-p20/p10 linkers are shown as dashed lines , and are drawn roughly to scale for their length .", "The number of possible pc-9 dimers in the active apoptosome may vary as a function of the number of pc-9 molecules tethered to the disk , with an odd number of zymogens creating parked catalytic domains for a single pc-9 .", "For each panel , the top figure shows the entire active complex while the bottom figure shows a zoomed in view of the disk and central hub .", "Left panels: apoptosome with 3 bound pc-9 molecules and a 7 CARD disk; right panels: an active complex with 4 bound pc-9 molecules and an 8 CARD disk .", "Note that 4 Apaf-1 CARDs are always present in the CARD disk .", "For clarity the Apaf-1 CARD-NBD linkers are not shown . DOI: http://dx . doi . org/10 . 7554/eLife . 17755 . 024 Procaspase-9 exists as a monomer in solution unlike most caspases which are constitutive dimers ( reviewed in Riedl and Shi , 2004 ) .", "However , pc-9 forms an unusual dimer when crystallized without the CARD , in which one substrate binding site is occluded and inactive , while a second site is in an active configuration and binds a peptide inhibitor ( Renatus et al . , 2001 ) .", "Close inspection of the pc-9 structure suggests that crystal contacts do not play a role in this asymmetry .", "Recent data have been interpreted to suggest a possible model for pc-9 activation that involves dimerization of p20-p10 catalytic domains ( Boatright et al . , 2003; Bratton and Salvesen 2010; Pop et al . , 2006; Renatus et al . , 2001 ) , which are attached to the apoptosome by flexible CARD-p20 linkers .", "Alternatively , tethered pc-9 catalytic domains may bind directly to the platform and/or the disk and be activated ( Yuan et al . , 2011a; Hu et al . , 2014; Yin et al . , 2006 ) .", "Recent computational modeling favors an allosteric mechanism wherein pc-9 catalytic domains bind to the apoptosome , but could not differentiate between monomers or dimers as the active species ( Würstle and Rehm , 2014 ) .", "However , recent experiments have shown that chimeric Apaf-1 CARD/ClpP or Apaf-1 CARD/GroEl complexes , which are thought to form ClpP and GroEl heptamers , respectively , are able to activate pc-9 to a similar extent as the Apaf-1 apoptosome using a fluorescence-based proteolysis assay and by monitoring procaspase-3 cleavage ( Hu et al . , 2014 ) .", "We have verified this remarkable observation for pc-9 added to chimeric ClpP molecules with N-terminal Apaf-1 CARDs ( not shown ) .", "When taken together , this data would argue against an activation mechanism that requires binding of pc-9 catalytic domains to the central hub formed by Apaf-1 molecules , since this region does not exist in chimeric complexes .", "These data also suggest that efficient procaspase-3 cleavage can proceed in chimeric complexes in the absence of the Apaf-1 platform .", "However , these studies do not rule out the possibility that procaspase-3 may interact with Apaf-1 at some point either during or after its activation ( Yuan et al . , 2011a and references therein ) .", "Our structure of an active apoptosome provides additional insights into the mechanism of pc-9 activation .", "One constraint is provided by stoichiometry measurements , which indicate that a range of 2 to 5 pc-9 molecules may be bound to the apoptosome ( Malladi et al . , 2009; Yuan et al . , 2011a; Hu et al . , 2014 ) .", "In line with this data , our structure suggests that 3 or 4 pc-9 molecules may be tethered to the disk through CARD-CARD interactions , with the caveat that a pc-9 molecule might also interact with Apaf-1 CARDs that are excluded from the disk .", "We also identified density in some particles for catalytic domains from a single pc-9 that are 'parked' on the NBD ring of the central hub ( this work , Yuan et al . , 2011a ) .", "The two 'most favored' positions for parked pc-9 catalytic domains are next to the pc-9 CARD at position 2p in the disk .", "Our analysis further suggests that the acentric position of the disk may create the binding site for pc-9 catalytic domains .", "However , it is unlikely that adjacent parking sites would be occupied at the same time due to steric restraints .", "One implication of this finding is that parked catalytic domains could be attached to pc-9 CARDs located at all possible positions in the disk , due to the length of the CARD-p20 linker .", "Hence , the most favored parking sites are generally available to all four pc-9 molecules that may be tethered to the disk .", "We hypothesize that parked pc-9 catalytic domains may be held in reserve when an odd number of zymogens are bound to the apoptosome .", "This in turn , may prevent entanglement of linkers between parked pc-9 catalytic domains and flexibly-tethered pc-9 dimers anchored to the disk , with the latter representing active proteolytic centers .", "There is no ordered density in our map for other pc-9 catalytic domains , and these domains can be released by a single clip in the CARD-p20 linker ( Yuan et al . , 2010 ) .", "Thus , we surmise that the remaining pc-9 catalytic domains are flexibly-tethered to the disk through CARD-p20 linkers .", "We propose that the active apoptosome is a dynamic proteolytic machine with flexibly-tethered pc-9 catalytic domains , whose nature may depend upon the number of pc-9 molecules that are bound at any instant .", "For example , a flexibly-tethered pc-9 dimer may form when three pc-9 molecules are anchored to form a 7 CARD disk , with the pc-9 catalytic domains that are odd man out occupying a parking spot on the central hub ( Figure 9 , left ) .", "However , if four pc-9 molecules are bound to the apoptosome they may form an 8-CARD disk with two flexibly-tethered pc-9 dimers , while leaving open parking spots on the central hub ( Figure 9 , right ) .", "By extension , if 5 pc-9 molecules were bound to the apoptosome then one pc-9 molecule might interact with an Apaf-1 CARD that is not incorporated into the disk .", "In this case , two flexibly-tethered pc-9 dimers may be formed on the apoptosome with an 8 CARD disk and a parked p20/p10 domain would also be present ( e . g . Figure 8—figure supplement 1B , C ) .", "Since the pc-9 CARD at position 8p in the disk is present at lower occupancy , it is clear that active apoptosomes with three bound pc-9 molecules were formed with a higher likelihood under our in vitro conditions .", "At the same time , the high protein concentration in our experiments may have aided the formation of an 8 CARD disk in particles with four pc-9 molecules , whereas apoptosomes with a 7 CARD disk and three pc-9 molecules may be favored in vivo .", "Moreover , the KCl concentration may affect the stability of the pc-9 CARD at position 8p .", "The lower KCl concentration used in Buffer A , relative to physiological conditions ( 20 mM versus ~150 mM ) , may favor electrostatic interactions that predominate at CARD-CARD interfaces .", "Hence , the transient association of a pc-9 CARD at position 8p may be more favorable in low salt buffer .", "Also note that it remains an open question whether each putative pc-9 dimer on the active apoptosome may contain one ( Renatus et al . , 2001 ) or possibly two active sites .", "Activation of pc-9 on the apoptosome has been proposed to follow the dictates of a molecular timer .", "In this model , auto-processing of pc-9 on the apoptosome triggers activation and subsequent release via exchange with unprocessed pc-9 in solution ( Malladi et al . , 2009 ) .", "The rapid nature of the auto-proteolysis reaction suggests that two-chain pc-9 molecules , as used in our structure ( Figure 1—figure supplement 1A ) , may account for most of the activity towards procaspase-3 ( Malladi et al . , 2009; Hu et al . , 2013 ) .", "Since there is no uncleaved pc-9 in our assembly reaction , we are not able to comment directly on the proposed turnover of pc-9 in the molecular timer hypothesis .", "However , the disposition of pc-9 on the apoptosome is similar for both unprocessed and cleaved pc-9 molecules .", "Indeed , a 3D map of apoptosomes with bound pc-9 CARDs , made by thrombinolysis of a single chain pc-9 with a triple point mutation ( E306A/D315A/D330A ) , also showed the disk , while flexibly-tethered catalytic domains were released from the complexes ( Yuan et al . , 2010 ) .", "Our structure of the active apoptosome suggests that flexibly-bound pc-9 catalytic domains are probably not able to affect the stability of the CARD-CARD disk .", "However , parked p20/p10 subunits of pc-9 could exert some effect on disk stability , to modulate the binding and release of pc-9 , although this is speculative .", "Intriguingly , pc-9 CARDs are located on the top surface of the disk , which would provide a route for pc-9 molecules to dis-associate from the complex , such as occurs at position 8p .", "This step would down-regulate apoptosome activity until a replacement pc-9 is bound from solution .", "The major variable in evaluating possible turnover of bound pc-9 molecules is the overall stability of the CARD disk .", "Given the multiple interactions of pc-9 CARDs in positions 2p , 4p , and 6p , it appears that a CARD at position 8p will be the most likely to exchange , as observed in our analysis .", "Hence , the turnover of pc-9 at position 8p in the disk would lead to an oscillation in the number of potentially active pc-9 dimers bound to the apoptosome .", "Finally , pc-9 molecules with shortened CARD-p20 linkers are not activated on the apoptosome as readily as wild type pc-9 ( Yuan et al . , 2011a ) .", "This suggests that the CARD-p20 linker may facilitate pc-9 activation by providing a suitable spacer between CARDs in the disk and the catalytic domains .", "Indeed , the requirement for a somewhat longer spacer may reflect the disposition of the four pc-9 CARD binding sites on the disk .", "Moreover , the CARD-p20 linker may also stabilize the pc-9 dimer when it is formed .", "In either case , a single clip in the linker releases the catalytic ( p20/p10 ) domains from the apoptosome and results in a complete loss of proteolytic activity ( Yuan et al . , 2010 , 2011a ) .", "This argues that putative pc-9 dimers are only stable when present at a high local concentration on the apoptosome .", "In summary , our structure of an active apoptosome suggests that most complexes will contain one ( or possibly two ) pc-9 dimers , which may be responsible for proteolytic activity and initiation of a cell death cascade .", "In a subsequent step , the cleavage and activation of procaspase-3 dimers may occur either in solution through a collisional process with flexibly-tethered pc-9 catalytic domains or the reaction may be facilitated by transient interactions with the Apaf-1 platform ( Yin et al . , 2006 ) .", "Additional experiments are needed to address this point in light of recent experiments with chimeric platforms that contain N-terminal Apaf-1 CARDs , as these complexes are able to activate procaspase-9 and cleave procaspase-3 efficiently ( Hu et al . , 2014 ) ." ], [ "His-tagged Apaf-1 was cloned into pFastBac , expressed in baculovirus infected sf9 cells and purified ( Acehan et al . , 2002; Zou et al . , 1999 ) .", "In brief , cells were harvested 48 hr post infection and lysed by homogenization in buffer T ( 20 mM tris pH 7 . 5 , 50 mM NaCl , 1 mM PMSF , 1 mM benzamidine , 1 mM beta-mercaptoethanol ) .", "The lysate was centrifuged ( 100 , 000 g for 30 min ) and the supernatant applied to a 2 ml Ni-NTA column; Apaf-1 was eluted in 250 mM Imidazole in Buffer T . Sample was dialyzed overnight at 4°C into 25 mM potassium phosphate ( pH 7 . 5 ) with 50 mM NaCl .", "The dialyzate was applied to 2 ml of hydroxyapatite ( BioRad HTP ) in batch and eluted with 250 mM potassium phosphate ( 7 . 5 ) and 50 mM NaCl .", "The Apaf-1 was dialyzed into buffer A without MgCl2 ( 10 mM Hepes pH 7 . 5 , 20 mM KCl , 1 mM EDTA , 1 mM EGTA ) and frozen at ~0 . 5 mg/ml until use .", "Clones for wild type and procaspase 9 mutants with a thrombin cleavage site in the CARD-p20 linker were expressed and purified as described ( Chao et al . , 2005; Yuan et al . , 2010 , 2011a ) .", "To induce holo-apoptosome assembly , Apaf-1 ( 120 µg ) in Buffer A without MgCl2 was mixed with a slight excess of two chain pc-9 ( ~1 . 5x ) , bovine cytochrome c ( ~10 µg ) , 1 mM dATP , 1 . 5 mM MgCl2 and incubated at 30°C for 5 min .", "The holo-apoptosome was concentrated in Buffer A with an Amicon Ultra ( 10K cutoff ) to ~4 mg/ml in a final volume of 30 µl .", "Concentrated sample ( 2 . 5 µl ) was pipetted onto a C-Flat 300 mesh R1 . 2/1 . 3 holey grid ( Protochips , Morrisville , North Carolina ) that was freshly glow discharged and blotted for 1 . 5 s in 100% humidity at 20°C and plunge frozen in liquid ethane using a Vitrobot Mark 3 ( FEI Company , Hillsboro , Oregon ) .", "Movie data were acquired on a Titan Krios electron microscope ( FEI Company ) operated at 300 kV , and equipped with a K2 Summit direct electron detector ( Gatan , Pleasanton , California ) , a spherical aberration corrector and a Gatan Image Filter ( GIF ) with a slit width of ~20eV .", "Super-resolution counting mode was used for movie data collection with SerialEM ( Mastronarde , 2005 ) at a nominal magnification of 81 , 000x , corresponding to 0 . 675 Å per super-resolution pixel , at a dose rate of ~10 . 2 electrons per physical pixel per second .", "Each total exposure of 40 electrons per Å2 was fractionated into 18 frames and lasted 7 . 2 s .", "Defocus ranged from –1 . 5 to –2 . 4 µm ( Figure 1—source data 1 ) .", "A total of 4900 movies were collected and stored in LZW compressed tiff format on the fly , with appropriate gain references .", "The data were uncompressed , corrected for the gain and binned 2X for processing with a script using IMOD ( Kremer et al . , 1996 ) giving a pixel size of 1 . 35 Å per pixel .", "The program UCSF MotionCorr was used to correct beam induced motions within each movie stack ( Li et al . , 2013 ) , the first frame of each movie was discarded , and a summed micrograph ( frames 2–18 ) was used for further processing .", "Contrast transfer function parameters were estimated by CTFFIND4 ( Rohou and Grigorieff , 2015 ) .", "In total , 134 , 970 particles were selected by automatic particle picking in RELION-1 . 3 ( Scheres , 2015 ) .", "A cleaned-up set of 92 , 867 particles was obtained by manual inspection , 2D and 3D classification in RELION-1 . 3 ( Scheres , 2012 ) .", "A previously published map ( EMD-1931 ) was low pass filtered to 60 Å and used as the starting model for 3D classification .", "Refinement with c7 symmetry gave a map at 4 . 7 Å resolution ( gold-standard FSC0 . 143 ) .", "Per particle corrections for beam-induced motion and B-factor weighting for radiation damage were carried out with statistical movie processing in RELION-1 . 3 ( Scheres , 2014 ) with frames 2–18 , a running average of 5 frames and a standard deviation of 1 pixel for translations .", "A soft mask was used along with correction for the modulation transfer function of the K2 Summit detector to produce a map at 4 . 1 Å resolution , which was sharpened using automatic B-factor estimation ( Rosenthal and Henderson , 2003 ) within RELION 1 . 3 .", "Local resolution was estimated using ResMap ( Kucukelbir et al . , 2014 ) .", "A focused 3D classification was used to improve the local resolution of the sensor domain in a single Apaf-1 subunit as described ( Zhou et al . , 2015 ) .", "In brief , the first Euler angle ( α ) from the alignment of each original particle image on the whole apoptosome was permutated cyclically by n times in 360°/7 increments , with n=1 to 6 , to create six additional 'virtual particle' line entries in a new alignment data star file .", "In effect , this rotates each spoke density region within a given 2D image into the mask in the aligned 3D reference volume .", "The 3D mask covered the HD2 , β-propellers , and cytochrome c in one Apaf-1 subunit .", "A focused 3D classification in RELION ( Scheres , 2012 ) was done without an alignment search and members of the best class were then used to calculate an improved 3D map using relion_reconstruct , which places each particle projection only in its most probable orientation .", "After excluding the HD2 , this gave a final sharpened map for the sensor domain with a resolution of 6 . 1 Å .", "The CARD disk is rotationally blurred during c7 alignments , and refinement with c1 symmetry failed to clarify the map due to the small size ( ~75–80 kD ) of the disk .", "Thus , we used a 3D classification that focused on the disk without local refinement .", "A soft mask was used that covered the disk and enclosed the NBD ring .", "In total , we identified seven classes with an asymmetric disk in different orientations , relative to the aligned c7 platform .", "After applying a soft mask to the disk , an estimated gold standard resolution of ~5 . 8 Å was obtained for each of the seven class maps .", "Sharpened 3D maps from each of the classes were then aligned and averaged in Chimera with vop_resample and equal weighting ( Pettersen et al . , 2004 ) to produce a final map of the disk .", "The following steps were used to produce a map of the pc-9 apoptosome with c1 symmetry .", "Coordinates of shiny particles ( 92867 ) from RELION were used to extract particles from the movement corrected micrographs in EMAN2 . 1 ( Tang et al . , 2007 ) and CTFFIND4 parameters ( Rohou and Grigorieff , 2015 ) were imported using a utility in EMAN2 . 1 .", "After 2D classification with e2refine2d . py , particles in the best classes were selected ( 82091 ) and further segregated by a supervised 3D classification using e2refinemulti . py with two references .", "The first 3D reference was EMD-1931 ( apoptosome with bound pc9 catalytic domains processed with c1 symmetry; Yuan et al . , 2011a ) , while the second reference was constructed by removing density for the parked pc9 catalytic domains from the first reference map using Chimera .", "Particles in the class with bound catalytic domains ( 39985 , ~48 . 7% ) were used for c1 refinement with EMD-1931 as a reference , which was low pass filtered to 30 Å .", "This produced a map with a gold standard FSC0 . 143 of 7 . 2 Å with α-helical rods resolved in the central hub ( not shown ) .", "However , density for the bound pc-9 catalytic domains was not resolved at α-helical resolution presumably due to local flexibility .", "A previously published model containing the NBD , HD1 , WHD , HD2 and β-propellers ( PDB 3J2T; Yuan et al . , 2010 ) was used for model building .", "The full Apaf-1 model was docked into the map as a rigid body in Chimera ( Pettersen et al . , 2004 ) , followed by Molecular Dynamics Flexible Fitting ( MDFF; Trabuco et al . , 2008 ) .", "The flexibly fitted model accounted well for most of the density , except for a loop between residues 349 to 364 in HD1 .", "The resulting model was manually rebuilt and adjusted in Coot ( Emsley et al . , 2010 ) with intervening cycles of real space refinement in PHENIX to insure proper geometry ( Adams et al . , 2010 ) .", "In the next stage , β-propellers were docked into the local map of the V-shaped domain using MDFF , while cytochrome c ( PDB 3J2T ) , and individual CARDs from a crystal structure ( Hu et al . , 2014; PDB 4RHW ) were docked with rigid body fitting in Chimera .", "We then calculated a model vs map FSC0 . 5 to obtain an estimate of the final resolution for each of the models .", "All molecular figures were made with Chimera ( Pettersen et al . , 2004; Goddard et al . , 2005 ) and Adobe Photoshop .", "Electron density maps and coordinates have been submitted to the Electron Microscopy Data Bank ( EMD-8178 ) and the Protein Data Bank ( 5JUY ) , respectively ." ] ]

[ "In response to cell death signals , an active apoptosome is assembled from Apaf-1 and procaspase-9 ( pc-9 ) .", "Here we report a near atomic structure of the active human apoptosome determined by cryo-electron microscopy .", "The resulting model gives insights into cytochrome c binding , nucleotide exchange and conformational changes that drive assembly .", "During activation an acentric disk is formed on the central hub of the apoptosome .", "This disk contains four Apaf-1/pc-9 CARD pairs arranged in a shallow spiral with the fourth pc-9 CARD at lower occupancy .", "On average , Apaf-1 CARDs recruit 3 to 5 pc-9 molecules to the apoptosome and one catalytic domain may be parked on the hub , when an odd number of zymogens are bound .", "This suggests a stoichiometry of one or at most , two pc-9 dimers per active apoptosome .", "Thus , our structure provides a molecular framework to understand the role of the apoptosome in programmed cell death and disease ." ]

[ "An adult human loses around 50–70 billion cells every day via a process termed apoptosis .", "The term arises from the Greek word that describes leaves “falling off” a tree , and the process entails damaged or unwanted cells essentially committing suicide in a controlled manner .", "As such , apoptosis keeps the number of cells in tissues and organs in check .", "It also allows components of old cells to be recycled to make new ones .", "In cells that are targeted to die , a protein called cytochrome c interacts with another protein , named Apaf-1 , together with a nucleotide triphosphate molecule .", "These steps work in concert to trigger the assembly of the apoptosome: a large wheel-like complex that contains seven copies each of Apaf-1 and cytochrome c .", "The central hub of the wheel then recruits a specific protein-cutting enzyme , which once activated sets in motion a cascade of events that dismantle the cell from the inside out .", "Cheng et al . now use an electron microscope to reveal the three-dimensional structure of the active human apoptosome , in enough detail to determine the positions of many of the amino acids that make up the complex .", "The three dimensional model provides new insights into how Apaf-1 changes shape as the complex assembles in the presence of cytochrome c and nucleotide triphosphate .", "Cheng et al . went on to reveal a disk-like structure made from the parts of four Apaf-1 proteins that interact with the protein-cutting enzymes .", "This disk forms a spiral that sits atop the central hub of the wheel-like apoptosome .", "Finally , the findings suggest that , although the wheel like complex has seven spokes , at any one time the active apoptosome may only need two ( or at most four ) copies of the protein-cutting enzyme to trigger the cascade of events that lead to cell death In the future , emerging technologies may provide high enough resolution to visualize fine details of the interactions between cytochrome c and Apaf-1 , and reveal more about the disk-like spiral as well .", "This in turn will give a better understanding of how the apoptosome assembles and how the protein-cutting enzyme becomes activated ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "cell biology", "structural biology and molecular biophysics" ]

[ [ "Motors of the kinesin family are ubiquitous enzymes essential for intracellular transport along microtubules in eukaryotes .", "The mechanism by which kinesin motor proteins convert the chemical energy of ATP hydrolysis into coordinated , long-range directional movement has fascinated cell biologists , biochemists , and engineers for many decades .", "Biophysical studies of kinesins have focused on conventional Kinesin-1 and established the ‘hand-over-hand’ model for the processive walking behavior of this type of motor ( Asbury et al . , 2003; Yildiz et al . , 2004; Kaseda et al . , 2003 ) .", "In analogy to other enzymes , the term ‘processivity’ describes the ability of individual motor molecules to undergo multiple catalytic cycles—and therefore translocate—before releasing from the microtubule .", "Kinesin-14 family members , exemplified by the Drosophila Ncd motor , are common examples for nonprocessive kinesins ( Case et al . , 1997; Foster and Gilbert , 2000 ) .", "They generate motility through the minus-end-directed rotational movement of a coiled-coil mechanical element that occurs upon ATP binding ( Endres et al . , 2006 ) .", "After each catalytic cycle , Ncd motors release from the microtubule lattice , meaning that to support microtubule sliding and crosslinking in the spindle , many Ncd motors must work together cooperatively in an ensemble ( Braun et al . , 2009; Fink et al . , 2009 ) .", "Budding yeast kinesin-14 Kar3 is distinct from other family members in its heterodimeric composition with either Cik1 or Vik1 ( Manning et al . , 1999 ) ( Figure 1A ) .", "High-resolution structural analysis has shown that these accessory proteins contain a motor homology domain that harbors a microtubule binding site but lacks the structural elements required to bind and hydrolyze ATP ( Allingham et al . , 2007 ) .", "Biochemical experiments have indicated that Cik1 and Vik1 modulate the interaction of Kar3 with microtubules ( Allingham et al . , 2007; Chen et al . , 2011; Rank et al . , 2012 ) .", "In vivo Kar3's heterodimeric composition governs its subcellular localization and function: Kar3 in complex with Vik1 crosslinks parallel microtubules in proximity to spindle pole bodies during mitosis ( Manning et al . , 1999 ) , whereas antiparallel microtubule sliding is powered by Cik1–Kar3 complexes that associate with the microtubule lattice and plus-ends during mitotic and meiotic events ( Maddox et al . , 2003; Gardner et al . , 2008 ) .", "In addition , Kar3 has been implicated in kinetochore capture and transport ( Middleton and Carbon , 1994; Tanaka et al . , 2005 , 2007 ) .", "The unusual composition of the Kar3 motor with the combination of a catalytic and a non-catalytic domain , as well as its key roles for diverse cellular processes in yeast , has made it a particularly interesting object of study both from a biophysical and cell biological point of view .", "The understanding of the mechanistic basis of Kar3 function , however , has remained incomplete , as biochemical experiments have been limited to ensemble assays using truncated or artificially dimerized proteins .", "On the basis of such experiments and the interpretation of in vivo phenotypes , it has been proposed that Cik1–Kar3 acts as a microtubule depolymerase ( Chu et al . , 2005; Sproul et al . , 2005; Allingham et al . , 2007 ) .", "We hypothesized that the presence of a non-catalytic domain may allow functionalities fundamentally different from conventional kinesin-14 homodimers .", "As the activity of individual full-length Kar3 motors had not been observed directly , we developed assays to investigate motors at the single molecule level and analyze the contribution of the non-catalytic domain . 10 . 7554/eLife . 04489 . 003Figure 1 . Purification and characterization of Cik1–Kar3 kinesin motors .", "( A ) Schematic representation of conventional Kinesin-1 in comparison to the kinesin-14 Cik1–Kar3 .", "( B ) Purification of recombinant Cik1–Kar3 from yeast extracts .", "Motors are covalently labeled with Tetramethylrhodamine ( TMR ) via a HaloTag on the amino-terminus of Kar3 .", "Coomassie-stained SDS-PAGE shows purity of the motor preparation and fluorescent labeling of the Kar3 subunit .", "( C ) Size-exclusion chromatography of Cik1–Kar3-Halo motors on a Superose 6 column .", "The void volume of the column ( V0 ) and the elution position of standard proteins with their respective stokes radii is indicated .", "( D ) SDS-PAGE analysis of Superose 6 fractions from ( C ) .", "( E ) Sucrose gradient centrifugation of Cik1–Kar3 motors .", "Consecutive fractions from top to bottom of a 5–25 ( wt/vol ) % sucrose gradient were analyzed by SDS-PAGE and Coomassie staining .", "The gradient positions of standard proteins are indicated together with their sedimentation coefficients .", "( F ) Low angle Pt/C rotary shadowing electron microscopy of Cik1–Kar3 motors obtained after size exclusion chromatography .", "Overview of Cik1–Kar3 motors , scale bar 100 nm .", "( G ) Gallery view of selected Cik1–Kar3 motors , scale bar 50 nm . DOI: http://dx . doi . org/10 . 7554/eLife . 04489 . 003" ], [ "To study Kar3 motors at the single molecule level , we developed a protocol to express and purify full-length kinesin-14 heterodimers from Saccharomyces cerevisiae using affinity tagged Cik1 and Kar3 fused COOH-terminally to a HaloTag that served as covalent attachment site for the fluorescent dye tetramethylrhodamine ( TMR ) .", "Purification and labeling yielded a homogenous preparation containing a heterodimer of Cik1 and TMR-labeled Kar3 ( Figure 1B ) .", "During size exclusion chromatography , Cik1–Kar3 motors eluted as a single major peak with a Stokes radius of ∼9 . 1 nm , well separated from the void volume of the column ( Figure 1C , D ) .", "Sucrose-gradient centrifugation revealed the presence of a single major species with an apparent sedimentation coefficient of ∼5 . 6S ( Figure 1E ) .", "Combining these hydrodynamic values yielded a native molecular weight of 214 kDa , close to the calculated molecular weight of a Halo-tagged Cik1–Kar3 heterodimer of 190 kDa .", "We further characterized the oligomeric state of full-length Cik1–Kar3 motors by performing low-angle Pt/C rotary shadowing electron microscopy on peak fractions from the gel filtration experiments .", "This analysis revealed individual well-defined , highly elongated molecules that were characterized by globular domains separated by a 61 ± 8 nm ( mean ± SD , n = 100 ) long coiled-coil ( Figure 1F ) .", "Typically , two closely spaced globular domains likely corresponding to catalytic and non-catalytic head domains were discernible at one end , while a single globular domain decorated the other ( Figure 1G ) .", "The highly elongated shape of the Cik1–Kar3 molecules can explain their early elution from the gel filtration column .", "Overall , the hydrodynamic analysis and the direct visualization of motor molecules by EM support the presence of heterodimeric Cik1–Kar3 molecules .", "We next observed the behavior of single motor molecules on surface-immobilized microtubules in vitro using time-lapse multi-color TIRF microscopy .", "Unexpectedly , and contrary to the classification of kinesin-14s as non-processive motors , Kar3 molecules displayed efficient ATP-dependent movement over several micrometers and accumulated at microtubule minus ends ( Figure 2A , Video 1 ) .", "Automated tracking of motors revealed a Gaussian velocity distribution of Cik1–Kar3 with a mean speed of 77 ± 23 nm/s ( mean ± SD; Figure 2B ) in range with reported microtubule gliding velocities for truncated Cik1–Kar3 molecules ( Chu et al . , 2005; Allingham et al . , 2007 ) .", "The motor is therefore approximately 10-fold slower than conventional Kinesin-1 , but similar in speed to yeast cytoplasmic Dynein , the major minus-end directed-motor in eukaryotes ( Reck-Peterson et al . , 2006 ) .", "The run-length histogram followed an exponential distribution and revealed that individual Cik1–Kar3 motors advanced processively for an average of 5 . 2 μm before detaching from the microtubule track ( Figure 2C ) .", "The motile parameters were highly sensitive to the ionic strength of the imaging buffer: at the same ATP concentration higher salt concentration increased the mean squared displacement of the motors ( Figure 2D ) , but decreased the on-rate , run length and minus-end dwell time ( Figure 2E ) .", "We additionally noticed that Cik1–Kar3 oscillated back-and-forth when entering microtubule overlap zones .", "Because Cik1–Kar3 is exclusively moving towards the minus-end on single MT filaments , we concluded that reversing motors encountered an antiparallel-oriented MT bundle .", "Individual motors were able to switch the track microtubule multiple times leading to a prolonged association with antiparallel bundles ( Figure 2F , Video 2 ) . 10 . 7554/eLife . 04489 . 004Figure 2 . Cik1–Kar3 motors move processively with a single catalytic domain .", "( A ) Kymograph showing two-color time lapse TIRF microscopy of Cik1–Kar3 ( red ) moving along taxol-stabilized microtubules ( blue ) .", "See Video 1 for example of Cik1–Kar3 motility .", "( B ) Histogram of velocities of Cik1–Kar3 molecules moving along taxol-stabilized microtubules ( fit with a Gaussian function , black line ) .", "The mean velocity is 77 ± 23 nm/s , n = 699 .", "( C ) Histogram of run lengths of Cik1–Kar3 molecules moving along taxol-stabilized microtubules , n = 209 .", "( D ) Mean-squared displacement analysis of wild-type Cik1–Kar3 at two different salt concentrations in the presence of ATP .", "( E ) Influence of ionic strength on the motile properties of Cik1–Kar3 molecules .", "Experiments were performed in BRB80-based imaging buffer containing the indicated concentrations of KCl .", "( F ) Behavior of Cik1–Kar3 in microtubule networks .", "Typical kymograph showing directional movement of Cik1–Kar3 on single microtubules vs repeated directionality switches of individual motors in antiparallel overlaps .", "The dashed line indicates the beginning of an overlap zone .", "See Video 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 04489 . 00410 . 7554/eLife . 04489 . 005Figure 2—figure supplement 1 . Characterization of Cik1–Kar3 motility .", "( A ) Photobleaching experiment in the absence of nucleotide .", "Kymograph representation showing that Cik-Kar3 motors do not exhibit displacement or diffusion in this state .", "( B ) Quantification of photobleaching .", "( C ) Example for single-step photobleaching event .", "( D ) Example for two-step photobleaching event .", "( E ) Mixing experiment combining TMR-labeled Cik1–Kar3 motors with Alexa488 labeled Cik1–Kar3 motors prior to imaging .", "Kymograph reveals individual traces for red-and green fluorescent motors , indicating that a single Cik1–Kar3 heterodimer is sufficient for movement .", "( F ) Microtubule-gliding experiment with varying concentrations of Cik1–Kar3 .", "In this assay , the motor is immobilized via anti-Halo antibodies to the coverglass and movement of microtubules is recorded by time-lapse TIRF microscopy .", "Kymographs indicate gliding velocity at different motor concentrations , the lower panel shows that gliding is ATP dependent .", "( G ) Movement of Cik1–Kar3 motors on dynamic microtubules .", "Dynamic extensions were grown from GMPCPP-stabilized microtubule seeds and imaged together with Cik1–Kar3 .", "Note movement of motors opposite to the directions of microtubule growth , indicating minus-end directed motility of Cik1–Kar3 .", "( H ) Quantitative analysis of MT shrinkage rate of taxol-stabilized MTs in the presence of 1 nM Cik1/Kar3 ( error bars represent SEM , n = 30 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 04489 . 00510 . 7554/eLife . 04489 . 006Figure 2—figure supplement 2 . A single Cik1–Kar3 heterodimer is sufficient to form a processive complex .", "( A ) Kymograph showing movement of Cik1–Kar3 .", "Position along the microtubule is depicted on the vertical axis while time changes along the horizontal axis .", "( B–C )", "Kymographs showing binding and movement of single Cik1–Kar3 complexes each composed of 1 ( B ) or 2 ( C ) heterodimers .", "( D–E )", "Records for background subtracted brightness vs times for complexes in B and C , respectively .", "( E ) Distribution of background subtracted brightness for Cik1–Kar3 complexes .", "The fit is with four peak Gaussian .", "( F ) Distribution of the initial size of processively moving complexes based on the brightness of a single fluorophore from E . ( G ) Histogram of moving motor complexes containing the indicated number of Cik1-Kar3 heterodimers .", "( H ) Model of oligomerization of Cik1–Kar3 complexes .", "Oligomer of two complexes is shown .", "Blue arrows show velocities of movement of each independent heterodimer .", "Interconnected tails act as a spring-like linker in between two moving heads .", "( I , J )", "Velocity of the movement and run length as a function of size .", "Blue squares are experimental data , red circles are results of theoretical modeling ( for parameters seeFigure 2—figure supplement 3 ) .", "The bars show average value and standard deviation . DOI: http://dx . doi . org/10 . 7554/eLife . 04489 . 00610 . 7554/eLife . 04489 . 007Figure 2—figure supplement 3 . Mathematical model of the cooperative kinesin movement .", "( A ) Schematics of the kinesin team .", "In the depicted case , the team consists of three kinesins and two of them are shown as bound to the microtubule and move at the velocities ν→1 and ν→3 , respectively .", "Since ν1>ν3 the elastic linkage between the dimers stretches generating the forces f1→ and f3→ .", "( B ) Representation of the force–velocity relation used in the model .", "( C ) Representation of the dependence of the unbinding rate on the applied force .", "( D ) Example of a simulation for a complex size with three dimers .", "The blue graph is the position of the center of mass of the complex as a function of time ( left axis ) , red graph shows the number of dimers attached to the microtubule at each time point .", "( right axis ) .", "( E–F )", "Velocity , run length and their standard deviations as a function of the complex size for different values of kstiff .", "Other parameters of the simulation are: V = 50 nm/s , σν = 20 nm/s; Fstall = 1 pN; kON = 0 . 55 s−1; kOFFo = 0 . 08 s−1; kOFFMAX = 4 s−1 .", "( G ) Comparison between experimental data ( blue ) and best fit ( see values of objective function in Table 5 ) of the simplified theoretical model without force dependence ( red ) .", "( H–I )", "Velocity , run length and their standard deviations as a function of the complex size for best parameters of the complete model .", "Data are shown for red—Fstall = 0 . 2 pN; kON = 0 . 57 s−1; kOFFMAX = 3 . 7 s−1; magenta—Fstall= 1 pN; kON = 0 . 55 s−1; kOFFMAX = 4 s−1; green—Fstall = 4 pN; kON = 0 . 49 s−1; kOFFMAX = 5 . 3 s−1; Other parameters for all simulations: V = 50 nm/s , σν = 20 nm/s; kOFFo = 0 . 08 s−1; kstiff = 0 . 03 pN/nm . DOI: http://dx . doi . org/10 . 7554/eLife . 04489 . 00710 . 7554/eLife . 04489 . 008Video 1 . Time-lapse two-color TIRF microscopy of Cik-Kar3-TMR motors ( red ) moving on taxol stabilized microtubules ( blue ) .", "100 frames were taken every 3 s .", "The video is played at 20 frames/s , scale bar: 5 μm .", "The video corresponds to Figure 2A . DOI: http://dx . doi . org/10 . 7554/eLife . 04489 . 00810 . 7554/eLife . 04489 . 009Video 2 . Behavior of Cik1–Kar3 in microtubule overlap zones .", "Two-color time-lapse TIRF video of Cik1–Kar3-TMR ( red ) moving on taxol-stabilized microtubules ( blue ) .", "Note back and forth movement of individual Cik1–Kar3 motors in microtubule overlap zones .", "100 frames were taken every 3 s , the video is played at 20 frames/s , scale bar 5 μm .", "The video corresponds to Figure 2F . DOI: http://dx . doi . org/10 . 7554/eLife . 04489 . 009 The processive movement of Kar3 may either be a property of individual heterodimers or alternatively require the formation of motor ensembles combining multiple catalytic domains that coordinate stepping .", "Photobleaching experiments in the absence of nucleotide , in which the motor is persistently bound to the microtubule , revealed that 43 out of 50 molecules lost fluorescence in a single step consistent with the presence of a single Cik1–Kar3 heterodimer ( Figure 2—figure supplement 1A–D ) .", "Mixing experiments combining TMR- with Alexa488 labeled Cik1–Kar3 molecules prior to imaging showed that the majority of moving complexes displayed exclusively either red or green fluorescence ( Figure 2—figure supplement 1E , Video 3 ) .", "To discriminate between processive and non-processive motility modes by an alternative approach , we varied the motor concentration in a standard microtubule gliding assay .", "Titration of Cik1–Kar3 over a 50-fold concentration range revealed no decrease in velocity for microtubule gliding , a characteristic feature of processive motility ( Hancock and Howard , 1998 ) ( Figure 2—figure supplement 1F ) .", "Contrary to previous reports , we did not observe substantial depolymerization of taxol-stabilized microtubules in the presence of Cik1–Kar3 .", "On dynamic microtubules motors moved towards the minus-ends , overall microtubule dynamics appeared unchanged in the presence of Cik1–Kar3 and catastrophes did not coincide with plus-end localization of the motor ( Figure 2—figure supplement 1G ) . 10 . 7554/eLife . 04489 . 010Video 3 . Mixing experiment to demonstrate processivity of individual Cik1–Kar3 heterodimers .", "Cik1–Kar3-TMR motors ( red ) were mixed with Cik1–Kar3-Alexa488 motors ( green ) and imaged by multi-color TIRF microscopy on taxol-stabilized microtubules .", "Red and green motors are seen moving in opposite directions because of closely spaced microtubules with opposite orientation .", "Frames were taken every 3 s for 100 frames , the video is played at 20 frames/s .", "Scale bar corresponds to 5 μm .", "The video corresponds to Figure 2—figure supplement 1E . DOI: http://dx . doi . org/10 . 7554/eLife . 04489 . 010 Quantification of TMR brightness of moving motors allowed us to compare the motile properties of Cik1–Kar3 heterodimers vs larger teams that consisted of two or more heterodimers ( Figure 2—figure supplement 2A–G ) .", "We found that Cik1–Kar3 velocity was largely independent of motor team size ( Figure 2—figure supplement 2H , I ) .", "The run length , however , increased with larger team size while the variance of the velocity decreased ( Figure 2—figure supplement 2J ) .", "These motile behaviors of Cik1–Kar3 complexes of different sizes can be quantitatively explained by a biophysical model in which individual motors influence each other through mechanical coupling with spring-like properties ( Figure 2—figure supplement 3 and Supplementary file 1 ) .", "Importantly , a key feature of the biophysical model is the ability of an individual Cik1–Kar3 heterodimer to move processively .", "We next sought to establish the molecular requirements for Kar3 motility: processive movement could be a property intrinsic to the head domains or require secondary microtubule interaction sites in the motor tails ( Gudimchuk et al . , 2013; Su et al . , 2013 ) .", "To distinguish between these possibilities , truncation constructs were designed , systematically eliminating parts of the tail , coiled-coil , or globular domains of either the motor or the partner protein ( Figure 3A ) .", "Truncation of the amino-terminal tail domain , either in Kar3 ( up to aa 174 ) or in Cik1 ( up to aa 250 ) allowed robust processive movement in the single molecule assay and also had little effect on multi-motor gliding velocity .", "This distinguishes Cik1–Kar3 from the homodimeric Drosophila kinesin-14 , Ncd , which has been shown to be capable of a weakly processive motion at very low ionic strength depending on its tail region that acts as an electrostatic tether to microtubules ( Furuta and Toyoshima , 2008 ) .", "Further truncations of the coiled-coil interfered with heterodimer formation ( not shown ) .", "In contrast , carboxyterminal truncations that either completely eliminated the predicted globular Cik1 motor homology domain ( Cik11–360 ) or removed a portion of the carboxyterminus ( Cik11–521 ) had severe effects and prevented directional movement .", "The specific nature of the defect was most apparent for the shorter truncation Cik11–521–Kar3 , which was able to bind microtubules under our standard conditions , but instead of smooth translocation it displayed erratic forward and backward displacements that did not lead to directional movement as revealed by kymographs ( Figure 3B , Videos 4 and 5 ) .", "The defect imposed by the Cik11–521 mutation was also readily apparent in multi-motor gliding assays , where microtubules frequently switched their direction of movement and displayed little net transport ( Figure 3C ) .", "The pronounced defect of the Cik11–521 mutant points to an essential role for the non-catalytic head in the motility mechanism .", "To corroborate this point , we also co-overexpressed Flag-tagged Kar3 with Kar3-Halo and purified fluorescently labeled Kar3 homodimers that can form in the absence of Cik1 ( Chu et al . , 2005 ) .", "We failed to observe processive movement of Kar3-Kar3 combining two catalytic domains or of Kar3-Kar3rigor complexes combining catalytically active and inactive Kar3 heads ( Figure 3A ) .", "These results point to a specific role of the non-catalytic Cik1 head that cannot be simply replaced by a second catalytically active or inactive Kar3 head . 10 . 7554/eLife . 04489 . 011Figure 3 . Molecular requirements for processivity and identification of a translocation-deficient Cik1 mutant .", "( A ) Schematic showing analyzed Cik1 and Kar3 truncation constructs with the corresponding results from TIRF assays and multi-motor gliding assays .", "All constructs contained the Halo-tag at the aminoterminus of Kar3 for fluorescent labeling with TMR .", "( B ) Kymographs of TMR-labeled Kar3 complexes containing either full-length Cik1 ( aa 1–594 ) or the carboxyterminal truncation mutant Cik11–521 .", "See Videos 4 and 5 .", "( C ) Kymograph of microtubule-gliding by full-length Cik1–Kar3 and the Cik11–521-Kar3 mutant .", "Note the impairment of directional translocation by the Cik11–521–Kar3 mutant . DOI: http://dx . doi . org/10 . 7554/eLife . 04489 . 01110 . 7554/eLife . 04489 . 012Video 4 . Wild-type Cik1–Kar3-TMR in the presence of 5 mM ATP moving on taxol-stabilized microtubules imaged at higher frame rate in single color with TIRF microscopy .", "Time between frames is 273 ms , the frames are numbered in the upper left corner , total length of the video is 54 s .", "The video is played at 20 frames/s .", "Scale bar is 3 μm .", "The video corresponds to Figure 3B . DOI: http://dx . doi . org/10 . 7554/eLife . 04489 . 01210 . 7554/eLife . 04489 . 013Video 5 . Cik1–Kar3-TMR with truncation of the non-catalytic Cik1 globular domain ( Cik1 1–521 ) interacting with taxol-stabilized microtubules in the presence of 5 mM ATP .", "Frame rate and imaging conditions as in Video 5 .", "Note non-directional movement of Cik11–521-Kar3 .", "Scale bar 3 μm .", "The video corresponds to Figure 3B . DOI: http://dx . doi . org/10 . 7554/eLife . 04489 . 013 To analyze the motile cycle in more detail , we imaged single Cik1–Kar3 motors in different nucleotide states by TIRF microscopy with high temporal resolution .", "In the absence of nucleotide ( +Apyrase ) and in the presence of the non-hydrolyzable ATP analog AMP-PNP Kar3 motors bound with high affinity to the microtubule but displayed no movement ( Figure 4A ) .", "Binding under these conditions was sensitive to ionic strength with on-rate and lifetime of motor–microtubule interactions decreasing as the salt concentration was raised from 30 mM to 100 mM KCl ( not shown ) .", "In the ADP state , motors displayed diffusive microtubule interactions ( D = 0 . 061 ± 0 . 003 μm2/s ) with short residence times ( τ = 0 . 6 ± 0 . 1 s ) ( Figure 4B , C ) . 10 . 7554/eLife . 04489 . 014Figure 4 . Single-molecule analysis of Kar3 motors in different nucleotide states .", "( A ) Kymographs showing single molecule TIRF microscopy of Cik1–Kar3 motors in different nucleotide states .", "Videos were taken with high temporal resolution ( 35 frames per second ) .", "Note the different motor concentrations and the directional displacement of Cik1–Kar3 molecules in the presence of ATP .", "( B ) Diffusive movement of individual Cik1–Kar3 molecules in the presence of ADP .", "( C ) MSD analysis of the diffusive movement of Cik1–Kar3 motors in the presence of ADP .", "Molecules with lifetimes between 0 . 5 and 5 s were analyzed .", "( D ) Typical kymographs of Cik11–360–Kar3 motors lacking the non-catalytic head domain in no-nucleotide ( Apyrase ) , AMPPNP and ATP states .", "Note the diffusive interactions of the motor with the microtubule in comparison to 3A .", "( E ) Mean-squared displacement ( MSD ) analysis of Cik1–Kar3 and Cik11–360–Kar3 motors in the presence of ATP .", "Data points were fitted to the formula <x2> = a·tn .", "n = 1 for Cik11–360–Kar3 indicates random diffusion without bias and constraints , while n = 2 for the wild-type motor indicates directional , processive movement .", "( F ) Quantitative comparison of microtubule interactions of wild-type vs Cik11–360–Kar3 motors in no nucleotide and AMPPNP states by mean squared displacement analysis .", "( G ) MSD analysis of wild-type vs Cik11–360–Kar3 motors in the ADP state .", "( H ) Summary of the diffusion coefficients obtained for wild-type vs Cik11–360-Kar3 motors in three different nucleotide states .", "Note the logarithmic scale on the y-axis .", "( I ) Typical kymographs of a monomeric Kar3 head construct ( residues 353–729 fused to an N-terminal Halo tag ) in the presence of ATP . DOI: http://dx . doi . org/10 . 7554/eLife . 04489 . 014 What is the contribution of the non-catalytic domain to the translocation mechanism ?", "To gain insights into this question , we quantitatively compared the microtubule-binding properties of single wild-type Cik1–Kar3 molecules with a mutant that lacks the globular Cik1 motor homology domain ( Cik11–360–Kar3 ) ( Figure 4D ) .", "In contrast to wild-type motors , the Cik11–360–Kar3 mutant displayed short-lived diffusive interactions with microtubules ( Figure 4D ) .", "MSD data obtained in ATP for wild-type and mutant were fitted to the equation <x2> = a·tn .", "n = 1 suggests standard diffusion and in this case , a = 2D , where D is the one-dimensional diffusion coefficient .", "For the mutant , an optimal fit was obtained with n = 1 . 01 ± 0 . 04 , indicating unconstrained diffusion with no directional component .", "By contrast , n = 1 . 96 ± 0 . 03 was obtained for the wild-type motor , consistent with directional processive movement ( Figure 4E ) .", "Further MSD analysis indicated that the mutant is about 20-fold more diffusive than the wild-type motor in the no nucleotide state ( D = 0 . 01 ± 0 . 0006 μm2/s vs 0 . 00045 ± 0 . 00011 μm2/s ) and about 30-fold more diffusive in the AMPPNP state ( D = 0 . 0036 ± 0 . 00011 μm2/s vs 0 . 00011 ± 0 . 00001 μm2/s ) ( Figure 4F ) .", "Further analysis indicated that the Cik11–360 mutation also increased diffusion in the ADP state , but the difference was less pronounced in comparison to the other states ( Figure 4G , H ) .", "We additionally constructed and purified a monomeric Kar3 head encompassing residues 353–729 with an N-terminal Halo-Tag .", "Under standard imaging conditions in the presence of ATP only very short-lived microtubule interactions without directional movement could be observed ( Figure 4I ) .", "Taken together , these results indicate only a heterodimer containing a non-catalytic Cik1 head is able to move processively and a key contribution of the non-catalytic domain is to promote tight , non-diffusive binding of the motor in no nucleotide and AMPPNP states .", "Having established that the non-catalytic partner is critical for achieving mechanically processive Kar3 , we sought to gain further insights into the underlying mechanism by studying the alternative Vik1–Kar3 motor .", "The non-catalytic proteins Cik1 and Vik1 are paralogs that display about 45% sequence similarity between each other .", "We purified Vik1–Kar3 complexes from yeast extracts and imaged their interaction with microtubules under the same conditions as previously employed for Cik1–Kar3 ( Figure 5A ) .", "Interestingly , Vik1–Kar3 also displayed highly processive movement , but with faster velocity compared to Cik1–Kar3 .", "Under standard assay conditions , Vik1–Kar3 complexes moved at 234 ± 29 nm/s towards microtubule minus-ends where they strongly accumulated ( Figure 5B , Video 6 ) .", "To ask how these differences might be determined by the non-catalytic partner , we constructed a chimeric protein in which the globular motor homology domain and the neck of Vik1 ( aa 351–647 ) were fused to the tail domain of Cik1 ( aa 1–353 ) ( Figure 5C ) .", "The Vik1–Cik1 chimera formed a stable heterodimer with Kar3-HALO and supported movement with 188 nm/s to the minus-end , significantly faster than Cik1–Kar3 and approaching the velocity of Vik1–Kar3 ( Figure 5D , E ) .", "Thus , the globular non-catalytic domain is a key determinant for setting the velocity of the motor .", "The engineered chimeric protein was able to functionally replace Cik1 in cells as demonstrated by its ability to rescue the phenotypes of a Cik1 deletion and to support growth at wild type level under all tested conditions ( Figure 5F ) . 10 . 7554/eLife . 04489 . 015Figure 5 . The non-catalytic head domain determines the velocity of the Kar3 motor .", "( A ) Schematic representation of the kinesin-14 Vik1–Kar3 and purification of recombinant Vik1–Kar3 from yeast extracts .", "Motors are covalently labeled with Tetramethylrhodamine ( TMR ) via a HaloTag on the amino-terminus of Kar3 .", "Coomassie-stained SDS-PAGE shows homogeneity of the motor preparation and fluorescent labeling of the Kar3 subunit .", "( B ) Kymographs of single color time lapse TIRF microscopy highlighting the velocity difference between Vik1–Kar3 and Cik1–Kar3 when moving along taxol-stabilized microtubules .", "( C ) Construction and purification of a chimeric motor in which the non-catalytic heads were switched .", "An asterisk denotes a proteolytic degradation product .", "( D ) Kymograph of typical single molecule runs by the chimeric Cik1Tail–Vik1Head motor .", "( E ) Comparison of velocities of the different Kar3 constructs .", "( F ) Serial dilution growth assay of the indicated yeast strains at different temperatures and conditions .", "Note that the integration of the chimeric Cik1Tail–Vik1Head fusion protein fully rescues the phenotypes of a Cik1 deletion . DOI: http://dx . doi . org/10 . 7554/eLife . 04489 . 01510 . 7554/eLife . 04489 . 016Video 6 . Vik1–Kar3 motors display processive movement and pronounced minus-end dwelling .", "Two-color time lapse TIRF microscopy of Vik1-Kar3-TMR ( red ) on taxol-stabilized microtubules ( blue ) .", "Note strong accumulation of motors at minus-ends at the end of the video .", "100 frames were taken every 3 s , video is played at 20 frames/s , scale bar is 5 μm .", "The video corresponds to Figure 5B . DOI: http://dx . doi . org/10 . 7554/eLife . 04489 . 016 In addition to directly controlling the motile characteristics of individual Kar3 motors , the non-catalytic partners may also provide functional diversification by allowing differential interactions with regulatory proteins or cargos .", "To identify such interactors , we performed affinity purifications of Cik1–FLAG at different cell cycle stages and analyzed associated proteins by mass spectrometry .", "In addition to Kar3 , we reproducibly mapped two plus-end tracking proteins Bim1 and Bik1 , the EB1 and CLIP-170 homologues of budding yeast , as well as the microtubule-binding Ndc80 kinetochore complex ( Figure 6A ) .", "By performing pull-down assays with recombinant proteins , we established that Cik1–Kar3 , but not Vik1–Kar3 , directly interacted with Bim1 ( Figure 6B ) .", "The binding interface involved the C-terminal EB homology domain of Bim1 and the aminoterminal tail domain of Cik1 ( Figure 6—figure supplement 1A–D ) .", "A physical interaction with Bim1 may account for the previously observed microtubule plus-end localization of Cik1–Kar3 in vivo ( Sproul et al . , 2005; Gardner et al . , 2008 ) .", "Live cell imaging showed that the localization of Kar3 to distinct foci along the yeast spindle , as well as to the tips of shmoo-tip-directed microtubules in alpha-factor-arrested cells ( Figure 6—figure supplement 1E ) , was abolished in a bim1Δ strain , pheno-copying a cik1 deletion ( Manning et al . , 1999 ) ( Figure 6C , D , E ) .", "Consistent with our biochemical experiments , spindle localization was maintained in the Cik11–360 and Cik11–521 mutants .", "Thus , the Cik1 tail domain specifies the differential localization of Kar3 in vivo by allowing a direct interaction with Bim1 . 10 . 7554/eLife . 04489 . 017Figure 6 . Cik1 specifies differential interactions of Kar3 motors to determine their subcellular localization .", "( A ) Affinity purification of the Cik1–Kar3 complex from different cell cycle states and identification of interaction partners by mass spectrometry .", "( B ) Pull-down assay with GST-Bim1 and Cik1- or Vik1-Kar3 .", "Only Cik1-Kar3 interacts with Bim1 in a dose-dependent manner .", "( C ) Localization of Kar3 in different deletion mutants in yeast investigated by live cell microscopy .", "( D ) Panel shows line scans of Kar3–GFP intensity along the spindle axis .", "Arrowheads point to Kar3 foci along the spindle .", "Scale bar: 3 μm .", "Both Cik1 and Bim1 are required for the localization to the anaphase spindle .", "( E ) Quantification of Kar3 foci on anaphase spindles in the indicated yeast strains .", "Kar3 spindle signals were never detected in a bim1Δ or cik1Δ strain .", "( F ) The Cik11–521 and Cik11–360 mutants elicit a temperature-sensitive phenotype in vivo .", "Growth assay was performed with serial dilutions of the indicated yeast strains at different temperature on rich medium ( YPD ) .", "( G ) Quantification of chromosome segregation in the indicated yeast strains at 25°C , n = 100 for all strains analyzed .", "Right panel shows representative fluorescent micrographs with segregation of fluorescently labeled Chromosome V . DOI: http://dx . doi . org/10 . 7554/eLife . 04489 . 01710 . 7554/eLife . 04489 . 018Figure 6—figure supplement 1 . Biochemical and genetic characterization of the Cik1-Kar3-Bim1 interaction .", "( A ) Analytical size exclusion chromatography of Cik1–Kar3 and Bim1 individually or in combination .", "Shift to earlier elution position indicates complex formation .", "( B ) Sequence alignment of the amino-termini of selected Cik1 and Vik1 proteins from diverse yeasts .", "Note the presence of an SxIP motif ( EB1 interaction motif ) in Cik1 proteins that is absent from Vik1 .", "( C ) Mutation of the SxIP motif in Cik1 does not impair binding of the Cik1–Kar3 complex to Bim1 in GST-pull down assays .", "( D ) The more extended amino-terminus of Cik1 is required for Bim1 binding .", "GST-Bim1 pull-down assays with indicated Cik1–Kar3 truncation mutants .", "Elimination of the first 250 amino acids in Cik1 impairs binding of the motor complex to Bim1 .", "( E ) Localization of Kar3 to the tips of shmoo-directed microtubules in alpha-factor arrested cells depends on Bim1 .", "Live cell microscopy of Kar3-3xGFP ( green ) in either wild-type ( upper panel ) or bim1-deletion mutant ( lower panel ) .", "Note the lack of microtubule plus-end localization of Kar3 in the bim1 deletion mutant .", "( F ) Mutation of the conserved SxIP motif in Cik1 does not cause a growth phenotype in vivo .", "Spot assays were carried out with the indicated strains either on YPD plates or on YPD plates supplemented with 100 mM Hydroxyurea .", "( G ) Binding of Cik1–Kar3 to Bim1 occurs via the cargo-binding domain .", "GST-pull down assays with full-length Bim1 or versions constituting the microtubule ( Lampert et al . , 2013 ) in the absence of the motor . DOI: http://dx . doi . org/10 . 7554/eLife . 04489 . 018 The insights into the localization determinants of the Kar3 motor allowed us to evaluate the key roles of Kar3 in the cell: yeast strains expressing Cik11–360 or Cik11–521 from the endogenous chromosomal locus displayed slow growth at 25°C and were inviable at 37°C , similar to a cik1 deletion ( Figure 6F ) .", "By contrast neither a bim1 deletion , which eliminates spindle localization , nor a vik1 deletion , removing spindle pole localization , displayed a pronounced growth phenotype ( data not shown ) .", "Together with our finding that the Vik1head–Cik1tail chimera fully supports viability , this implies that the key function of Kar3 in vegetatively growing yeast cells requires motility—supported by any of the two non-catalytic heads—and the Cik1 tail domain , but does not involve spindle localization via Bim1 .", "Based on previously characterized mutants and on our finding that the Ndc80 complex co-purifies with Cik1 , this supports a key role for Kar3 at the kinetochore .", "Consistent with this notion , we found that 80% of Cik11–521 cells arrested as large budded cells upon shift to the restrictive temperature , indicative of mitotic checkpoint activation .", "Analysis of chromosome segregation at the semi-permissive temperature of 25°C by fluorescent labeling of chromosome V showed that 50% of large budded Cik1 mutant cells attempted nuclear division without proper bi-orientation of sister chromatids ( Figure 6G ) .", "We conclude that the key contribution of the non-conventional Cik1 motility mechanism for cell proliferation lies in the promotion of sister chromatid bi-orientation in mitosis .", "To ask whether Cik–Kar3 can act as a transport motor , we first performed bulk in vitro recruitment assays in the absence of nucleotide using purified motor proteins and recombinant , fluorescently labeled Ndc80 complex as the candidate kinetochore binding partner revealed in the mass spectrometry experiments .", "In the absence of motor , the Ndc80 complex only weakly associated with taxol-stabilized microtubules under standard conditions ( Figure 7A , B ) .", "Vik1–Kar3 motors strongly decorated microtubules in the absence of ATP , but had no effect on the Ndc80 complex .", "By contrast , combining Cik1–Kar3 and the Ndc80 complex resulted in decoration of microtubules with both molecules .", "Upon lowering protein concentrations , Cik1–Kar3 and the Ndc80 complex co-localized to distinct spots on the microtubule where their intensities were correlated ( Figure 7C ) .", "Inclusion of ATP initiated co-transport of the Ndc80 complex and Cik1–Kar3 towards the minus end ( Figure 7D , Video 7 ) .", "While transporting the Ndc80 complex , the average speed of the motor was 76 ± 15 nm/s ( mean ± SD ) , indicating that cargo binding did not significantly impede movement .", "The value is also in range with the reported velocity for kinetochore transport in vivo ( Tanaka et al . , 2005 ) .", "Microtubule binding by the Ndc80 complex was not required for efficient transport , as shown by effective recruitment and translocation of the microtubule-binding defective calponin-homology domain mutant K122E K204E ( Lampert et al . , 2013 ) ( Figure 7—figure supplement 1 ) .", "This result is in agreement with the observation that Kar3 localizes to unattached kinetochores prior to microtubule binding .", "We conclude that the Ndc80 complex can be transported by the Kar3 motor in vitro and that in addition to promoting processive motility a key function of Cik1 may lie in the differential binding of this complex . 10 . 7554/eLife . 04489 . 019Figure 7 . Processive transport of the Ndc80 kinetochore complex by Cik1–Kar3 motors in vitro .", "( A ) Cik1–Kar3 , but not Vik1–Kar3 recruits the Ndc80 kinetochore complex to taxol-stabilized microtubules in vitro .", "The experiment was performed with TMR-labeled Kar3 motors and fluorescently labeled recombinant Ndc80 complex in the absence of nucleotide .", "( B ) Quantification of the recruitment experiment by analyzing fluorescence intensity of microtubule-bound Ndc80-eGFP complex in the presence of different motor constructs .", "Error bars denote standard error of the mean ( s . e . m . ) ( C ) Co-localization of the Ndc80 kinetochore complex and Cik1–Kar3 motors to distinct signals on the microtubule lattice .", "Right panel shows line scans of fluorescent intensity of Kar3 motors and Ndc80 complex along the length of a microtubule .", "( D ) Kymograph showing triple-color time-lapse TIRF microscopy demonstrating that in the presence of ATP , Ndc80 complexes are processively transported towards microtubule minus ends by Cik1–Kar3 motors . DOI: http://dx . doi . org/10 . 7554/eLife . 04489 . 01910 . 7554/eLife . 04489 . 020Figure 7—figure supplement 1 . Cik1–Kar3 transports a mutant Ndc80 kinetochore complex that is unable to bind MTs . Schematic and kymographs of an effective recruitment and translocation of the microtubule-binding defective calponin-homology domain mutant K122E K204E Ndc80 complex by the motor Cik1–Kar3 .", "The experiment was performed with TMR-labeled Kar3 motors , Alexa647-taxol stabilized MTs , and fluorescently labeled recombinant Ndc80 complex in the presence of ATP . DOI: http://dx . doi . org/10 . 7554/eLife . 04489 . 02010 . 7554/eLife . 04489 . 021Video 7 . Transport of the Ndc80 kinetochore complex by Cik1–Kar3 motors along taxol-stabilized microtubules .", "Triple-color TIRF video showing Ndc80-Alexa488 complex ( green ) , Cik1–Kar3-TMR motors ( red ) , and Alexa647-Taxol stabilized microtubules ( blue ) in the presence of 5 mM ATP .", "Frames were taken every 3 s .", "Note co-transport of Ndc80 complexes and Cik1–Kar3 complexes towards the end of the microtubule .", "Video is played at 20 frames/s , scale bar is 5 μm .", "The video corresponds to Figure 7D . DOI: http://dx . doi . org/10 . 7554/eLife . 04489 . 021" ], [ "Our study provides direct evidence for the processivity of Kinesin-14 motors by analyzing full-length yeast Kar3 motors on the single-molecule level .", "Contrary to other kinesin-14s , we demonstrate that individual Cik1–Kar3 motors can move processively towards the minus-end and show that the non-catalytic Cik1 head domain is functionally required for this activity .", "We further demonstrate that the combination of Kar3 with two different non-catalytic partners generates motors with different motile properties and also allows the differential binding of partner proteins such as Bim1 and the Ndc80 complex ( Figure 8A ) . 10 . 7554/eLife . 04489 . 022Figure 8 . Model for Kar3 motility and function .", "( A ) Functional contributions of the different domains of heterodimeric Kar3 motors analyzed in this study .", "( B ) Model for Cik1–Kar3 motility depending on a non-catalytic head domain .", "Upon ATP uptake a conformational change occurs with the stalk rotating towards the minus end .", "A key role for the non-catalytic domain is to prevent diffusion in this state but allow one-dimensional diffusion in the subsequent ADP state with a bias towards the minus end . DOI: http://dx . doi . org/10 . 7554/eLife . 04489 . 022 The demonstration of Cik1–Kar3 processivity presented here provides evidence that effective translocation along microtubules does not strictly require the conventional hand-over-hand walking mechanism that has been established for conventional Kinesin-1 .", "So far this point has been most clearly demonstrated for the Dynein motor , as recent studies have shown that cytoplasmic dynein is capable of processive movement in vitro despite inactivating mutations in one of the two force-generating AAA ring domains ( DeWitt et al . , 2012; Qiu et al . , 2012 ) , or even as a combination of one active head with a second passive microtubule-binding domain ( Cleary et al . , 2014 ) .", "Also several kinesin motors have been reported to move in violation of the hand-over-hand model: monomeric constructs of the kinesin-3 KIF1A have been shown to move along microtubules in vitro using a biased diffusion mechanism ( Okada and Hirokawa , 1999 ) , but the in vivo function of these motors probably involves dimeric molecules for effective cargo transport ( Tomishige et al . , 2002; Endres et al . , 2006 ) .", "We could not detect evidence for processive movement of monomeric Kar3 , making it unlikely that it works via a KIF1A-type mechanism .", "Among the kinesin-14 family , Ncd can exhibit processive motility but requires at least two homodimers coupled together ( Furuta et al . , 2013 ) .", "A number of studies have shown that inactivating mutations in one of the two heads of conventional kinesin can still allow residual processivity ( Subramanian and Gelles , 2007; Thoresen and Gelles , 2008; Kaseda et al . , 2003 ) .", "We note , however , that in these examples processive movement of the mutant heterodimeric kinesins was compromised compared to wild-type homodimers .", "By contrast , Kar3 does not simply tolerate the loss of a second active head but instead it has evolved a specific requirement for the non-catalytic domain in order to move processively .", "In other words , Kar3 can only walk with a combination of an active and an inactive ‘leg’ .", "A precise elucidation of the stepping mechanism of Kar3 motors will require further biophysical experiments .", "Based on the results obtained in this and in previous studies , we propose a working model that describes the non-catalytic head as a ‘foothold’ for Kar3 .", "Based on recent structural data Kar3 and its partner non-catalytic head may be located on adjacent protofilaments ( Rank et al . , 2012 ) , where the non-catalytic domain could contribute to hold the motor complex in place and prevent diffusion .", "During its ATP cycle , the motor goes through rounds of tightly and weakly bound states .", "When ADP is bound , the motor is in its weakly bound state which allows for one-dimensional diffusion along the microtubule lattice .", "On its own , movement in this state would lack directionality and an additional mechanism providing minus-end directed bias is required .", "We speculate that the presence of Cik1 in combination with the kinesin-14 typical stalk rotation upon ATP binding by Kar3 ( Endres et al . , 2006; Gonzalez et al . , 2013 ) might provide such a mechanism either by shifting the overall position of the molecule or by allowing subsequent directed binding/unbinding ( Figure 7C ) .", "Because of the combination of one foot on the track and a diffusive movement to the next binding site , we refer to this possible mechanism as the ‘skateboard’ model .", "This model can explain a number of our experimental observations: the effect of raising the ionic strength is that initially motor movement occurs with faster velocity , probably by increasing the diffusion term .", "At some point however , stable binding of the non-catalytic domain is prevented and directional displacement is disrupted , similar to the situation in which the motor lacks the motor homology domain .", "In addition , diffusive movement in the ADP state and the observed large variation in movement rates are compatible with the model .", "The biochemical properties of the non-catalytic head binding to the microtubule will have profound effects on the motile characteristics as evidenced by the different properties of Cik1–Kar3 and Vik1–Kar3 motors .", "One of the open questions regarding the mechanism of processivity is how the motor homology domain can provide ‘footing’ in no nucleotide and AMPPNP states yet allow effective diffusion in the ADP state .", "In agreement with previous work , this suggests that similar to conventional kinesins , a form of head-to-head communication must occur in Kar3 motors such that the catalytic head influences the microtubule affinity of the motor homology domain ( Allingham et al . , 2007; Chen et al . , 2011; Duan et al . , 2012; Rank et al . , 2012 ) .", "Such coordination may involve intramolecular strain communicated by mechanical elements between the heads as has been reported for conventional kinesin ( Yildiz et al . , 2008 ) .", "Based on our experiments and the previously reported rates of microtubule shortening in the presence of Cik1–Kar3 , we consider it unlikely that Kar3 functions as a microtubule depolymerase in vivo .", "We note that none of the Kar3 functions poses a strict requirement for such an activity and indeed recent experiments analyzing Kar3 function during karyogamy support the view that its primary function is transport , not shortening ( Gibeaux et al . , 2013 ) .", "What are the implications of Kar3's processivity ?", "The ability to move processively may be used in the cell to enrich Kar3 motors at minus-ends .", "In addition , Kar3 could work more effectively in small teams if it can undergo multiple catalytic cycles before releasing from the microtubule .", "This may be especially important during kinetochore transport , where Ndc80 complexes can provide only a limited number of binding sites for the motor .", "Kar3 heterodimers have probably evolved from conventional Kinesin14-homodimers that performed Ncd-like roles in spindle organization .", "We speculate that with the exclusion of dynein from the budding yeast nucleus additional functional requirements for minus-end directed motility in spindle assembly and kinetochore function arose that could be fulfilled with the ‘invention’ of the Cik1 and Vik1 non-catalytic domains .", "While commonly perceived as conferring a loss of functionality , the pseudo-motor domains have the ability to convert a kinesin-14 into a processive motor and additionally create functional diversity through allowing differential interactions with partners .", "Together this allows Kar3 to function as a processsive sliding and transport motor that substitutes for key roles of dynein in the yeast nucleus .", "Other examples for heterodimeric motors exist in the Kinesin-2 family with different subunits conferring distinct activities ( Brunnbauer et al . , 2010 ) .", "Kar3 motors may therefore constitute an extreme example for such diversification strategies that could be more widely used in other motor families .", "More generally , we note that pseudoenzymes have emerging roles in other cellular contexts , for example in the form pseudo-GTPases during human kinetochore assembly ( Basilico et al . , 2014 ) , or as pseudo-kinases and pseudo-phosphatases in cell signaling ( Boudeau et al . , 2006; Tonks , 2009 ) .", "This may suggest that the use of catalytically inactive enzyme derivatives could be a more widespread strategy employed by the cell .", "The establishment of an in vitro assay for Ndc80 transport serves as a starting point for a detailed analysis of the function and regulation of kinetochore motility .", "Furthermore , the unique design principle of the Kar3 motor , which allows the same catalytic domain to be paired with different non-catalytic heads , generates functional diversity that may also be exploited in nanotechnological applications ." ], [ "The protein coding sequences of S . cerevisae ( S288c ) full-length Kar3 , Cik1 , and Vik1 were amplified by PCR and cloned into the overexpression pESC-TRP ( Agilent Technologies , Santa Clara , CA ) vector .", "The non-catalytic motor subunit was tagged C-terminally with 1xFLAG and the motor itself was fused to a HaloTag ( DHA , Promega ) at the 5′ end of the coding sequence , separated by a 13 amino acid linker .", "Mutated and truncated versions of Cik1 and Kar3 were generated by site-directed mutagenesis PCR ( Phusion , Thermo Scientific ) .", "To determine the TMR-labeling efficiency , Halo-Kar3 was fused with enhanced green fluorescent protein ( eGFP ) , creating Halo-eGFP-Kar3 .", "All motor proteins were overexpressed in budding yeast using the pESC vectors ( Agilent Technologies ) following the manufacturer's instructions .", "In brief , yeast cells harboring the respective pESC plasmid were induced with 2% Galactose at OD600 = 1 . 0 for 7–9 hr , harvested by centrifugation , washed , and frozen as droplets in liquid nitrogen .", "Lysis was conducted in liquid nitrogen using a freezer mill ( Biospec Inc . ) .", "The cell powder was resuspended in lysis buffer ( 25 mM Hepes [7 . 4] , 300 mM NaCl , 1 mM MgCl2 , 5% glycerole , 0 . 1 mM EDTA , 0 . 5 mM EGTA , 0 . 1% Tween-20 , 0 . 01 mM ATP , 0 . 1 mM PMSF supplemented with PhosSTOP Phosphatase Inhibitor Cocktail [Roche] ) .", "The lysed cells were centrifuged twice , first at 43 . 146×g for 20 min and afterwards at 125 . 749×g for 1 hr .", "The resulting supernatant was incubated with M2 affinity agarose ( Sigma–Aldrich ) for 1 hr , gently rotating at 4°C .", "The agarose resin was washed five times with lysis buffer ( adjusted to 150 mM NaCl , 0 . 09% Tween-20 , omitting the PhosSTOP reagent ) .", "Elution of the kinesin heterodimeric constructs from M2 agarose was achieved by applying one resin volume of 3xFLAG peptide at final 2 mg/ml in lysis buffer ( adjusted to 250 mM NaCl , 1 mM DTT , 0 . 09% Tween-20 , omitting ATP and PhosSTOP ) .", "If needed , the elution was loaded onto a cation exchange chromatography ( MonoS 5/50 GL , GE Healthcare ) in running buffer ( 10 mM Hepes [pH 7 . 2] , 150 mM NaCl , 1 mM MgCl2 , 1 mM DTT , 1 mM EGTA ) to remove the FLAG peptide .", "Afterwards a linear salt gradient eluted a single peak , pure motor fraction at 250 mM salt .", "Elution fractions were supplemented with glycerol ( final concentration: 5% ) , snap-frozen in liquid nitrogen and stored at −80°C .", "The protein concentration was measured using the DC Assay kit ( Bio-Rad ) .", "All proteins were pre-cleared by centrifugation using a 0 . 1-μm spin filter ( Millipore ) to remove aggregates before each experiment .", "Labeling of the NH2-terminus of Kar3 with the HaloTag TMR ligand or HaloTag Alexa488 ligand ( Promega , Madison , WI ) was performed during the above-described purification before eluting the kinesin heterodimer from the M2 affinity agarose: the proteins were incubated with 10 μM HaloTag ligand for 3 hr .", "Extensive washing removed unbound TMR ligand and the kinesin was eluted as described before .", "In order to assess the labeling efficiency the Halo-eGFP-Kar3 construct was expressed , purified , and TMR-labeled .", "Observing the motor in our multi-color single-molecule imaging setup in the absence or presence of ATP revealed that >90% of eGFP-labeled kinesins also had a TMR-ligand covalently bound to the HaloTag .", "To obtain a stoichiometric heterodimer of Cik1 and Kar3 , the motor was subjected to analytical SEC .", "The purified motor was loaded onto a Superose 6 PC 3 . 2/30 column ( GE Healthcare ) , and 100 μl fractions were collected and separated by SDS-PAGE .", "Proteins were stained with Coomassie Brilliant blue R250 .", "Expression and purification of the full-length and mutant Ndc80 complex ( Ndc80p-6xHis/Nuf2p-EGFP and Spc24p/6xHis/Spc25p ) was performed as described previously ( Lampert et al . , 2013; Lampert et al . , 2010 ) .", "Bim1 was expressed and purified as described previously ( Zimniak et al . , 2009 ) .", "Peak fractions of motor from the gel filtration experiments were diluted to a final concentration of 60 μg/ml in spraying buffer , containing 100 mM ammonium acatate and 30% ( vol/vol ) glycerol , pH adjusted to 7 . 4 .", "After dilution , the samples were sprayed onto freshly cleaved mica chips and immediately transferred into a Bal-Tec MED020 high vacuum evaporator equipped with electron guns .", "After drying in the vacuum , the rotating samples were coated with 0 . 6 nm Platinum at an elevation angle between 5° and 6° , followed by 9 . 5 nm Carbon at 90° .", "The produced replicas were floated off from the mica chips and picked up on 400 mesh Cu/Pd grids .", "The grids were inspected in an FEI Morgagni 268D TEM operated at 80 kV .", "Electron micrographs were acquired using an 11 megapixel Morada CCD camera from Olympus-SIS .", "Images were examined and analyzed using ImageJ .", "Single-step purified Cik1–Halo–Kar3 was loaded on top of a 4 . 4 ml 5–25% linear sucrose gradient and spun at 50 , 000 rpm for 16 hr using an Sorvall TH-660 rotor and a Sorvall Discovery 90SE centrifuge .", "Fractions ( 270 μl ) were collected and analyzed by SDS-PAGE and Coomassie Brilliant Blue R250 staining .", "The sedimentation value for Cik1-Halo-Kar3 was defined by comaring the mobilities of the motor with linear plots of mobility standards .", "The single molecule motor assays were conceptually designed as described previously ( Korten et al . , 2011; Lampert et al . , 2010; Bieling et al . , 2010 ) .", "Biotin-PEG-SVA- and mPEG-SVA-functionalized coverslips ( Laysan Bio ) were prepared as described ( Lampert et al . , 2010; Jain et al . , 2012 ) .", "Coverslips were assembled onto passivated glass slides using double-sided tape creating a flow chamber .", "First , a solution of 1 mg/ml avidin DN ( Vector Laboratories ) was applied to the chamber for 30 min and exchanged for 1% pluronic F-127 ( Sigma–Aldrich ) in BRB80 ( Zimniak et al . , 2009; Nitzsche et al . , 2010 ) .", "Porcine-derived HiLyte-647-labeled , biotinylated and taxol-stabilized microtubules ( MTs ) were immobilized ( labeled and biotinylated tubulin source: Cytoskeleton Inc . , unlabeled tubulin was purified from pig brains as described previously [Ashford and Hyman , 2006] ) and excess of MTs was washed out with BRB80 buffer supplemented with 5 μM taxol , 0 . 5% ( vol/vol ) β-mercaptoethanol , 4 . 5 μg/ml glucose , 200 μg/ml glucose-oxidase , and 35 μg/ml catalase .", "Single molecule imaging was performed by introducing the TMR- or Alexa488-labeled kinesins at low nanomolar range in assay buffer ( BRB80 , 0 . 33 mg/ml casein , 16 . 6 μM taxol , 0 . 13% [vol/vol] methylcellulose , 0 . 5% [vol/vol] β-mercaptoethanol , 4 . 5 μg/ml glucose , 200 μg/ml glucose-oxidase and 35 μg/ml catalase , 0 . 06% [vol/vol] Tween-20 , the indicated amount of KCl and the respective amount of nucleotide ) into the flow cell .", "Time-lapse videos were recorded at 28°C in 3 s intervals between frames ( if not annotated differently ) using a TIRF microscopy setup described previously ( Lampert et al . , 2010 ) .", "Multi-color imaging was achieved by the use of an external filterwheel ( Ludl Electronic Products Ltd . ) .", "Each channel ( excitation: 488 nm , 561 nm , 639 nm ) was exposed for 100 ms at every time-interval , recorded by a Cascade II EMCCD camera and projected to two-dimensional images ( software: Metamorph [Molecular Devices] , ImageJ ) .", "For photobleaching analysis , the oxygen-scavenger mix was omitted ( β-mercaptoethanol , glucose , glucose-oxidase , catalase ) , and images were recorded at maximum laser power .", "Videos are represented as kymographs ( time-space plot ) or as example single frame ( software: MetaMorph [Molecular Devices] ) .", "High temporal resolution recordings ( as presented in Figure 2 ) have been obtained using custom-made TIRF microscope based on Olympus IX-71 body and Coherent CUBE lasers in a temperature stabilized room ( 21 ± 0 . 1°C ) .", "Images were acquired using a Andor iXon3 897 EMCCD camera and subsequently analyzed using custom-made software written in MATLAB ( MathWorks , Inc ) .", "The microtubule gliding assay was designed as described before ( Nitzsche et al . , 2010 ) .", "For this assay , hydrophobic coverslips were prepared according to the following scheme: sonication in acetone for 15 min was followed by sonication for 15 min in ethanol .", "Coverslips were incubated 1 hr in boiling Piranha solution and rinsed with a lot of water afterwards .", "Then rinsed with 0 . 1 M KOH , MilliQ and dried with nitrogen and immersed in 5% dichlorodimethylsilane in heptane for 1 hr at room temperature .", "Coverslips were rinsed again with MilliQ and sonicated for 5 min , sonicated in chloroform for 5 min and air-dried .", "Motor proteins were attached to the hydrophobic coverslips via application of 0 . 2–20 μg/ml anti-Halo antibody ( Promega ) to the flow chamber .", "After the excess of antibody was washed out with BRB80 supplemented with 0 . 5 mg/ml casein , motors were introduced to the chamber and incubated for 5 min .", "Finally a microtubule-containing solution supplemented with 5 mM ATP and oxygen scavenger mix was perfused into the chamber .", "Microtubules used for this experiment were assembled from porcine tubulin mixed together with HiLyte-647-labeled tubulin ( Cytoskeleton Inc . ) in the presence of 10 μM taxol .", "Time-lapse videos were recorded at 28°C in 3-s intervals between frames using a TIRF microsopy setup described previously ( Lampert et al . , 2010 ) .", "MetaMorph ( Molecular Devices ) software was used to compile images into videos and create kymographs out of individual moving microtubules .", "Velocity of gliding microtubules was calculated based upon the slope of the kymographs .", "The tracking of the proteins was performed automatically using the Definiens Software Suite .", "Prior to the analysis , the image data were processed performing a shading correction and smoothing .", "This was done by applying an 11 × 11 pixel ( 1463 × 1463 nm ) kernel median filter and dividing the original raw image by the filtered image data .", "The resulting image was smoothed , using a 3 × 3 ( 399 × 399 nm ) kernel Gaussian filter .", "The processed image data were searched and segmented for fluorescent signal .", "The signal area was searched for local intensity maxima within a search range of 532 nm .", "Circular objects with a radius of 332 nm were created on the found maxima and used for tracking the identified proteins .", "The tracking of the movement of the TMR- , Alexa-488- , or GFP-labeled proteins was done through linking the proteins frame by frame by direct overlap , using the best fitting overlap .", "Cik1 protein sequences of Saccharomyces cerevisiae ( accession number NP_013925 . 1 ) , Saccharomyces kudriavzevii ( EJT42048 . 1 ) , Saccharomyces arboricola ( EJS44163 . 1 ) and Vik1 sequences from S . cerevisiae ( NP_015070 . 1 ) , S . kudriavzevii ( EJT43871 . 1 ) , S . arboricola ( EJS41496 . 1 ) were retrieved from the National Center for Biotechnology Information ( NCBI ) .", "The Saccharomyces bayanus protein sequences of Cik1 ( WashU_Sbay_Contig651 . 30 ) and Vik1 ( WashU_Sbay_Contig637 . 18 ) were retrieved from the Saccharomyces Genome Database ( SGD ) .", "The sequences were aligned with MAFFT ( version 6 , L-INS-I method ) and further processed with Jalview and colored according to ClustalW .", "Varying amounts ( 0 . 1–1 μM ) of recombinant bait protein ( GST , GST-Bim1FL , GST-Bim10–185 , GST-Bim1185–344 ) were immobilized on 30 μl glutathione sepharose ( GE Healthcare ) in 0 . 5 ml binding buffer ( 25 mM Hepes pH 7 . 2 , 250 mM NaCl , 1 mM MgCl2 , 1 mM EGTA , 0 . 5 mM DTT , 0 . 05% NP-40 ) .", "The binding partner was added at a constant concentration between 0 . 5 and 1 μM and incubation lasted for 1 hr at 4°C .", "Afterwards beads were washed three times with 0 . 5 ml binding buffer and analyzed by SDS-PAGE and Coomassie Brilliant blue R250 staining .", "All modifications were performed in the S288C background ( Supplementary file 2 ) .", "Genetic modifications were introduced by using standard procedures .", "For the spot assay , the desired strains were grown overnight in YPD medium .", "The following day cells were diluted to OD600 = 0 . 6 which was the starting point of a 1:4 dilution series and spotted on YPD or 100 mM hydroxyurea ( HU ) plates .", "Plates were incubated at the indicated temperatures to 2–3 days .", "Imaging strains were grown in synthetic medium containing 2% glucose and imaged on concanavalin A-coated culture dishes ( Matek ) at ambient temperature .", "Eight z stacks with planes 0 . 3 μm apart were acquired at 30 s intervals on an Axiovert 200M microscope ( Carl Zeiss ) using an UPlanSApo 100× NA 1 . 40 oil immersion objective lens ( Olympus ) and a Coolsnap HQ CCD camera ( Photometrics ) .", "Images were projected to two-dimensional images ( SoftWoRx software ) and further analyzed by MetaMorph ( Molecular Devices ) .", "The linescans showing the fluorescence intensity for Kar3-3xGFP on spindles were plotted using ImageJ .", "Desired strains were grown to OD600 = 1 . 2 in YPD , centrifuged , drop frozen in liquid nitrogen , and lysed by freezer mill treatment .", "5 g of yeast powder was dissolved in 10 ml buffer A ( 25 mM Hepes pH 8 . 0 , 2 mM MgCl2 , 0 . 5 mM EGTA pH 8 . 0 , 0 . 1 mM EDTA , 0 . 1% NP-40 , 15% glycerole , 150 mM KCl , 0 . 01 mM ATP , 0 . 1 mM PMSF , 1× protease inhibitor cocktail set IV [Calbiochem] ) .", "The lysed cells were centrifuged twice , first at 43 . 146×g for 20 min and afterwards at 125 . 749×g for 1 hr .", "The resulting supernatant was incubated for 2–3 hr with 100 μl Dynabeads ( Life Technologies ) that were coupled to 50 μl anti-FLAG M2 antibody ( Sigma Aldrich ) .", "Beads were washed three times with buffer A and four times with buffer B ( 25 mM Hepes pH 8 . 0 , 150 mM KCl ) .", "Elution was achieved by using 1 beads volume 2 mg/ml 3xFLAG peptide in buffer B . The elution fractions were analyzed by SDS-PAGE and silver stain .", "For mass spectrometry analysis , an on-bead digest replaced the elution procedure: 500 ng LysC protease was added per 50 μl dynabeads in ammonium bicarbonate buffer and incubated at 37°C overnight .", "The supernatant was filtered and applied to mass spectrometry analysis , which was performed on three independent preparations ." ] ]

[ "Motor proteins of the conserved kinesin-14 family have important roles in mitotic spindle organization and chromosome segregation .", "Previous studies have indicated that kinesin-14 motors are non-processive enzymes , working in the context of multi-motor ensembles that collectively organize microtubule networks .", "In this study , we show that the yeast kinesin-14 Kar3 generates processive movement as a heterodimer with the non-motor proteins Cik1 or Vik1 .", "By analyzing the single-molecule properties of engineered motors , we demonstrate that the non-catalytic domain has a key role in the motility mechanism by acting as a ‘foothold’ that allows Kar3 to bias translocation towards the minus end .", "This mechanism rivals the speed and run length of conventional motors , can support transport of the Ndc80 complex in vitro and is critical for Kar3 function in vivo .", "Our findings provide an example for a non-conventional translocation mechanism and can explain how Kar3 substitutes for key functions of Dynein in the yeast nucleus ." ]

[ "Molecules can be transported around a cell by so-called motor proteins that move along a network of filaments called microtubules .", "Many motor proteins—including the kinesin family of these proteins—can only move in one direction along a microtubule .", "In most cells , kinesins tend to transport other molecules away from the center and towards the cell edge .", "Kinesins can have different structures , but most are made up of two subunits that are joined and work together to create a walking-like movement .", "Each subunit has a region called a motor domain ( also known as its ‘head’ ) that can bind to the microtubule and to a molecule called ATP , which provides the energy required for the motor to step forward .", "Kinesins can be classed either as processive or non-processive motors .", "Processive motors can walk continuously along a microtubule for several hundred steps , whereas non-processive motors fall off after just a few steps .", "A motor protein called Kar3 belongs to a group of non-processive kinesins .", "Kar3 is unusual; unlike most of the motors in this group ( which work together in pairs ) , Kar3 motor protein subunits each bind to and work with non-motor protein subunits , including one called Cik1 .", "The head of the non-motor protein cannot bind to ATP , although it can bind to microtubules .", "This means that the non-motor protein subunits are not provided with the energy to make a stepping motion; this raises questions about how the Kar3 motor protein moves along the microtubule , and whether this affects the roles the motor performs .", "Mieck et al . studied how a molecular motor made up of Kar3 and Cik1 moves along microtubules using sensitive microscopy that allows single molecules to be observed .", "This revealed that , contrary to what is expected from a non-processive motor , Kar3–Cik1 moves long distances on microtubules without detaching from them .", "Further investigation showed that Cik1 provides a ‘foothold’ for the motor , binding it to the microtubule in such a way that allows it to move along the microtubule in the opposite direction to most kinesins .", "In addition , Mieck et al . found that the Kar3–Cik1 motor binds to and transports a protein complex that is crucial for separating chromosomes during cell division .", "A challenge for the future is to understand in even greater detail how the movement of such a motor occurs .", "If it doesn't ‘walk’ like other motors , then how can its motion be explained ?", "The benefits for the cell that underlie why this mechanism evolved also remain to be discovered ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "chromosomes and gene expression", "cell biology" ]

[ [ "To facilitate partitioning of the genome during cell division , dynamic spindle microtubules capture chromosomes at sites called kinetochores , forming end-on attachments .", "These attachments are extremely important , as they allow chromosomes to harness the forces generated by microtubule dynamics to drive movements crucial for proper segregation ( Rago and Cheeseman , 2013; Cheerambathur and Desai , 2014; Cheeseman , 2014 ) .", "During prometaphase , chromosomes use these forces to align at the center of the spindle in a process called congression and subsequently in anaphase , kinetochores couple separating chromosomes to depolymerizing microtubules , powering segregation ( Walczak and Heald , 2008; Kops et al . , 2010 ) .", "Despite this fundamental role for the kinetochore in modulating chromosome dynamics , a recent study reported the surprising discovery that kinetochores are not required for chromosome segregation in Caenorhabditis elegans female reproductive cells ( oocytes ) ; the authors found that kinetochore components are removed from chromosomes during anaphase , and when they perturbed kinetochore assembly , chromosomes still segregated relatively normally ( Dumont et al . , 2010 ) .", "The mechanisms driving chromosomes apart in C . elegans oocytes are not understood but are important to uncover , as they are a departure from the canonical kinetochore-driven mode of segregation and therefore represent a new strategy for controlling chromosome dynamics during cell division .", "The divisions of oocytes differ from other cell types in two major respects .", "First , reproductive cells undergo a single round of DNA replication followed by two meiotic divisions where homologous chromosomes ( in Meiosis I ) and then sister chromatids ( in Meiosis II ) segregate away from one another , generating haploid gametes .", "Second , oocytes of most species lack centrosomes , which in other cells duplicate and then nucleate and organize microtubules , forming the two spindle poles ( Manandhar et al . , 2005 ) .", "Therefore , microtubules are not nucleated from pre-defined poles in oocytes and instead are sorted and organized into a bipolar structure around the chromosomes ( Dumont and Desai , 2012 ) .", "This difference influences the way chromosomes initially associate with spindle microtubules and the mechanism by which they achieve metaphase alignment .", "Work in mouse oocytes has demonstrated that homologous chromosome pairs ( bivalents ) lack end-on kinetochore-microtubule interactions during prometaphase ( Brunet et al . , 1999 ) , and stable attachments are not achieved until they have already congressed to the metaphase plate ( Kitajima et al . , 2011 ) .", "Similarly , we previously found that C . elegans oocytes utilize a unique congression mechanism .", "C . elegans chromosomes are holocentric , so kinetochore proteins form cup-like structures around the ends of bivalents ( in Meiosis I ) and sister chromatid pairs ( in Meiosis II ) ( Monen et al . , 2005 ) ( Figure 1A , orange ) .", "However , we found that microtubule density is low at chromosome ends in prometaphase and instead lateral microtubule bundles run along the sides of chromosomes ( Figure 1A , green ) .", "We also found that the microtubule motor KLP-19 forms a ring around the center of each bivalent ( in Meiosis I ) and at the sister chromatid interface ( in Meiosis II ) ( Figure 1A , red ) and provides a plus-end directed force on the chromosomes .", "Therefore , chromosome movement on the C . elegans acentrosomal spindle during congression is mediated by movement of chromosomes along laterally associated microtubule bundles , facilitated by plus-end directed forces operating on the chromosomes ( Wignall and Villeneuve , 2009 ) .", "The discovery that chromosome segregation in these cells does not require kinetochores ( Dumont et al . , 2010 ) raised the possibility that lateral microtubule associations might also drive chromosome separation , which would represent a new strategy for segregation . 10 . 7554/eLife . 06462 . 003Figure 1 . Poles broaden but remain intact during anaphase , creating open channels .", "( A ) Chromosome organization during C . elegans meiosis .", "Homologous chromosomes ( blue ) undergo recombination to link them together and then reorganize to form a cruciform bivalent with long and short arms .", "This structure condenses further and a holocentric cup-like kinetochore ( orange ) forms around the bivalent ends and a complex of proteins ( red ) forms a ring around the center .", "Following homolog separation , the two resulting chromosomes each form a similar structure , with kinetochores cupping the chromosome ends and a ring around the sister chromatid interface .", "Bundles of microtubules ( green ) associate laterally with chromosomes during both meiotic divisions .", "( B ) Oocyte spindles stained for tubulin ( green ) , DNA ( blue ) , and spindle pole marker KLP-18 ( red ) .", "KLP-18 is at focused poles in metaphase ( top ) and remains as poles broaden in mid-anaphase ( middle ) .", "Chromosomes move past the poles in late anaphase ( bottom ) .", "( C ) Live imaging of worms expressing mCherry-histone ( red ) and GFP-tubulin ( green ) .", "Full projections of entire spindles are shown on the left and partial projections of insets chosen to highlight particular features of spindle organization are on the right .", "Lateral bundles associate with and extend beyond segregating chromosomes in mid-anaphase ( top row ) and maintain association with chromosomes as they move past the poles ( bottom ) .", "( D ) Super-resolution image of an early anaphase oocyte spindle stained for DNA ( blue ) and tubulin ( green ) .", "End-on views show a rotated 3D rendering and highlight the six open channels between segregating chromosomes .", "( E ) Super-resolution image of a late anaphase oocyte spindle stained for DNA ( blue ) and tubulin ( green ) showing microtubule bundles splitting around the chromosomes as they reach microtubule ends .", "Bars = 2 . 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 06462 . 003 Here , we use super-resolution microscopy to reveal that chromosomes remain associated with lateral microtubule bundles during anaphase on C . elegans acentrosomal spindles , and we define a mechanism that facilitates segregation in the context of this organization .", "We find that a balance of forces mediates chromosome dynamics during congression and segregation , where plus-end directed forces originating in the rings are countered by progressive accumulation of minus-end directed forces on the chromosomes .", "Removal of the ring at the metaphase to anaphase transition then shifts this balance , triggering poleward movement .", "Therefore , we have defined a novel mode of chromosome segregation in oocyte meiosis , facilitated by opposing chromosomal forces driving movement along lateral microtubule bundles ." ], [ "When chromosome separation in C . elegans oocytes was demonstrated to be kinetochore-independent , a model was proposed that spindle poles disassemble at anaphase onset and a new microtubule array polymerizes between segregating chromosomes to push them apart; an outward-pushing force generated by these microtubules on the inside surfaces of chromosomes was suggested to be the primary candidate for driving separation ( Dumont et al . , 2010 ) .", "To test this model , we used high-resolution imaging to visualize a component of acentrosomal spindle poles as the spindle reorganized during anaphase ( Figure 1B ) .", "Consistent with previous studies , we found that a number of events occur concomitantly at the metaphase to anaphase transition: ( 1 ) the spindle rotates until it is perpendicular to the cortex , ( 2 ) the spindle shrinks dramatically , and ( 3 ) the poles broaden .", "Then , as anaphase progresses , the spindle elongates again ( Albertson and Thomson , 1993; Yang et al . , 2003; McNally and McNally , 2005; McNally et al . , 2006 ) .", "However , the spindle pole marker KLP-18 remained associated with microtubules throughout anaphase progression despite these morphological changes; the chromosomes moved towards the KLP-18-marked poles and then moved past them in late anaphase ( Figure 1B ) .", "This finding is consistent with earlier work showing that another pole marker , ASPM-1 , also remains at poles during anaphase ( van der Voet et al . , 2009; McNally and McNally , 2011 ) and with previous lower resolution imaging of KLP-18 localization ( Segbert et al . , 2003 ) .", "Therefore , spindle poles broaden but remain intact during anaphase , suggesting that the metaphase microtubule array is reorganized but does not disassemble .", "Since we previously showed that lateral microtubule bundles run alongside chromosomes during congression ( Wignall and Villeneuve , 2009 ) ( Figure 1A ) , we wanted to determine whether these lateral contacts were present during anaphase , potentially to facilitate segregation .", "High-resolution live imaging of oocytes expressing GFP-tubulin and mCherry-histone ( to mark microtubules and chromosomes , respectively ) revealed parallel bundles of microtubules running alongside and extending beyond pairs of separating chromosomes during early- and mid-anaphase ( Figure 1C ) .", "In partial projections ( focused on particular sets of separating chromosomes ) , bundles of microtubules could be observed on both sides of the chromosomes , with a lower density of microtubules in between the separating pair ( Figure 1C , top inset ) , suggesting that chromosomes may move through ‘channels’ as they segregate .", "To better visualize this organization , we used super-resolution imaging to obtain a stack of images through the entire spindle , then made a 3D rendering and rotated the image to achieve end-on views from each pole .", "This imaging revealed six regions of low-microtubule density in the anaphase spindle , representing the six sets of separating homologous chromosomes in C . elegans; this organization was clear in both early- ( Figure 1D , Video", "1 ) and mid-anaphase ( Figure 2A , Videos 2–5 ) .", "Therefore , each separating chromosome pair sits within its own channel in the microtubule array , and each of these channels is open from pole to pole .", "In late anaphase , chromosomes are found at the extreme ends of the microtubules at the spindle poles and the microtubule bundles in the center of the spindle converge , closing the channels ( Figure 1C , E ) .", "However , even at this stage , lateral microtubule bundles maintain contact with the chromosomes , splitting around them at the poles ( Figure 1C , E , insets ) .", "Taken together , these results reveal that chromosomes associate with lateral bundles of microtubules during anaphase and are inconsistent with a model in which a new array of microtubules polymerized between separating chromosomes provides a pushing force on their inside surfaces to drive separation . 10 . 7554/eLife . 06462 . 004Video 1 . Super-resolution imaging revealing six open channels in the early anaphase spindle . Super-resolution image of a fixed oocyte spindle stained for tubulin .", "Video is a rotation of a 3D volume rendering generated using Softworx software ( Applied Precision ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06462 . 00410 . 7554/eLife . 06462 . 005Figure 2 . Ring structures are located within the microtubule channels during anaphase .", "( A , B )", "Super-resolution imaging of an anaphase oocyte spindle stained for DNA ( blue ) , tubulin ( green ) , and ring marker MPM-2 ( red ) .", "( A ) The first column shows a single z-slice highlighting an open channel containing a separating chromosome pair with a flattened ring in between .", "Columns 2 , 3 , and 4 are end-on views ( each a single z-slice , depicting the region denoted by the corresponding dotted line on the first image ) , showing the chromosomes and disassembling rings within microtubule channels .", "( B ) Rings dissociate from the chromosomes; although most of the structures appear flattened , partial rings can still be observed ( ‘asterisks’ ) .", "( C ) Oocyte spindles stained for DNA ( blue ) , ring marker MPM-2 ( red in left column ) , SEP-1/separase ( middle column , red in right column ) , and tubulin ( green ) .", "SEP-1 localizes to cup-like structures and filaments within the spindle during metaphase , consistent with kinetochore localization ( top row ) .", "In anaphase ( bottom row ) , SEP-1 localizes to the rings .", "Bars = 2 . 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 06462 . 00510 . 7554/eLife . 06462 . 006Figure 2—figure supplement 1 . Ring structures are gone in late anaphase . Late anaphase oocyte spindles stained for DNA ( blue ) , ring markers ( SEP-1 , MPM-2 , BUB-1 , respectively; red ) , and tubulin ( green ) .", "At this stage , ring components are no longer detectable in distinct structures .", "Bar = 2 . 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 06462 . 00610 . 7554/eLife . 06462 . 007Video 2 . Super-resolution view of spindle organization in mid-anaphase . Super-resolution image of a fixed oocyte spindle stained for DNA ( blue ) , tubulin ( green ) , and ring marker MPM-2 ( red ) .", "Video shows a 3D volume rendering , generated using Imaris software ( Bitplane ) and the Orthogonal Slice View function is used to show individual slice views within the spindle .", "Individual components blink in and out to highlight particular features of spindle organization; specifically that each pair of separating chromosomes is located within an open microtubule channel , with a disassembling ring structure in between . DOI: http://dx . doi . org/10 . 7554/eLife . 06462 . 00710 . 7554/eLife . 06462 . 008Video 3 . Super-resolution view of spindle organization in mid-anaphase . Super-resolution image of a fixed oocyte spindle stained for DNA ( blue ) , tubulin ( green ) , and ring marker MPM-2 ( red ) .", "This is the same video as Video 2 , but without individual components blinking in and out . DOI: http://dx . doi . org/10 . 7554/eLife . 06462 . 00810 . 7554/eLife . 06462 . 009Video 4 . Super-resolution view of spindle organization in mid-anaphase . Super-resolution image of a fixed oocyte spindle stained for DNA ( blue ) and tubulin ( green ) .", "This is the same sequence as Video 3 , but with only DNA and tubulin displayed . DOI: http://dx . doi . org/10 . 7554/eLife . 06462 . 00910 . 7554/eLife . 06462 . 010Video 5 . Super-resolution view of spindle organization in mid-anaphase . Super-resolution image of a fixed oocyte spindle stained for tubulin ( green ) and ring marker MPM-2 ( red ) .", "This is the same sequence as Video 3 , but with only tubulin and the rings displayed . DOI: http://dx . doi . org/10 . 7554/eLife . 06462 . 010 We previously found that a set of proteins forms a ring around the mid-region of each chromosome pair during congression ( Figure 1A ) ; this ring includes the kinesin motor KLP-19 and the chromosome passenger complex ( CPC ) , which contains the kinase AIR-2/Aurora B ( Wignall and Villeneuve , 2009 ) .", "While AIR-2/Aurora B colocalizes with microtubules in anaphase ( Schumacher et al . , 1998; Speliotes et al . , 2000; Romano et al . , 2003 ) , KLP-19 and a few other ring components stay as rings and then elongate into bar-shaped structures between separating chromosomes ( Dumont et al . , 2010 ) .", "We used super-resolution imaging to further assess the morphology of these structures and to examine their organization in relation to the microtubule channels .", "First , we found that the MPM-2 antibody ( raised against mitotic phospho-proteins [Davis et al . , 1983] ) , which had been previously shown to stain the mid-region of meiotic bivalents ( Kitagawa and Rose , 1999 ) , marked the ring structures in both metaphase and anaphase ( Figure 2A–C ) .", "During anaphase , the MPM-2-marked rings completely dissociated from chromosomes and remained in the center of the spindle ( Figure 2B ) .", "In mid-anaphase , these structures appeared elongated but retained a ring-like appearance ( Figure 2B , asterisk ) , suggesting that the rings flatten as anaphase progresses , forming the bar-shaped structures previously described ( Dumont et al . , 2010 ) .", "Importantly , these structures were located within the six microtubule channels; each open channel contained a pair of separating chromosomes with a disassembling ring between them ( Figure 2A , Videos 2–5 ) .", "In late anaphase , at the stage where microtubule bundles in the central spindle converge ( Figure 1C ) , MPM-2 staining was diffuse and other ring components were also not discernable as distinct structures , suggesting that the rings had disassembled ( Figure 2—figure supplement 1 ) .", "To determine the timing of ring release from chromosomes , we next co-stained with MPM-2 and an antibody against separase ( SEP-1 ) , a protease that cleaves a subunit of the cohesin complex to release sister chromatid cohesion and allow homologous chromosome separation ( Siomos et al . , 2001; Kudo et al . , 2006 ) .", "We found that when the rings were associated with chromosomes in metaphase , SEP-1 localized to cup-like structures surrounding bivalents and to filamentous structures found within the spindle ( Figure 2C ) , a pattern also exhibited by meiotic kinetochore components ( Monen et al . , 2005 ) , consistent with an earlier study ( Bembenek et al . , 2007 ) .", "However , during anaphase , SEP-1 localized to the ring structures; this dramatic relocalization correlated with anaphase onset , as SEP-1 was found on the rings in early anaphase , when the spindle had rotated but chromosomes had barely separated ( Figure 2C ) .", "Therefore , separase likely redistributes to the mid-region of meiotic chromosomes at anaphase onset to cleave cohesin and then remains associated with the ring structures during anaphase , suggesting that ring removal from chromosomes is coordinated with cohesion release .", "Given the organization of the anaphase spindle and the finding that kinetochores are not required for chromosome segregation ( Dumont et al . , 2010 ) , we hypothesized that segregation could be mediated by minus-end directed movement of chromosomes along lateral microtubule bundles .", "To examine this idea , we first assessed chromosome behavior during anaphase after inducing monopolar spindle formation; we previously showed that microtubule bundles make lateral contacts with chromosomes in both bipolar and monopolar spindles ( Wignall and Villeneuve , 2009 ) , enabling us to compare chromosome behavior in these two contexts .", "Assessing monopolar anaphase is particularly interesting because the organization of microtubules in these spindles ( with minus ends in the center and plus ends out ) makes them ideal for dissecting the contributions of plus- and minus-end-directed forces .", "In this case , if minus-end-directed movement drives segregation we would expect to observe movement of separated chromosomes towards the central pole .", "Live imaging of monopolar spindle dynamics demonstrated that in Meiosis I the six bivalents move away from the central pole , oscillate , and then coordinately move inwards to the pole ( Figure 3A , Video 6 ) ; in most cases , polar body extrusion failed so all chromosomes were retained in the cell .", "This behavior was then repeated in Meiosis II , with the 12 separated chromosomes moving out as the monopolar spindle forms , oscillating , and then moving back in ( Figure 3B , Video 7 ) .", "Poleward movement of chromosomes in monopolar spindles has been previously described and was interpreted to represent capture of chromosomes by microtubules followed by a congression-like process that drew them to the center ( Connolly et al . , 2014 ) .", "However , we found that separase colocalizes with ring components when the chromosomes are located near the central pole ( Figure 3C ) , demonstrating that this configuration represents anaphase and not congression . 10 . 7554/eLife . 06462 . 011Figure 3 . Chromosomes move towards microtubule minus ends during monopolar anaphase .", "( A , B )", "Montages of time-lapse imaging of a strain expressing GFP-histone; GFP-tubulin following klp-18 ( RNAi ) to generate monopolar spindles during Meiosis I ( A ) and II ( B ) .", "Chromosomes move away from the pole during monopolar spindle formation and then coordinately move polewards during anaphase .", "( C ) klp-18 ( RNAi ) monopolar oocyte spindles stained for DNA ( blue ) , SEP-1 ( red ) , ring marker MPM-2 ( green in third column ) , and tubulin ( green in fourth column ) .", "When chromosomes are away from the pole in metaphase , SEP-1 localizes to kinetochores and then moves to the ring structures in anaphase .", "( D ) klp-18 ( RNAi ) monopolar oocyte spindles stained for tubulin ( green ) , DNA ( blue ) , and ring marker MPM-2 ( red ) .", "In the top row , MCAK/KLP-7 was also depleted , creating a larger spindle .", "When separated chromosomes move to the pole in monopolar anaphase , the rings stay out .", "Bars = 2 . 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 06462 . 01110 . 7554/eLife . 06462 . 012Figure 3—figure supplement 1 . MCAK inhibition causes larger monopolar spindles . Monopolar oocyte spindles generated by RNAi-mediated depletion of KLP-18 , stained for DNA ( blue ) and microtubules ( green ) .", "The top panel shows a normal klp-18 ( RNAi ) monopolar spindle , and the bottom panel highlights the increase in spindle size following RNAi depletion of MCAK/KLP-7 .", "Bar = 2 . 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 06462 . 01210 . 7554/eLife . 06462 . 013Video 6 . Dynamics of monopolar Meiosis I . Time-lapse imaging of a strain expressing GFP-histone; GFP-tubulin following klp-18 ( RNAi ) to generate monopolar spindles .", "In monopolar Meiosis I , the 6 bivalents move away from the central pole , oscillate , and then coordinately move inwards to the pole .", "Frames were acquired every 10 s and displayed at 5 frames per second . DOI: http://dx . doi . org/10 . 7554/eLife . 06462 . 01310 . 7554/eLife . 06462 . 014Video 7 . Dynamics of monopolar Meiosis II . Time-lapse imaging of a strain expressing GFP-histone; GFP-tubulin following klp-18 ( RNAi ) to generate monopolar spindles .", "In monopolar Meiosis II , the 12 separated chromosomes move away from the central pole , oscillate , and then coordinately move inwards to the pole .", "Frame acquisition same as Video 6 . DOI: http://dx . doi . org/10 . 7554/eLife . 06462 . 014 Though our results are consistent with the idea that separated chromosomes display minus-end directed movement on monopolar spindles , because the anaphase spindle shrinks ( Albertson and Thomson , 1993; Yang et al . , 2003; McNally and McNally , 2005; McNally et al . , 2006 ) another possibility is that chromosomes move towards the central pole as a result of the microtubules shortening and the chromosomes moving inwards passively .", "To investigate this possibility , we induced monopolar spindle formation in a strain that had also been depleted of the microtubule depolymerizing kinesin MCAK/KLP-7 , as we found that MCAK depletion increased spindle size in both metaphase ( Figure 3—figure supplement", "1 ) and anaphase ( Figure 3D ) .", "Following MCAK depletion , monopolar spindles remained large in anaphase , but chromosomes still moved in ( Figure 3D , top row ) , indicating that spindle shrinkage and microtubule depolymerization is not responsible for the inward movement .", "Importantly , we found that while chromosomes move inwards in monopolar anaphase , the ring structures could be observed out towards the periphery of the monopolar spindle , where the plus ends are located .", "We obtained the same result without MCAK depletion; although these monopolar spindles were smaller due to spindle shortening , the ring structures could still be found near the microtubule plus ends when the chromosomes were found at the central pole ( Figure 3C , D ) .", "Therefore , similar to bipolar anaphase , where the rings are left behind in the center of the spindle , in monopolar anaphase they are also removed from chromosomes and remain near the plus ends , presumably where the chromosomes were located when anaphase was induced .", "Our data so far are consistent with the idea that during anaphase in oocyte spindles , minus-end directed forces drive chromosome movement through open microtubule channels to spindle poles .", "This would represent a switch in the dominant force operating on chromosomes since during congression , plus-end directed forces ( including KLP-19 in the ring [Wignall and Villeneuve , 2009] ) promote movement to the metaphase plate .", "To better understand how this switch occurs , we next wanted to determine whether minus-end forces only act on chromosomes in anaphase , or whether they also act on chromosomes during congression , counterbalancing the plus-end forces .", "Therefore , we imaged monopolar spindles that had been arrested in metaphase using a temperature-sensitive mutant in the anaphase promoting complex ( APC ) ( emb-27ts ) ; following shift to the restrictive temperature , oocytes continue to be ovulated , but then arrest at metaphase I ( Golden et al . , 2000 ) .", "Therefore , fixed imaging yields a mixture of oocytes that have been arrested for varying amounts of time .", "Intriguingly , we observed multiple phenotypes in our experiments , potentially reflecting differences in the length of APC arrest .", "First , a significant fraction of structures ( 48/119 ) looked indistinguishable from unarrested prometaphase/metaphase monopolar spindles , with bivalents located away from the central pole and a ring around the center of each ( Figure 4A , top ) .", "In these monopolar spindles , chromosomes tended to be oriented such that the long axis of the bivalent was parallel to the direction of the microtubule bundles , as they are found in unarrested monopolar spindles ( Wignall and Villeneuve , 2009 ) .", "However , we also observed many monopolar spindles ( 42/119 ) where the rings appeared dramatically stretched in the direction of the microtubule plus ends ( Figure 4A ) .", "Some rings appeared to be stretching off the bivalents on both sides ( arrows ) , while others were predominantly stretched off one side ( arrowheads ) ; in the latter case , the bivalents were sometimes turned 90° from their usual orientation .", "These observations are consistent with the interpretation that under metaphase arrest , a plus-end directed force acts on the rings , causing them to stretch , and a counterbalancing minus-end force is exerted on the chromosome , resisting the stretching force; when the ring predominantly stretches off of one side of the bivalent , this tug-of-war can cause the bivalents to rotate .", "As further evidence that minus-end-directed forces act on chromosomes under metaphase arrest , we also observed structures that had chromosomes at the center of the microtubule aster similar to normal monopolar anaphase ( 29/119 ) ; in most of these cases , the rings had come off the chromosomes and were located near the microtubule plus ends , though in a minority of cases the rings were dramatically stretched but still on the chromosomes .", "Inward chromosome movement was not the result of premature release from the metaphase arrest , as the kinetochore component KNL-3 still localized around the bivalents in these structures ( Figure 4B ) ( despite the fact that kinetochore components are normally removed in anaphase [Monen et al . , 2005] ) , and two-cup like kinetochores could be observed on each bivalent , confirming that the homologous chromosomes had not separated and the cells had not initiated anaphase .", "Moreover , the rings often appeared broken ( forming fragments of varying sizes , Figure 4A , asterisks ) , suggesting that they had been aberrantly removed from bivalents without the release of cohesion .", "Taken together , these results indicate that minus end forces act on chromosomes prior to anaphase induction .", "The fact that we observe a mixed population of structures suggests that these forces accumulate under metaphase arrest , causing the rings to stretch and then ultimately be removed from the chromosomes as these increasing forces drive chromosomal movement to minus ends . 10 . 7554/eLife . 06462 . 015Figure 4 . Chromosomes are subjected to minus-end directed forces prior to anaphase .", "( A ) Monopolar oocyte spindles arrested at Metaphase I stained for DNA ( blue ) , tubulin ( green ) , and ring marker MPM-2 ( red ) .", "Top row shows intact rings .", "Rows 2 and 3 show examples of rings stretching , but still near chromosomes ( ‘arrowheads’ show rings predominantly coming off one side of the bivalent , ‘arrows’ show rings off both sides ) .", "Bottom three rows show examples of chromosomes at the pole and ring fragments ( ‘asterisks’ ) at microtubule plus ends or off the spindle .", "( B ) Monopolar oocyte spindles arrested at Metaphase I stained for DNA ( blue ) , kinetochore protein KNL-3 ( green ) , and ring component AIR-2 ( red ) .", "The kinetochore remains intact in spindles with stretched rings , indicating that the cells have not initiated anaphase .", "Bars = 2 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 06462 . 015 Next , we wanted to identify factors that contribute to the minus-end force operating on chromosomes .", "As dynein is an important minus-end-directed motor that plays diverse roles during mitosis and meiosis ( Raaijmakers and Medema , 2014 ) , we set out to determine whether it could also help drive chromosome movement on acentrosomal spindles .", "Previously , dynein has been shown to localize diffusely to the oocyte spindle during prometaphase and metaphase and then further concentrate at poles as the spindle rotates in preparation for anaphase ( Ellefson and McNally , 2009; van der Voet et al . , 2009 ) .", "We confirmed this localization pattern , but we also detected a population of dynein that cupped meiotic chromosomes .", "This chromosomal pool of dynein was difficult to distinguish on prometaphase and metaphase bipolar spindles , potentially due to the fact that dynein localized all over the spindle as well , which could obscure a faint chromosomal pool .", "However , chromosomal localization was more clearly visible on monopolar spindles .", "Since chromosomes are spread out in these structures , the spindle pool of dynein was more separated from the chromosomal pool , allowing visualization of dynein cupping the bivalent ends ( Figure 5A ) .", "On these chromosomes , dynein was often seen asymmetrically localized , with stronger staining on the end of the bivalent pointing outwards towards the microtubule plus ends ( asterisks ) , raising the possibility that dynein may preferentially load from this direction .", "However in many cases , dynein could be observed on both faces of the chromosomes ( arrowheads ) , suggesting either that dynein loading is not always asymmetric , or that asymmetrically loaded dynein can ultimately spread to the entire chromosomal surface .", "In anaphase , we observed dynein at the poleward surfaces of separating chromosomes , again cupping the chromosomal surface ( Figure 5B ) , but undetectable on the inside surfaces ( in the region where chromosomes would have been previously associated before release of cohesion ) .", "These findings are in line with previous work that reported that dynein moves from spindle poles towards the outside surfaces of chromosomes in anaphase ( Dumont et al . , 2010 ) .", "Therefore , dynein localizes to meiotic chromosomes both prior to and during anaphase , placing it in a location where it could provide a minus-end directed force during congression and segregation . 10 . 7554/eLife . 06462 . 016Figure 5 . Dynein contributes to chromosome segregation on oocyte spindles .", "( A ) Monopolar oocyte spindles stained for tubulin ( green ) , DNA ( blue ) , and DHC-1/dynein ( red ) .", "The tubulin and DNA channels were deconvolved , but the dynein channel was not , to show a faint pool of dynein surrounding chromosomes .", "Dynein cups chromosomes , sometimes found enriched on the side facing away from the pole ( ‘asterisks’ ) and sometimes around the entire chromosome ( ‘arrowheads’ ) ; partial projections are shown to highlight particular chromosomes .", "( B ) Dynein localization on anaphase spindles with the same colors as ( A ) , except the dynein channel was deconvolved .", "Dynein localizes adjacent to the outside surface of separating chromosomes .", "( C ) Images of monopolar spindles in a strain expressing GFP-histone; GFP-tubulin following partial dynein RNAi .", "Spindles are larger with chromosomes often located at the extreme ends microtubules; graph depicts chromosome positions on monopolar spindles in dhc-1 ( RNAi ) ( black bars ) compared to control ( gray bars ) .", "( D ) Images of monopolar anaphase spindles in a strain expressing GFP-histone; GFP-tubulin with no treatment ( top row ) or following incubation with 5 μM ciliobrevin for 20 min ( bottom row ) .", "Following dynein inhibition , lagging chromosomes were observed in a significant fraction of spindles ( 15/21 ) .", "( E ) Images of bipolar anaphase spindles following dynein inhibition; DNA is shown in blue and microtubules in green .", "The dynein temperature-sensitive mutant dhc-1 ( ct76ts ) was shifted to the restrictive temperature for 5 ( top ) , 7 . 5 ( middle ) , or 10 min ( bottom ) ; spindles with lagging chromosomes were observed under all three conditions .", "Bars = 2 . 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 06462 . 01610 . 7554/eLife . 06462 . 017Figure 5—figure supplement 1 . Strong dynein inhibition causes spindle defects . Images of a strain expressing GFP-histone and GFP-tubulin to show chromosomes and microtubules , respectively .", "Top row: Strong dhc-1 ( RNAi ) causes defects in oocyte spindle morphology .", "Bottom row: Incubation of worms in high concentrations of the dynein inhibitor ciliobrevin ( 30μM for 30 minutes ) leads to mitotic defects consistent with dynein inhibition , such as errors in pronuclear fusion ( bottom left; [Gonczy et al . , 1999] ) , and monopolar mitotic spindles ( bottom right; [Vaisberg et al . , 1993] ) , showing that ciliobrevin works to inhibit dynein in C . elegans .", "Bars = 2 . 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 06462 . 017 To test whether dynein functionally contributes to chromosome movements , we used multiple inhibition strategies and then assessed the effects on chromosome positioning and segregation .", "These experiments were complicated by the fact that full dynein inhibition ( either by strong dhc-1 RNAi or high concentrations of the dynein inhibitor ciliobrevin [Firestone et al . , 2012] ) resulted in spindle defects ( Figure 5—figure supplement 1 ) .", "Therefore , we used partial or conditional dynein inhibition , first finding conditions where spindle morphology looked normal and then assessing chromosome behavior on those spindles .", "To determine if dynein provides a minus-end directed force on chromosomes , we first assessed whether partial dynein depletion altered chromosome position on monopolar spindles .", "During normal monopolar spindle formation , chromosomes move out away from the pole and oscillate ( Figure 3A , B , Videos 6 , 7 ) ; in fixed images , we found that 34% of chromosomes were found near the plus ends of microtubules , reflecting this dynamic positioning ( Figure 5C , gray bars ) .", "However , significantly more chromosomes ( 60% ) were found in this location following dynein depletion ( Figure 5C , black bars ) , suggesting that dynein generates a minus-end directed force on chromosomes that affects their positioning .", "Consistent with this finding , we also observed lagging chromosomes on anaphase monopolar spindles following partial dynein inhibition .", "While chromosomes usually move coordinately to the pole in monopolar anaphase ( Figure 3A , B ) , a significant fraction of spindles had some chromosomes towards microtubule plus ends upon treatment with low concentrations of ciliobrevin ( Figure 5D; 15/21 monopolar anaphases ) , suggesting that dynein provides a minus-end directed force during anaphase as well as during congression .", "Next , we assessed the effects of dynein inhibition on chromosome segregation on bipolar spindles .", "To reduce the possibility that lagging chromosomes were the result of altered spindle morphology , we took advantage of a temperature-sensitive mutant ( dhc-1 ( ct76 ) ) .", "A previous study reported that dynein is inactivated within a few minutes in this mutant and used short temperature shifts show that dynein plays a role in mitotic chromosome congression , independent of any effects on the spindle ( Schmidt et al . , 2005 ) .", "Therefore , we used the same strategy , shifting worms to the restrictive temperature for short periods and then assessing chromosome segregation .", "Following brief dynein inactivation using this strategy , we observed a significant fraction of anaphase spindles that had lagging chromosomes ( 2/12 after a 5 min shift , 15/20 after a 7 . 5 min shift , and 15/21 after a 10 min shift ) , supporting a role for dynein in chromosome segregation ( Figure 5E ) .", "Integrating our findings , our data support the model that there are opposing forces acting on oocyte chromosomes during congression and segregation , with plus-end directed forces originating in the rings countered by accumulating minus-end forces .", "To further test this idea , we assessed the behavior of chromosomes lacking rings by analyzing mutants where particular sets of homologous chromosomes are unable to pair ( him-8 ( me4 ) and zim-2 ( tm574 ) , where the X chromosome ( Phillips et al . , 2005 ) and chromosome V ( Phillips and Dernburg , 2006 ) are affected , respectively ) , resulting in five bivalents and two unpaired chromosomes ( univalents ) .", "These univalents were not recognized by antibodies against MPM-2 or the CPC component AIR-2/Aurora B , indicating that they do not form ring structures ( Figure 6A , asterisks ) , and consequently each spindle had only five rings .", "However , the univalents were able to load kinetochore components , as BUB-1 , which localizes to both the kinetochore and the ring ( Monen et al . , 2005; Dumont et al . , 2010 ) , coated their outer surfaces ( Figure 6A ) . 10 . 7554/eLife . 06462 . 018Figure 6 . Univalents lacking rings exhibit congression and segregation errors .", "( A ) Oocytes from him-8 ( me4 ) and zim-2 ( tm574 ) worms stained for DNA ( blue ) , AIR-2 or BUB-1 ( red ) , and MPM-2 ( green ) ; each of these strains has two univalents ( ‘asterisks’ ) .", "Rings are not detected on univalents , but kinetochore staining is present ( BUB-1; bottom two rows ) .", "( B ) Metaphase-arrested him-8 ( me4 ) bipolar and monopolar spindles , stained for DNA ( blue ) , AIR-2 ( red ) , and tubulin ( green ) .", "During congression , univalents are randomly positioned on the bipolar spindle ( top two rows ) , but were located near the pole on every monopolar spindle we observed ( bottom ) .", "For quantification of bipolar spindles , univalents were scored as being at the center of the metaphase plate ( zone 1 ) , partially overlapping with the bivalents ( zone 2 ) , or not overlapping ( zone 3 ) .", "Examples of each category are indicated on the images .", "( C ) him-8 ( me4 ) anaphase spindles stained for DNA ( blue ) , MPM-2 ( red ) , and tubulin ( green ) .", "During segregation , univalents are either found near other segregating chromosomes ( bottom ) or lag behind in the center ( top ) .", "Bars = 2 . 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 06462 . 018 Importantly , we found that univalents lacked a plus-end directed force , since in monopolar spindles both univalents were located near the pole in every spindle we quantified ( 40/40 spindles; 80/80 univalents ) ( Figure 6B , bottom panel ) .", "Moreover , we found that univalents exhibited congression defects on bipolar spindles .", "This phenotype was especially apparent following metaphase arrest , as most bivalents are aligned in those conditions , allowing us to more clearly quantify univalent position with relation to the metaphase plate ( Figure 6B ) .", "For quantification , we assessed whether the univalent was found in the middle region of the spindle where the bivalents were located ( Zone 1 ) , close to but not completely overlapping with the bivalents ( Zone 2 ) , or in the regions closer to the poles ( Zone 3 ) .", "Univalents were fairly evenly distributed between these three categories , consistent with the univalents being positioned at random on bipolar spindles .", "Next , we assessed the behavior of univalents during anaphase .", "First , we found that univalents remained as intact units during anaphase and did not release cohesion between sister chromatids .", "This observation is consistent with our finding that separase relocalizes to the rings at the metaphase to anaphase transition ( Figure 2C ) ; since univalents lack ring components ( Figure 6A ) , separase is likely not targeted to them , preventing cohesion release .", "Most often , we found that these intact univalents lagged during anaphase ( Figure 6C , top row; 76% ) .", "However , 24% of univalents did not lag and instead appeared to segregate with the rest of the chromosomes ( Figure 6C , bottom row ) .", "These findings are consistent with the interpretation that the position of the univalent when anaphase is triggered determines its behavior during segregation .", "Univalents that were located close to a pole prior to segregation ( zone", "3 ) likely remain close to that same pole during anaphase; minus-end forces could properly act on that univalent and therefore it would appear to segregate with the rest of the chromosomes .", "However , a univalent located close to the chromosomes ( zones 1 and", "2 ) would become trapped in the center , as it would be caught between overlapping bundles of microtubules , with minus-end forces acting on both sides of the univalent pulling it in opposite directions .", "Therefore , the behavior of univalents during congression and segregation supports a model in which a balance between opposing plus- and minus-end directed forces dictate chromosome position on the acentrosomal spindle and would represent a case in which the plus-end force is eliminated ." ], [ "Taken together , our data support a model in which chromosome segregation on the C . elegans acentrosomal spindle is driven by minus-end directed movement along lateral microtubule bundles ( Figure 7A ) , revealing a new kinetochore-independent strategy for partitioning chromosomes .", "The spindle reorganizes at the metaphase to anaphase transition to enable this mode of segregation , by broadening the spindle poles and thereby creating channels that are open from pole to pole .", "Chromosomes move through these channels until they are located at the tips of the microtubules at the minus ends , at which point the channels close and the spindle disassembles .", "Coupling spindle pole broadening to anaphase onset could potentially serve a dual purpose in regulating chromosome dynamics in the context of our model .", "First , pole broadening would be important in anaphase , as it would serve to open a path for chromosomes to move all the way to the microtubule minus ends .", "Moreover , we speculate that having the poles more focused in prometaphase could serve the opposite purpose; since chromosomes move along lateral microtubule bundles during congression as well , having the poles ‘closed’ at that stage could bias chromosomal movement towards the metaphase plate . 10 . 7554/eLife . 06462 . 019Figure 7 . A balance of forces controls chromosome movement on the acentrosomal spindle .", "( A ) Model depicting microtubules ( green ) , chromosomes ( blue ) , rings ( red ) , and spindle pole proteins ( yellow ) .", "During metaphase , poles are focused and chromosomes are aligned between overlapping microtubule bundles .", "During anaphase , poles broaden creating open channels and separated chromosomes move along lateral bundles until they are past the poles; at this stage , the channels close .", "In anaphase , rings are removed from chromosomes and then flatten and disassemble .", "( B ) Model depicting microtubules ( green ) , chromosomes ( blue ) , rings ( red ) , and dynein ( orange ) .", "During congression , KLP-19 and potentially other ring components provide a plus-end directed force ( ‘red arrows’ ) , moving the chromosome toward the metaphase plate; during this time dynein begins to accumulate on chromosomes , resulting in weak minus-end forces ( ‘small orange arrows’ ) .", "At metaphase , the plus- and minus-end directed forces are each balanced as the chromosome associates with microtubules in an overlap zone .", "Ring removal at the metaphase to anaphase transition shifts this balance , triggering poleward movement .", "In anaphase , more dynein has accumulated on chromosomes , resulting in stronger minus-end forces ( ‘large orange arrows’ ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06462 . 019 We further propose that chromosome movement on acentrosomal spindles is mediated through a balance of plus- and minus-end directed forces operating on chromosomes ( Figure 7B ) .", "During congression , the ring provides a plus-end directed force that promotes movement to the metaphase plate and during the same period minus-end directed forces begin to accumulate on chromosomes .", "As chromosomes then align at the metaphase plate , the plus- and minus-end directed forces would each be balanced , since at that stage the chromosomes are positioned within microtubule bundles of overlapping polarity , with both plus- and minus-ends on each side .", "Since our data support the idea that minus-end forces increase as metaphase progresses , we infer that these forces are likely weak early in congression ( allowing plus-end forces to be dominant ) and then accumulate in preparation for anaphase .", "Once anaphase is triggered , ring removal alters the balance of forces , enabling minus-end forces to propel separated chromosomes to the poles .", "Importantly , our proposed mechanism would directly couple key essential events at the metaphase to anaphase transition , as cleavage of cohesin by separase would also facilitate ring removal from chromosomes , thereby also removing a plus-end directed force .", "Consequently , the outcome would be coordinated poleward movement , driving chromosome segregation .", "A previous study proposed a different model for kinetochore-independent segregation , where the metaphase spindle disassembles from the poles and then a new microtubule array assembles between separating chromosomes , stimulated by ring component CLASP ( CLS-2 ) .", "In this model , a force provided by polymerizing microtubules pushing against the inside surface of separating chromosomes is the primary candidate driving segregation ( Dumont et al . , 2010 ) .", "Our findings are not consistent with key features of this model , since spindle poles do not appear to disassemble and polymerization of a new microtubule array coupled to the inside surfaces of separating chromosomes would be predicted to close the microtubule channels that we observe .", "Moreover , the previous model also does not account for the behavior of chromosomes during monopolar anaphase , where separated chromosomes do not move apart , pushed from the center , but instead move together to the same pole , leaving the ring behind .", "However , the major length changes that occur in the C . elegans oocyte spindle ( with it shrinking and then elongating as anaphase progresses ) make it likely that spindle elongation also contributes to segregation , potentially driven by CLASP-dependent microtubule polymerization as this previous study proposed .", "This process may be analogous to Anaphase B in mitotically dividing cells ( where spindle pole separation drives sets of separating chromosomes further apart [Walczak and Heald , 2008] ) but it would have to occur within the context of the unique spindle organization that we have now defined , with lateral microtubule bundles extending alongside separating chromosome pairs .", "However , even if spindle elongation contributes to segregation , our studies have now revealed another mechanism that operates in parallel , with forces operating on the chromosomes themselves driving poleward movement .", "This segregation mechanism could be viewed as analogous to Anaphase A , but it would be mechanistically distinct from what occurs in mitotically dividing cells , as the chromosome to pole movement that we have described does not require kinetochores and is instead mediated by motor-driven movement along lateral microtubule bundles .", "Our work raises a number of intriguing questions that remain to be addressed .", "First , our current data do not completely explain how chromosomes escape the zone of overlapping microtubules in the center of the spindle to ensure that they move to the correct poles .", "Although dynein is asymmetrically localized on separating chromosomes ( as we do not detect it on the inside surface ) , it is still possible that some motors could associate with microtubule bundles oriented towards the incorrect pole ( Figure 7B ) .", "Therefore , we speculate that an additional mechanism may exist to give the separating chromosomes an initial push apart; this mechanism could involve a morphological change in the ring structure as it is removed ( a constriction or elongation ) , a small amount of CLASP-dependent microtubule polymerization , or a currently undiscovered mechanism .", "It is also possible that the rings could simply serve as physical barriers between separating chromosomes , blocking the center of the channel and therefore preventing chromosomes from moving in the wrong direction , giving motors associated with properly oriented microtubule bundles an advantage .", "Another related question that remains to be addressed is the polarity of microtubules within the bundles and the exact length of the overlap zone , which would rely on careful analysis by electron microscopy .", "We favor the idea that microtubules within the laterally associated bundles are organized primarily , if not completely , with the polarity depicted in Figure 7 , as bundles organized with a defined polarity would best explain the opposing forces we observe acting on rings and chromosomes on monopolar spindles , and the phenotype of dynein depletion on these spindles ( where chromosomes are then positioned closer to the outside of the aster ) .", "However , our studies do not rule out the possibility that the spindle may also contain non-bundle associated microtubules of mixed polarity that could affect chromosome behavior .", "Our studies have also revealed a role for dynein in mediating chromosome dynamics on the acentrosomal spindle .", "First , we found that dynein exhibits chromosomal localization .", "In prometaphase and metaphase bipolar spindles , chromosomal dynein is faint and often difficult to distinguish above the spindle-localized pool , suggesting that there is only a small amount of the motor on the chromosomes .", "However , anaphase localization is more clearly apparent , supporting the idea that dynein accumulates on chromosomes in preparation for driving anaphase movements .", "Pre-anaphase chromosomal dynein localization can be more clearly visualized on monopolar spindles , where the motor is often seen more concentrated on the end of the chromosome facing away from the pole .", "We speculate that this asymmetric pattern could reflect the mechanism by which dynein loads onto the chromosomes , for example , if it walks towards the chromosomal surface from the microtubule plus ends .", "This loading mechanism could possibly facilitate accumulation of the motor on the outer surfaces of chromosomes prior to anaphase , since when chromosomes are at the metaphase plate with overlapping plus ends nearby , dynein loading via this mechanism might be especially efficient , allowing the motor to accumulate on chromosomes in preparation for anaphase initiation .", "We also observed chromosome segregation defects following dynein inhibition , with lagging chromosomes in a significant fraction of both monopolar and bipolar anaphase spindles .", "However , these phenotypes were not completely penetrant , as some chromosomes did exhibit poleward movement in each of these cases .", "One possibility is that this movement is due to residual dynein activity; since dynein also plays a role in spindle assembly , we had to use partial inactivation or a fast-acting temperature-sensitive mutant and each of these strategies could potentially leave a significant pool of active dynein .", "A second possibility is that there are additional factors contributing to the minus-end force operating on chromosomes ( for example , poleward flux or other minus-end directed motors ) .", "This second possibility could also apply to the plus-end directed force; we previously demonstrated that the kinesin motor KLP-19 in the ring provides a polar ejection force using our monopolar spindle assay ( Wignall and Villeneuve , 2009 ) , but it is possible that other factors operate in parallel to promote congression .", "However , even if there are multiple components of either the plus- or minus-end directed forces acting on chromosomes , the mechanism that we propose for segregation , with a hand-off between these forces , could still operate as proposed ( Figure 7B ) .", "Another interesting observation that emerged from our studies is that partial dynein inhibition results in especially large monopolar spindles ( Figure 5C , the spindles shown are approximately 50% larger than normal monopolar spindles ) , suggesting that dynein also regulates spindle length .", "A full understanding of this phenotype will require further experimentation , though our observation is consistent with work in other systems , where dynein depletion has also been reported to cause larger spindles ( Gaetz and Kapoor , 2004; Morales-Mulia and Scholey , 2005 ) .", "However , relevant to this study , larger monopolar spindles were particularly useful in assessing the contribution of opposing forces on the chromosomes; with longer microtubules , the positioning of chromosomes at the extreme plus ends was particularly clear , providing support for the idea of a minus-end directed force provided by dynein acting on the chromosomes .", "In conclusion , our work defines a new mechanism of kinetochore-independent chromosome segregation and highlights a role for lateral microtubule–chromosome associations in driving chromosome movements .", "Notably , lateral interactions have been previously described in mitotically dividing cells and have been shown to play a role in chromosome congression .", "For example , kinetochores associate laterally with microtubules before forming end-on attachments ( Tanaka , 2012 ) , a class of kinesins on chromosome arms promotes congression ( Vanneste et al . , 2011 ) , and chromosomes can congress under experimental conditions where kinetochore-fiber formation is inhibited ( Cai et al . , 2009 ) .", "However , our work now demonstrates that these associations can be the primary drivers of chromosome movement in an unperturbed system , and that in addition to congression , they can also provide the force to power chromosome segregation .", "Interestingly , our findings also have parallels with previous work on the mammalian oocyte spindle .", "First , it has been shown that acentrosomal spindles in mouse oocytes don't have end-on kinetochore microtubule-attachments prior to achieving metaphase alignment ( Brunet et al . , 1999 ) , and fluorescence imaging of prometaphase is suggestive of the presence of lateral microtubule bundles surrounding the chromosomes ( with a lower density of microtubules at the ends ) ( Schuh and Ellenberg , 2007 ) .", "While kinetochore-microtubule attachments are established after metaphase alignment ( Brunet et al . , 1999; Kitajima et al . , 2011 ) and have been demonstrated during anaphase ( FitzHarris , 2012 ) , there is also evidence for kinetochore-independent mechanisms .", "An intriguing study reported that DNA-coated beads injected into mouse oocytes formed acentrosomal spindles and that when anaphase was induced , these beads exhibited dynein-dependent poleward movements ( Deng et al . , 2009 ) .", "Therefore , we suggest that the mechanism that we describe could also operate in mammalian oocytes during anaphase .", "Although the primary driver of chromosome-to-pole movement in these cells may be the canonical kinetochore-driven mechanism , lateral microtubule associations established to promote congression could remain intact in anaphase , and dynein localized on the chromosomes could provide a force to help move chromosomes along these microtubules towards minus ends .", "Therefore , by studying anaphase in C . elegans oocytes , we have revealed a new mechanism driving chromosome segregation on acentrosomal spindles that may be widely applicable to understanding chromosome behavior during oocyte meiosis ." ], [ "‘Wild type’ refers to N2 ( Bristol ) worms , and ‘control’ refers to RNAi vector control , temperature-sensitive strains grown at the permissive temperature , or worms not treated with inhibitor .", "A chart showing all strains used is shown in the Supplementary material ( Supplementary file 1 ) .", "From a feeding library ( Fraser et al . , 2000; Kamath et al . , 2003 ) , individual RNAi clones were picked and grown overnight at 37°C in LB with 100 μg/ml ampicillin .", "Saturated cultures were spun down , plated on NGM ( nematode growth media ) plates supplemented with 100 μg/ml ampicillin and 1 mM IPTG ( isopropyl-β-d-thiogalactopyranoside ) , and dried overnight .", "L1 worms ( synchronized by bleaching gravid adults and hatching overnight without food ) were then plated on RNAi plates and grown to adulthood at 15° for 5 days .", "For partial dhc-1 ( RNAi ) , worms were grown until the L4 stage on regular NGM/OP50 plates and then transferred to the RNAi plate 24–48 hr before imaging .", "Metaphase arrest was achieved by either RNAi-mediated depletion of emb-30 or by shifting emb-27 ( g48 ) worms to 25°C for 4–5 hr .", "Dynein was inhibited by dhc-1 partial RNAi ( described above ) , by shifting dhc-1 ( ct76 ) worms to 26°C for 5–15 min , or by soaking worms for 20 min in 5 μM ciliobrevin ( HPI-4 , Sigma , St . Louis , MO ) .", "Immunofluorescence was performed as previously described ( Oegema et al . , 2001 ) , with slides fixed in methanol for 40–45 min .", "The following antibodies were used: rabbit anti-AIR-2 ( 1:500; gift from Jill Schumacher ) , rabbit anti-BUB-1 ( 1:2800; gift from A Desai ) , rabbit anti- DHC-1 ( 1:100 , gift from P Gönczy ) , rabbit anti-GFP ( 1:500; AbCam , Cambridge , UK ) , mouse anti-GFP ( 1:500; AbCam ) , rabbit anti-KLP-18 ( 1:10 , 000 , gift of O Bossinger ) , rabbit anti-KNL-3 ( 1:3800; gift from A Desai ) , mouse anti-MPM-2 ( 1:500; AbCam and Millipore , Billerica , MA ) , rabbit anti-SEP-1 ( 1:200; gift from A Golden ) , mouse anti-α-tubulin-FITC ( 1:500; Sigma ) .", "Alexa-flour-conjugated secondary antibodies ( Invitrogen , Carlsbad , CA ) were used at 1:500 .", "For most imaging , a Deltavision deconvolution microscope with a 100× objective was used ( Applied Precision , part of GE Healthcare , Piscataway , NJ ) .", "This microscope is housed in the Northwestern Biological Imaging Facility supported by the NU Office for Research .", "Image stacks were obtained at 0 . 2 μm z-steps for fixed samples and deconvolved using SoftWoRx ( Applied Precision ) .", "For images denoted as super-resolution , a Deltavision OMX 3D-SIM with an Olympus ( Shinjuku , Tokyo , Japan ) 100× UPlanSApo 1 . 4 NA objective was used ( Applied Precision ) , located in the Light Microscopy Imaging Center ( Indiana University ) .", "Images were captured at 0 . 125 μm z-steps and processed using SoftWoRx ( Applied Precision ) and IMARIS 3D imaging software ( Bitplane , Zurich , Switzerland ) .", "Control and RNAi worms were processed in parallel , and image exposure times were kept constant within each experiment .", "For live imaging , GFP- or mCherry-expressing worms were picked into a solution of tricaine ( 2% ) and tetramisole ( 0 . 4% ) , and incubated for 40 min .", "Worms were then pipetted onto a 3% agar pad , covered with a coverslip , and imaged immediately .", "Image stacks were obtained at 1 μm z-steps at 10-s intervals using 2 × 2 binning , and then deconvolved .", "Video images are full projections of data stacks encompassing the entire meiotic spindle ." ] ]

[ "During cell division , chromosomes attach to spindle microtubules at sites called kinetochores , and force generated at the kinetochore-microtubule interface is the main driver of chromosome movement .", "Surprisingly , kinetochores are not required for chromosome segregation on acentrosomal spindles in Caenorhabditis elegans oocytes , but the mechanism driving chromosomes apart in their absence is not understood .", "In this study , we show that lateral microtubule–chromosome associations established during prometaphase remain intact during anaphase to facilitate separation , defining a novel form of kinetochore-independent segregation .", "Chromosome dynamics during congression and segregation are controlled by opposing forces; plus-end directed forces are mediated by a protein complex that forms a ring around the chromosome center and dynein on chromosome arms provides a minus-end force .", "At anaphase onset , ring removal shifts the balance between these forces , triggering poleward movement along lateral microtubule bundles .", "This represents an elegant strategy for controlling chromosomal movements during cell division distinct from the canonical kinetochore-driven mechanism ." ]

[ "An animal's genetic material is packaged into structures called chromosomes .", "Most animals have two sets of chromosomes: one from each parent .", "Sperm and egg cells must contain half the number of chromosomes compared to other cells in the body , so that when they fuse , the resulting embryo receives a full complement of chromosomes .", "Egg and sperm cells are made via a type of cell division called meiosis .", "In meiosis , the genetic material of a cell is copied once but then the cell divides twice .", "Therefore , at the end of the two divisions , the resulting sperm or egg cells contain half the number of chromosomes as the original cell .", "During cell division , the genetic material is separated by a structure called the spindle apparatus .", "The spindle is made of protein filaments called microtubules .", "At each end of the spindle , there is a cluster of microtubule ends , known as a ‘pole’ .", "The other ends of the microtubules extend out towards the center of the spindle , where they overlap with the microtubules from the opposite pole .", "The chromosomes line up in the center of the spindle and then the chromosomes are separated , with half moving to one spindle pole , and half to the other .", "In most forms of cell division , the microtubules attach to the chromosomes via sites called kinetochores .", "However , it was recently discovered that kinetochores are not required to separate chromosomes to make egg cells in the worm C . elegans , suggesting that these chromosomes associate with the spindle in a different way .", "Muscat , Torre-Santiago et al . have now used high-resolution imaging to look at this chromosome separation process in more detail and to figure out how the chromosomes separate when C . elegans forms egg cells .", "The experiments revealed that the chromosomes move within the spindle along parallel microtubule bundles , much like trains moving along a track .", "The chromosomes are moved into position at the center of the spindle by a ring-shaped group ( or ‘complex’ ) of proteins that forms around the center of each chromosome .", "The protein complex comes off the chromosomes as they separate , and a motor protein called dynein walks along the microtubules to pull the separated chromosomes to the poles .", "Muscat , Torre-Santiago et al . 's findings thus show that meiosis in C . elegans during the production of egg cells works in a very different way to other types of cell division .", "In the future , it will be important to understand how dynein and the ring-shaped complex are regulated , as this may shed light on what causes mistakes in the separation of genetic material during meiosis , which can lead to infertility , miscarriages , and birth defects in humans and other animals ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "neuroscience" ]

[ [ "Neuropathic pain—pain arising from damage to or dysfunction of the nervous system ( Merskey and Bogduk , 1994 ) —is notoriously difficult to treat .", "Effective treatments have eluded discovery despite intense research and many promising leads ( Woolf , 2010 ) .", "Proposed explanations for the lack of clinical translation have focused on preclinical animal models ( Rice et al . , 2008; Mogil , 2009 ) and on clinical trial design ( Dworkin et al . , 2011; Mao , 2012 ) .", "An alternative possibility is that degeneracy within the pain system allows the pathogenic process to circumvent single-target-drugs .", "If true , the single-target-drug paradigm is bound to fail no matter how good the animal models or clinical trials are , and a new paradigm is needed .", "Degeneracy refers to multiple ‘different’ mechanisms conveying equivalent function ( Edelman and Gally , 2001 ) ; by comparison , redundancy refers to multiple instantiations of the ‘same’ mechanism .", "Both convey robustness to complex systems ( Kitano , 2004a ) .", "But if a pathogenic process were to hijack degeneracy , the pathological state could itself become robust , or in other words resistant to treatment .", "Accordingly , degeneracy is recognized as an important factor for cancer and other complex diseases ( Kitano , 2004a , 2004b; Tian et al . , 2011 ) , including epilepsy ( Klassen et al . , 2011 ) but has yet to inform pain research or analgesic drug development .", "That said , degeneracy has been recognized in neural systems ( Prinz et al . , 2004 ) and its existence and functional implications are gaining increasing attention ( Grashow et al . , 2009 , 2010; Marder , 2011; Amendola et al . , 2012; Zhao and Golowasch , 2012; Gutierrez et al . , 2013; O’Leary et al . , 2013; Ransdell et al . , 2013 ) .", "A degenerate basis for neuropathic pain is suggested by basic research findings .", "For example , increased function of the sodium channel Nav1 . 8 is sufficient to produce the hypersensitivity associated with neuropathic pain ( Bierhaus et al . , 2012; Wu et al . , 2012 ) .", "Nav1 . 8-targetted manipulations predictably fail in Nav1 . 8 knock-out animals , but those animals can nonetheless develop injury-induced hypersensitivity ( Nassar et al . , 2005 ) .", "This indicates that injury-induced changes in other ion channels—and indeed many such changes occur ( for reviews , see Campbell and Meyer , 2006; Woolf and Ma , 2007; Basbaum et al . , 2009; Marchand et al . , 2009; Gold and Gebhart , 2010 ) —are also sufficient to cause neuropathic pain .", "To directly explore degeneracy in the context of neuropathic pain , we tested whether multiple distinct molecular pathologies are sufficient to produce the cellular hyperexcitability associated with neuropathic pain .", "Hyperexcitability characterized by quantitative changes such as reduced threshold and qualitative changes such as altered spiking patterns ( see below ) develops at multiple points along the neuraxis ( Costigan et al . , 2009 ) .", "This includes primary somatosensory afferents whose spontaneous spiking and exaggerated responsiveness are thought to underlie spontaneous pain and hypersensitivity , respectively ( for review , see Devor , 2005 ) .", "Notably , peripheral input helps drive central sensitization ( Devor , 1991; Gracely et al . , 1992; Koltzenburg et al . , 1994 ) , meaning increased peripheral input is amplified rather than attenuated centrally .", "Injury-induced hyperexcitability is not limited to nociceptors; on the contrary , hyperexcitability also develops in myelinated afferents that normally convey innocuous information but that contribute to mechanical allodynia ( hypersensitivity ) under neuropathic conditions ( Campbell et al . , 1988; Koltzenburg et al . , 1994; Devor , 2009; King et al . , 2011 ) .", "Hyperexcitability in myelinated afferents is characterized by a triad of qualitative changes that include repetitive spiking , membrane potential oscillations ( MPOs ) , and bursting ( Liu et al . , 2000; Herzog et al . , 2001; Xing et al . , 2001; Liu et al . , 2002; Ma and LaMotte , 2007; Fan et al . , 2011; Xie et al . , 2011; Song et al . , 2012 ) .", "We refer to this qualitatively altered excitability as ‘neuropathic’ .", "We recently showed through mathematical modeling that all three neuropathic changes arise from a switch in spike initiation dynamics ( Rho and Prescott , 2012 ) ; there is , therefore , a one-to-many mapping between altered spike initiation dynamics and qualitative changes in cellular excitability .", "On the other hand , we found a many-to-one mapping between parameter values ( whose variations represent injury-induced molecular changes ) and cellular excitability; moreover , continuous parameter variations led to a discontinuous change in spike initiation dynamics .", "The many-to-one mapping constitutes degeneracy and the discontinuity , or tipping point , reflects criticality .", "The results identify spike initiation as a key nonlinear process whose qualitative alteration explains multiple features of cellular hyperexcitability on the basis of several possible molecular pathologies .", "In the current study , we experimentally tested our theory .", "After identifying the activation properties of currents affecting spike initiation , we used nonlinear dynamical analysis of our mathematical model to define the system’s tipping point .", "From this , we generated several predictions as to how that tipping point could be crossed .", "To experimentally test those predictions , we applied different manipulations designed to force neurons in one or the other direction across their tipping point , therein acutely reproducing neuropathic excitability in primary afferent neurons from naive animals or acutely reversing neuropathic excitability in neurons from nerve-injured animals .", "Manipulations involved decreasing and/or increasing subthreshold currents via pharmacology and/or dynamic clamp , respectively , in identified myelinated primary afferents .", "All predictions were confirmed , therein supporting our theory that primary afferent hyperexcitability arises through a critical transition whose molecular basis is highly degenerate ." ], [ "According to our previous theoretical work , spike initiation occurs through a time- and voltage-dependent competition between net fast-activating inward current and net slower-activating outward current ( Prescott et al . , 2008 ) .", "Because multiple types of ion channels contribute to each net current—currents with similar kinetics sum linearly ( Kepler et al . , 1992 ) —injury-induced changes in any one of the contributing ion channels can bias the competition ( Rho and Prescott , 2012 ) .", "Using a Morris–Lecar model that comprises only two variables whose interaction is sufficient to produce spikes , we adjusted parameters ( ‘Materials and methods’ ) to give a spike threshold approximating that observed in the soma of myelinated afferents , which is about −35 mV .", "The parameters of this base model were thereafter unchanged .", "Next , we derived the voltage-dependency and kinetics ( Figure 1A ) for an additional conductance ( Equations 1–4 in ‘Materials and methods’ ) which , when added to our base model , modulates its spike initiation dynamics .", "This conductance corresponds to either a sodium or potassium channel depending on its associated reversal potential and , according to our analysis , must be active at subthreshold voltages .", "To be clear , the parameters for this conductance were chosen to modulate spike initiation dynamics , not to model specific ion channel types that are known to be altered by nerve injury .", "That said , although our determination of parameters was agnostic to molecular identities , parameter values resemble those of known channel types , as noted in subsequent sections .", "We then varied the maximal conductance of the additional conductance ( s ) .", "Based on two-parameter bifurcation analysis , we determined the combinations of sodium conductance ḡNa and potassium conductance ḡK required to give repetitive spiking at different stimulus thresholds ( Figure 1B ) .", "Somata of myelinated primary afferents normally generate a single spike at stimulus onset , which corresponds to the gray-shaded region , whereas a subset of those neurons spike repetitively after nerve injury ( e . g . , Liu et al . , 2002 ) , which corresponds to the colored region . 10 . 7554/eLife . 02370 . 003Figure 1 . Modeling and theory .", "( A ) Voltage-dependency and kinetics of subthreshold conductance .", "In bottom panel , τ = ( α+β ) −1 where α and β are defined in Equations 3 and 4 .", "Equivalent activation parameters were used to implement either a sodium or potassium current based on reversal potential .", "( B ) Two-parameter bifurcation analysis in which ḡNa and ḡK were co-varied to determine the ḡNa:ḡK ratio for a given threshold for repetitive spiking .", "Gray shading shows parameter regime associated with onset-only spiking .", "Changes in excitability are explained by a switch in spike initiation dynamics ( Figure 1—figure supplement 1 ) .", "( C ) Predictions ( indicated by numbered arrows ) of how manipulating ḡNa and/or ḡK may force the system across a tipping point , thus reproducing or reversing neuropathic excitability .", "Position of the tipping point depends on many other parameters including leak conductance ( D ) , voltage-dependency of ḡNa and ḡK ( E ) , and potassium reversal potential ( F ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 02370 . 00310 . 7554/eLife . 02370 . 004Figure 1—figure supplement 1 . Spike initiation dynamics differ between normal and neuropathic conditions . In bifurcation analysis , a parameter such as stimulus intensity Istim is systematically varied to determine at what value the system qualitatively changes behavior , which is to say it undergoes a bifurcation .", "We repeated this analysis in a model with the ḡNa:ḡK ratio set to give normal excitability ( ḡNa = 2 . 0 µS/cm2; ḡK = 2 . 5 µS/cm2 ) or neuropathic excitability ( ḡNa = 2 . 5 µS/cm2; ḡK = 2 . 0 µS/cm2 ) .", "Bifurcation diagrams ( left ) illustrate how the system behaves at different Istim: a stable fixed point corresponds to quiescence whereas a stable limit cycle corresponds to repetitive spiking .", "A subcritical Hopf bifurcation is evident in the bottom ( Neuropathic ) bifurcation diagram but not in the top ( Normal ) bifurcation diagram , suggesting that repetitive spiking is only possible in the former condition .", "In both conditions , the Istim range in which a single spike is produced at stimulus onset is indicated with gray shading .", "Onset-only spiking is achieved through a quasi-separatrix crossing , independent of any bifurcation .", "Dotted arrows indicate Istim values for sample traces on the right .", "Membrane potential oscillations ( MPOs ) occur when the fixed point is in proximity to a Hopf bifurcation; after being perturbed by noise , the system relaxes towards its nearly-unstable fixed point following a loose spiral trajectory , which corresponds to MPOs on the time series .", "In the absence of a Hopf bifurcation , the fixed point is more stable and MPOs are negligible .", "Repetitive spiking occurs when Istim exceeds the intensity required for the Hopf bifurcation .", "Repetitive spiking can also occur in the Istim range between the Hopf bifurcation and where the stable and unstable limit cycles meet ( at a saddle-node bifurcation of limit cycles ) ; that region also contains a stable fixed point , meaning that the system is bistable .", "The neuron will remain in one or the other stable state unless a slow process like adaptation sweeps the system across the bistable region , in which case the neuron will remain quiescent when sweeping rightward toward the Hopf bifurcation , while spiking repetitively when sweeping leftward toward the saddle-node bifurcation of limit cycles , thereby producing bursts as a consequence of hysteresis .", "Bistability and adaptation are both required for bursting . DOI: http://dx . doi . org/10 . 7554/eLife . 02370 . 004 Neuropathic changes in excitability arise from a switch in spike initiation dynamics ( Rho and Prescott , 2012; Figure 1—figure supplement 1 ) .", "In brief , the colored region of Figure 1B corresponds to a parameter regime in which a subcritical Hopf bifurcation occurs when stimulus intensity Istim reaches a critical value I* .", "A Hopf bifurcation represents destabilization of the ‘resting’ state: repetitive spiking occurs when Istim exceeds I* , membrane potential oscillations ( MPOs ) arise in the presence of noise when Istim approaches I* , and bursting occurs when adaptation Iadapt causes Istim−Iadapt to sweep back and forth across I* .", "Outside the colored region of Figure 1B , spike initiation is effectively limited to a quasi-separatrix crossing; according to this mechanism , the resting state remains stable but a single spike is produced if the system transiently escapes from that state during a stimulus transient .", "In the absence of a Hopf bifurcation , repetitive spiking , MPOs and bursting are simply not possible .", "The boundary between gray and blue regions in our colored ‘excitability’ diagram ( Figure 1B ) , thus represents the critical tipping point separating normal and neuropathic parameter regimes .", "That tipping point is represented by a simple curve in subsequent excitability diagrams .", "We predict that a cell with normal excitability can be converted to neuropathic excitability ( i . e . , forced across its tipping point ) by a decrease in ḡK , an increase in ḡNa , or some combination thereof ( arrows 1–3 on Figure 1C ) .", "However , balanced conductance changes may offset one another , resulting in no qualitative alteration of excitability ( arrow 4 ) .", "Contrariwise , the inverse changes are predicted to normalize excitability in a hyperexcitable cell by forcing the system in the opposite direction across its tipping point ( arrows 5 and 6 ) .", "Each arrow corresponds to a prediction tested in the experimental portion of this study .", "Changes in other conductances , in parameters other than maximal conductance ( e . g . , activation properties ) , or in parameters not strictly connected to conductance ( e . g . , reversal potential ) can all affect where the tipping point lies ( Figure 1D–F , respectively ) .", "It is not feasible to experimentally test all parameter variations and combinations thereof , and hence we focused on predictions outlined in Figure 1C .", "But one should bear in mind that the tipping point in a real neuron will depend on numerous parameters and thus correspond to a boundary within a high-dimensional space .", "The important considerations are ( 1 ) that that boundary exists ( which implies criticality ) , and ( 2 ) that there are many ways to cross it ( which implies degeneracy ) .", "In other words , our theory predicts that excitability can change abruptly on the basis of many different molecular changes when and if those molecular changes reach a tipping point .", "Our next step was to test our predictions experimentally .", "Unlike typical experiments in which excitability is compared between different neurons before and after nerve injury , and where it is impossible to account for all injury-induced molecular changes and between-cell differences , we compared excitability in the same neuron before and after strictly controlled manipulations dictated by our simulation results .", "Furthermore , rather than trying to reproduce a complete set of injury-induced molecular pathologies , our manipulations were designed to reproduce one or two of those pathologies at a time .", "This approach reveals which molecular pathologies , alone or together , are sufficient to reproduce neuropathic excitability without unaccounted for co-variations in other parameters .", "Furthermore , by applying manipulations acutely , our approach avoids the confounding influence of compensatory changes that might develop over longer time scales .", "We targeted myelinated afferents ( somatic diameter ≥30 µm ) because they are directly implicated in allodynia and because they have been reported to develop the triad of neuropathic excitability changes described in the ‘Introduction’ .", "We initially targeted myelinated cutaneous afferents retrogradely labeled by DiI injected intradermally into the hindpaw ( ‘Materials and methods’ ) because this population comprises low-threshold mechanosensors that may subserve mechanical allodynia; we subsequently tested labeled muscle afferents , as reported at the end of the ‘Results’ section .", "In keeping with previous studies , neurons harvested from naive , uninjured rats responded to a square pulse of current injection with onset-only spiking , even at high stimulus intensities ( Figure 2A; n = 23 cutaneous afferents ) .", "Neurons could spike again in response to subsequent increments in stimulus intensity ( Figure 2A , inset ) , thus ruling out complete sodium channel inactivation as the basis for non-repetitive spiking .", "This spiking pattern places naive neurons within the gray-shaded region of our excitability diagram . 10 . 7554/eLife . 02370 . 005Figure 2 . Reproduction of neuropathic excitability in naive cutaneous neurons .", "( A ) Medium- to large-diameter ( putative myelinated ) cutaneous afferents from naive rats respond to depolarization with a single spike at stimulus onset .", "Neurons can respond to increments in stimulus intensity ( inset ) , which demonstrates that sodium channels are not completely inactivated after the initial spike .", "( B ) Prediction 1 was tested by blocking potassium current by application of 1 . 5–5 mM 4-AP .", "By repeating stimulation during wash-in and/or wash-out of the drug , we observed repetitive spiking and MPOs ( in the order expected based on the change in drug concentration ) in response to equivalent stimulation .", "Gray-shaded windows show enlarged views of voltage traces , with colors corresponding to the power spectra shown at the top .", "( C ) Prediction 2 was tested by adding virtual sodium conductance via dynamic clamp .", "As predicted , MPOs and repetitive spiking developed as virtual sodium conductance was increased , but bursting was absent .", "Note that a conductance density of 1 nS/pF corresponds to 1 mS/cm2 assuming a specific membrane capacitance of 1 μF/cm2 , which means that the conductance densities added by dynamic clamp are the same order of magnitude as those predicted by modeling in Figure 1B .", "( D ) Bursting was achieved in repetitively spiking neurons by introducing an AHP-type adaptation current via dynamic clamp . DOI: http://dx . doi . org/10 . 7554/eLife . 02370 . 005 Our first prediction was that decreasing subthreshold potassium conductance would reproduce neuropathic changes in excitability , consistent with injury-induced reduction of Kv1 channels ( Everill and Kocsis , 1999; Ishikawa et al . , 1999; Kim et al . , 2002; Hammer et al . , 2010; Zhao et al . , 2013 ) .", "To test this , we applied 4-aminopyridine ( 4-AP ) with a maximal concentration between 1 . 5 mM and 5 mM to decrease the total potassium current activated at subthreshold voltages .", "As drug concentration increased during wash-in , MPOs developed followed by a switch to repetitive spiking during stimulation; the reverse sequence occurred during washout ( Figure 2B ) .", "The broad peak in the power spectrum ( Figure 2B , inset ) shows that pharmacologically induced MPOs are consistent with previous descriptions of injury-induced MPOs ( Song et al . , 2012 ) and with a noise-dependent mechanism ( Rho and Prescott , 2012 ) .", "In 6 of 11 neurons tested , MPOs and repetitive spiking co-developed during wash-in of 4-AP , consistent with their common connection with the subcritical Hopf bifurcation .", "Bursting was not observed ( see below ) .", "Using 5 nM α-dendrotoxin , which more selectively blocks Kv1 channels , we obtained equivalent results in 2 of 4 additional neurons ( data not shown ) .", "Reproduction of neuropathic excitability via potassium channel blockade in a total of 8 of 15 neurons represents a significantly higher conversion rate than expected by chance ( i . e . , 0 of 23 neurons , as determined from the excitability at the start and end of recordings in each neuron; p<0 . 001; Fisher’s exact test ) .", "Our second prediction was that increasing subthreshold sodium conductance would reproduce neuropathic excitability , consistent with injury-induced increase of Nav1 . 3 ( Waxman et al . , 1994; Kim et al . , 2001; Fukuoka et al . , 2008; Huang et al . , 2008 ) .", "To test this , we added a virtual sodium conductance with the activation properties described in Figure 1A .", "As expected , increasing the virtual sodium current produced MPOs and repetitive spiking ( Figure 2C ) .", "Power spectral analysis ( Figure 2C , inset ) shows that dynamic clamp-induced MPOs are comparable to injury- and pharmacologically-induced MPOs .", "In 9 of 19 neurons tested , MPOs and repetitive spiking co-developed when sufficient virtual sodium conductance was inserted; this conversion rate is significantly higher than expected by chance ( p<0 . 001; Fisher’s exact test ) .", "Of the 10 neurons that did not develop repetitive spiking , five nonetheless developed MPOs .", "Again , bursting was not observed .", "Before proceeding to test predictions 3 and 4 , we sought to explain the absence of bursting .", "Our modeling indicated that although the subcritical Hopf bifurcation is necessary to create a stimulus range in which the system is bistable ( i . e . , in which quiescence or repetitive spiking are both possible ) , bursting also requires a slow process like spike-dependent adaptation to sweep the system back and forth across the bistable region , thus allowing hysteresis to manifest bursting; in other words , bursting requires a subcritical Hopf bifurcation and adaptation ( Rho and Prescott , 2012 ) .", "We therefore hypothesized that our repetitively spiking neurons , although capable of bursting , lacked the required adaptation .", "To test this , we inserted a virtual adaptation current ( ‘Materials and methods’ ) .", "As expected , this enabled bursting under conditions associated with repetitive spiking in all three cells tested ( Figure 2D ) but had no effect in the same three cells under normal conditions with onset-only spiking ( data not shown ) .", "These data confirm that bursting would have co-developed with repetitive spiking and MPOs if adaptation currents had been present , and suggest that adaptation currents are upregulated after nerve injury ( possibly as a compensatory measure ) rather than existing occultly in naive neurons whose spiking is already transient .", "In a subset of neurons , neither decreasing Kv1 potassium conductance nor increasing Nav1 . 3-like sodium conductance reproduced neuropathic excitability .", "Larger manipulations may have succeeded in forcing neurons across their tipping point but technical considerations ( e . g . , off-target effects at high drug concentrations and recording instability for large virtual conductances ) limited the magnitude of manipulations .", "But as explained in Figure 1 , variation of a single conductance is not the only way to force a neuron across its tipping point; instead , small changes in more than one conductance may combine to produce a net conductance change that is large enough to force the neuron across its tipping point .", "As an aside , if multiple small changes can recombine in different ways ( which is plausible under conditions in which dozens of different ion channels are up- or down-regulated ) , then degeneracy could exist on the basis of many different combinations being sufficient to produce neuropathic excitability; in other words , no one set of combined changes would be uniquely necessary just as no single change is uniquely necessary .", "To explore how conductance changes combine , we proceeded with testing predictions 3 and 4 .", "Our third prediction was that manipulations tested independently in predictions 1 and 2 could combine to reproduce neuropathic changes in excitability; indeed , in 8 neurons in which neither decreasing potassium conductance nor increasing sodium conductance was sufficient to produce repetitive spiking and MPOs , we tested the two manipulations together and found that the combination was sufficient to produce hyperexcitability in 7 of them ( Figure 3A ) .", "We also observed that the minimum virtual sodium conductance needed to reproduce neuropathic excitability was reduced during 4-AP application ( Figure 3B ) .", "We quantified the degree to which potassium channel blockade had moved the system towards its tipping point by comparing the minimum virtual sodium conductance needed to force the system across its tipping point without vs with 4-AP .", "The median ( and 25–75 percentile range ) virtual sodium conductance was significantly reduced from 0 . 51 ( range 0 . 47–0 . 63 ) nS/pF without 4-AP to 0 . 23 ( range 0 . 13–0 . 40 ) nS/pF with 4-AP ( p<0 . 001; Mann–Whitney U test ) ( Figure 3C ) .", "Non-parametric statistics were used because of the non-Gaussian distribution of data points . 10 . 7554/eLife . 02370 . 006Figure 3 . Additivity and subtractivity of conductance changes .", "( A ) Example in which neither virtual sodium conductance nor potassium channel blockade reproduced neuropathic excitability , whereas the combination of manipulations did , thus confirming prediction 3 , that manipulations can be additive .", "( B ) Example in which the minimum virtual sodium conductance required to reproduce neuropathic excitability was reduced when combined with potassium channel blockade .", "( C ) Even when 4-AP application did not , on its own , produce repetitive spiking , it nonetheless moved the neuron significantly closer to criticality as evidenced by comparing the minimal virtual sodium conductance needed to produce repetitive spiking without vs with 4-AP ( *p<0 . 001; Mann–Whitney U test ) .", "Points represent data from individual neurons and boxes represent median and 25–75 percentile range .", "Repetitive spiking caused by 4-AP application ( D ) or by virtual sodium conductance ( E ) was reversed by insertion of virtual potassium conductance .", "Panel E confirms prediction 4 , that manipulations can be subtractive . DOI: http://dx . doi . org/10 . 7554/eLife . 02370 . 006 Our fourth prediction was that different manipulations could offset one another , resulting in no net change in excitability .", "To test this , we first tested whether inserting a virtual potassium conductance could reverse the hyperexcitability induced by blockade of native potassium conductance by 4-AP .", "Results confirmed our prediction in 3 of 3 neurons tested ( Figure 3D ) , thus demonstrating the specificity of the 4-AP effect; in other words , even if channels other than Kv1 were blocked by 4-AP , the effect of 4-AP on excitability is attributable to blockade of subthreshold potassium current given that reintroducing that type of current reverses the altered excitability .", "More interesting is the observation that hyperexcitability caused by insertion of a virtual sodium conductance was reversed by insertion of a virtual potassium conductance in 3 of 3 neurons tested ( Figure 3E ) .", "These data confirm prediction 4 and demonstrate the subtractivity of different manipulations .", "In our final two predictions , we sought to move neurons in the opposite direction across their tipping point , which required neurons rendered hyperexcitable by nerve injury ( ‘Materials and methods’ ) .", "Surprisingly , only 1 of 9 injured cutaneous afferents exhibited any degree of repetitive spiking defined here as at least three spikes during sustained stimulation , which is not a significant change compared to chance ( p=0 . 28; Fisher’s exact test compared to spontaneous conversion rate of 0 in 23 cutaneous afferents ) .", "By comparison , 6 of 9 injured neurons not labeled by intradermal injection of DiI exhibited neuropathic excitability , an example of which is shown in Figure 4A ( p<0 . 001; Fisher’s exact test compared to 1 in 9 cutaneous ) .", "However , because the identity of unlabeled neurons is uncertain and given previous studies showing that muscle afferents are in fact more prone than cutaneous afferents to becoming grossly hyperexcitable ( Michaelis et al . , 2000; Liu et al . , 2002 ) , we retrogradely labeled muscle afferents .", "11 of 20 injured muscle afferents exhibited neuropathic excitability ( Figure 4B ) , which is a significantly higher proportion than 1 in 9 injured cutaneous afferents ( p<0 . 05; Fisher’s exact test ) and the spontaneous conversion rate of 0 in 27 muscle afferents from naive animals ( p<0 . 001; Fisher’s exact test ) .", "Neuropathic excitability was reversed by insertion of virtual potassium conductance in 10 of 10 neurons tested or by reduction of sodium conductance via application of 10 µM riluzole in 4 of 4 neurons tested , thus confirming predictions 5 and 6 , respectively ( Figure 4A , B ) .", "In the injured muscle afferents whose neuropathic excitability was reversed by insertion of virtual potassium conductance , the median conductance ( and 25–75 percentile range ) needed to cause reversal was only 0 . 14 ( range 0 . 12–0 . 15 ) nS/pF .", "This suggests that the neuropathic state lies close to the tipping point , which is consistent with our 100% success rate in reversing neuropathic excitability by manipulating either potassium or sodium conductance ( see above ) . 10 . 7554/eLife . 02370 . 007Figure 4 . Reversal of neuropathic excitability in neurons from nerve-inured rats .", "( A ) Typical response of an injured unlabeled primary afferent ( top ) .", "Repetitive spiking and MPOs were abolished by insertion of a potassium conductance ( middle ) or by blockade of sodium channels using 10 μM riluzole ( bottom ) , thus confirming predictions 5 and 6 , respectively .", "( B ) Typical response of an injured muscle afferent whose neuropathic excitability was similarly reversed by increased potassium conductance and reduced sodium conductance .", "( C ) Although only 1 of 9 injured cutaneous afferents exhibited repetitive spiking and MPOs , those afferents were nonetheless closer to criticality insofar as they required significantly less virtual sodium conductance than uninjured cutaneous afferents to reach criticality ( *p<0 . 05; Mann–Whitney U test ) .", "Points represent data from individual neurons and boxes represent median and 25–75 percentile range . DOI: http://dx . doi . org/10 . 7554/eLife . 02370 . 007 The concept of measuring proximity to the tipping point prompted us to ask whether cutaneous afferents , only one of which exhibited grossly abnormal excitability after nerve injury , were nonetheless closer to their tipping point after nerve injury than under control conditions .", "In the four injured cutaneous afferents to which we added virtual sodium conductance , only 0 . 30 ( range 0 . 14–0 . 44 ) nS/pF of sodium conductance was required to reproduce neuropathic excitability , which is significantly less than the 0 . 51 ( range 0 . 47–0 . 63 ) nS/pF required to reproduce neuropathic excitability in uninjured cutaneous afferents ( p<0 . 05; Mann–Whitney test ) ( Figure 4C ) .", "Hence , nerve injury leads to qualitatively altered excitability in only a subset of cells ( i . e . , ones that cross their tipping point ) but the remaining cells are nonetheless quantitatively more excitable ( i . e . , closer to their tipping point ) .", "Using neurons from naive rats , we subsequently verified that muscle afferents , like cutaneous afferents described in Figure 2 , normally exhibit onset-only spiking ( n = 27 muscle afferents ) .", "Neuropathic excitability was reproduced in 16 of 20 muscle afferents by insertion of virtual sodium conductance ( Figure 5A ) , which is a significantly greater conversion rate than 9 of 19 cutaneous afferents ( p<0 . 05; Fisher’s exact test ) .", "Moreover , we found that the median sodium conductance required to reproduce neuropathic excitability was 0 . 31 ( range 0 . 25–0 . 41 ) nS/pF in muscle afferents compared with 0 . 51 ( range 0 . 47–0 . 63 ) nS/pF in cutaneous afferents , indicating that naive muscle afferents are significantly closer to their tipping point than naive cutaneous afferents ( p<0 . 001; Mann–Whitney test ) ( Figure 5B ) .", "Because we cannot isolate the density of native NaV1 . 3 cannels , we cannot gauge the relative change that this absolute virtual conductance represents .", "Furthermore , our measurement represents the distance to tipping point along only one dimension; if criticality exists along a boundary in a high-dimensional space ( Figure 1 ) , then the distance to tipping point may differ significantly along different dimensions .", "But notably , input resistance did not differ significantly between cutaneous and muscle afferents ( p=0 . 2; unpaired t test; 282 ± 47 MΩ in cutaneous afferents vs 377 ± 57 MΩ in muscle afferents ) .", "Overall , these data suggest that muscle afferents are more prone to developing neuropathic excitability after nerve injury because they operate closer to their tipping point than do cutaneous afferents . 10 . 7554/eLife . 02370 . 008Figure 5 . Reproduction of neuropathic excitability in naive muscle afferents .", "( A ) As predicted , repetitive spiking developed as virtual sodium conductance was increased .", "( B ) Uninjured muscle afferents operate significantly closer to criticality than uninjured cutaneous afferents based on the minimum virtual sodium conductance needed to produce repetitive spiking ( ***p<0 . 001; Mann–Whitney U test ) .", "In B and D , points represent data from individual neurons and boxes represent median and 25–75 percentile range .", "These data suggest that muscle afferents are more prone to develop neuropathic excitability after nerve injury because they start off closer to their tipping point , and not necessarily because injury induces a larger change in membrane conductances .", "( C ) Based on the observation that 4-AP and dendrotoxin ( DTX ) produced repetitive spiking in only 1 of 12 muscle afferents , we measured the DTX-sensitive current .", "The persistent outward current activated at perithreshold voltages ( −50 to −30 mV ) and blocked by 10 nM DTX was significantly smaller in muscle afferents than in cutaneous afferents ( **p<0 . 01; two-way ANOVA and post-hoc Student-Newman-Keuls test ) .", "( D ) Although 4-AP produced repetitive spiking in only 1 of 12 muscle afferents , it nonetheless moved neurons closer to criticality as evidenced by the minimal virtual sodium conductance needed to cause hyperexcitability after 4AP application ( *p<0 . 05; Mann–Whitney U test ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 02370 . 008 We also tested whether neuropathic excitability could be reproduced in muscle afferents by reduction of potassium conductance by application or 4-AP or α-dendrotoxin .", "Contrary to the expectations , 4-AP and dendrotoxin reproduced neuropathic excitability in only 1 of 12 muscle afferents , which is a significantly lower conversion rate than 8 of 15 cutaneous afferents ( p<0 . 05; Fisher’s exact test ) .", "We hypothesized that this was either ( 1 ) because muscle afferents are further than cutaneous afferents from their tipping point or ( 2 ) because KV1 expression is lower in muscle afferents .", "Because hypothesis 1 is inconsistent with data in Figure 5B , we tested hypothesis 2 by comparing the density of dendrotoxin-sensitive current in muscle and cutaneous afferents measured in voltage clamp after blockade of sodium channels using 1 μM tetrodotoxin; dendrotoxin was used for these experiments because it is a more selective KV1 blocker than 4-AP .", "As predicted , the sustained outward current activated at perithreshold voltages ( −50 mV to −30 mV ) and blocked by 10 nM dendrotoxin was significantly smaller in muscle afferents than in cutaneous afferents ( p<0 . 01; two-way ANOVA and post-hoc Student-Newman-Keuls test; 1 . 7 ± 0 . 6 pA/pF in muscle afferents vs 4 . 2 ± 0 . 6 pA/pF in cutaneous afferents after removing variance attributable to voltage ) ( Figure 5C ) .", "With fewer KV1 channels available to be blocked , 4-AP and dendrotoxin naturally have a smaller impact on neuronal excitability .", "However , amongst muscle afferents in which neuropathic excitability was not reproduced by application of 4-AP and in which virtual sodium conductance was subsequently inserted , significantly less sodium conductance was needed to reproduce neuropathic excitability with 4-AP ( 0 . 22 , range 0 . 14–0 . 27 nS/pF ) than without 4-AP ( 0 . 31 , range 0 . 25–0 . 41 nS/pF ) ( p<0 . 05; Mann–Whitney U test ) ( Figure 5D ) .", "This indicates that 4-AP moves muscle afferents toward their tipping point but simply not far enough to reach it , reminiscent of the effects of nerve injury on cutaneous afferents ( Figure 4C ) .", "Moreover , although many factors contribute to regulating excitability ( ‘Introduction’ ) , the relatively low expression of Kv1 channels in muscle afferents likely contributes to them existing closer to their tipping point , as shown in Figure 5B ." ], [ "Using computer simulations and experiments , we have demonstrated that primary afferent hyperexcitability associated with neuropathic pain arises through a switch in spike initiation dynamics that occurs when neurons cross a tipping point .", "Furthermore , there are many different ways to cross that tipping point .", "The first observation demonstrates criticality , whereas the latter demonstrates degeneracy .", "Criticality and degeneracy are both important concepts for understanding how complex systems normally operate and how they fail in disease states , but neither concept has been previously applied to help decipher the pathogenesis of neuropathic pain .", "We will discuss each concept in turn , and then address their relevance for understanding neuropathic pain .", "Nonlinear dynamical analysis of our mathematical model predicted that qualitative neuropathic changes in primary afferent excitability—repetitive spiking , MPOs , and bursting—co-develop when subthreshold inward and outward currents become sufficiently unbalanced .", "According to our theory , the tipping point represents a qualitative change in spike initiation dynamics .", "By pharmacologically decreasing and/or electrophysiologically increasing candidate conductances , we were able to force primary afferent neurons in different directions across their tipping point , thereby reproducing or reversing neuropathic changes in excitability .", "These data clearly demonstrate criticality and support our attribution of it to a switch in spike initiation dynamics .", "Our theory further predicted , and our experimental data confirmed , that there are multiple ways in which spike initiation dynamics can be altered .", "To be clear , the tipping point constitutes a unique switch in spike initiation dynamics but there are many different ways to reach that tipping point .", "Multiple bases for the same outcome constitute degeneracy ( Edelman and Gally , 2001; Marder and Taylor , 2011 ) .", "Degeneracy prevents emergent network and cellular behaviors from being ascribed to unique ion channel combinations , but therein allows for a disturbance in one type of ion channel to be offset by compensatory changes in other ion channels so that network and cellular function can be robustly maintained ( Flake et al . , 2004; Howard et al . , 2007; Marder , 2011 ) .", "We focused on manipulating two factors , maximal conductance of subthreshold sodium and potassium conductances , while maintaining all other factors unchanged .", "Our data clearly demonstrate the degeneracy of neuropathic excitability insofar as distinct manipulations produced equivalent outcomes .", "In contrast to our simplified testing paradigm , nerve injury induces numerous molecular changes within primary afferents as evidenced by gene expression profiling ( Costigan et al . , 2002; Xiao et al . , 2002; Valder et al . , 2003; Hammer et al . , 2010; LaCroix-Fralish et al . , 2011 ) .", "Some of those changes increase excitability , others are compensatory , and most are irrelevant for ( although nonetheless correlated with ) hyperexcitability and instead bear on other aspects of cellular function .", "That said , alterations in cellular excitability cannot be definitively explained without accounting for all ‘relevant’ changes .", "This is an insurmountable task if one considers that each neuron experiences different molecular changes and that those changes occur on different molecular backgrounds .", "However , one can ascertain how close or far a neuron sits from its tipping point based on whether controlled manipulations succeed in forcing the neuron across that point .", "This requires a theoretical understanding of tipping points ( i . e . , their nonlinear dynamical basis ) to identify telltale signs by which they can be inferred experimentally .", "It also requires a testing paradigm in which controlled manipulations can be applied , and where unknown changes ( differences ) do not occur ( exist ) .", "It is for these reasons that we compared excitability within the same neuron before and after artificial manipulations rather than comparing excitability before and after nerve injury; notably , the latter approach would have necessitated comparison of excitability in separate neurons , each of which is distinct , and is plagued by countless injury-induced changes , not all of which can be simultaneously measured .", "Moreover , the abruptness of our manipulations precludes compensatory changes from developing .", "Thus , our approach represents an innovative alternative to experiments using nerve injury models or molecular–genetic techniques in which channel expression is more slowly up- or down-regulated .", "Our results do not reveal a cure for neuropathic pain but , instead , suggest that a paradigm shift is needed in how we approach this task .", "Criticality and degeneracy may explain features of neuropathic pain that have hitherto eluded explanation .", "For instance , neuropathic symptoms often develop at long and highly variable delays after the inciting injury or disease .", "Criticality can explain this insofar as underlying molecular changes can develop occultly , without obvious outward manifestations , until a tipping point is reached , whereupon symptoms develop abruptly because of gross changes in excitability .", "Furthermore , once near the tipping point , symptoms could wax or wane because of the system moving forward and backward across its tipping point due to subtle fluctuations in underlying factors .", "From a therapeutic perspective , an obvious goal is to move the system from the ‘abnormal’ side of the tipping point back to the ‘normal’ side .", "Because of degeneracy , there are many ways to cross the tipping point in the forward direction , and an equally large number of ways to cross back in the reverse direction .", "This last observation is promising , as is the observation that the neuropathic state seems to lie close to the tipping point .", "But why then is neuropathic pain so notoriously difficult to treat ?", "The answer to the last question hinges on the answer to another question: Why does neuropathic excitability develop in the first place ?", "Degeneracy ought to convey robustness to the regulation of neuronal excitability since effects of pathological molecular changes can be mitigated by compensatory changes in any one of a multitude of other ion channels .", "But gross changes in excitability do occur , at least in some neurons , and although those changes were easily reversed by our acute manipulations , prolonged therapies in patients and animal models often have only transient effects .", "One possible explanation is that the homeostatic regulation of excitability is itself deranged , in effect maintaining the system at an incorrect ‘set point’ and hijacking degeneracy to paradoxically maintain hyperexcitability .", "In that scenario , therapeutically manipulating only one misregulated conductance will fail to sustainably reverse hyperexcitability if a deranged homeostatic control mechanism can fall back on other conductances to achieve its misguided goal .", "Instead , all misregulated conductances need to be coordinately targeted .", "If epilepsy has a similarly degenerate molecular basis ( Klassen et al . , 2011 ) , it is no wonder that multi-target drugs tend to work well or that polypharmacy with single-target drugs is beneficial ( Kwan et al . , 2001 ) ; indeed , combination pharmacotherapy also offers benefits in the treatment of neuropathic pain ( Chaparro et al . , 2012 ) .", "But , alternatively , the deranged control mechanism itself could be commandeered or its set point adjusted .", "The idea that neuropathic pain reflects the maladaptive homeostatic response to injury rather than being a direct consequence of injury has started to gain traction ( Costigan et al . , 2009 ) .", "Our data emphasize the importance of considering the context in which that plasticity occurs , namely , that it occurs within a system that is both complex and degenerate .", "To conclude , we reproduced and reversed neuropathic excitability in primary afferent neurons using manipulations designed to force the system across the tipping point that separates normal and neuropathic excitability .", "Existence of that tipping point demonstrates ‘criticality’ and speaks to the importance of the nonlinear interactions that give rise to system complexity .", "Observation that multiple different manipulations can force the system across that tipping point demonstrates ‘degeneracy’ .", "Criticality and degeneracy are fundamentally important concepts for understanding how complex systems fail , and how to therapeutically intervene to prevent or reverse those failures .", "Our theory suggests that treatment strategies for neuropathic pain must move away from single-target-drugs and towards a systems-based approach , capitalizing on degeneracy rather than being thwarted by it ." ], [ "Starting with a two-dimensional Morris–Lecar ( ML ) model , which is sufficient to produce spikes , we added Hodgkin–Huxley ( HH ) style conductances for the express purpose of modulating spike initiation dynamics in the ML model .", "For ML equations , see Rho and Prescott ( 2012 ) .", "Parameter values were as follows: C = 2 μF/cm2 , ENa = 50 mV , EK = −100 mV , Eleak = −70 mV , φw = 0 . 15 , ḡfast = 20 mS/cm2 , ḡslow = 20 mS/cm2 , gleak = 2 mS/cm2 , βm = −1 . 2 mV , γm = 14 mV , βw = −10 mV , γw = 10 mV , and Istim was varied .", "HH conductances were modeled according to ( 1 ) INa , K=g¯Na , Km ( V−ENa , K ) , ( 2 ) dmdt=α ( 1−m ) −βm , ( 3 ) α=kα ( V−Vαsα ) e ( V−Vαsα ) −1 , ( 4 ) β=kβe ( V−Vβsβ ) , where , Vα , β = −24 mV , sα , β = −17 mV , and kα , β was between 1 and 1 . 5 ms−1 .", "These parameters were chosen to give activation in the perithreshold voltage range ( based on the threshold determined by ML parameters ) .", "In the interest of simplicity , the activation variable m was not raised to any exponent and nor was inactivation included .", "The Na+ and K+ currents differed only their reversal potential , where ENa = +50 mV and EK = −100 mV .", "Maximal conductances ḡNa and/or ḡK were varied in order to modulate spike initiation dynamics and thereby test our hypotheses .", "Where indicated , we also included a virtual slow AHP current modeled according to ( 5 ) IAHP=g¯AHPz ( V−EK ) , ( 6 ) dzdt= ( 11+eβz−Vγz−z ) /τz , where .", "βz = 0 mV , γz = 5 mV , τz = 300 ms . Setting βz to a suprathreshold voltage ensures a spike-dependent form of adaptation ( Prescott and Sejnowski , 2008 ) .", "Gaussian noise with standard deviation 0 . 3 μA/cm2 and 0 mean was also added , where indicated .", "Simulations were conducted in XPP and bifurcation analysis was conducted with AUTO using the XPP interface ( Ermentrout , 2002 ) .", "All experiments were carried out on adult ( 200–340 g ) male Sprague–Dawley rats ( Harlan , Indianapolis , IN or Charles River , Montreal ) and were approved by the University of Pittsburgh Institutional Animal Care and Use Committee ( protocol number 1108600 ) and by The Hospital for Sick Children Animal Care Committee ( protocol number 22919 ) .", "To retrogradely label cutaneous or muscle afferent cell bodies in the dorsal root ganglion ( DRG ) , animals were anesthetized with isofluorane ( 4% for induction; 2 . 5% for maintenance ) and DiI ( Invitrogen , Carlsbad , CA ) dissolved in DMSO ( 170 mg/ml; Sigma–Aldrich , St . Louis , MO ) and diluted 1:10 in 0 . 9% sterile saline was injected into either the hindpaw skin or gastrocnemius muscle .", "Specifically , to label cutaneous afferents , 10 μl of DiI solution was injected intradermally in 5 sites ( 2 μl/site ) over an area of ∼5 mm2 into the glabrous skin of the left hindpaw .", "To label muscle afferents , 10 μl of DiI solution was slowly injected into five sites of the gastrocnemius muscle of the left leg ( 2 μl/site ) ; to exclude spurious labeling of cutaneous afferents along the needle track , 10 μl of Fast Blue ( 1% in sterile saline ) was injected intradermally around the intramuscular injection site .", "Only cells labeled with DiI and not Fast Blue were considered muscle afferents .", "Neurons were harvested 10–20 days after injections .", "A subset of animals received spinal nerve ligation ( SNL ) ( Kim and Chung , 1992 ) 2–5 days before terminal experiments .", "Under isoflurane anesthesia , paraspinal muscles of the lower lumbar and sacral level were separated to access the area around the L6 process .", "The L6 process was carefully removed and the L5 spinal nerve was tightly ligated with 6-0 silk suture .", "All nerve-injured animals maintained motor function but developed neuropathic pain as inferred by guarding of the affected paw .", "To collect DRG neurons , rats were deeply anesthetized by subcutaneous injection of anesthetic cocktail ( 1 ml/kg of 55 mg/ml ketamine , 5 . 5 mg/ml xylazine , and 1 . 1 mg/ml acepromazine ) or by isoflurane inhalation ( 4% for induction; 2 . 5% for maintenance ) .", "DRG were surgically removed ( L4 and L5 in naive animals; L5 in nerve-injured animals ) , enzymatically treated , mechanically dissociated , plated on glass coverslips previously coated by a solution of 0 . 1 mg/ml poly-D-lysine , and incubated in MEM-BS at 37°C , 3% CO2 , and 90% humidity for 2 hr .", "After incubation , coverslips were transferred to a HEPES-buffered L-15 media containing 10% BS and 5 mM glucose and stored at room temperature .", "Neurons were studied 2–28 hr after harvesting .", "Coverslips with cultured cells were transferred to a recording chamber constantly perfused with room temperature , oxygenated ( 95% O2 and 5% CO2 ) artificial cerebral spinal fluid containing ( in mM ) 126 NaCl , 2 . 5 KCl , 2 CaCl2 , 2 MgCl2 , 10 D-glucose , 26 NaHCO3 , 1 . 25 NaH2PO4 .", "Using gradient contrast optics , neurons with somatic diameter ≥30 μm were targeted for patching as these give rise to myelinated fibers ( Harper and Lawson , 1985 ) .", "Cutaneous or muscle afferents were targeted based on epifluorescent illumination of DiI labeling ( and exclusion of Fast Blue labeling in the case of muscle afferents ) .", "Cells were recorded in the whole-cell configuration with >70% series resistance compensation using an Axopatch 200B amplifier ( Molecular Devices; Palo Alto , CA ) .", "Electrodes ( 2–5 MΩ ) were filled with a recording solution containing ( in mM ) 125 KMeSO4 , 5 KCl , 10 HEPES , 2 MgCl2 , 4 ATP , 0 . 4 GTP , as well as 0 . 1% Lucifer Yellow; pH was adjusted to 7 . 2 with KOH and osmolality was between 270 and 290 mosmol/l .", "The pipette shank was painted with Sylgard to reduce pipette capacitance .", "Responses were low-pass filtered at 2 KHz , digitized at 20 KHz using a CED 1401 computer interface ( Cambridge Electronic Design , Cambridge , UK ) , and analyzed offline .", "Membrane potential ( after correction for the liquid junction potential of −9 mV ) was adjusted to −65 mV through tonic current injection .", "To block native conductances , various drugs were bath applied as reported in the ‘Results’ .", "To add virtual conductances , the dynamic clamp technique was applied using Signal 5 software ( Cambridge Electronic Design ) .", "Conductances were modeled using the same equations as in computer simulations , as described above .", "To account for differences in cell size , values of inserted conductances were converted to densities by normalizing by membrane capacitance C , which was measured from responses to small ( ≤50 pA ) hyperpolarizing current steps , where C = τm/Rin , τm is the membrane time constant , and Rin is input resistance .", "Notably , given a specific membrane capacitance of 1 μF/cm2 , a conductance density of 1 mS/cm2 ( as reported for simulations ) corresponds to 1 nS/pF ( as reported for dynamic clamp experiments ) ." ] ]

[ "Neuropathic pain remains notoriously difficult to treat despite numerous drug targets .", "Here , we offer a novel explanation for this intractability .", "Computer simulations predicted that qualitative changes in primary afferent excitability linked to neuropathic pain arise through a switch in spike initiation dynamics when molecular pathologies reach a tipping point ( criticality ) , and that this tipping point can be reached via several different molecular pathologies ( degeneracy ) .", "We experimentally tested these predictions by pharmacologically blocking native conductances and/or electrophysiologically inserting virtual conductances .", "Multiple different manipulations successfully reproduced or reversed neuropathic changes in primary afferents from naïve or nerve-injured rats , respectively , thus confirming the predicted criticality and its degenerate basis .", "Degeneracy means that several different molecular pathologies are individually sufficient to cause hyperexcitability , and because several such pathologies co-occur after nerve injury , that no single pathology is uniquely necessary .", "Consequently , single-target-drugs can be circumvented by maladaptive plasticity in any one of several ion channels ." ]

[ "Although the pain associated with an injury is unpleasant , it normally serves an important purpose: to make you avoid its source .", "However , some pain appears to arise from nowhere .", "Frustratingly , this type of pain , known as neuropathic pain , does not respond to common painkillers and is thus very difficult to treat .", "The neurons that transmit pain and other sensory information do so using electrical signals .", "In response to a stimulus , ions travel through channels in the membrane of a neuron , which leads to a change in the electrical potential of the membrane .", "When this change is large enough , a voltage spike is produced: this signal is ultimately transmitted to the brain .", "When certain neurons fire too easily or too often , neuropathic pain can arise .", "This hyperexcitability can make something painful feel even worse , or it can make things hurt that shouldn’t .", "To prevent this , extensive research has been devoted to identify drugs that target particular types of ion channels and block them .", "However , despite the discovery of many promising drugs , those drugs have been frustratingly ineffective in clinical trials .", "Using simulations and experiments , Ratté et al . have examined the behavior of a type of neuron that normally conducts information about touch , but the brain sometimes misinterprets this information as pain .", "Increasing the flow of ions through the cell membrane in these simulations eventually causes a ‘tipping point’ to be crossed , which triggers a dramatic , discontinuous change in spiking pattern .", "However , as several different types of ion channels contribute to the current , there are several different ways in which the tipping point can be crossed .", "This ability to produce the same result by multiple means is a common feature of complex systems .", "Known as degeneracy , it makes systems more robust , as a given result can still be achieved if one particular attempt to achieve this result fails .", "The work of Ratté et al . helps to explain why drugs that target just one type of ion channel may fail to relieve neuropathic pain: maladaptive changes in any one of several other ion channels may circumvent the therapeutic effect ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "biochemistry and chemical biology", "tools and resources", "microbiology and infectious disease" ]

[ [ "Complex gut microbiome phenotypes shape human nutrition ( Martens et al . , 2014; Sonnenburg et al . , 2016; Bretin et al . , 2018 ) , therapeutic drug responses ( Guthrie et al . , 2017; Haiser et al . , 2013; Koppel et al . , 2017 ) and disease susceptibility ( Koeth et al . , 2013 ) .", "Multi’omic studies suggest that the human gut microbiota can be discretized at the resolution of microbial enzymes ( Guthrie et al . , 2017; Tang and Hazen , 2014 ) , species ( Haiser et al . , 2013; Haiser et al . , 2014 ) , guilds ( Joossens et al . , 2011; Wu et al . , 2013 ) or metabolites ( Clayton et al . , 2009 ) to characterize a range of human health and disease states .", "Gut microbial mediated biochemical transformations have consequences for drug treatment efficacy ( Koppel et al . , 2017; Spanogiannopoulos et al . , 2016; Alexander et al . , 2017; Wilson and Nicholson , 2017 ) and the etiology of inflammatory gastrointestinal diseases ( Tilg et al . , 2018; Arthur et al . , 2014; Belcheva et al . , 2014; Brennan and Garrett , 2016 ) , however despite many examples there exist few unifying principles that govern microbiome impacts on human health .", "Some microbiome/drug interactions have been characterized in detail .", "For example , the inactivation and decreased bioavailability of digoxin , a cardiac glycoside inhibitor , is linked to cgr operon expression levels in a single species , E . lenta ( Haiser et al . , 2013 ) .", "Microbial β-glucuronidases mediate the reactivation of the key therapeutic metabolite of irinotecan , a chemotherapeutic prodrug used in the treatment of colorectal cancer , causing toxicity in some patients ( Guthrie et al . , 2017; Wallace et al . , 2010 ) .", "Notably , diet-derived compounds that are conjugated to glucuronic acid in the human liver and excreted via the biliary route into the GI tract are known substrates for microbial β-glucuronidases ( O'Leary et al . , 2003; Sakurama et al . , 2014; Maathuis et al . , 2012 ) .", "Many other gastrointestinally-routed drugs share overlapping chemical properties with diet-derived compounds .", "We understand in detail species-specific metabolism of some discrete chemical structures in dietary compounds , particularly polysaccharides ( Martens et al . , 2008 ) ; however we know little about the potential spectrum of drug metabolism by the microbiome .", "Beyond the role of the microbiome in therapeutic drug treatment efficacy and polysaccharide metabolism , we have some mechanistic insight into how microbial metabolism contributes to host immunity .", "Microbial enzymes mediate the conversion of tryptophan into indole ( Sasaki-Imamura et al . , 2010 ) and indole derivatives ( Arora and Bae , 2014 ) that shape human host immune responses ( Levy et al . , 2017; Blacher et al . , 2017 ) .", "Microbe produced indole 3-aldehyde functions as an activating ligand for human host aryl hydrocarbon receptors which are expressed by immune cells ( Zelante et al . , 2013 ) .", "Indole binding induces IL-22 secretion by innate lymphoid cells , promoting the secretion of antimicrobial peptides that protects the host from pathogenic infection by Candida albicans ( Zelante et al . , 2013 ) .", "Microbial production of short chain fatty acids ( SCFAs ) from dietary fiber also shapes host immunity , contributing to both innate and adaptive immune system functions ( Fukuda et al . , 2011; Donohoe et al . , 2011; Smith et al . , 2013 ) .", "Host-microbe interactions and phenotypes , ranging from host drug response to host immune response , are thus intimately connected to gut chemical signaling .", "Beyond these few well understood examples lie a vast space of uncharacterized microbe-drug-diet-phenotype interactions .", "We propose three key requirements to characterize the dynamics of the gut chemical space and its impact on health .", "The first is predicting which compounds microbes can metabolize , the second is connecting the chemistry of gut microbes to host phenotypes , and the third is linking gut chemistry to microbial ecology .", "Towards the goal of systematically mapping the gut microbial chemistry that contributes to the metabolism of xenobiotics , including therapeutic drugs , recent efforts have used chemical structure-centric approaches to enable high-throughput computational predictions of gut microbe metabolism of drugs ( Sharma et al . , 2017; Mallory et al . , 2018 ) .", "These tools represent an important first step towards ecological and mechanistic insights into gut microbiota driven biotransformation of foods and drugs .", "The second requirement , which has not yet been achieved , is to connect the known and predicted chemistry of gut microbes to host phenotypes .", "To date , information on human responses to therapeutic drugs is available in disparate databases and formats including FDA Adverse Report System ( FAERs ) ( Burkhart et al . , 2015 ) , the Side Effect Resource ( SIDER ) ( Kuhn et al . , 2016 ) and DrugBank ( Law et al . , 2014 ) .", "The third requirement , also lacking , is to systematically link gut microbe chemistry to microbial ecology to understand how the distribution of enzymes in populations of microbes facilitates ecological interactions that structure the human gut .", "Here , we develop MicrobeFDT , a resource encompassing this 3-step framework that connects compound structure , enzyme function , taxonomy , and toxicity to characterize microbe-diet-drug-phenotype interactions .", "We organize ~10 , 000 food , drug , and endogenous compounds by structural similarity .", "We then link toxicity , enzyme interactions , and the propensity for gut microbes to carry out metabolism on each compound to the structural similarity network .", "We validate MicrobeFDT computationally by demonstrating that structural similarity is a reasonable proxy for toxicity , enzyme sharing , and coarse-grained functional similarity .", "We propose , and experimentally validate , active gut microbiome demethylation of an ovarian cancer drug , altretamine , a metabolism that we propose may drive toxicity of this drug .", "All data is available in the MicrobeFDT database ( MicrobeFDT; Guthrie , 2019; copy archived at https://github . com/elifesciences-publications/microbeFDT-neo4j ) ." ], [ "The foundation of the MicrobeFDT resource is a chemical similarity network linking 10 , 822 food , drug , and endogenous compounds with PubChem compound identifier ( CIDs ) ( Kim et al . , 2016 ) .", "In the network , nodes designate compounds and edges are weighted by pairwise chemical substructure similarity quantified by comparing PubChem fingerprints ( Kim et al . , 2016 ) using the Tanimoto score ( Bajusz et al . , 2015 ) ( Figure 1 ) .", "The Tanimoto score prioritizes overlap between compounds that share substructures over compounds with shared co-absences ( Bajusz et al . , 2015 ) .", "We hypothesized that compounds with overlapping substructure and physiochemical properties , in which one compound is a known substrate of an enzyme , will be more likely to serve as substrates for the same enzyme .", "Recent in silico approaches to predict enzymatic reactions of drugs in the context of human enzyme catalyzed reactions also employ this hypothesis ( Niu et al . , 2013; Yu et al . , 2018 ) .", "Substructure-based clustering thus serves as a first step towards synthesizing publicly available information on gut compound chemical diversity and gut microbiome biochemistry .", "To validate that our network can identify shared metabolism , we developed an in silico prediction model to assign a probability of shared metabolism between compounds based on substructure overlap and the following physiochemical categories: geometry , functional groups , amino acid composition , polarity and hydrophobicity .", "We find that the probability estimates of compound-pairs sharing an enzyme based on substructure and physiochemical parameters , increase as the substructure overlap score between compound pairs increases ( Figure 2 ) .", "Weighting compound pair chemical similarity relationships based on substructure similarity is thus a reasonable filtering step to identify compounds that may share metabolism .", "As an example of how the network can reveal shared metabolism we selected compounds in the network with substructure overlap with digoxin , a cardiac glycoside inhibitor .", "Reduction of digoxin by a human microbiome reductase inactivates the drug , contributing to poor bioavailability in some individuals ( Haiser et al . , 2013; Haiser et al . , 2014; Lindenbaum et al . , 1981 ) .", "Koppel et al . , biochemically characterized the capacity of a single flavin- and [4Fe-4S] cluster-dependent reductase , cgr2 , to reduce various substrates with a range of substructure similarity to digoxin ( Koppel et al . , 2018 ) .", "We identified the substructure overlap between digoxin and compounds in the Koppel et al . study that were evaluated as substrates of Cgr2 enzyme .", "Among the biochemically assayed compounds ( Koppel et al . , 2018 ) that are present in the MicrobeFDT network , compounds with substructure similarity scores greater than 0 . 8 are also substrates for Cgr2 .", "This assessment suggests that for the cgr enzyme substructure based clustering can distinguish experimentally characterized substrates from non-substrates ( Figure 3 ) .", "Previous studies have found that structural similarity predicts both toxicity and drug target similarity ( Campillos et al . , 2008 ) .", "To evaluate whether our network also recapitulates shared drug toxicity we fit a linear regression and computed the effect size to assess the association between substructure similarity and toxicity similarity for therapeutic drugs in our network .", "We find that structural similarity moderately positively predicts toxicity similarity for therapeutic drug pairs linked by structural similarity overall in the network ( r = 0 . 03116 , p<2 . 2e-16 ) ( Figure 4 ) .", "Finally , we evaluated how well our compound clustering recapitulates structure-based chemical taxonomy as defined by the ClassyFire ( Djoumbou Feunang et al . , 2016 ) resource , a comprehensive chemical classification schema , at the level of superclass taxonomy .", "We found that substructure-based compound clustering , significantly groups compounds within a ClassyFire superclass based on a comparison of the MicrobeFDT network with a randomized network with the same number of nodes and edges ( p<8 . 06×10–15 , Wilcoxon rank-sum test ) .", "Compound-pairs at higher substructure similarity share Superclass membership at higher substructure values and at a greater frequency than randomized pairs , indicating that the MicrobeFDT substructure similarity metric can capture established chemical classifications ( Figure 5 ) .", "In the network , therapeutic drug structural diversity is embedded within food-derived chemical diversity .", "For example , drugs share structural similarity with food-derived compounds from a diverse range of classes including benzenoids , lipids , nucleosides and phenylpropanoids ( Figure 6 ) .", "Food derived compounds also contributed significantly greater molecular structure diversity ( Figure 6—figure supplement", "1 ) and higher self-similarity than therapeutic drug compounds ( two-sample K-S test 0 . 49 , p value=4 . 7395e-06 ) .", "Metabolic functions are not necessarily equally distributed across microbes in the microbiome .", "For example , as described above , inactivation of digoxin , a cardiac glycoside inhibitor , is linked to cgr operon expression levels in a single species , E . lenta ( Haiser et al . , 2013; Koppel et al . , 2018 ) .", "In contrast , the deconjugation and resulting reactivation of SN-38 , the active metabolite of the chemotherapeutic colorectal cancer drug irinotecan , is linked to a phylogenetically diverse guild of microbial β-glucuronidase carrying microbes ( Guthrie et al . , 2017; Pollet et al . , 2017; Wallace et al . , 2015 ) .", "The question arises , how many microbes can perform specific enzymatic functions ?", "Knowing the taxonomic distribution of a function can guide approaches to validate hypotheses of microbiota driven modification of specific therapeutic drug or food compounds .", "More broadly , addressing this question informs therapeutic approaches for targeting specific enzymes to modulate patient responses to drugs and foods .", "In MicrobeFDT , we quantify how many taxa have the capacity to carry out a specific function by applying a modified Simpson index function to compute an Enzyme Commission number-specific dominance ( ECsD ) score for all enzymes present in the network .", "ECsD scores are based on the abundance of enzymes annotated at the species level across healthy human metagenomes from the Integrative Human Microbiome Project ( iHMP ) ( Proctor et al . , 2014 ) and are normalized between 0 and 1 .", "Functions carried out by small numbers of species have values closer to 0 while functions carried out by taxonomically diverse groups have functions closer to 1 .", "Thus , the ECsD indicates how broadly distributed a function is , a crucial metric for ( 1 ) understanding how to modify a function in the microbiome and ( 2 ) predicting how disruptive to the community modifying a function might be .", "To validate ECsD scores we first identified biochemical pathways containing enzymes with high and low taxonomic dominance in the literature .", "Bacterial synthesis of various B group vitamins including biotin , cobalamin and riboflavin vary in the number of potential producers at the Phylum level ( Magnúsdóttir et al . , 2015 ) .", "The most commonly synthesized B vitamin across diverse microbial taxa is riboflavin while vitamin B12 is dominated by Fusobacteria ( Magnúsdóttir et al . , 2015 ) .", "The ECsD scores of cobalt-precorrin-2 C ( 20 ) -methyltransferase ( 0 . 305502 ) from the anaerobic Vitamin B12 synthesis pathway and riboflavin synthase ( 0 . 691618 ) from the riboflavin synthesis pathway in MicrobeFDT agree with the prior systematic genome assessment and experimental results of Magnúsdóttir et al . ( 2015 ) ( Figure 7 ) .", "While most bacteria do not synthesize sphingolipids , sphingolipid biosynthetic capacity has been identified in Sphingomonas spp , Bacteroides and human intestinal pathogens that synthesize and incorporate sphingolipids into their membranes or target host sphingolipids as a point of entry into host cell types ( Heaver et al . , 2018; Heung et al . , 2006; Olsen and Jantzen , 2001 ) .", "The low ECsD score of phosphatidate phosphatase ( 0 . 007353 ) , an enzyme involved in sphingolipid biosynthesis and metabolism ( Olsen and Jantzen , 2001 ) , mirrors the limited distribution of the sphingolipid biosynthetic capacity across gut microbes .", "To provide a practical example of using multiple features of MicrobeFDT to identify uninvestigated microbiota-driven drug toxicity , we searched the network for compounds with high structural and toxicity similarity .", "Among these compounds were the ovarian cancer drug altretamine ( Lee and Faulds , 1995 ) and the environmental contaminant melamine ( Figure 8 ) .", "Both melamine and altretamine have toxicity profiles that include diarrhea and renal toxicity ( Rose et al . , 1996; Zheng et al . , 2013 ) .", "Melamine , an industrial compound , has experimentally validated microbiome-mediated toxicity ( Zheng et al . , 2013 ) .", "Altretamine toxicity , however , has not previously been linked to an individual’s gut microbiota .", "Approximately half of patients taking altretamine orally experience various forms of gastrointestinal toxicity including diarrhea , nausea and/or vomiting ( Keldsen et al . , 2003 ) .", "Within the network altretamine is linked to microbial N-demethylase enzymes which may remove methyl groups from this compound , potentially leading to similar toxic effects as seen with melamine .", "We found no published experimental evidence of gut microbiota mediated conversion of altretamine .", "However , N-demethylases in Pseudomonas putida CBB5 enable this microbe to grow on caffeine and other purine alkaloids as the sole carbon and nitrogen source; thus annotated N-demethylases in P . putida CBB5 can act on compounds that are structurally similar to altretamine ( Summers et al . , 2012 ) .", "Furthermore , we identify hypothetical proteins homologous to Pseudomonas putida CBB5 N-demethylases in a subset of healthy human guts ( Figure 9—figure supplement 1 ) .", "We hypothesized that gut microbial N-demethylases may partially or completely N-demethylate altretamine , converting it into metabolites that contribute to patient toxicity .", "A first step in validating this hypothesis is to demonstrate that the gut microbiome can demethylate altretamine .", "We incubated altretamine in a pooled fecal slurry generated from three healthy individuals and monitored altretamine and potential metabolites using LC-MS .", "We controlled for the formation of spontaneous N-demethylation of altretamine , which has been reported in the literature ( Damia and D'Incalci , 1995 ) , and found that a metabolite that is structurally identical to pentamethylmelamine , a demethylated altretamine metabolite , increases in active fecal microcosms over 48 hr ( Figure 9 ) .", "In active fecal biotic conditions the metabolite continually increased between time 0 and 48 hr .", "Killed controls demonstrated an increase in metabolite between 0 and 24 hr , though to a lesser extent than in active fecal microcosms .", "Notably there was little metabolite formation after 24 hr , indicating that in addition to abiotic N-demethylation , active gut microbes demethylate altretamine to the putative metabolite pentamethylmelamine .", "MicrobeFDT suggests an unrecognized role for bile acid-like foods and drugs in altering the composition of the human gut .", "Conjugated primary bile acids ( BA ) function as potent detergents and antimicrobial agents capable of dissolving microbial membranes and causing intracellular acidification; bile acid function is linked to specific structural features of these compounds ( Jones et al . , 2008; Begley et al . , 2006 ) .", "Taurochenodeoxycholic acid ( TCDCA ) is a taurine conjugated primary bile acid with a diet-tunable concentration in the gut ( Ridlon et al . , 2016 ) .", "Energy drinks , animal protein and fish are rich sources of taurine while vegetarian and vegan diets dominated by fruits , vegetables , legumes and soy are poor sources ( Ridlon et al . , 2016 ) .", "Taurine conjugated bile acids are hypothesized to contribute to the etiology of colorectal cancer by generating hydrogen sulfide during microbial mediated de-conjugation of taurine conjugates ( Ridlon et al . , 2016 ) .", "Conjugated primary bile acids have demonstrated in vitro activity as antimicrobial compounds , for example glycocholic and taurocholic conjugated bile acids are bacteriostatic , inhibiting S . aureus growth by decreasing intracellular pH and disrupting the proton motor force ( Sannasiddappa et al . , 2017 ) .", "Using MicrobeFDT , we identified therapeutic drug and food compounds that are structurally similar to TCDCA; we propose these compounds might have similar antimicrobial effects on the microbiome and we discuss studies from other groups that support this hypothesis ( Figure 10a ) .", "Bile salt hydrolase ( BSH ) mediated bile salt deconjugation is one mechanism that gut microbes use to detoxify conjugated primary bile acids ( Begley et al . , 2006 ) ; thus BSH activity may support gut bacterial persistence in face of frequent contact with primary BAs .", "We first subdivided TCDCA-like antimicrobial compounds based on BSH enzyme susceptibility .", "BSH enzymes are phylogenetically diverse and abundant across healthy human fecal metagenomes ( Figure 10—figure supplement 1 ) .", "Among the BSH-susceptible therapeutic drug compounds , we identified known antibiotics such as clindamycin and lincomycin , as well as non-antibiotic prescribed therapeutics such as finasteride , which is used for the treatment of androgenetic alopecia ( Manabe et al . , 2018 ) and benign prostatic hyperplasia ( Chau et al . , 2015 ) , and the oral antidiabetic drug saxagliptin ( Men et al . , 2018 ) ( Figure 10b ) .", "Notably , in a Wistar rat model of chronic bacterial prostatitis ( CBP ) , finasteride reduces bacterial infection as a single agent and has a synergistic effect with ciprofloxacin through an unknown mechanism ( Lee et al . , 2011 ) .", "Through in vitro studies , Chavex-Dozal and colleagues propose a role for finasteride in the prevention of Candida albicans biofilm formation and filamentation ( Chavez-Dozal et al . , 2014 ) .", "These experimental results support the hypothesis that finasteride may have unrecognized off-target antibiotic effects .", "Most TCDCA-like compounds in MicrobeFDT are non-BSH susceptible food-derived compounds .", "Among the TCDCA-like non-BSH susceptible compounds are oral steroid medications , including dexamethasone and betamethasone ( Figure 10c ) .", "The immunomodulatory activities of glucocorticoids , including dexamethasone , involve the activation of genes related to anti-inflammatory cytokines such as IL-10 and proteins that inhibit the pro-inflammatory NFκB signaling pathway ( Coutinho and Chapman , 2011; Huang et al . , 2015 ) .", "Dexamethasone has known anti-microbial properties .", "For example , dexamethasone has dose-dependent anti-microbial activity against clinically isolated Streptococcus milleri , Aspergillus flavus , and Aspergillus fumigatus in culture , while not killing Staphylococcus aureus ( Neher et al . , 2008 ) .", "Pseudomonas aeruginosa was found to be susceptible to dexamethasone at high concentrations ( Neher et al . , 2008 ) .", "Cortisone , which also has significant structural overlap with TCDCA , has been linked to a variety of opportunistic infections by enteric bacterial pathogens , for example an increase in gastrointestinal parasites ( Nair et al . , 1981 ) and reactivation of Chlamydia pneumoniae ( Laitinen et al . , 1996 ) .", "Food-derived TCDCA-like compounds include steviol , lanosterol and tomatidine .", "Steviol is a component of stevia which has antimicrobial properties against Borrelia burgdorferi in vitro ( Theophilus et al . , 2015 ) , and lanosterol derivatives have antifungal activities ( Shingate , 2013 ) .", "Tomatidine was recently identified as an antibiotic molecule that inhibits ATP synthesis against Staphylococcus aureus ( Lamontagne Boulet et al . , 2018 ) ; we hypothesize that the antimicrobial activity of this compound may include intracellular acidification given its structural overlap with TCDCA ( Figure 10d ) .", "The network thus identifies compounds with known anti-microbial properties in addition to proposing additional , structurally related compounds with uncharacterized effects .", "We propose that in addition to modulating immune responses , bile salt-like compounds may selectively alter human microbiomes , again , with unknown consequences for treatment outcomes and health .", "We next applied MicrobeFDT to identify diet-derived substrates of a gut carbohydrate active enzyme , β-glucuronidase .", "β-glucuronidases play a major role in the toxicity of the colorectal cancer chemotherapeutic prodrug irinotecan ( CPT-11 ) , whose active form , SN-38 , is inactivated by hepatic glucuronidation and excreted into the gut as the inactive metabolite SN-38 glucuronide ( SN-38G ) ( Wallace et al . , 2010; Sparreboom et al . , 1998 ) .", "Microbial β-glucuronidases hydrolyze the glucuronide group , releasing the aglycone SN-38 into the intestinal environment ( Figure 11a ) .", "Deconjugation promotes epithelial damage and severe diarrhea in some patients and in mouse models ( Wallace et al . , 2010; Sparreboom et al . , 1998; Slatter et al . , 2000 ) .", "We previously demonstrated that individual human fecal samples have variable capacities to deconjugate SN-38G ( Guthrie et al . , 2017 ) .", "Identifying the full substrate pool of β-glucuronidases is thus important for", "1 ) understanding how diet contributes to β-glucuronidase abundance and expression levels in the gut and", "2 ) to enable novel therapeutic strategies such as nutritional competition .", "Some food compounds may be preferred substrates for microbiome β-glucuronidases which would otherwise deconjugate SN-38G .", "If true , one could potentially alleviate toxicity associated with the deconjugation of SN-38G via nutritional competition with a preferred substrate .", "Therefore , we scanned the chemical similarity module containing SN-38G for dietary compounds that may serve as alternative substrates for microbial β-glucuronidases .", "Most compounds identified as significantly similar to SN-38G were food derivatives or other constituents ( Figure 11b ) .", "Among these targets were flavonoids such as baicalin and scutellarin which are widely distributed in plants ( Kumar and Pandey , 2013 ) ( Figure 11c ) .", "We propose that these compounds may compete with SN-38G for turnover by microbial β-glucuronidases and are a potential avenue for decreasing the adverse drug responses associated with irinotecan administration ." ], [ "The chemical space of the human gastrointestinal tract ecosystem is shaped by host dietary intake , xenobiotic exposure , and host and gut microbiome derived products .", "In turn , diet shapes the composition and potential niches of organisms within human gut microbiomes .", "A combination of compound , host , and microbiome features influence potential microbial metabolism .", "Examining these features individually cannot reliably infer clinical phenotypes associated with microbiome/compound interactions .", "Two molecules may have the same toxicity profile but very different biochemistry , for example .", "Automated enzyme annotation may be incorrect , and compound structural similarity is often insufficient to predict substrate preferences .", "Finally , enzymes that carry out a reaction associated with a patient phenotype may be unevenly distributed across microbes and across human microbiomes .", "MicrobeFDT is designed to overcome some of these limitations by enabling a more holistic analysis of toxicity , structure , metabolism and ecology .", "We used a combination of network features to successfully predict the novel microbial metabolism of the cancer drug altretamine .", "Metabolomics data indicate active demethylation of altretamine by fecal slurries but cannot propose a mechanism by which microbial activity metabolizes this compound .", "MicrobeFDT suggests that altretamine is a putative substrate of microbial N-demethylases .", "Microbe-mediated N-demethylation reactions , and the subsequent release of N-methyl groups , occur as a part of amino acid and nucleotide metabolism ( D’Mello and International , 2017 ) .", "Notably , diet is a source of amino acids which are derived in part from metabolism of dietary choline , carnitine and legumes , and have physiological functions for bacteria including osmoprotection and incorporation into bacterial flagellin proteins and lipid membranes ( Goldfine and Hagen , 1968 ) .", "Amino acid-specific bacterial N-demethylases have been identified but are poorly characterized ( Wargo , 2017 ) .", "Additionally , fecal and species specific N-demethylation has been observed for other therapeutic drugs and commonly ingested compounds such as caffeine , which clusters with altretamine in the network due to its structural similarity ( Summers et al . , 2012; Caldwell and Hawksworth , 1973; Clark et al . , 1983; Colombo et al . , 1982 ) .", "N-demethylases can act on chemically diverse substrates ( Wargo , 2017; Burnet et al . , 2000 ) .", "Given this body of evidence , we propose N-demethylases may demethylate altretamine partially or completely , creating metabolites that are toxic to patients .", "Human gut metagenomic data indicate that Rieske family oxidative N-demethylases are carried by a small , phylogenetically conserved set of gut taxa , with notable inter-personal variation .", "That these enzymes require oxygen may make them more relevant during disruptions to gut homeostasis when oxygen becomes available , such as colonic crypt hyperplasia caused by injuries to the intestinal epithelia ( Litvak et al . , 2018 ) .", "Finally , we note that N-demethylation in the gut may be relevant for differences in individual metabolism of numerous other compounds such as the cancer drug tamoxifen , the widely used antihistamine diphenhydramine , and theobromine , a plant alkaloid found in foods .", "While is possible that N-demethylation is enzyme independent or that enzymes annotated with other functions are responsible for this activity , MicrobeFDT provides a clear path forward for mechanistic studies of N-demethylation in the gut .", "Beyond predicting the toxicity or function of gut compounds , MicrobeFDT identifies the larger substrate pool for enzymes involved in drug metabolism .", "For example , shared conjugation patterns may represent a clinically relevant way to group compounds that share microbial enzymatic processing .", "As an example , compounds inactivated by glucuronidation are susceptible to microbial β-glucuronidase-mediated reactivation .", "We used MicrobeFDT to identify compounds structurally similar to the conjugated , detoxified irinotecan metabolite SN-38G and found dietary substrates that may interact with similar β-glucuronidases that this drug interacts with .", "Structurally similar compounds may act competitively – via inhibition of SN-38G turnover by higher priority β-glucuronidase substrates or synergistically – via substrate inducible transcriptional upregulation of β-glucuronidase enzymes .", "A person consuming a large amount of the plant-based compound scutellarin as part of a supplement , for example , might be inadvertently modulating the effects of their cancer therapy .", "Outside of drug metabolism , β-glucuronidases mediate deconjugation and enterohepatic circulation of estrogens , impacting the human host total estrogen burden ( Shapira et al . , 2013; Kwa et al . , 2016 ) .", "It has been hypothesized that β-glucuronidase deconjugation may result in greater absorption of estrogens and thus influence the development of estrogen-driven cancers including breast , ovarian and endometrial cancers ( Shapira et al . , 2013; Kwa et al . , 2016 ) .", "Our network is useful for developing mechanistic hypotheses targeting how diet and the microbiome jointly act as moderators of estrogen-driven cancers , and to suggest opportunities for diet-based modulation of total estrogen levels .", "An important step towards characterizing the role of the gut microbiome in shaping individual responses to foods and drugs is identifying how gut microbiome metabolism varies from compound to compound and how this metabolism relates to inter-personal variation in diet or drug responses to specific compounds .", "To tackle this challenge , we add the context of taxonomic diversity to the predicted impact of microbial on specific targets by quantifying enzyme specific taxonomic dominance and diversity with a novel metric , the ECsD score .", "This score distinguishes enzymatic activities carried out by single species or few taxa , such as N-demethylase activity , from those where many taxa may contribute , such as β-glucuronidase and bile salt hydrolase activity .", "The ECsD score is a readout of potential substrate metabolism at the community level that can be linked to inter-personal variation in gut function and phenotypic outcomes .", "The structural similarity network that underlies MicrobeFDT could be improved by using compound atom and bond connectivity information as an additional filtering step for compounds of interest , for example by using information from the SMARTS molecular pattern matching language ( Chepelev et al . , 2012 ) .", "SMARTS can be used to specify sub-structural patterns in molecules; these patterns could be added to MicrobeFDT as an additional information source indicating potential active moieties in compounds .", "MicrobeFDT does not predict substrate specificity for microbiome enzymes; available data and methods are not sufficient to achieve this goal .", "Enzyme promiscuity also shapes the probability that two chemically overlapping compounds will be processed by the same enzyme .", "A future improvement to our resource could extract data from resources like RetroRules ( Duigou et al . , 2019 ) , which uses SMARTS strings to define reaction rules , or utilize the Promis server measure of enzyme multifunctionality ( Carbonell and Faulon , 2010 ) to further support a user’s ranking of hypothesized compound-enzyme interactions .", "It must be noted that the set of diet-derived and xenobiotic compounds that form the basis of the network is a non-exhaustive representation of the gut chemical landscape .", "Efforts to characterize the gut chemical space using metabolomics approaches including mass spectrometry and nuclear magnetic resonance spectroscopy will play key roles in elucidating a fuller gut chemical landscape ( Vernocchi et al . , 2016; Wishart , 2012 ) .", "MicrobeFDT does not address the issue of compound concentrations in the gut , which are vital to assess likely physiological effects .", "Lastly , MicrobeFDT is limited to enzymes in KEGG , and does not address the many hypothetical enzyme sequences identified through metagenomic sequencing .", "Despite these limitations , MicrobeFDT highlights areas of known gut chemical space for which our understanding of microbial processing is limited and is a powerful tool to guide mechanistic investigations into diet-drug-microbiota interactions ." ], [ "The MicrobeFDT graph database encodes heterogeneous information on the interactions between compounds and microbial enzymes in the gut chemical landscape , highlighting the following four relationships across 13 , 440 nodes ( 10 , 822 xenobiotic , diet-derived and human gut endogenous compounds , 2062 microbial enzymes and 525 therapeutic drug use labels ) defined from publicly available data directly or computed: ( 1 ) compound-compound substructure similarity; ( 2 ) compound-compound toxicity similarity; ( 3 ) microbial enzyme-compound interactions; and ( 4 ) drug-indication associations .", "The database is implemented in Neo4j ( https://neo4j . com/ ) and can be queried through the Cypher Query Language .", "Through graph-based searches users can query the network based on node or relationship features .", "MicrobeFDT can be accessed here ( Guthrie , 2019 ) .", "The SIDER 4 . 1 side effect resource is a database of approved medicines and their known adverse reactions ( Kuhn et al . , 2016 ) .", "Drugs from this database with pharmacokinetic profiles that involve entry into the gastrointestinal tract were identified through literature mining and manual curation and indexed by their PubChem CID identifier ( Kim et al . , 2016 ) .", "Drug use annotations were based on the WHO Anatomical Classification System ( Skrbo et al . , 2004 ) .", "FooDB ( http://foodb . ca/ ) ( Wishart , 2018 ) , a database containing raw food component structures , biological interactions and chemical properties was the source of food components linked to PubChem CID identifiers .", "ClassyFire was used to annotate all xenobiotic and food derived compounds with a shared chemical taxonomy ( Djoumbou Feunang et al . , 2016 ) .", "To link microbial enzymes to the set of compounds they metabolize we used KEGGREST ( v1 . 14 . 1 ) to retrieve KEGG compound identifiers with links to Enzyme Commission numbers , metabolic modules and pathways , and presence in either organisms listed as microbial or Homo sapiens ( Tenenbaum , 2019 ) .", "Enzyme abundance data across human metagenomes were determined based on the total abundance of each enzyme in the healthy participants of the Human Microbiome Project .", "This data was extracted from the Integrated Microbial Genomes database ( Markowitz et al . , 2012 ) .", "Enzyme specific dominance scores ( ECsD ) , which is a measure of the number of different species that carry a specific enzyme , were computed based on species-specific enzyme abundance data from healthy individuals from the Integrative Human Microbiome Project ( Proctor et al . , 2014 ) .", "For each enzyme , we computed an enzyme commission number-specific dominance ( ECsD ) score .", "This score is an application of the Simpson’s index , which is particularly sensitive to sample evenness ( DeJong , 1975 ) , and describes the dominance and diversity profile of species carrying the enzyme ( Ofaim et al . , 2017 ) .", "The taxa-specific enzyme abundance information is based on data collected as a part of the integrative Human Microbiome Project ( iHMP ) ( PRJNA306874 ) ( Proctor et al . , 2014 ) .", "ECsD scores are reported as Simpson index measure ( Simpson , 1949 ) subtracted from one , as implemented in the phyloseq R package ( McMurdie and Holmes , 2013 ) .", "In this implementation , the Simpson dominance index per enzyme defined by its enzyme commission number ( D ( EC ) ) is computed such that n is number of individuals of each species that carry the enzyme and N is the total number of individuals of all species that carry the enzyme ( 1 ) .", "For better interpretability , the dominance scores are subtracted from 1 ( 2 ) .", "( 1 ) D ( EC ) = Σ n ( n-1 ) N ( N-1 ) ( 2 ) ECSD=1−D ( EC ) Thus , enzyme functions carried out by small numbers of microbes have values closer to 0 while functions carried out by taxonomically diverse groups have functions closer to 1 ." ] ]

[ "Microbes are nature’s chemists , capable of producing and metabolizing a diverse array of compounds .", "In the human gut , microbial biochemistry can be beneficial , for example vitamin production and complex carbohydrate breakdown; or detrimental , such as the reactivation of an inactive drug metabolite leading to patient toxicity .", "Identifying clinically relevant microbiome metabolism requires linking microbial biochemistry and ecology with patient outcomes .", "Here we present MicrobeFDT , a resource which clusters chemically similar drug and food compounds and links these compounds to microbial enzymes and known toxicities .", "We demonstrate that compound structural similarity can serve as a proxy for toxicity , enzyme sharing , and coarse-grained functional similarity .", "MicrobeFDT allows users to flexibly interrogate microbial metabolism , compounds of interest , and toxicity profiles to generate novel hypotheses of microbe-diet-drug-phenotype interactions that influence patient outcomes .", "We validate one such hypothesis experimentally , using MicrobeFDT to reveal unrecognized gut microbiome metabolism of the ovarian cancer drug altretamine ." ]

[ "Microbes in the human gut can play helpful roles by producing vitamins or breaking down complex carbohydrates .", "Collectively , gut microbes carry out these roles using a large toolkit of enzymes that catalyze a diverse range of chemical reactions , some of which cannot be carried out by human enzymes .", "However , these microbial enzymes can also cause harm if they alter drugs in a way that makes them toxic or prevents them from working .", "Little is known about which microbial enzymes interact with which foods and drugs , or how these interactions affect human health .", "Guthrie et al . have now developed and tested a tool called MicrobeFDT that can help researchers to understand these complex interactions .", "In MicrobeFDT , 10 , 000 compounds produced by the human body or found in food or drugs are grouped based on their structure .", "Compounds are linked to the microbial enzymes that interact with them and drugs are annotated with information on known toxicities .", "The result is a network where compounds with similar structure are linked to each other .", "If a microbial enzyme interacts with one compound in a group , it may interact with related compounds as well , potentially causing similar effects on human health .", "The network makes it easier for researchers to work out which compounds are affected by particular gut microbes .", "For example , MicrobeFDT suggested how gut microbes might alter the structure of an ovarian cancer drug called altretamine , which can cause diarrhea and kidney damage as side effects .", "Experiments confirmed that the predicted structural change does occur in human feces .", "MicrobeFDT may increase how quickly researchers can assess harmful interactions between gut microbes , food , and drugs .", "It also may help them to develop new strategies to improve human health based on how microbial enzymes interact with food and drugs ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "biochemistry and chemical biology", "cancer biology" ]

[ [ "The pharmaceutical industry must deliver safe and effective medicines while simultaneously limiting the costs associated with drug development , and a central part of this process is de-risking potential safety liabilities at an early stage ( Morgan et al . , 2018 ) .", "In many cases , lack of mechanistic understanding about how compounds cause toxicity hampers predictions of adverse drug reactions ( ADRs ) , and more information about the specific substructures of drug molecules that cause ADRs is required .", "Such information can then be used to populate machine learning algorithms to generate adverse outcome pathways ( AOPs ) that predict likely outcomes ( Dey et al . , 2018 ) from off-target toxicities and that can be used in early phase drug design ( Allen et al . , 2018 ) .", "It is widely accepted that disruption of mitochondrial function is a common cause of ADRs and it has been proposed that mitochondrial toxicity , which has a major role in idiosyncratic drug toxicity ( Uetrecht and Naisbitt , 2013 ) , is responsible for up to 50% of post-market drug withdrawals ( Will and Dykens , 2014; Dykens and Will , 2007 ) .", "Mitochondrial toxins often have a differential effect on tissue function due to organ-specific variations in the mitochondrial proteome and its function ( Johnson et al . , 2007a ) ; primarily biosynthetic and metabolic in liver , and energy production in muscle ( Johnson et al . , 2007b; Calvo and Mootha , 2010 ) .", "For example , cardiomyocytes , which have high energy requirements and are rich in mitochondria ( El-Hattab and Scaglia , 2016 ) , are particularly sensitive to mitochondrial toxins that alter ATP production ( Kolwicz et al . , 2013 ) .", "Mitochondria are central to many cell-wide processes so mitochondrial toxicity affects bioenergetics , metabolism , signalling and oxidative stress ( Meyer et al . , 2018 ) in addition to impacting stemness , differentiation and apoptosis ( Guerra et al . , 2017 ) .", "Due to these pleiotropic roles , ‘off-target’ drug toxicity resulting in impairment of mitochondrial function can easily be misattributed to other targets and cellular processes .", "Given the relative paucity of mechanistic knowledge relating specific drug substructures to ADRs we carried out a detailed chemical dissection of mubritinib , which was reported to act as a tyrosine kinase ( HER2 ) inhibitor and which has been trialled as a treatment for a range of cancers ( Nagasawa et al . , 2006; Ouchida et al . , 2018 ) .", "However , mubritinib has also been shown to affect energy status ( Baccelli et al . , 2019; Sridhar et al . , 2003 ) , and it was recently demonstrated to have an ‘off-target’ effect on mitochondrial function through inhibition of respiratory complex I in cells derived from patients with Acute Myeloid Leukaemia ( AML ) , leading to a new therapeutic option ( Baccelli et al . , 2019 ) .", "Here , we show that mubritinib does not bind directly to HER2 and that its inhibitory effect on complex I negatively impacts on cardiomyocyte function , an important clinical consideration .", "Through the use of a focussed chemical library , we show that modifying the 1H-1 , 2 , 3-triazol-1-yl moiety present in mubritinib substantially alters both the inhibition of complex I and the toxicity to cardiomyocytes; identifying a heterocyclic 1 , 3-nitrogen motif as being key to its complex I inhibitory action .", "A search of chemical space was then undertaken for the same substructure , and led to the drug carboxyamidotriazole ( CAI ) , which has also been trialled as an anti-cancer agent ( Sridhar et al . , 2003; Omuro et al . , 2018; Azad et al . , 2009; Johnson et al . , 2008 ) .", "We show that , like mubritinib , CAI does not directly inhibit its reported target ( calcium channels ) and is also a potent complex I inhibitor .", "Furthermore , like mubritinib , chemically altering the triazole ring moiety ablated toxicity .", "In both cases we show that mitochondrial toxicity is directly linked to anti-cancer activity , as chemical manipulation of the toxicophoric heterocycle ablates the desired effects of both compounds on cancer-cell proliferation and apoptosis .", "Thus , we have identified a novel toxicophoric motif that is mechanistically linked to an adverse cardiac cell event .", "Our data demonstrate that caution must be taken when attributing a drug mechanism of action without detailed structure activity relationship ( SAR ) analysis , since inhibition of mitochondrial function has such cell-wide effects ." ], [ "Previous data have reported that mubritinib inhibits phosphorylation of HER2 in breast cancer cell lines that express high levels of this receptor ( Nagasawa et al . , 2006 ) .", "Therefore , the HER2-overexpressing cell line , BT474 , was treated with increasing doses of mubritinib and the effect on HER2 phosphorylation was analysed by western blotting , with the specific HER2 inhibitor lapatinib ( Brandão et al . , 2018 ) as a positive control ( Figure 1A , Figure 1—figure supplement 1A ) .", "Surprisingly , there was only a small decrease in HER2 phosphorylation in the presence of mubritinib , in contrast to lapatinib .", "Furthermore , recent data have further shown that mubritinib , in common with known inhibitors of mitochondrial respiratory complex I ( Sica et al . , 2020 ) , alters the phosphorylation status of proteins that sense changes in energy status and stimulate cellular proliferation , such as mTOR ( Leibovitch and Topisirovic , 2018 ) .", "In agreement with these data , we show that treatment of cells with mubritinib ( Figure 1B ) alters the phosphorylation status of proteins downstream of the energy sensor AMPK ( e . g . acetyl-CoA carboxylase ) and impacts on mTOR signalling ( e . g . phosphorylation of RPS6 , Figure 1—figure supplement 1B ) .", "Moreover , similar effects were also observed with treatment by the known mitochondrial inhibitors , rotenone and antimycin A ( Figure 1B and Figure 1—figure supplement 1Ci and ii ) .", "In contrast , lapatinib , which is a specific HER2 inhibitor , blocks signalling downstream from mTOR with minimal effect on ACC phosphorylation ( Figure 1B , Figure 1—figure supplement 1Ci and ii ) , but a large effect on RPS6 ( Figure 1—figure supplement 1Ci and ii ) .", "An inactive mubritinib analogue ( compound 5 , see below ) was used as a negative control .", "An in vitro tyrosine kinase activity assay was then carried out with increasing concentrations of mubritinib , in which recombinant human HER2 was incubated with radioactively labelled 32P-ATP .", "Mubritinib did not decrease 32P incorporation , even at 10 μM ( Figure 1C and D ) , demonstrating that it does not inhibit HER2 phosphorylation .", "Furthermore , consistent with our data ( Figure 1E ) , mubritinib has been reported to display no activity against almost 300 other kinases screened ( Anastassiadis et al . , 2011 ) .", "Because mubritinib affects signalling pathways associated with a decrease in cellular energy , and because inhibiting mitochondrial respiration could have particularly deleterious effects on tissues with high energy demand such as the heart , we tested the effect of mubritinib on ATP production in H9c2 cardiomyoblasts ( Kimes and Brandt , 1976 ) and human embryonic stem cell derived cardiomyocytes ( hESC-CM , GE Healthcare ) using the glucose/galactose system .", "In galactose-containing media cells are predominantly reliant on mitochondria for the production of cellular ATP , allowing mitochondrial liabilities that are masked in glucose to be revealed ( Marroquin et al . , 2007; Rana et al . , 2011 ) .", "Exposure of H9c2 cells to 2 μM mubritinib for 2 hr in galactose-containing media led to a 50% decrease in ATP levels ( Figure 1F ) .", "Furthermore , prolonged exposure depleted ATP levels to 10% of the control value ( Figure 1F ) and induced cell death ( Figure 1G ) .", "Importantly , inhibition of ATP production in galactose ( but not glucose ) containing media by mubritinib and other inhibitors of oxidative phosphorylation was also observed in hESC-CMs ( Figure 1H ) and had a profound effect on cell beat rate ( Figure 1I ) .", "It has been shown recently that mubritinib targets respiratory complex I and inhibits growth of cancer cells from patients with AML , which are highly dependent on oxidative phosphorylation for survival ( Baccelli et al . , 2019 ) .", "Therefore , to understand likely toxicities associated with such treatments , we investigated whether complex I is similarly affected in cardiomyocytes exposed to mubritinib ( 1 ) , and used a focussed chemical library of mubritinib derivatives ( 2-8 ) to identify the potential toxicophore ( Figure 2A ) .", "The mubritinib variants were synthesised with modifications in two regions of the molecule; the aryl trifluoromethyl group ( compounds 2–4 ) and the triazole group ( compounds 5–8 ) .", "The oxygen consumption rates ( OCR ) of H9c2 and hESC-CM cells treated with mubritinib ( 1 ) were measured to determine whether the decreased ATP content in galactose containing media ( Figure 1F and H ) were due to direct inhibition and/or uncoupling of the respiratory chain ( Felser et al . , 2013 ) .", "As expected , oligomycin A , which inhibits ATP synthase , had no effect on the rotenone-sensitive OCR in the presence of the uncoupling agent FCCP ( carbonyl cyanide-4- ( trifluoromethoxy ) phenylhydrazone ) .", "However , in H9c2 or hESC-CMs cells exposed to mubritinib the rotenone-sensitive OCR decreased by ~50% ( Figure 2B and Figure 2—figure supplement 1A and B ) .", "Taken together , these data suggest that mubritinib ( 1 ) inhibits the mitochondrial respiratory electron transport chain in cardiomyocytes .", "The effects of mubritinib ( 1 ) on complex I and II linked respiration were then determined in plasma-membrane permeabilised H9c2 cells .", "First , cells were pre-treated with increasing doses of mubritinib ( 1 ) , or the complex I inhibitor rotenone or the complex III inhibitor antimycin A . As expected , all inhibitor treatments decreased the OCR ( Figure 2C ) .", "Cells were then treated with plasma membrane permeabiliser followed by addition of ADP .", "Pyruvate/malate and succinate were used to drive respiration from complex I and complex II , respectively and , in untreated cells , they both stimulated the OCR .", "All three inhibitors inhibited pyruvate/malate-driven respiration but , crucially , the inhibition of only mubritinib and rotenone was alleviated by subsequent treatment with succinate ( Figure 2C ) , suggesting that mubritinib is a complex I inhibitor .", "Mitochondrial membranes were then used to assess the effect of mubritinib ( 1 ) on complex I and complex II driven respiration directly .", "In agreement with the data obtained from cell lines , mubritinib ( 1 ) showed a dose-dependent decrease in the rate of NADH oxidation and no effect on succinate oxidation ( Figure 2D and E ) .", "The NADH oxidation data were then fit to the standard dose-effect relationship and yielded an IC50 value of 19 . 2 nM ( Figure 2D ) .", "Purified complex I was then used to confirm inhibition of complex I unambiguously , and to dissect whether mubritinib ( 1 ) inhibits it at its NADH or ubiquinone binding site .", "NADH oxidation was coupled to reduction of either the ubiquinone-10 analogue decyclubiquinone ( dQ ) or to reduction of an artificial electron acceptor ( APAD+ or ferricyanide , FeCN ) that reoxidises the flavin in the NADH binding site directly ( Birrell et al . , 2009; Yakovlev and Hirst , 2007 ) , without the involvement of ubiquinone ( Figure 2E; Birrell et al . , 2009 ) .", "While APAD+ and FeCN reduction were unaffected at 500 nM mubritinib ( 1 ) , the rate of ubiquinone reduction was essentially abolished ( Figure 2E ) .", "These data confirm that mubritinib inhibits complex I directly by inhibiting ubiquinone reduction , most likely by binding in the ubiquinone-binding site .", "The variants of mubritinib were then tested for their ability to inhibit complex I in mitochondrial membranes ( Figure 2F and Figure 2—figure supplement 2 ) .", "Compounds 2–4 ( Figure 2A ) , which have the same N1-linked triazole moiety as mubritinib but contain either no substituent ( 2 ) or a mild ( 3 ) or strong ( 4 ) electron donating group in the para-position of the phenyl ring , retain the ability to inhibit complex I , similarly to mubritinib ( Figure 2F ) .", "In contrast , compound 6 , which has the 1 , 2 , 3-triazol-1-yl moiety substituted for an isomeric 1 , 2 , 3-triazol-2-yl group , is a much weaker inhibitor ( Figure 2F ) .", "Complete removal of the triazole group in 5 and modification of the triazole to an N-linked pyrrole in 8 , also resulted in compounds that no longer inhibited NADH oxidation ( Figure 2F ) .", "Most interestingly , modification of the triazole to the N-linked imidazole 7 retained the inhibitory activity .", "These data provide strong evidence that a 1 , 3-amidine-like motif , housed within the 1H-1 , 2 , 3-triazol-1-yl substituent , is required for complex I inhibition .", "The same pattern of inhibition was observed for ATP production in cells grown in media containing galactose ( Figure 2G ) .", "Therefore , inhibition of ATP production by mubritinib results from the inhibition of complex I , and depends strongly upon its 1 , 2 , 3-triazol-1-yl moiety and the embedded toxicophore .", "Based on the 1H-1 , 2 , 3-triazol-1-yl moiety being critical for the function of mubritinib we initially searched for other compounds that contain a 1 , 2 , 3-triazol-1-yl or similar moiety that might display analogous toxicity profiles .", "A structural similarity search carried out on the ChEMBL database revealed a range of terminal 1 , 2 , 3-triazole-containing drugs as putative complex I inhibitors ( Supplementary file", "1 ) including carboxyamidotriazole ( 9 , Figure 3A ) , which has been trialled widely as an anticancer agent in single and combination therapies to treat glioblastoma , ovarian cancer and non-small cell lung cancer ( Azad et al . , 2009; Johnson et al . , 2008 ) .", "Data from a number of studies have suggested that the anti-proliferative and anti-metastatic properties of CAI ( 9 ) are mediated through the inhibition of non-voltage gated Ca2+ channels in non-excitable cells ( Hupe et al . , 1990 ) , which in turn modulates downstream phosphorylation events ( Bauer et al . , 2000 ) , however this has not been demonstrated directly .", "Calcium binding assays performed here show clearly that there is no significant binding of CAI ( 9 ) to non-voltage gated Ca2+ channels ( Supplementary file 2 ) .", "It has also been shown that CAI ( 9 ) affects mitochondrial calcium import and local calcium clearance , which is essential for the maintenance of capacitative calcium entry ( Mignen et al . , 2005 ) and it was proposed that this then inhibited oxidative phosphorylation ( Ju et al . , 2016 ) .", "These data suggest that the toxicophore , in the context of CAI ( 9 ) , might act in a similar way to mubritinib , and that the effects on mitochondrial calcium release could be the secondary effects of complex I inhibition .", "To explore the effect of the heterocycle in CAI ( 9 ) on mitochondrial function we generated three variants .", "Two compounds , which we predicted by analogy with our mubritinib variants that would be inactive , contained isomeric pyrazoles ( 10 and 11 ) .", "One further compound was synthesised where the triazole ring in CAI was replaced with an imidazole ring ( 12 ) , which we predicted would retain mitochondrial toxicity ( Figure 3A ) .", "The set of compounds was then tested in H9c2 cardiomyoblasts for their effects on oxygen consumption rates and ATP production ( Figure 3B and C ) .", "CAI ( 9 ) and compound 12 inhibited ATP production in galactose containing media much more strongly than compounds 10 and 11 ( Figure 3B ) .", "Similarly , CAI ( 9 ) and compound 12 decreased oxygen consumption rates much more effectively than compounds 10 and 11 ( Figure 3C and Figure 3—figure supplement 1 ) .", "Given that CAI has been trialled as an anti-cancer therapeutic against lung cancer ( Johnson et al . , 2008 ) , CAI ( 9 ) and compounds 10 and 11 were tested in the lung cancer derived cell line , A549 , to determine whether the effects on mitochondrial function observed in the H9c2 cardiomyoblasts were replicated .", "The data show that CAI ( 9 ) inhibits ATP production in A549 cells grown in galactose , whereas 10 and 11 have minimal effect ( Figure 3D ) with no difference observed , as expected , in glucose containing media ( Figure 3—figure supplement 1C ) .", "Moreover , the basal and maximal OCR of cells treated with CAI ( 9 ) were significantly reduced , with a much smaller decrease observed with compounds 10 and 11 ( Figure 3E and Figure 3—figure supplement 1 ) .", "To confirm that CAI inhibited complex I , A549 cells were treated with PMP followed by addition of ADP , pyruvate and malate ( Figure 3F ) .", "In untreated cells , or cells treated with 10 and 11 , there was a large increase in oxygen consumption as expected , however , oxygen consumption was inhibited in cells treated with CAI ( 9 ) , antimycin A or piericidin .", "Following addition of succinate and ADP there was an increase OCR in CAI ( 9 ) and piericidin-treated cells ( but not in cells treated with antimycin A ) , strongly suggesting that CAI ( 9 ) is a complex I inhibitor .", "Importantly , given that compounds 10 and 11 only have a small effect , in this chemical context the triazolyl toxicophore contributes in a similar manner to mubritinib .", "To confirm these data , mitochondrial membranes were used to assess the impact of each compound on complex I-driven respiration .", "As expected , both CAI ( 9 ) and 12 inhibited complex I-driven respiration whereas compounds 10 and 11 have essentially no effect ( Figure 3G ) , as reflected by the measured IC50 values for each compound ( Figure 3—figure supplement 2 ) .", "Similar to mubritinib , inhibition of complex I with CAI also affects signalling pathways downstream of the energy sensor AMPK , such as increased ACC phosphorylation and inhibition of mTOR signalling reducing RPS6 phosphorylation ( Figure 3—figure supplement 3 ) .", "Importantly these pathways are unaffected by the non-specific calcium channel inhibitor bebridil hydrochloride or the inactive CAI variant compound 11 .", "To confirm that the toxicophore we have identified is directly linked to the reported anti-proliferative/cancer chemotherapeutic properties of mubritinib ( 1 ) and CAI ( 9 ) , the degree of cell death following treatment with these compounds was measured in a range of cancer derived cell lines .", "The cells lines used were representative of AML ( HL60 ) , glioblastoma ( M059K ) , lung ( A549 ) , osteosarcoma ( U-20S ) , in addition to HeLa cells since these cancers display varying degrees of dependence on glycolysis versus oxidative phosphorylation for energy production and to provide key metabolites required for tumour cell survival ( Molina et al . , 2018; Shi et al . , 2019 ) .", "Cells were grown in galactose or glucose containing media in the presence of mubritinib ( 1 ) , CAI ( 9 ) , or a corresponding inactive variant , 5 or 11 respectively , and the degree of cell death was measured using DRAQ7 staining and Annexin-V-FITC labelling ( Figure 4 and Figure 4—figure supplement 1 ) .", "The data show that treatment of the AML derived cell line with either CAI ( 9 ) or mubritinib ( 1 ) caused cell death in both glucose and galactose , while the analogue compounds which lacked the heterocyclic 1 , 3-nitrogen motif had no effect ( Figure 4A and B , Figure 4—figure supplement 1 ) .", "The other cell lines used showed no cell death in the presence of glucose ( Figure 4D and F and Figure 4—figure supplement 1D , F and G ) , however again there was a correlation between the presence of the toxicophore and cell death in galactose ( Figure 4C and E and Figure 4—figure supplement 1C , E and H ) .", "To explore whether the toxicophore had similar effects on cell growth , BT474 ( Figure 4G ) and A549 ( Figure 4H ) cells were grown in glucose and proliferation measured using xCELLigence RTCA DP instrument .", "Again , the data show a direct correlation between the presence of the 1 , 2 , 3-triazole and cell growth inhibition as both mubritinib ( 1 ) and CAI ( 9 ) slowed cell growth , whereas the analogues , which lacked the heterocyclic 1 , 3-nitrogen motif , had reduced or no effect .", "Taken together these data establish that the presence of the triazole and its embedded heterocyclic 1 , 3-nitrogen toxicophore is essential for the parent drug effects on tumour cell growth of otherwise chemically distinct mubritinib and CAI ." ], [ "Our data show the value of using SARs to probe the molecular signatures that potentially trigger toxicity pathways and , through the identification of a novel toxicophore , have implications for drug development programmes ( Figure 5 ) .", "The toxicophore in the context of mubritinib and CAI is the embedded 1 , 3-nitrogen motif of the 1H-1 , 2 , 3-triazol-1-yl heterocycle and we have shown that this nitrogen atom disposition appears critical for both mitochondrial toxicities ( Figures 1–4 ) .", "In a preliminary screen of compounds that inhibited complex I , we identified two antifungal agents , ketoconazole and terconazole , which also contained the heterocyclic 1 , 3-nitrogen motif embedded within a 1H-1 , 2 , 4-triazol-1-yl or 1H-imidazol-1-yl substituent respectively ( Supplementary file 3 ) .", "Interestingly , we also found that rufinamide ( Supplementary file 1 ) , which contains a chemically similar core scaffold to CAI , displayed no complex I inhibitory activity ( Serreli , 2018 ) .", "However , there are key structural differences between these two drugs and in particular , rufinamide lacks an anilino nitrogen in the 5-position of the 1 , 2 , 3-triazole , the para-chlorobenzoyl moiety and there is a chloro to fluoro switch with regards the halogen substituents on the N-benzyl group ( alongside the switch in position from 3 , 5 to 2 , 6 ) .", "These observations are therefore coupled with a difference in the logP of rufinamide ( 1 . 3 ) when compared to CAI ( 3 . 1 ) which indicates a significant decrease in its overall lipophilicity and may therefore reflect a compromised pharmacokinetic-driven target engagement .", "Which of these differences drive the observed loss in complex I inhibition by rufinamide is a focus of ongoing work .", "It is of serious concern that while both mubritinib and CAI are trialled as part of anti-cancer therapies ( Baccelli et al . , 2019 ) , neither directly bind their reported targets , HER2 and Ca2+ channels respectively ( Figure 1 and Supplementary file 2 ) .", "Mitochondria play a central role in cell-wide processes in addition to bioenergetics and metabolism by providing a signalling hub that controls stemness , differentiation and apoptosis ( Guerra et al . , 2017 ) , therefore disruption of mitochondrial function can easily be misattributed to other targets , such as receptors involved in cell signalling .", "Cardiac cells are especially sensitive to mitochondrial toxicants that alter ATP production and our data suggest that both mubritinib and CAI have the potential to affect these cell types in situations when glucose is limiting ( Figures 1 , 2 and 4 ) .", "It would therefore be important to monitor patients treated with such agents for changes in cardiac function during future clinical trials of CAI or mubritinib .", "In terms of cancer treatment , mubritinib has been used to chemo-sensitise tumour cells to other cytotoxic agents .", "For example , in combination with AC220 ( quizartinb ) , mubritinib reduces the viability of ovarian derived cell lines ( Ouchida et al . , 2018 ) .", "However , sole inhibition of complex I is also a viable treatment option for some cancer types .", "Thus while drugs that target the coordinated upregulation of glycolysis that is often associated with tumorigenesis are being developed , several studies have shown that many cancer cell subpopulations are particularly dependent upon OXPHOS for bioenergetic and biosynthetic processes ( Vazquez et al . , 2013; Viale et al . , 2014; Birsoy et al . , 2015 ) .", "Compounds that target the mitochondria , either complex I including mubritinib ( Baccelli et al . , 2019 ) and IACS-010759 ( Molina et al . , 2018 ) , or the ATP synthase such as Giboxin ( Shi et al . , 2019 ) , have been shown to have efficacy in glycolysis–deficient tumour cells derived from patients with AML and glioblastoma ( Baccelli et al . , 2019; Bauer et al . , 2000; Shi et al . , 2019 ) .", "Interestingly , IACS-010759 ( Bauer et al . , 2000 ) contains a 1H-1 , 2 , 4-triazole suggesting a similar mode of action of this drug since this heterocycle also possesses the amidine-like nitrogen substitution pattern .", "Since the efficacy of both mubritinib and CAI ( Figure 4 ) is dependent upon an amidine-like nitrogen substructure our data suggest that new drug development programmes using this substructure within the correct chemical context could be employed to devise therapies for glycolysis deficient tumours , providing that cardiac liabilities are assessed and evaluated .", "Therefore , a more detailed examination of potential molecular recognition of these and other substructures by complex I is ongoing within our laboratories , coupled with consideration of target access from a pharmacokinetic perspective to allow repurposing of a number of drugs for their anti-cancer properties ." ], [ "All cell lines were obtained from ATCC , except hESC-cardiomyocytes which were obtained from GE healthcare ( Cytiva Plus , GE Healthcare ) .", "Glucose containing media consists of glucose-containing DMEM ( Life Technologies , Gibco 41966 ) supplemented with 10% FBS .", "Galactose containing media consists of glucose-free DMEM ( Life Technologies , Gibco ) supplemented with 10 mM galactose , 2 mM L-glutamine , 1 mM sodium pyruvate and 10% FBS .", "Prior to treatments , cells were grown in their respective media overnight .", "hESC-cardiomyocytes were cultured in RPMI-1640 media supplemented with either glucose ( 11 mM ) or galactose ( 10 mM ) .", "All cell lines were routinely tested to ensure that they were mycoplasma free .", "Whole cell lysates were prepared in lysis buffer ( 50 mM Tris pH 7 . 5 , 150 mM sodium chloride , 1% Triton X-100 , 0 . 1% SDS , 0 . 5% sodium deoxycholate , 1X Roche protease inhibitor cocktail and 1X Roche PhosStop phosphatase inhibitor cocktail ) .", "Protein amount was quantified using Pierce BCA protein assay kit ( ThermoFisher scientific ) and 25 μg protein was separated using SDS-PAGE and transferred to PVDF membranes .", "Primary antibodies used: phospho-HER2 ( Y1221/1222 ) ( CST , #2243 ) , HER2 ( CST , #2165 ) , phospho-ACC ( S-79 ) ( CST , #3661 ) , ACC ( CST , #3676 ) , phospho-RPS6 ( S240/244 ) ( CST , #2215 ) , RPS6 ( CST , #2217 ) , β-tubulin ( CST , #2146 ) .", "Secondary antibodies used: IR-dye labelled α-rabbit ( CST , #5366S ) .", "Fluorescent signal was detected using LI-COR Odyssey imaging system ( LI-COR biosciences ) and images analysed with LI-COR image studio software ( package version 5 . 2 . 5 ) .", "H9c2 rat cardiomyoblast cells and hESC-cardiomyocytes were seeded in 96 well plates at a density of 7 × 103 and 3 . 6 × 105 cells per well respectively , whereas A549 cells were seeded at a density of 1 × 104 cells per well .", "ATP concentrations were measured using the Promega Cell Titer Glo assay according to manufacturer’s protocol .", "An Agilent XF Seahorse Analyzer was used to measure respiration and glycolysis in intact cells in real time .", "Cells were seeded in Seahorse Biosciences XF24 plates .", "H9c2 , hESC-CM ( Cytiva Plus , GE Healthcare ) and were seeded at 9 × 104 , and 6 × 104 cells per well ( coated with 25 μl fibronectin prior to seeding ) .", "Prior to the assay , medium was replaced with DMEM , containing either glucose or galactose .", "Drugs were dissolved in DMSO and injected from pre-loaded ports pneumatically .", "Cholesterol-dependent permeabilser XF Plasma Membrane Permeabiliser ( PMP ) ( Seahorse Biosciences ) , was used as described ( Salabei et al . , 2014 ) .", "Mitochondrial assay buffer or mannitol and sucrose buffer ( MAS , pH 7 . 2 ) ( 220 mM mannitol , 70 mM sucrose , 10 mM KH2PO4 , 5 mM MgCl2 , 2 mM HEPES , 1 mM EGTA , 4 mg/ml BSA ) was used .", "Cells were seeded at 5 × 104 per well in XF Cell Culture plates in standard culture medium .", "Cells were washed once with MAS before addition of pre-warmed MAS at a final volume of 675 μl .", "Pyruvate ( 5 mM ) , malate ( 2 . 5 mM ) and ADP ( 1 mM ) were added for measurement of complex I-driven respiration and succinate ( 10 mM ) and ADP ( 1 mM ) for complex II-driven respiration .", "Mitochondrial membranes and complex I were prepared from bovine ( Bos taurus ) heart mitochondria as described previously ( Blaza et al . , 2014; Jones et al . , 2016 ) .", "Assays were performed in 10 mM Tris-SO4 ( pH 7 . 5 ) , 250 mM sucrose at 32°C .", "For measurement of NADH:O2 oxidoreduction , 20–30 μg mL−1 of membranes , 1 or 3 μM horse heart cytochrome c , and 120 or 200 μM NADH were added and the absorbance of NADH measured at 340–380 nm using linear regression , once steady-state was reached .", "Succinate oxidation was determined using a coupled enzymatic assay ( Jones and Hirst , 2013 ) in the presence of 5 mM succinate .", "NADH:decylubiquinone ( dQ ) , NADH:3-acetylpyridine adenine dinucleotide ( APAD+ ) , and NADH:ferricyanide ( FeCN ) oxidoreduction by complex I were measured using 0 . 5 μg mL−1 complex I , 100 μM NADH , 0 . 075% soy bean asolectin ( Avanti Polar Lipids ) , 0 . 075% 3-[ ( 3-Cholamidopropyl ) dimethylammonio]−1-propanesulfonate ( CHAPS , Merck Chemicals Ltd . ) and 100 μM dQ , 500 µM APAD+ or 1 mM FeCN , respectively .", "MEA plates ( Axion Biosystems , M768-KAP-48 ) contain 48 wells , each with 16 electrodes .", "hESC-CM were grown on these plates and incubated with the doses of mubritinib shown and recordings were taken over a time course up to 72 hr .", "AxIS ( Version 2 . 0 . 2 . 9 ) cardiac beat detector which uses an inflection search algorithm was used to detect changes in cardiac action potential .", "For analysis of cell death and apoptosis , cells were harvested and FITC-conjugated Annexin-V antibody and far-red DNA stain DRAQ7 were added to pellets resuspended in Annexin-V Binding Buffer ( BD Pharmingen ) .", "Samples were analysed by flow cytometry using the FITC and APC channels .", "Ion channel binding assays were carried out by Eurofins Panlabs Discovery Services Taiwan Ltd .", "The binding affinity of compounds to ion channels was measured using an in vitro radioligand binding assay ( [3H] 1 , 4 , 5-IP3 ) in rat cerebellum , following incubation at 25°C for 10 min .", "All data are displayed show a percentage binding of each compound relative to control 1 , 4 , 5-IP3 .", "A radiometric kinase assay was carried out by Eurofins Ltd .", "In brief , the effect of mubritinib on recombinant human HER2 activity was determined by measuring the incorporation of radioactive 32P-ATP using concentrations from 10 nM to 10 μM .", "Activity values represent the percentage relative to the positive control .", "Mubritinib was also tested for activity against EGFR , ErbB4 , Flt1 and PDGRα at a concentration of 1 μM .", "Lapatinib was tested against HER2 as a positive control .", "The experiments were carried out in triplicate and the counts per minute ( CPMs ) were normalised to the control .", "Cell proliferation assays were performed in a standard CO2 incubator using the xCELLigence RTCA DP instrument ( ACEA Biosciences ) according to manufacturer’s instructions .", "Microelectrode sensor arrays embedded on the base of the E-plate 16 ( ACEA Biosciences ) measure changes in impedance as cells attach and proliferate , enabling label free quantification of cell proliferation .", "A549 or BT474 cells were seeded on an E-plate 16 in a total volume of 150 µl media .", "Cells were allowed to attach to the plate and enter log phase growth ( ~20 hr ) before treatment with indicated compounds of interest .", "Cell proliferation was monitored for 96 hr post treatment and all treatments were performed in at least technical duplicate and biological triplicate .", "For synthesis of mubritinib and CAI variants see supplementary material ." ] ]

[ "Disruption of mitochondrial function selectively targets tumour cells that are dependent on oxidative phosphorylation .", "However , due to their high energy demands , cardiac cells are disproportionately targeted by mitochondrial toxins resulting in a loss of cardiac function .", "An analysis of the effects of mubritinib on cardiac cells showed that this drug did not inhibit HER2 as reported , but directly inhibits mitochondrial respiratory complex I , reducing cardiac-cell beat rate , with prolonged exposure resulting in cell death .", "We used a library of chemical variants of mubritinib and showed that modifying the 1H-1 , 2 , 3-triazole altered complex I inhibition , identifying the heterocyclic 1 , 3-nitrogen motif as the toxicophore .", "The same toxicophore is present in a second anti-cancer therapeutic carboxyamidotriazole ( CAI ) and we demonstrate that CAI also functions through complex I inhibition , mediated by the toxicophore .", "Complex I inhibition is directly linked to anti-cancer cell activity , with toxicophore modification ablating the desired effects of these compounds on cancer cell proliferation and apoptosis ." ]

[ "The pharmaceutical industry needs to make safe and effective drugs .", "At the same time this industry is under pressure to keep the costs of developing these drugs at an acceptable level .", "Drugs work by interacting with and typically blocking a specific target , such as a protein in a particular type of cell .", "Sometimes , however , drugs also bind other unexpected targets .", "These “off-target” effects can be the reason for a drug’s toxicity , and it is important – both for the benefit of patients and the money that can be saved when developing drugs – to identify how drugs cause toxic side effects .", "The earlier researchers detect off-target effects , the better .", "Recent data has suggested that an anti-cancer drug called mubritinib has off-target effects on the compartments within cells that provide the cell with most of their energy , the mitochondria .", "This drug’s intended target is a protein called HER2 , which is found in large amounts on the surfaces of some breast cancer cells .", "Yet if mubritinib has this off-target effect on mitochondria , it may be harmful to other cells including heart cells because the heart is an organ that needs a large amount of energy from its mitochondria .", "Stephenson et al . have now performed experiments to show that mubritinib does not actually interact with HER2 at all , but only targets mitochondria .", "The effect of mubritinib as an anti-cancer drug is therefore only due to its activity against mitochondria .", "Digging deeper into the chemistry revealed the small parts of its chemical structure that was responsible for mubritinib’s toxicity against heart cells , the so-called toxic substructure .", "Another anti-cancer drug called carboxyamidotriazole also has the same toxic substructure .", "Carboxyamidotriazole is supposed to stop cells from taking up calcium ions , but a final set of experiments demonstrated that this drug also only acts by inhibiting mitochondria .", "Often there is not enough information about many drugs’ substructures , meaning off-target effects and toxicities cannot be predicted .", "The pharmaceutical industry will now be able to benefit from this new knowledge about the toxic substructures within some drugs .", "This research may also help patients who take mubritinib or carboxyamidotriazole , because their doctors will have to check for side effects on the heart more carefully ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "developmental biology", "cell biology", "tools and resources" ]

[ [ "We sought to overcome the image analysis bottleneck that exists for complex tissues and organs by creating easy to use , automated image analysis tools able to accurately identify cell types and determine their distributions and division rates in 3D , over time within intact tissues .", "To date , challenging image analysis tasks of this sort have largely depended on slow , painstaking manual analysis , or the bespoke development or modification of dedicated specialised tools by an image analyst with significant programming skills ( Chittajallu et al . , 2015; Schmitz et al . , 2014; Stegmaier et al . , 2014; Homem et al . , 2013; Myers , 2012; Meijering , 2012; Meijering et al . , 2012; Rittscher , 2010 ) .", "Of the current freely available automated tools , amongst the most powerful are Ilastik and the customised pipelines of the FARSIGHT toolbox and CellProfiler ( Padmanabhan et al . , 2014; Sommer and Gerlich , 2013; Sommer , 2011; Roysam et al . , 2008 ) .", "However , these three approaches require advanced knowledge of image processing , programming and/or extensive manual annotation .", "Other software such as Advanced Cell Classifier are targeted at analysis of 2D data , whilst programs such as RACE , SuRVoS , 3D-RSD and MINS are generally tailored to specific applications ( Luengo et al . , 2017; Stegmaier et al . , 2016; Lou et al . , 2014; Cabernard and Doe , 2013; Homem et al . , 2013; Arganda-Carreras et al . , 2017; Logan et al . , 2016; Gertych et al . , 2016 ) .", "Recently , efforts to make deep learning approaches easily accessible have made great strides ( Falk et al . , 2019 ) ; such implementations have the potential to increase access to these powerful supervised segmentation methods , but at present hardware and installation requirements are likely to be too complex for the typical biologist .", "In general , we find that existing tools can be powerful in specific examples , but lack the flexibility , speed and/or ease of use to make them effective solutions for most biologists in the analysis of large time-lapse movies of 3D developing tissues .", "In developing CytoCensus , we sought to design a widely applicable , supervised machine leaning-based image analysis tool , addressing the needs of biologists to efficiently characterise and quantitate dense complex 3D tissues at the single-cell level with practical imaging conditions .", "This level of analysis of developing tissues , organoids or organs is frequently difficult due to the complexity and density of the tissue arrangement or labelling , as well as limitations of signal to noise .", "We therefore aimed to make CytoCensus robust to these issues but also to make it as user friendly as possible .", "In contrast to other image analysis approaches that require the user to define the cell boundaries , CytoCensus simply requires the user to point-and-click on the approximate centres of cells .", "This single click training need only be carried out on a few representative 2D planes from a large 3D volume , and tolerates relatively poor image quality compatible with extended live cell imaging .", "To make the task very user friendly , we preconfigured most algorithm settings leaving a few , largely intuitive , parameters for the user to set .", "To improve performance , we enabled users to define regions of interest ( ROIs ) which exclude parts of a tissue that are not of interest or interfere with the analysis .", "We also separated the training phase from the analysis phase , allowing efficient batch processing of data .", "For the machine learning , we choose a variation of Random Forests with pre-calculated image features , which allows for much faster training compared to neural networks on typical computers , and with a fraction of the user annotation .", "A similar approach is taken by the image analysis software Ilastik ( Berg et al . , 2019 ) .", "Using machine learning , CytoCensus then determines the probability of each pixel in the image being the centre of the highlighted cell class in 3D , based on the characteristics of the pixels around the site clicked .", "This proximity map is used to identify all of the cells of interest .", "Finally , to increase the ease of adoption , we designed CytoCensus to be easily installed and work on multiple platforms and computers with standard specifications , including generically configured laptops without any pre-requisites .", "Collectively , these improvements make CytoCensus an accessible and user-friendly image analysis tool that will enable biologists to analyse their image data effectively , increase experimental throughput and increase the statistical strength of their conclusions ." ], [ "To extend our ability to study stem cell behaviour in the context of the intact Drosophila brain , we modified the methods of Cabernard and Doe ( 2013 ) , revised in Syed et al . ( 2017 ) , to produce a convenient and effective protocol optimising tissue viability for long-term culture and quantitative imaging .", "We first developed an isolation procedure incorporating scissor-based dissection of second or third-instar larvae , in preference to solely tweezer or needle-based dissection which can damage the tissue .", "We then simplified the culture medium and developed a convenient brain mounting technique that immobilises the organ using agar ( Figure 1A; Materials and methods ) .", "We also made use of bright , endogenously expressed fluorescently tagged proteins Jupiter::GFP and Histone::RFP marking microtubules and chromosomes respectively , to follow the developing brain ( Figure 1B ) .", "We chose generic cytological markers as these are more consistent across wild-type ( WT ) and different mutants than more specific markers , such as Deadpan ( Dpn ) , Asense ( Ase ) or Prospero ( Pros ) , commonly used to identify NBs , GMCs and neurons .", "Finally , we optimised the imaging conditions to provide 3D data sets of sufficient temporal and spatial resolution to follow cell proliferation over time without compromising viability ( see Materials and methods ) .", "Significantly , to maximise temporal and spatial resolution without causing damage , we reduced photo-damage by decreasing the laser excitation power by approximately 10 fold ( see Materials and methods ) and subsequently restoring image quality using patch-based denoising ( Carlton et al . , 2010 ) , developed by Kervrann and Boulanger ( 2006 ) .", "This approach allowed us to follow the lineage and quantitate the divisions of NBs and GMCs in the intact brain in 3D ( Figure 1C , D ) .", "To assess whether our culturing and imaging protocol supports normal development , we used a number of criteria .", "We found that by all the criteria we measured , brain development is normal in our ex vivo conditions .", "First , the cultured ex vivo brains do not show signs of damage during preparation , which can be easily identified as holes or lesions in the tissue that expand with time in culture .", "Second , our cultured larval brains consistently increase in size as they progress through development ( Figure 1—figure supplement 1 ) .", "Third , using our approach , we recorded average division rates of 0 . 66 divisions/hour ( ~90 min per cycle , Figure 1C ) for the Type 1 NB of the central brain ( Figure 1—figure supplement 1A′ ) , at the wandering third instar larval stage ( wL3 ) , as previously published ( Homem et al . , 2013; Bowman et al . , 2008; Figure 1—video 1; Figure 4—video 1 ) .", "We note here that experiments were performed at 21°C , which differs from some developmental studies performed at 25°C .", "Type I NBs were identified by location according to Homem and Knoblich ( 2012 ) .", "Fourth , we rarely observed excessive lengthening or arrest of the cell cycle in NBs over a 22 h imaging period , which is approximately the length of the wL3 stage ( Figure 1C ) .", "With longer duration culture and imaging , up to 48 hr , we observe an increase in cell cycle length , which might be expected for wL3 brains transitioning to the pupal state ( Homem et al . , 2014 ) .", "Finally , we observed normal and sustained rates of GMC division throughout the imaging period that correspond to the previously described literature in fixed brain preparations ( Bowman et al . , 2008; Figure 1D; Figure 4—video 2 ) .", "We conclude that our ex vivo culture and imaging methods accurately represent development of the Drosophila brain and support high time and spatial resolution imaging for quantitation of cell numbers and division rates .", "Progress in elucidating the molecular mechanisms of regulated cell proliferation during larval brain development has largely depended on the characterisation and quantification of mutant phenotypes by painstaking manual image analysis ( for example , Neumüller et al . , 2011 ) .", "However , the sheer volume of image data produced by whole brain imaging experiments means that manual assessment is impractical .", "Therefore , we attempted to use freely available image analysis tools in an effort to automate the identification of cell types .", "We found that none of the available off-the-shelf image analysis programs perform adequately on our complex 3D datasets , in terms of ease of use , speed or accuracy ( Table 1 ) .", "Neuroblast nuclei are large and diffuse , which means that conventional spot detectors ( e . g . TrackMate ) struggle to identify them .", "Ilastik Density Counting ( which takes a related approach to CytoCensus ) was promising to count NB in 2D , but is not designed to work in 3D nor to detect cell centres ( Berg et al . , 2019 ) .", "Similarly , image segmentation tools ( such as RACE , Ilastik Pixel Classification and WEKA ) struggle to segment NB marked by microtubule labels as they vary significantly in appearance with the cell cycle and cell boundaries may appear incomplete .", "To overcome these limitations , we developed CytoCensus , an easily deployed , supervised machine learning-based image analysis software ( Figure 2; Figure 2—figure supplement 1 ) .", "CytoCensus facilitates automated detection of cell types and quantitative analysis of cell number , distribution and proliferation from time-lapse movies of multichannel 3D image stacks even in complex tissues .", "A full technical description of the algorithm is found in Materials and methods and a User Guide is available in the Supplemental Information .", "To optimise its effectiveness , we developed CytoCensus with a minimal requirement for supervision during the training process .", "We developed an implementation of supervised machine learning ( see Materials and methods ) , in which the user trains the program in 2D on a limited number of images ( Figure 2 ) .", "In this approach , the user simply selects , with a single mouse click , the approximate centres of all examples of a particular cell type within small user-defined regions of interest in the image .", "This makes CytoCensus is more convenient and faster than other machine learning-based approaches , such as FIJI-WEKA ( Arganda-Carreras et al . , 2017 ) or Ilastik Pixel Classification , ( Sommer , 2011 ) , which require relatively extensive and time consuming annotation of the cells by their boundaries .", "However , this simple training regime requires assumptions of roundness , which precludes direct analysis of cell shape .", "We explore the extent of this limitation in subsequent sections .", "To further optimise the training , our training workflow outputs a ‘proximity’ map , similar to those described in Fiaschi et al . ( 2012 ) ; Swiderska-Chadaj et al . ( 2018 ) ; Liang et al . ( 2019 ) ; Höfener et al . ( 2018 ) .", "These approaches all focus on 2D proximity maps , while CytoCensus utilises proximity maps in 3D .", "One may think of the proximity map as a probability of how likely it is that a given pixel is at the center of one of the cells of interest .", "Using this proximity map the user can assess the accuracy of the prediction and , if necessary , provide additional training ( Figure 2 ) .", "This proximity map and the predicted locations of cell centres across the entire volume and time-series are saved and may be conveniently passed to ImageJ ( FIJI ) , or other programs ( Schindelin et al . , 2012 ) for further processing ( Figure 2; Figure 2—figure supplement 1; Figure 3—figure supplement 1 ) .", "After this initial phase of manual user training , the subsequent processing of new unseen data is automated and highly scalable to large image data sets without any further manual user training .", "To determine the required training , the impact of training level ( number of regions used in the training ) was assessed on live imaging data sets ( Materials and methods ) .", "The results show that detection accuracy was optimised even with a modest levels of training ( Figure 3—figure supplement 2 ) .", "We assessed the performance of CytoCensus at cell identification on challenging live imaging data sets that were manually annotated by a user to generate ‘ground-truth’ results .", "Before comparison between applications , algorithm parameters were optimised for the different approaches to prevent overfitting ( Materials and methods ) .", "In our tests CytoCensus outperformed the machine learning based approaches Fiji-WEKA ( p=0 . 005 , t-test , n = 3 ) and Ilastik Pixel Classification ( p=0 . 007 , t-test , n = 3 ) , and other freely available approaches in the accuracy of NB detection , speed and simplicity of use ( Figure 3A; Table 1 ) .", "We calculated a metric of performance , intuitively similar to accuracy , which is known as the F1-score , with a maximum value of 1 . 0 ( Materials and methods; Table 1 ) .", "We found that the best performing approaches on our complex datasets were Ilastik Pixel Classification and CytoCensus , which are machine learning based .", "It is likely that both approaches might be further improved with additional bespoke analysis , specific to each data set , however this would limit their flexibility and ease of use .", "To further critically assess the performance of CytoCensus , we used an artificially generated ‘neutral challenge’ 3D dataset , which facilitates fair comparison ( Figure 3B ) .", "We used a dataset of 30 images of highly clustered synthetic cells , in 3D , with a low signal-to-noise ratio ( SNR ) , obtained from the Broad Bioimage Benchmark Collection ( Materials and methods ) .", "We selected this dataset because it has similar characteristics to our live imaging data .", "Using this dataset , we directly compared the abilities of Ilastik Pixel Classification ( Figure 3B′′ ) and CytoCensus ( Figure 3B′′′ ) , to identify cell centres in 3D .", "In both cases , we trained on a single image , optimised parameters on five images , and evaluated performance on the remaining 25 images .", "We found that CytoCensus ( Table 2 , F1-score: 0 . 98 ± 0 . 05 ) outperforms Ilastik Pixel Classification in the accuracy of cell centre detection ( Figure 3B ) even after the Ilastik Pixel Classification results were post-processed to aid separation of touching objects ( Table 2 , F1-score: 0 . 88 ± 0 . 09 ) .", "We conclude that CytoCensus is significantly more accurate than Ilastik at identifying cells when both are tested out-of-the-box on neutral challenge data ( Figure 3B′′′′ p<0 . 001 , Welch’s t-test , n = 25 ) .", "For a more general comparison to other detection and segmentation methods , we applied CytoCensus to 3D data from the Cell Tracking Challenge Segmentation Benchmark ( Ulman et al . , 2017; Maška et al . , 2014 ) .", "To properly participate in this challenge , we trained CytoCensus as normal , and then applied a simple post-processing of the CytoCensus centres , using a small number of iterations of MorphACME ( Marquez-Neila et al . , 2013 ) , an active contour segmentation method , in order to get a segmentation .", "The results of CytoCensus are shown in Figure 3—figure supplement 2B , along with the results of the top 3 ranked algorithms at the time of publication .", "CytoCensus in general performs well at detection , achieving top 3 performance on 3/6 datasets tested ( Figure 3—figure supplement 2B′′ ) .", "CytoCensus with post-processing performs surprisingly well at the segmentation aspect of the challenge , despite primarily being aimed at detection , achieving top 3 in 2/6 datasets ( Figure 3—figure supplement 2B′′′ ) .", "Unsurprisingly , datasets such as N3DH-CE , which have cells with highly variable cell size and shape , were challenging for CytoCensus .", "However , CytoCensus performed particularly well on datasets with lower signal to noise and a high density of cells ( e . g . N3DH-SIM+ , N3DH-DRO , N3DH-TRIC [Jain et al . , 2019] ) , illustrating where CytoCensus is best applied .", "In general CytoCensus performs competitively on the tested datasets , leveraging a small amount of training to achieve good results without needing dataset specific algorithms for denoising or object separation .", "We conclude that CytoCensus represents a significant advance over the other current freely available methods of analysis , both in ease of use and in ability to accurately and automatically analyse cells of interest in the large volumes of data resulting from live imaging of an intact complex tissue such as a brain .", "This will greatly facilitate the future study of subtle or complex mutant developmental phenotypes .", "To demonstrate the power and versatility of using CytoCensus in the analysis of a complex brain mutant phenotype , we characterised the brain overgrowth phenotype of syncrip ( syp ) knockdown larvae ( Figure 4A ) .", "SYNCRIP/hnRNPQ , the mammalian homologue of Syp , is a component of RNA granules in the dendrites of mammalian hippocampal neurons ( Bannai et al . , 2004 ) .", "Syp also determines neuronal fate in the Drosophila brain ( Ren et al . , 2017 ) , NB termination in the pupa ( Yang et al . , 2017; Samuels et al . , 2020b ) and is required for neuromuscular junction development and function ( McDermott et al . , 2014; Halstead et al . , 2014; Titlow et al . , 2020 ) .", "syp has previously been identified in a screen for genes required for normal brain development ( Neumüller et al . , 2011 ) , although the defect was not characterised in detail .", "In light of these studies , we wanted to understand the defect caused by syp on brain development in more detail .", "We therefore examined syp - / - brains ( eliminating Syp expression in the NB lineages ) and found that in early wL3 , brains were significantly enlarged compared to WT larvae at the same stage of development ( p<0 . 0001 , t-test , Figure 4A , Figure 4—figure supplement 1A ) .", "syp brain lobes exhibit a 23% increase in diameter ( WT 206 . 5 µm ± 5 . 0 , n = 10 , syp 253 . 7 µm ± 11 . 0 , n = 5 ) , and a 35% increase in central brain ( CB ) volume .", "Significantly , a more specific RNAi knockdown of syp driven under the inscuteable promoter , which is expressed primarily in NBs and GMCs , demonstrates a similar increase in CB diameter ( p=0 . 002 , 13% larger than WT; 234 µm ± 17 . 0 , n = 12; Figure 4—figure supplement 1A ) .", "Our data raises the question as to how the removal of syp from the neural lineages causes such a significant increase in central brain size .", "We tested whether this brain overgrowth is caused by additional ectopic NBs , as has been previously described for other mutants ( Bello et al . , 2006 ) .", "We used CytoCensus to accurately determine the total number of NBs in the CB of fixed syp knockdown verses WT wL3 brains .", "Our results show that wL3 brains with syp RNAi knockdown have no significant difference in ventral NB number compared to WT ( Figure 4B; WT 45 . 6 ± 1 . 3 , n = 22 , syp RNAi 44 . 1 ± 2 . 1 , n = 15 ) .", "We conclude that a change in NB number is not the underlying cause of brain enlargement observed in syp RNAi and hypothesise that a change in NB division rate or that of their progeny might be responsible .", "To investigate whether an increase in NB division rate contributes to the brain overgrowth observed in syp knockdown larvae , we examined the rate of NB division in living brains using our optimised culturing and imaging methods , followed by CytoCensus detection and tracking .", "First , we perform 3D NB detections using CytoCensus ( as shown previously in Figure 3A ) , and we fed this input into TrackMate , a simple tracking algorithm .", "Without the CytoCensus detections , TrackMate spot detection performs poorly on the raw data ( F1 score 0 . 11 ± 0 . 09 ) , and tracking is all but impossible .", "Applying TrackMate to the proximity maps generated by CytoCensus dramatically improves TrackMate detections ( F1 score 0 . 92 ± 0 . 02 , Figure 5—figure supplement 1A ) .", "As a result , 16 out of 17 NBs were successfully and accurately tracked for over 20 h in our tests ( Figure 4C′ , Figure 5—figure supplement 1AV ) .", "In order to follow the NB cell cycle , we next showed CytoCensus can accurately identify individual dividing NBs in live image series , both in WT ( Figure 4C′′ ) and in RNAi brains ( Figure 5—figure supplement 1B ) .", "We detected dividing NBs by training on NBs with visible spindles using CytoCensus , and used this output to create plots of division for each NB ( Figure 4C′′′ , Figure 5—figure supplement 1C–D ) .", "Using these plots , we measured the cell cycle length of NBs in wild type and syp RNAi brains and found that , on average , syp RNAi NBs have a 1 . 78-fold shorter cell cycle compared to WT ( p=0 . 02 , Welch’s t-test , WT N = 7 , syp RNAi n = 5 brains; Figure 4C′′′′ ) .", "We propose that this shorter cell cycle length ( i . e . an increased division rate ) in the syp knockdown is the primary cause of its increased brain size .", "These results illustrate the potential of CytoCensus to analyse the patterns of cell division in a complex , dense tissue , live , in much more detail than conventional methods in fixed material .", "We also investigated GMC behaviour in the CB region of syp RNAi and WT larval brains , to test whether an aberrant behaviour of mutant GMCs could also contribute to a brain enlargement phenotype .", "Given that GMCs are morphologically indistinguishable from their immature neuronal progeny ( which makes them particularly difficult to assess ) we had to identify GMCs by tracking them from their birth in a NB division to their own division into two neurons .", "To achieve this goal required us to use high temporal resolution imaging and patch based denoising ( Materials and methods ) , which allowed us to confirm that normal , symmetric GMC divisions occurred with the correct timing and resulted in two daughter cells ( which did not regrow or divide further ) , both in WT and syp RNAi ( Figure 4D ) .", "Using our refined culture and imaging conditions , we trained CytoCensus to successfully detect GMCs in denoised images ( Figure 4E′-E′′ ) and , similarly to NBs , track them with a trackpy based script ( Allan et al . , 2012 , see Materials and methods ) .", "Unlike in the case of NB tracking , GMCs do not go through repeated cycles of division , so following automated detection , for each GMC , we manually identified the birth and final division and additionally corrected any tracking errors .", "This semi-automated tracking allowed us to compare the cell cycle length of GMCs in multiple brains over 12 h time-lapse movies for the first time ( Figure 4E′′′ ) .", "In syp RNAi , we find a small but significant shortening ( p=0 . 01 , Welch’s t-test ) of the cell cycle compared to WT ( 8 . 00 h ± 0 . 89 , n = 8 WT; 6 . 25 h ± 1 . 45 , n = 8 syp RNAi ) .", "However , while we conclude that GMC cell cycle length is decreased by 20% , GMCs terminally divide normally ( representative example , Figure 4D ) , and we see no evidence of further divisions in the neurons .", "We take this to mean that no additional cells are produced by GMC or neuron division and therefore brain size is not significantly affected .", "We conclude that the cause of the enlarged brain size in syp RNAi brains is an increase in NB division rate resulting in more GMCs and their progeny than in WT .", "Most current methods for measuring NB division rates produce an average rate for whole brains rather than providing division rates for individual NBs .", "It has previously been shown that NB lineages give rise to highly variable clone size ( 30–150 neurons for Type I neuroblasts ) .", "The origin of this diversity has primarily been attributed to patterned cell death ( Yu et al . , 2013; Pinto-Teixeira et al . , 2016 ) , but the importance of NB division rate in determining clone size is less well understood .", "Using live imaging and CytoCensus , however , we were able to quantitate the behaviour of multiple individual NBs over time within the same brain to investigate whether cell division rates are constant or variable across the population .", "Interestingly , we found that each NB has a constant cell cycle period ( Figure 5A ) , matching observations in vitro ( Homem et al . , 2013 ) .", "However , there is considerable variation in cell cycle length between NBs within the same brain lobe ( Figure 5A ) .", "Given the scale of this variation , which covers more than twofold difference in rate , we expect that the regulation of NB division rate is a key factor that contributes to the observed variation in NB lineage size .", "By comparing the distribution of division rates in individual WT and syp RNAi brains , we found that syp knockdown NBs have a more consistent division rate in individual NBs ( Figure 5B ) and between brains ( Figure 4C′′′ ) , which suggests a role for syp in the regulation of NB division rate .", "Future work using CytoCensus and live imaging would allow one to explicitly link individual NB division rates to atlases of neural lineages and explain the contribution of division rate to each neural lineage .", "We conclude that analysing live imaging data with CytoCensus can provide biological insights into developmental processes that would be difficult to obtain by other means .", "However , it was important to establish the use of CytoCensus in other situations outside Drosophila tissues , especially in vertebrate models of development .", "To test the utility of CytoCensus for the analysis of complex vertebrate tissue , we first analysed Zebrafish tissue , an outstanding model for studying development with many powerful tools , such as the Spectrum of Fates ( SoFa ) approach ( Almeida et al . , 2014 ) , which marks cells from different layers of the Zebrafish retina by expression of distinct fluorescent protein labels .", "Previously published work by Eldred et al . ( 2017 ) studying eye development in artificial Zebrafish organoids , provided an excellent example of material that was previously analysed using bespoke MATLAB image analysis software that measured only the cumulative fluorescence at different radii from the organoid centre .", "While this was sufficient for a summary of organoid organisation , future research will require the ability to examine organoids at the single-cell level , particularly in cases where layers are formed from a mixture of cell types or cell types are defined by combinations of markers .", "We deployed CytoCensus to this end , without the need for bespoke image analysis , in directly locating and counting cells ( Figure 6A ) .", "Using CytoCensus , we trained multiple models on subsets of the raw data ( Figure 6A′ , gift from the William Harris lab ) , corresponding to each of the different cell types .", "Applying our models to the remainder of the dataset , CytoCensus was able to identify individual cells ( Figure 6A , bottom panels ) , allowing an analysis of cellular distribution that would not be possible from cumulative fluorescence measurements .", "We then calculated the number of cells found at different distances from the center of the organoid ( Materials and methods , Figure 6A′′-A′′′ ) .", "Using this approach , we reproduced the previously published analysis ( Eldred et al . , 2017 ) , mapping the different cell distributions in the presence and absence of retinal pigment epithelium cells .", "We show that CytoCensus produces similar results to Figure 2 of Eldred et al . ( 2017 ) , but with identification of individual cells and without the need for a dedicated image analysis pipeline ( Figure 6A′′-A′′′ ) .", "In particular , we are able to produce an estimate of the distribution of the photoreceptor ( PR ) cell class , which is defined by a combination of markers ( Crx::gapCFP , Ato7::gapRFP ) that could not be separated from other cell types in the original analysis .", "Given that the SoFa markers support the study of live organoid development , and CytoCensus can be used to identify cells based on the SoFa markers , we expect CytoCensus could easily be used to analyse live organoid development along similar lines to our Drosophila analysis .", "We conclude that CytoCensus is an effective tool to investigate the distribution of cell types in the assembling retinal organoid , with the potential to analyse other complex Zebrafish tissues .", "Mouse models are widely used to understand developmental processes in the early embryo .", "In such work , genetic studies have been fundamental in understanding the molecular mechanisms underlying important lineage decisions ( Piliszek et al . , 2016; Arnold and Robertson , 2009 ) .", "However , assessment of changes in cell numbers and distribution frequently relies on manual counting and qualitative estimation of phenotypes .", "We tested the ability of CytoCensus to provide quantitative data on the number of transcription factor positive cells in the early post-implantation embryo for each of the transcription factors Brachyury , Lhx1 and Sox2 .", "Using CytoCensus , we quantitated the number of cells that express each of these transcription factors in two regions of interest: the visceral endoderm ( VE ) and the proximal posterior epiblast ( PPE ) , where primordial germ cells ( PGCs ) are specified .", "We also analysed the distribution of Blimp1-mVenus in membranes in both the VE and PGCs ( Ohinata et al . , 2008; Vincent et al . , 2005; Figure 6B′ , B′′ ) .", "Using CytoCensus , we identified all Blimp1 expressing cells and mapped them to structures of interest using a 3D ROI ( Figure 6B′ marked regions ) .", "We then used CytoCensus to identify cells expressing both Blimp1 and Brachyury in the proximal posterior epiblast ( PPE ) ( Figure 6B′′ and insert ) .", "We note that CytoCensus could be used to successfully detect cells of the VE and PGCs , despite the fact that they are frequently far from round .", "CytoCensus is able to detect these cells , almost as well as truly round cells , by integrating information from the nuclear and membrane markers to produce robust cell centre detections .", "Our analysis highlights the enrichment of Brachyury in the developing PGCs and their almost complete absence from the VE , which matches well with manual 2D quantification ( Figure 6B′′′ ) .", "Repeating this analysis for the transcription factors Sox2 and Lhx1 highlights a differential expression of the transcription factors ( Figure 6B′′′′-V ) .", "These proportions match well with qualitatively reported expression patterns in the field ( Piliszek et al . , 2016 ) .", "Our results demonstrate how CytoCensus can be used to produce a robust and detailed quantitation of cell type and TF expression in specific complex mouse tissues using standard markers , improving on the standard qualitative analysis .", "Taking our results in their entirety , in Drosophila , Zebrafish and mouse , we illustrate the wide applicability of CytoCensus to transform the quantitative analysis of any complex tissue .", "CytoCensus makes it possible without bespoke programming to quantitate cell numbers and their divisions in complex living or fixed tissues in 3D ." ], [ "Progress in understanding the development and function of complex tissues and organs has been limited by the lack of effective ways to image cells in their native context over extended developmentally relevant timescales .", "Furthermore , a major hurdle has been the difficulty of automatically analysing the resulting large 4D image series .", "Here , we describe our development of culturing and imaging methods that support long term high resolution imaging of the cells in intact living explanted Drosophila larval brains .", "This progress relies on optimised dissection and mounting protocols , a simplified culture medium for extending brain viability and the use of patch-based denoising algorithms to allow high resolution imaging at a tenth of the normal illumination intensity .", "We next describe our development of CytoCensus: a convenient and rapid image analysis software employing a supervised machine learning algorithm .", "CytoCensus was developed to identify neural stem cells and other cell types , both in order to quantitate their numbers and distribution and to enable analysis of the rate of division on an individual cell level , from complex 3D and 4D images of cellular landscapes .", "We demonstrate the general utility of CytoCensus in a variety of different tissues and organs .", "To image all the cell types in an explanted brain , we used very bright generic markers of cellular morphology , which offer major advantages over specific markers of cell identity , as they are more abundant and brighter , allowing the use of low laser power to maximise viability .", "Markers of cell morphology can also be used in almost all mutant backgrounds in model organisms , unlike specific markers of cell identity , whose expression is often critically altered in mutant backgrounds .", "However , imaging cells in a tissue or organ with generic markers leads to complex images , in which it is very challenging to segment individual cells using manual or available image analysis tools .", "In contrast to other approaches , we demonstrate that CytoCensus allows the user to teach the program , using only a few examples , by simply clicking on the cell centres .", "CytoCensus outperforms , by a significant margin , the other freely available approaches that we tested , so represents a step change in the type and scale of datasets that can be effectively analysed by non-image analysis experts .", "Crucially , CytoCensus analysis combined with cell tracking in extensive live imaging data allows parameters such as cell cycle length to be determined for individual cells in a complex tissue , rather than conventional methods that provide snapshots or an ensemble view of average cell behaviour .", "The image analysis approach we have developed depends critically on the use of ‘supervision’ or training regimes which are , by definition , subjective and user dependent .", "Supervised machine learning methods ( Luengo et al . , 2017; Arganda-Carreras et al . , 2017; Logan et al . , 2016; Chittajallu et al . , 2015; Sommer , 2011 ) require the user to provide training examples by manually identifying ( annotating ) a variety of cells or objects of interest , often requiring laborious ‘outlining’ of features to achieve optimal results .", "Where extensive training data , appropriate hardware and expertise are available , users should consider the use of NN such as those described in Falk et al . ( 2019 ) because of their superior ability to make use of large amounts of training data .", "However , our use of a 2D ‘point-and-click’ interface ( Figure 2—figure supplement 1 ) , to simplify manual annotation , with a 3D proximity map output , and choice of fast machine learning algorithm , makes it quick and easy for a user to train and retrain the program with minimal effort .", "Using our approach , a user can rapidly move from initial observations to statistically significant results based upon bulk analysis of data .", "We show the value of CytoCensus in three key exemplars .", "In Drosophila , we measure cell cycle lengths ex vivo in two key neural cell types , revealing the significant contribution of neuroblast division rate to the syp RNAi overgrowth phenotype .", "This complements a study some of the authors of this paper published while this paper was being revised ( Samuels et al . , 2020a ) .", "Samuels et al . show that Syncrip exerts its effect on NB by inhibiting Imp , which in turn regulates the stability of the mRNA of myc a proto-oncogene that regulates size and division .", "In Zebrafish organoids , we illustrate that CytoCensus is generally applicable and compatible with other cell types and live imaging markers .", "We show it is possible to easily characterise organoid organisation at the cellular level , including analysis of cell type which was not previously quantified ( Eldred et al . , 2017 ) .", "Finally , we quantify TF expression in images of mouse embryos , illustrating how qualitative phenotypes can be straightforwardly converted into quantitative characterisations , even in epithelial tissue which differs from the typical assumptions of round cells .", "A technical limitation of our ‘point-and-click’ strategy is that the program ‘assumes’ a roughly spherical cell shape .", "This means that cellular projections , for instance axons and dendrites of neurons , would not be identified , and other programs ( e . g . Ilastik , etc . ) may be more appropriate to answer specific questions that require knowledge of cell shape or extensions .", "However , we find that the robustness of the CytoCensus cell centres , even with irregular or extended cells can be a useful starting point for further analysis .", "To this end , we configured the output data from CytoCensus to be compatible with other programs , such as FIJI ( ImageJ ) , allowing a user to benefit from the many powerful plug in extensions available to facilitate further extraction of information for defined cell populations from bulk datasets .", "With the increased availability of high-throughput imaging , there is a greater unmet need for automated analysis methods .", "Ideally , unsupervised methods will remove the need for manual annotation of datasets , but at present , the tools required are in their infancy .", "In this context , methods that require minimal supervision , such as CytoCensus are desirable .", "Machine learning approaches , such as CytoCensus , offer the potential to analyse larger datasets , with statistically significant numbers of replicates , and in more complex situations , without the need for time-consuming comprehensive manual analysis .", "Easing this rate limiting step will empower researchers to make better use of their data and come to more reliable conclusions .", "We have demonstrated that analysis of such large live imaging datasets with CytoCensus can provide biological insights into developmental processes in Drosophila that would be difficult to obtain by other means , and that CytoCensus has a great potential for the characterisation of complex 4D image data from other tissues and organisms ." ], [ "Stocks were raised on standard cornmeal-agar medium at either 21°C or 25°C .", "To assist in determining larval age , Bromophenol Blue was added at 0 . 05% final concentration in cornmeal-agar medium .", "The following Drosophila fly strains were used: [Wild-Type Oregon-R]; [Jupiter::GFP;Histone::RFP ( recombination on the third ) ]; [AseGal4 >UAS-MCD8-GFP]; [w11180;PBac ( PB ) sype00286/TM6B]; [Bloomington 9289 , w11180 ( homozygote syp Null ) ]; [Df ( 3R ) BSC124/TM6B ( crossed to BL 9289 for syp Null ) ]; [syp RNAi lines - w11180; P{GD9477}v33011 , v33012] .", "Refer to Simon et al . ( 2017 ) for details on mouse embryo preparation .", "Flies of both genders were raised as described above and larvae from second instar to pre-pupal stages collected and dissected directly into fresh 4% EM grade paraformaldehyde solution ( from a 16% stock . Polysciences ) in PBS with 0 . 3% TritonX-100 then incubated for 25 min at room temperature ( RT ) .", "Following fixation , samples were washed 3 times for 15 min each in 0 . 3% PBST ( 1x PBS containing 0 . 3% Tween ) and blocked for 1 hr at RT in Immunofluorescence blocking buffer ( 1% FBS prepared in 0 . 3% PBST ) .", "Samples were incubated with primary antibody prepared in blocking buffer for either 3 hr at RT or overnight at 4°C .", "Subsequently , samples were washed three times for 20 min each with 0 . 3% PBST followed by incubation with fluorescent labelled secondary antibodies prepared in blocking buffer for 1 hr at RT .", "For nuclear staining , DAPI was included in the second last wash .", "Samples were mounted in VECTASHIELD ( Vector Laboratories ) for examination .", "For details on the preparation and labelling of mouse embryos , refer to Simon et al . ( 2017 ) .", "Brains were dissected from 3rd instar larvae in Schneider’s medium according to https://www . youtube . com/watch ?", "v=9WlIoxxFuy0 and placed inside the wells of a pre-prepared culturing chamber ( Figure 1A ) .", "To assemble the culturing chamber , 1% low melting point ( LMP ) agarose ( ThermoFischer ) was prepared as 1:1 v/v ratio of 1 x PBS and Schneider’s medium ( ThermoFisher 21720024 ) then pipetted onto a 3 cm Petri dish ( MatTek ) dish and allowed to solidify .", "After solidification , circular wells were cut out using a glass capillary ~2 mm diameter .", "To secure the material in place , a 0 . 5% LMP solution [1% LMP solution brain diluted 1:1 with culturing medium ( BCM ) ] was pipetted into the wells to form a cap .", "Finally , the whole chamber was flooded with BCM .", "BCM was prepared by homogenising ten 3rd instar larvae in 200 μl of Schneider’s medium and briefly centrifuge to separate from the larval carcasses .", "This lysate was added to 10 ml of 80% Schneider’s medium , 20% Foetal Bovine Serum ( GibcoTM ThermoFisher ) , 10 μl of 10 mg/ml insulin ( Sigma ) .", "A lid is used to reduce evaporation .", "For GMC imaging we used a solid-agar cap ( 1–2% LMP agarose ) placed directly on top of the brains , which we found was more consistent at holding brains against the coverslip than our earlier approach .", "We note that care must be taken not to flatten brains during this process , as it appears to result in a higher rate of stalled NB divisions which are likely artefacts .", "This approach reduced movement in brains significantly , but did not eradicate it - it seems likely remaining movement is the primarily the result of thermal drift of the microscope focus , and is well corrected using image registration .", "Confocal , live imaging of Drosophila was performed using an inverted Olympus FV3000 six laser line spectral confocal fitted with high sensitivity gallium arsenide phosphide ( GaAsP detectors ) , x30 SI 1 . 3 NA lens .", "The confocal pinhole was set to one airy unit to optimise optical sectioning with emission collection .", "Images were collected at 512 × 512 pixels using the resonant scanner ( pixel size 0 . 207 μm ) and x2 averaging ) .", "The total exposure time per Z stack ( 60 ) frames was ~20 s .", "For live culture and imaging , the sample was covered with a lid at 21 ± 1°C .", "Imaging of the GMC cell cycle required increased temporal and spatial resolution , compared to imaging NB: 2 min . time-lapse with 0 . 2 × 0 . 2×0 . 5 µm resolution .", "Initial tests indicated that the resulting increased light dosage reduce the number of GMC divisions over time , which we consider to be a sign of phototoxicity .", "Therefore , we reduced the laser power by approximately a factor of 10 ( to ~12µW at the objective for 488 nm , and 7µW for 561 nm ) , and used post-acquisition patch-based denoising NDSAFIR , by Kervrann and Boulanger ( 2006 ) , implemented as part of PRIISM , with adapt = 0 , island = 4 , zt mode and iterations = 3 by Carlton et al . ( 2010 ) to restore image quality .", "For details on imaging of mouse embryos ( Figure 6 ) refer to Simon et al . ( 2017 ) .", "Details of organoid imaging can be found in Eldred et al . ( 2017 ) .", "Additional live imaging was carried out on a GE Deltavision Core widefield system with a Lumencor 7-line illumination source , Cascade-II EMCCD camera and x30 SI 1 . 3 NA lens .", "For imaging of fixed Drosophila material , either an Olympus FV1200 or FV1000 confocal was used with x20 0 . 75 dry or x60 1 . 4 NA .", "lenses .", "Settings were adjusted according to the labelling and were kept consistent within experiments .", "For brightfield imaging ( Figure 1—figure supplement 1; Figure 4—figure supplement 1; Figure 4 ) , a GE Deltavision Core widefield system , Cascade-II EMCCD camera and x30 SI 1 . 3 NA lens was used .", "Measurements of brain diameters were performed by hand in OMERO .", "Reported measurements are the average of one measurement along the longest axis of a brain lobe ( passing through the central brain and optic lobe ) , and another at right angles to that ( typically across the medulla ) .", "All programs used for image analysis were installed on a MacBook Pro11 , 5; Intel Core i7 2 . 88 GHz;16 GB RAM .", "Basic image handling and processing was carried out in FIJI ( ImageJ V1 . 51d; http://fiji . sc; Schindelin et al . , 2012 ) .", "The CytoCensus software , and additional scripts were written in Python , a detailed technical description is given below .", "With the development of CytoCensus , we aim to make identification of cell types in multi-dimensional image sets as straightforward as possible .", "We do so by asking the user to identify cell centres for a small number of 2D image slices .", "Using the cell centre annotation and the estimated size of the cells , we create an initial ‘proximity map’ of cell centres , similar in concept to density kernel estimation approaches ( Waithe et al . , 2015; Waithe et al . , 2016; Fiaschi et al . , 2012; Lempitsky and Zisserman , 2010 ) .", "In particular , the Ilastik Cell Counting module ( Berg et al . , 2019 ) performs 2D density counting , a related method to CytoCensus , but provides only 2D count estimates .", "On most biological tissues , including the 3D tissues that we have tested , 2D counting is insufficient to accurately count cells in 3D , because applying it to many slices results in repeated detections of the same cells in multiple slices .", "More recently , using modified density maps as an intermediate for detecting ( and not just counting ) cells has become more popular .", "Such intermediate maps are variously described as proximity maps ( our preferred term ) , probability density maps , and Pmaps , they are well reviewed in Höfener et al . ( 2018 ) .", "The advantage of using ‘proximity map’ based methods to detect and count cells has been previously documented , but these methods have not been extended to the case of 3D cell centres with 2D annotations ( Kainz et al . , 2015; Waithe et al . , 2016 ) .", "Once we have the proximity maps of the cell centres , we then apply a series of image filters , which pull out image features such as edges , and try to use these features to predict a new proximity map of cell centres .", "We generate this new proximity map using a machine learning algorithm known as an ‘ensemble of decision trees’ ( Breiman , 2001; Breiman et al . , 1984 ) , which creates a series of ‘decision trees’ that individually predict poorly , but averaged together are a strong predictor .", "Once we have the new proximity maps , the location of cell centres in 3D is inferred from the 2D predictions by applying a 3D Hessian filter ( see later ) , which enhances the detections and resolves their coordinates in the additional dimension .", "The CytoCensus software has three main components in its workflow: The 2D training and evaluation algorithm , the 3D object finding algorithm and the 3D ROI drawing and interpolation algorithms .", "The software is written in python and includes a Graphical User Interface ( GUI ) written using the PyQt library .", "The 2D training and evaluation algorithm utilises an ensemble of random decision trees and a bank of filters which utilise the matplotlib , scipy , scikit-learn and scikit-image libraries ( Jones et al . , 2001; Hunter , 2007; Pedregosa , 2011; van der Walt et al . , 2014 ) .", "For the 2D training and evaluation algorithm , the user must provide suitable images and make annotations indicating the locations of features , objects or cells of interest within defined regions .", "The user annotates 2-D sections of 3D image volumes and defines rectangular regions which encapsulate areas containing cells or features of interest or just background .", "There are N image volumes ( Ii=1 , I2 , I3 , … , IN ) in the training set and M annotation sections where M > 1 ( Aj=1 , A2 , A3 , … , AM ) .", "Each annotation contains a region of interest ( Rj=1 , R2 , R3 , … , RM ) and also a set of corresponding points ( Pj=1 , P2 , P3 , … , PM ) with one or more dot/points Pj = {ptc=1 , pt2 , … , ptC} or no points if the region only contains background .", "It is worth noting that providing sufficient area that does not contain cells is important for minimising false positives .", "As the model is designed to distinguish cells from the background it maybe appropriate to annotate regions as empty so as to acclimatise the model to the background .", "The points and regions are supplied by the user as they label the centroid locations of cells or objects within the image plane of interest .", "For each annotation , we produce a centre-of-mass representation ( Fj=1 , F2 , F3… , FM ) which for each pixel ( p ) is defined as the maximum value of all the Gaussian kernels ( N ) centred on dot annotations which overlap this pixel:∀p∈Rj , Fj0 ( p ) =max[𝒩 ( p;pt , σ212×2 ) , ∀pt∈Pj]and σ = [σx , σy] .", "The kernel is isotropic ( σx = σy ) as long the features or cells of interest are roughly spherical .", "For this application , we recommend choosing a sigma which is smaller than the radius of the cells or features .", "The Gaussian will weight pixels in the centre of cells more highly than those towards the edges or in the background .", "Finding the maximum pixel , rather than summing pixels amongst all the overlapping Gaussians , ensures that pixels at the edges of objects , but overlapping , are not more highly weighted than pixels that are central and represent the centre of the cells , allowing better separation of close objects .", "For each pixel in the annotation region , we calculate a feature vector which describes the corresponding image pixels .", "Each descriptor of the feature vector is created through processing of the input image or volume with one of a bank of filters which includes: Gaussian , Gaussian Gradient Magnitude , Laplacian of Gaussian , and the minimum and maximum eigenvalues of curvature ( Fiaschi et al . , 2012 ) .", "These filter kernels are applied at multiple scales ( sigma = 1 , 2 , 4 , 8 , 16 ) to aggregate data from the surrounding pixels into the feature descriptor at that specific pixel .", "This scale range was appropriate for all the cases used in this study and were not changed .", "Once training data has been supplied by the user and the pixel features calculated , an ensemble of random decision trees is used to learn the association between input pixels and the ‘proximity map’ centre-of-mass representation ( Geurts et al . , 2006 ) .", "The decision tree framework was parameterised as follows: the data was sampled at a rate of 1/5 from the input regions , with 30 trees generated during training , with a depth of 10 levels and a minimum split condition of 20 samples for each node .", "At each node n/3 features were considered .", "Once trained , the decision tree framework can be applied to unseen images ( without user annotation ) , requiring only input features to be calculated .", "Evaluation of images produces a centre-of-mass representation of where the cell centres are located , highly similar to the representation used during training .", "The 3D object finding algorithm is applied to the output images of the random decision tree framework and involves multiple steps .", "First , the output images of the decision framework are rearranged into a 3D volume , this provides a representation of the proximity of cell centres in 3D .", "To facilitate the object identification , we next apply a determinant of Hessian blob detector which smooths our signal and also enhances objects of a specific size ( Lindeberg , 1994 ) .", "Using this filter greatly simplifies our cell identification procedure , although some idea of the size of the object is required , h = [hx , hy , hz] ( where hx = hy if the object is spherical in two dimensions and hx = hy = hz if the object is spherical in three dimensions ) .", "Finally , a 3D maxima finding algorithm is used to identify the centroid locations of the enhanced objects present in the Hessian filtered image ( Gao and Kilfoil , 2009 ) .", "A simple threshold is used to set the sensitivity to detected maxima .", "To allow for selective application of the 3D counting algorithm in distinct regions of a tissue ( for instance the primitive streak in Figure 6 ) , and over time , a novel Region Of Interest ( ROI ) interpolation algorithm was introduced .", "The user defines a ROI by clicking points around an area of interest in a single image ( e . g . top of tissue region ) .", "The user then defines another region either at the other end of the object ( e . g . bottom of tissue region ) or partially through the region .", "The algorithm can then interpolate between these user-defined ROI to create a ROI for each frame in the image-volume .", "The User can then repeat this process in subsequent time-frames , and the algorithm will interpolate the ROI between frames creating a smooth transition which can be tweaked through the addition of further user defined regions to smoothly follow a 3D region of the tissue over time .", "The interpolation is performed using bilinear interpolation of points sampled uniformly along the user defined ROI .", "Objects or cells with a centroid position within the tissue region can then be filtered from the image volume allowing for selective counting and location of cells over-time within the defined region .", "Validation of algorithm performance is critical in developing an effective tool .", "We used both real and artificial data sets to assess performance .", "For our baseline performance tests of CytoCensus , we quantified the number and location of NBs identified in five time-points from a movie sequence .", "We then compared the output from CytoCensus to that of other algorithms applied to the same test dataset .", "In each case , we attempted to optimise the parameters used , based , whenever possible , on the published information on two time-points that were not used for final evaluation .", "For TrackMate detections we used detection diameter of 20 pixels , and five conservatively set filters ( standard deviation , max intensity , mean intensity , contrast ) .", "For RACE , we used the histone ( nuclear ) marker for the seeds , and set parameters at default except for ( Max segmentation area 100 , min 3D area 20 , Closing 2–6 , Threshold 0 . 0002 , H-maxima 30 ) .", "For Ilastik , we used features from ( sigma 0 . 3 , 1 . 0 , 3 . 5 , 10 ) for color , edge and intensity .", "For FIJI WEKA , we used default parameters , followed by filtering to remove small objects .", "For CytoCensus we used default parameters , except for object size , which was set at radius 8 .", "To carry out a direct comparison of algorithm performance , we used artificial ‘neutral challenge’ datasets of highly clustered ( 75% ) synthetic cells , in 3D , with a low signal-to-noise ratio ( SNR ) , obtained from the Broad Bioimage Benchmark Collection ( image set BBBC024vl: Svoboda et al . , 2009 ) .", "This data has an absolute ground truth and provides a good measure of comparison for the performance of different algorithms .", "As CytoCensus is designed to identify cell centres , the ground truth for the Neutral Challenge data and Ilastik segmentation results were adapted to report estimated cell centres ( centroids ) rather than segmented boundaries , before carrying out comparison of algorithm performance .", "In both cases , Ilastik and CytoCensus were trained on a single image , parameters optimised over five image , and performance evaluated over the remaining ( 25 ) images .", "For the neuroblast dataset , detections within 1/2 a neuroblast radius were considered correct .", "For the BBBC dataset , detections were considered correct if they were within a stricter four pixel radius ( ~1/8 cell size ) .", "At the more generous 1/2 a cell size , CytoCensus reached perfect precision and recall , but Ilastik’s F1-score remained below 0 . 4 primarily due to the problem of merged cells .", "To quantitate how CytoCensus analysis of complex multidimensional image data is performing and to compare performance to other freely available programs , we made use of the weighted mean of the true and false positive identification rates , known as the F1-score ( maximum value 1 . 0; Chinchor , 1992; Figure 3 and Figure 3—figure supplement 1 , Table 1 and Table 2 ) .", "F1-score is intuitively similar to accuracy: strictly it is the harmonic mean of the fraction of detections that were correction ( precision ) and the fraction of cells correctly identified ( recall ) .", "This metric , therefore , takes into account both false positives and false negatives .", "Such analysis is particularly useful in parameter determination for optimum algorithm performance in applying to experimental data sets and in optimising algorithms for systematic comparison on test data .", "To apply CytoCensus to the Cell Tracking Challenge Segmentation Benchmark datasets we downsampled all Figures 2–4x before processing to increase speed of processing , although better results might be achieved without downsampling .", "We trained CytoCensus on 2–4 frames selected ( from the training set ) to capture the imaging variation between datasets and selected appropriate object sizes for each of the images .", "Following object detection , we create a crude segmentation by creating a sphere of corresponding size around each detected object , and refined this segmentation using a small number of iterations ( 2-6 ) of MorphACWE ( Marquez-Neila et al . , 2013 ) , an active contour segmentation method , followed by marker-based watershed ( Meyer and Beucher , 1990 ) from the CytoCensus centres in order to separate detected objects .", "This approach is highly dependent on the quality of the detected centres to determine the number of objects and assumes cells are approximately round , so is not well suited to tasks with extended , misshapen objects .", "The code for the cell tracking challenge , including dataset specific parameters , is available at github . com/hailstonem/CTC_CytoCensus .", "The algorithm underlying CytoCensus requires a range of parameters to be set , described in detail above .", "To simplify usage , we set most most parameters to reasonable default values .", "Parameters , such as the threshold setting , were assessed systematically , using the objective measures of performance described above with various data sets .", "This approach helped us to define which parameters should be fixed and which need to be user-modified .", "Details of user defined parameters and how to assess and set appropriate values are documented in the User Manual .", "The value of Sigma , which sets the scale of the object of interest , is particularly critical for detection .", "Optimum Sigma value was assessed systematically and the optimum found to be slightly smaller than the size of the cell type of interest ( Figure 3—figure supplement 1A′′ ) , this parameter was subsequently defined as ‘Object Size’ ( in pixels ) .", "The level of training required is also important in supervised machine-learning approaches .", "We assessed the level of training required to achieve good detection of cell types of interest with different datasets ( Figure 3—figure supplement 1A′ ) .", "In all cases tested , successful identification of NBs or progeny required minimal user training ( of the order of tens of examples on only a few image planes ) and increasing training gave only marginal improvement ( Figure 3—figure supplement 1A’ ) .", "This is advantageous for quickly annotating datasets , but may limit the flexibility for learning in particularly difficult and complex cases .", "To facilitate development of CytoCensus for the study of the larval brain , we initially tested performance on multichannel 3D image datasets of fixed material where NBs and GMC’s were defined by specific immunostaining ( for Ase and Dpn; Neumüller et al . , 2011; Bayraktar et al . , 2010; Boone and Doe , 2008; Figure 3—figure supplement 1A , B ) .", "We confirmed that , given these ideal markers , NB’s and GMC’s could be identified .", "After this initial development , we extended the application of CytoCensus to our live cell imaging data with generic cytological markers .", "To show that cell types could be recognised correctly with the generic marker combination used for live imaging , we carried out specific immunostaining on Jupiter::GFP/Histone::RFP expressing larval brains .", "Manual and automated annotations , first based upon generic labels alone , were scored against identification using the specific labelling ( Figure 3—figure supplement 1C ) .", "The results of this assessment show that , for NB ( 96% ± 4 Dpn positive , n = 12 , three repeats ) and their progeny ( 92% ± 2 Pros positive , n = 189 , three repeats ) , our imaging of the generic labels supports identification of NB and progeny by CytoCensus after training ( Figure 3—figure supplement 1D ) .", "Using a combination of these different datasets , we refined the workflow of CytoCensus and optimised the key parameters of the algorithm .", "CytoCensus outputs proximity map images and object centre XYZ co-ordinates , both of which may be used as the starting points for further data analysis pipelines , for example as seeds in watershed segmentation to determine cell areas , volumes or quantitate fluorescence intensity .", "CytoCensus outputs ( tif files ) which can easily be passed to Fiji ( ImageJ ) or custom analysis scripts .", "In this study , we illustrate several examples of extending data analysis using outputs from CytoCensus .", "In our analysis of NB division rates , we generate plots of cell division over time for individual NB from the map output and NB centre coordinates ( Figure 5A , B and Figure 5—figure supplement 1 ) .", "Here , we used a custom trackpy based python script to track individual NBs over time , however , similar results can be achieved simply , using ImageJ ROI tools , or at scale using TrackMate ( Figure 5—figure supplement 1A ) .", "For each of these tracks , we follow the changes in the dividing NB proximity map that correspond to division .", "Robustness of the division plots is further improved by subtracting a moving average over about 20 image frames , which removes spatial differences in the background of the probability density maps .", "For analysis of long imaging series , such as multiple NB divisions , it is important to follow individual cells over time .", "For such analysis , it is necessary to spatially register the individual Z stacks across time , to correct for image drift due to movements in culture , prior to applying CytoCensus .", "This was achieved with an ITK based python script ( http://www . simpleitk . org/SimpleITK/resources/software . html ) , but similar results can be achieved using the Correct 3D drift plugin in ImageJ ( it is crucial to use a high noise threshold , ignoring low value pixels , as this approach is sensitive to noise ) .", "Similar approaches were employed in our analysis of GMC cell cycle length ( Figure 4D ) .", "In the challenging case of GMCs , it was necessary to explicitly track cells as they moved significantly during development of the brain .", "To achieve this , estimated GMC centres were passed to a trackpy ( Allan et al . , 2016 ) , based custom python script to perform linkage analysis and track each individual GMC over time .", "Similar results may be achieved using TrackMate in FIJI ( Tinevez et al . , 2017 ) .", "Python scripts for further analysis are available on the CytoCensus Github page .", "Mutant comparisons were performed using an appropriate test in GraphPad Prism ( see Figure legends for specific tests ) , typically a Student’s T test , following Shapiro-Wilk test to test normal distribution of the data .", "Appropriate tests were selected depending on data type and normality: ( Figure 1: one-way ANOVA to determine differences between any time-point , Figure 3A: one-way RM-ANOVA with post-hoc t-tests to determine difference between CytoCensus performance and the other programs , Figure 3B: Welch’s t-test ( unequal variance ) to determine if there is difference between CytoCensus and Ilastik performance Figure 4: t-test or Welch’s t-test ( following F-test for variance ) to determine differences in NB number or cell cycle lengths respectively .", "Figure 5: F-test to determine difference in variance between syp RNAi and WT .", "Figure 6: t-test to determine difference between CytoCensus and manual annotation ) .", "A p-value of <0 . 05 was considered significant .", "Numbers of replicates typically refer to the number of independent brains and are detailed in the figure legends and main text .", "Measurements of cell cycle lengths/division rates are the average of 2–7 measurements ( NB that only divided once were excluded ) from 5 to 10 NB per brain ( i . e . each NB contributes one measurement ) .", "NB Numbers were limited by the number of visible NB within the imaged region .", "For live imaging , sample sizes were as large as reasonably practical given the capture and processing time .", "For the purposes of Figures 1 and 4 , independent brains were considered biological replicates , and NB considered technical replicates .", "For the purpose of comparing variation in division rates , in Figure 5 , each NB is considered as a biological replicate , and each measurement of cell cycle length is a technical replicate .", "Unless otherwise stated , error bars shown are standard deviation ." ] ]

[ "A major challenge in cell and developmental biology is the automated identification and quantitation of cells in complex multilayered tissues .", "We developed CytoCensus: an easily deployed implementation of supervised machine learning that extends convenient 2D ‘point-and-click’ user training to 3D detection of cells in challenging datasets with ill-defined cell boundaries .", "In tests on such datasets , CytoCensus outperforms other freely available image analysis software in accuracy and speed of cell detection .", "We used CytoCensus to count stem cells and their progeny , and to quantify individual cell divisions from time-lapse movies of explanted Drosophila larval brains , comparing wild-type and mutant phenotypes .", "We further illustrate the general utility and future potential of CytoCensus by analysing the 3D organisation of multiple cell classes in Zebrafish retinal organoids and cell distributions in mouse embryos .", "CytoCensus opens the possibility of straightforward and robust automated analysis of developmental phenotypes in complex tissues ." ]

[ "There are around 200 billion cells in the human brain that are generated by a small pool of rapidly dividing stem cells .", "For the brain to develop correctly , these stem cells must produce an appropriate number of each type of cell in the right place , at the right time .", "However , it remains unclear how individual stem cells in the brain know when and where to divide .", "To answer this question , Hailstone et al . studied the larvae of fruit flies , which use similar genes and mechanisms as humans to control brain development .", "This involved devising a new method for extracting the brains of developing fruit flies and keeping the intact tissue alive for up to 24 hours while continuously imaging individual cells in three dimensions .", "Manually tracking the division of each cell across multiple frames of a time-lapse is extremely time consuming .", "To tackle this problem , Hailstone et al . created a tool called CytoCensus , which uses machine learning to automatically identify stem cells from three-dimensional images and track their rate of division over time .", "Using the CytoCensus tool , Hailstone et al . identified a gene that controls the diverse rates at whichstem cells divide in the brain .", "Earlier this year some of the same researchers also published a study showing that this gene regulates a well-known cancer-related protein using an unconventional mechanism .", "CytoCensus was also able to detect cells in other developing tissues , including the embryos of mice .", "In the future , this tool could aid research into diseases that affect complex tissues , such as neurodegenerative disorders and cancer ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "developmental biology" ]

[ [ "Acinar cells are essential to the function of exocrine organs including the pancreas , tracheonasal glands and salivary glands .", "These cells produce a mucin and/or protein-rich fluid that is transported through an interconnected ductal network to the external surface where it serves multiple roles including the protection and function of the epithelial mucosa .", "Although there have been extensive studies on the mechanisms of branching morphogenesis ( reviewed in Mattingly et al . , 2015 ) , the process by which most mammalian exocrine organs develop , very little is known about the mechanisms that control acinar cell development .", "Moreover , the factors required for the establishment of the acinar lineage in the developing salivary gland are not known .", "As for the majority of exocrine organs , salivary gland acinar and duct cells form through the expansion and differentiation of a simple cuboidal epithelium .", "In the submandibular salivary gland ( SMG ) , the most characterized of the three major salivary glands ( considerably less is known about the development of the sublingual ( SLG ) and parotid ( PG ) ) , this begins with invagination of the epithelium into the condensing mesenchyme to form a single end bud containing SOX10-positive pre-acinar cells by embryonic day ( E ) 12 ( Lombaert et al . , 2013 ) .", "Branching of the single bud into 3–5 KIT-positive end buds occurs between E12 . 5 and E13 . 0 and is followed by rapid expansion and further differentiation between E13 . 5-E15 . 0 ( Lombaert et al . , 2013; Walker et al . , 2008 and Figure 1A ) .", "Acinar cell differentiation can be monitored by temporal expression kinetics of genes including aquaporin ( AQP ) 5 at E14 ( Figure 1B ) , Basic Helix-Loop-Helix Family Member A15 ( MIST1 ) ( Pin et al . , 2001 ) and demilune cell and parotid protein ( DCPP ) at E15 . 0; submandibular gland protein C ( SMGc ) /mucin 19 ( MUC19 ) and parotid secretory protein ( PSP ) at E16 . 0 , and MUC10 at E17 . 0 ( Nelson et al . , 2013 ) .", "In conjunction with acinar cell development is formation of the ductal lineage , which is marked initially by KRT19 at E12 . 0 , followed by KRT7 at E13 . 5 ( before lumenization ) and prominin-1 at E14 . 0 ( Nedvetsky et al . , 2014; Walker et al . , 2008 ) .", "Both acinar and duct cells are derived from a reservoir of undifferentiated basal KRT5+ progenitors that are essential to SG development ( Knox et al . , 2010 ) .", "However , the mechanisms regulating specification of KRT5+ cells toward these two lineages or how the proportion of duct to acinar cells is controlled are unknown . 10 . 7554/eLife . 26620 . 003Figure 1 . SOX2 marks a progenitor cell population in the salivary gland that gives rise to acinar and duct cells .", "( A ) Schematic showing markers of acinar and duct cells at E12 and E15 .", "( B ) E14 SLG stained for AQP5 , SOX2 and E-cadherin ( ECAD ) .", "Scale bar is 20 µm .", "( C ) Immunostaining of E14 SLG for SOX2 , nerves ( TUBB3 ) and Ecadherin-positive epithelial cells ( ECAD ) .", "( D and E )", "Recombination was induced in Sox2CreERT2;Rosa26mTmG mice at E10 . 5 and SMG+SLG traced until E17 . 5 or P0 .", "( E ) SLG were immunostained for KRT5 ( red ) and nuclei .", "( F and G )", "Representative images of P0 Sox2eGFP ( F ) and adult wild-type ( G ) SLG immunostained for the acinar marker AQP5 , ECAD and/or SOX2 .", "Scale bars in F and G are 20 µm , ( C ) and E are 50 µm and D is 100 µm; Images in B , E , F and G are 2 µm confocal sections and the image in C is a 30 µm projection of 4 µm sections . DOI: http://dx . doi . org/10 . 7554/eLife . 26620 . 003 In addition to the known growth factor pathways that regulate epithelial branching morphogenesis ( e . g . FGF10/FGFR2b [Jaskoll et al . , 2005] ) , we recently discovered that neurotransmitters released from intraglandular parasympathetic nerves also control multiple aspects of SG development .", "The SMG and SLG receive innervation from the parasympathetic submandibular ganglion that forms at E12 . 0 through coalescence of neuronal cell bodies around the primary duct .", "The ganglion begins to innervate newly forming end buds and ducts from E12 . 5 ( Knox et al . , 2010 ) , with unidirectional axon outgrowth from the ganglion toward the end buds being mediated by neurturin/GFRa2 signaling ( Knox et al . , 2013 ) .", "Acetylcholine is produced as soon as the ganglion forms ( Coughlin , 1975 ) and activates muscarinic receptors on the epithelium to maintain an undifferentiated stem cell population marked by KRT5 ( Knox et al . , 2010 ) .", "In addition to this function , muscarinic activation also induces epithelial branching ( Knox et al . , 2013 , 2010 ) but whether parasympathetic nerves are specifically required for the generation of the acinar lineage is unknown .", "Using the acini-ductal network of developing murine and human salivary glands in combination with in vivo and ex vivo studies , we reveal that SOX2 is specifically required to generate the acinar cells necessary for the creation of a secretory organ .", "We show that SOX2 is essential for the establishment and survival of acinar cells and that its expression and the proliferative expansion of SOX2-positive cells depend on neuronal acetylcholine signaling by parasympathetic nerves .", "As such , we identify SOX2 as a master regulator of the salivary acinar cell lineage and uncover a conserved peripheral nerve-based mechanism for selectively generating the secretory acinar cell lineage during organogenesis ." ], [ "SOX2 is an important transcriptional regulator of development and maintenance in multiple organs including trachea , oesophagus and intestine ( Arnold et al . , 2011; Gontan et al . , 2008; Okubo et al . , 2006; Que et al . , 2009 , 2007 ) .", "In the murine E13 embryo , SOX2 is expressed by the invaginating oral epithelium ( Figure 2—figure supplement 1B ) , throughout the epithelium of the SLG ( Figure 1C ) and the proximal duct of the SMG and the developing parasympathetic ganglion ( Figure 1C; Lombaert and Hoffman , 2010 ) .", "Genetic lineage tracing at E10 . 5 indicated that SOX2-positive cells are progenitors giving rise to both duct and acinar cells of the SMG and SLG ( Figure 1D , E Arnold et al . , 2011 ) .", "However , we found that SOX2 becomes restricted to a subpopulation of AQP5+ acinar cells in the postnatal and adult SLG , the most poorly understood of the three major salivary glands ( Figure 1F and G ) , suggesting that SOX2 might function in the expansion and/or maintenance of the acinar lineage .", "To study the role of Sox2 in salivary gland morphogenesis , a tamoxifen-inducible Cre under the control of the epithelial Krt14 promoter was used to ablate Sox2 prior to the onset of gland ontogenesis ( E10 . 5; see extensive loss of SOX2 expression in epithelia but not the ganglion in Figure 2—figure supplement 1B ) .", "Unexpectedly , despite SOX2-positive cells giving rise to both acinar and duct cells , genetic ablation of Sox2 had a profound effect on the generation of acini but not ducts .", "The number of end buds in the SMG and SLG was significantly reduced from early stages of development and onwards , with the SLG exhibiting little to no branching ( E13 to E16 . 5; Figure 2A–B and Figure 2—figure supplement 1A–E ) .", "Conversely , duct morphology , the number of KRT19-positive ductal cells and ductal gene transcripts Krt7 , Krt19 and Egfr , which are required for ductal development ( Häärä et al . , 2009; Knox et al . , 2010 ) , were similar to wild-type controls ( Figure 2A–D and Figure 2—figure supplement 1C ) .", "Moreover , by E16 . 5 , the number of SOX10-positive progenitors and differentiated AQP5-positive and MIST1-positive acinar cells were severely reduced ( Figure 2C–E , Figure 2—figure supplement 1C ) .", "Further analysis of changes in gene expression profiles between wild-type control and Sox2-deficient SMG+SLG by RNA-seq and qPCR validation confirmed the reduction in genes associated with differentiated acinar cells ( Smgc , Pip , Spdef , Rab3d; Figure 2C , Figure 2—figure supplement 1G ) .", "To rule out the phenotype being a cause of a difference in recombination efficiency between acini and ducts , we confirmed that Sox2 ablation efficiency is comparable between AQP5+/SOX10+ acini and KRT19+ ducts ( Figures 1E and 2F ) using a lineage-tracing model ( Krt14CreERT2;Sox2fl/fl;Rosa26mTmG ) . 10 . 7554/eLife . 26620 . 004Figure 2 . SOX2 is essential for establishing SOX10+ acini during organogenesis .", "( A–D )", "SMG+SLG from Krt14CreERT2; Sox2fl/fl and wild-type ( WT ) embryos in which recombination was induced before gland ontogenesis at E10 . 5 and 11 . 5 .", "( A ) Representative brightfield images of Krt14CreERT2; Sox2fl/fl and wild-type ( WT ) SMG+SLG at E16 . 5 .", "Scale bar is 1 mm . ( B ) Representative images of SMG+SLG immunostained for nerves ( TUBB3 ) and epithelium ( Ecadherin , ECAD ) .", "Scale bar is 1 mm .", "SMG = submandibular gland , SLG = sublingual gland .", "Dashed white line denotes SLG .", "( C ) qPCR analysis of E16 . 5 Krt14CreERT2; Sox2fl/fl and wild-type ( WT ) SMG+SLG for genes involved in acinar differentiation , ductal differentiation and innervation , with expression normalized to Rsp29 .", "Red dashed line = WT .", "n = 3 embryos per genotype .", "Data are means+s . d . and were analyzed using a one-way analysis of variance with post-hoc Dunnett’s test .", "*p<0 . 05 , **p<0 . 01 .", "( D ) Quantification of cells expressing acinar or ductal markers , cleaved caspase-3 or Ki67 in acini of E16 . 5 in Krt14CreERT2; Sox2fl/fl and wild-type ( WT ) .", "n = 2–4 glands/genotype and cells were counted in 3–4 acini/gland .", "Data are means+s . d . and were analyzed using a Student’s t-test , ***p<0 . 001 .", "( E and F )", "SMG+SLG from Krt14CreERT2; Rosa26mTmG and Krt14CreERT2; Rosa26mTmG; Sox2fl/fl in which recombination was induced at E10 . 5 and 11 . 5 were immunostained for SOX10 and AQP5 ( E ) and GFP+ cells expressing SOX10 and AQP5 were quantified ( F ) .", "n = 3 glands/genotype and cells were counted in 3–4 acini/gland .", "Data were subjected to a Student’s t-test , ***p<0 . 001 .", "Scale bar in E is 20 µm .", "Arrowheads indicate double positive cells .", "( G ) qPCR for enrichment of Sox10 in SOX2 ChIP .", "n = 20 pooled SLG , average three experiments , *p<0 . 05 .", "Additional data for this figure in Figure 2—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 26620 . 00410 . 7554/eLife . 26620 . 005Figure 2—source data 1 . Source data relating to Figure 2C . qPCR analysis of E16 . 5 Krt14CreERT2; Sox2fl/fl and wild-type ( WT ) SMG+SLG for genes involved in acinar differentiation , ductal differentiation and innervation , with expression normalised to Rsp29 and the WT .", "n = 3 embryos per genotype .", "s . d . = standard deviation . DOI: http://dx . doi . org/10 . 7554/eLife . 26620 . 00510 . 7554/eLife . 26620 . 006Figure 2—source data 2 . Source data relating to Figure 2D . Quantification of cells expressing acinar or ductal markers , cleaved caspase-3 or Ki67 in acini of E16 . 5 in Krt14CreERT2; Sox2fl/fl and wild-type ( WT ) .", "n = 2–4 glands/genotype and cells were counted in 3–4 acini/gland .", "s . d . = standard deviation . DOI: http://dx . doi . org/10 . 7554/eLife . 26620 . 00610 . 7554/eLife . 26620 . 007Figure 2—source data 3 . Source data relating to Figure 2F . SMG+SLG from Krt14CreERT2; Rosa26mTmG and Krt14CreERT2; Rosa26mTmG; Sox2fl/fl were immunostained for SOX10 and AQP5 and GFP+ cells expressing SOX10 and AQP5 were quantified and expressed as a percentage of total positive cells .", "n = 3 glands/genotype and cells were counted in 3–4 acini/gland .", "s . d . = standard deviation . DOI: http://dx . doi . org/10 . 7554/eLife . 26620 . 00710 . 7554/eLife . 26620 . 008Figure 2—source data 4 . Source data relating to Figure 2G . qPCR for enrichment of Sox10 in SOX2 ChIP .", "n = 20 pooled SLG , average three experiments .", "s . d . = standard deviation . DOI: http://dx . doi . org/10 . 7554/eLife . 26620 . 00810 . 7554/eLife . 26620 . 009Figure 2—figure supplement 1 . Acini are depleted in the absence of Sox2 . Representative images of Krt14CreERT2; Sox2fl/fl and wild-type ( WT ) SMG+SLG at E13 ( A; scale bar is 100 μm ) , E13 . 5 ( B; top and bottom panels; scale bars are 100 and 50 μm , respectively ) and E16 . 5 ( C; scale bar is 200 µm ( top panels ) , 50 µm ( second and fourth panels ) and 10 µm ( third panels ) ) .", "SMG = submandibular gland , SLG = sublingual gland .", "E13 . 5 SGs ( B ) were immunostained for SOX2 and E-cadherin ( ECAD ) and E16 . 5 SMG+SLG ( C ) for KRT19 , MIST1 , SOX10 , AQP5 , KRT5 , Ecadherin ( ECAD ) and nuclei .", "Dashed white lines outline acini .", "Lower panel in B highlights the oral epithelium of the E13 . 5 SG .", "( D–E )", "Quantification of acini at E13 . 5 and E16 . 5 is shown in D ( n = 3–7 ) and E ( n = 3 ) , with WT set to 100% .", "Data in D and E were analyzed using a Student’s t-test , *p<0 . 05 , **p<0 . 01 .", "( F and G ) qPCR analysis of gene expression of SG from Krt14CreERT2; Sox2fl/fl versus wild-type littermate ( WT ) at E13 . 5 ( F ) and E16 . 5 ( G ) .", "Red dashed line = WT .", "Data are means+s . d . of 3–4 SMG+SLG per genotype .", "Data in F and G were analyzed using a one-way analysis of variance with a post-hoc Dunnett’s test .", "*p<0 . 05 , **p<0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 26620 . 00910 . 7554/eLife . 26620 . 010Figure 2—figure supplement 1—source data 1 . Source data relating to Figure 2—figure supplement 1D . Quantification of acini in Krt14CreERT2; Sox2fl/fl and wild-type ( WT ) glands at E13 . 5 , with WT set to 100% .", "n = 3–7 .", "s . d . = standard deviation . DOI: http://dx . doi . org/10 . 7554/eLife . 26620 . 01010 . 7554/eLife . 26620 . 011Figure 2—figure supplement 1—source data 2 . Source data relating to Figure 2—figure supplement 1E . Quantification of acini in Krt14CreERT2; Sox2fl/fl and wild-type ( WT ) glands at E16 . 5 , with WT set to 100% .", "n = 3–7 .", "s . d . = standard deviation . DOI: http://dx . doi . org/10 . 7554/eLife . 26620 . 01110 . 7554/eLife . 26620 . 012Figure 2—figure supplement 1—source data 3 . Source data relating to Figure 2—figure supplement 1F . qPCR analysis of gene expression in Krt14CreERT2; Sox2fl/fl and wild-type ( WT ) glands at E13 . 5 .", "Data were normalized to Rsp29 and WT .", "n = 3–4 SMG+SLG per genotype .", "s . d . = standard deviation . DOI: http://dx . doi . org/10 . 7554/eLife . 26620 . 01210 . 7554/eLife . 26620 . 013Figure 2—figure supplement 1—source data 4 . Source data relating to Figure 2—figure supplement 1G . qPCR analysis of gene expression in Krt14CreERT2; Sox2fl/fl and wild-type ( WT ) glands at E16 . 5 .", "Data were normalized to Rsp29 and WT .", "n = 3–4 SMG+SLG per genotype .", "s . d . = standard deviation . DOI: http://dx . doi . org/10 . 7554/eLife . 26620 . 013 SOX2 co-localizes with the acinar progenitor marker SOX10 in acini of developing murine glands ( Figure 2E , left panel ) .", "Given SOX10 expression was lost upon ablation of Sox2 , we performed lineage tracing in conjunction with Sox2 ablation to determine if SOX10 was specifically lost in cells derived from those in which Sox2 was ablated ( Krt14CreERT2;Sox2fl/fl;Rosa26mTmG ) .", "In this model , cells in which Cre recombinase has been activated ( and consequently Sox2 ablated ) will be labeled with membrane-bound GFP+ ( green ) , whereas cells where no recombination has occurred will retain membrane-bound Tomato ( mT , red ) .", "We found that only ~5% of end bud cells deficient in Sox2 expressed SOX10 or AQP5 in comparison to control glands ( Krt14CreERT;Rosa26mTmG ) in which >90% express SOX10 or AQP5 ( Figure 2E and F ) .", "We then determined whether SOX2 could directly regulate Sox10 using ChIP-qPCR analysis of E17 . 5 SG .", "This time point was chosen due to the small size of the tissue limiting our analysis at earlier time points .", "We found significant enrichment of SOX2 within a specific region of the Sox10 promoter ( promoter region A; Figure 2G ) , suggesting that SOX2 may promote the generation of the acinar lineage via transcriptional control of Sox10 .", "Combined , these data indicate that the maintenance of SOX10-positive acinar progenitor cells and the generation of differentiated AQP5-positive acinar cells are dependent on SOX2 .", "Previous studies in other organ systems show that a deficiency in SOX2 results in a cell fate switch ( 23–26] ) .", "However , we did not find any evidence that Sox2-deficient cells in the end buds divert toward a duct cell fate ( Figure 3A , GFP+ cells do not express early ductal marker KRT19 ) , indicating that the reduction in epithelial branching was not due to the accumulation of the ductal lineage .", "Therefore , we addressed if loss of end bud cells and reduced morphogenesis in Sox2-deficient SMG+SLG were due to apoptosis .", "In the absence of Sox2 , there were activated Caspase-3-positive cells in the end buds ( Figures 2D and 3B ) and increased expression of pro-apoptotic genes ( Bax , Bbc3 and Pmaip1; Figure 2—figure supplement 1G ) at E16 . 5 .", "Apoptosis was specific to end bud cells as no apoptosis was observed in the ducts ( Figure 3B ) , suggesting a preferential requirement for SOX2 in end bud cell survival .", "In addition to increased apoptosis , the proliferation of end bud cells was decreased ~70% in E16 . 5 salivary glands lacking Sox2 ( Figures 2D and 3B; Ki67+ cells ) .", "Apoptosis and reduced proliferation were not due to a loss in FGF signaling that has previously been shown to be required for epithelial survival and acinar cell expansion ( Lombaert et al . , 2013; Matsumoto et al . , 2016 ) as the expression of downstream targets of FGF signaling transduction ( Etv4 , Etv5 ) and FGF ligands ( Fgf1 , Fgf10 , Fgf7 ) were not reduced compared to wild-type controls ( Figure 2—figure supplement 1G ) . 10 . 7554/eLife . 26620 . 014Figure 3 . Regulation of the acinar lineage by SOX2 is independent of its function in cell survival .", "( A–C )", "Representative images of SMG+SLG from WT , Krt14CreERT2; Sox2fl/fl; Rosa26mTmG or Krt14CreERT2; Sox2fl/fl immunostained for the ductal marker KRT19 , apoptotic marker CASP3 , proliferation marker Ki67 , SOX2 , ECAD and/or nuclei .", "Scale bars are 50 µm .", "Images in A , B and lower panel C are 1–2 µm confocal sections; images in C upper panel are 20 µm projections of 1 . 5 µm sections .", "Recombination was induced at E10 . 5 and E11 . 5 for images in A and B , and E10 . 5 for images in C . ( D ) Representative images of E11 . 5 mandibles from Krt14CreERT2; Sox2fl/fl and wild-type ( WT ) cultured for 60 hr in the presence of DMSO or the pan-caspase inhibitor ZVAD-FMK ( 50 µM ) .", "SMG+SLG ( lower panels ) were immunostained for Ecadherin ( ECAD ) , nuclei and cleaved caspase-3 ( CASP3 ) .", "White dashed line ( upper panels ) denotes SMGs branching off the tongue .", "Red dashed line indicates tongue .", "Scale bars are 200 µm ( upper panels ) and 25 µm ( lower panels ) .", "( E , F )", "Quantification of the number of CASP3+ cells ( E ) and acini ( F ) .", "n = 3 glands per treatment and cells were counted in 3–4 end acini per gland ( E ) .", "Data in E and F are means+s . d . of three biological replicates and two experiments .", "Data were analyzed using a Students t-test .", "*p<0 . 05 , **p<0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 26620 . 01410 . 7554/eLife . 26620 . 015Figure 3—source data 1 . Source data relating to Figure 3E . Quantification of the number of CASP3+ cells in acini of E11 . 5 Krt14CreERT2; Sox2fl/fl and wild-type ( WT ) glands cultured for 60 hr ± Z-VAD-FMK .", "n = 3 glands per treatment and cells were counted in 3–4 acini per gland .", "Data are the mean of three biological replicates and two experiments .", "s . d . = standard deviation . DOI: http://dx . doi . org/10 . 7554/eLife . 26620 . 01510 . 7554/eLife . 26620 . 016Figure 3—source data 2 . Source data relating to Figure 3F . Quantification of the number of acini of E11 . 5 Krt14CreERT2; Sox2fl/fl and wild-type ( WT ) glands cultured for 60 hr ± Z-VAD-FMK .", "n = 3 glands per treatment .", "Data are means of three biological replicates and two experiments .", "s . d . = standard deviation . DOI: http://dx . doi . org/10 . 7554/eLife . 26620 . 016 As both apoptosis and reduced proliferation were apparent at E16 . 5 , and there was reduced branching and increased apoptotic markers at E13-E13 . 5 ( Figure 2—figure supplement 1A , B and F ) to further delineate between mitosis and cell death as a cause of reduced growth , we analyzed tissue at E11 . 5 ( Cre induction at E10 . 5 by injection of tamoxifen ) .", "At this stage of development , the undifferentiated oral epithelium has begun to invaginate into the condensing mesenchyme .", "We observed an increase in apoptotic cells in the invaginating mutant epitheliums compared to wild-type controls ( arrows indicate cleaved CASP3-p cells ) but proliferation was not perturbed , suggesting that cell death rather than reduced proliferation impairs organ growth ( Figure 3C ) .", "To test if inhibiting apoptosis could rescue epithelial branching that is , cell death is a causative factor in the loss of morphogenesis , we cultured E11 . 5 mandibles from wild-type and Sox2-deficient embryos ex vivo ( Knosp et al . , 2015 ) with the pan-caspase inhibitor Z-VAD-FMK ( 50 µM , Nedvetsky et al . , 2014 ) ; Figure 3D ) for 60 hr .", "However , despite a significant reduction in CASP3+ cells , extensive epithelial cell survival and growth of the epithelium , we did not measure an increase in the number of acini in Sox2-deficient SMG+SLG cultured with the cell death inhibitor ( Figure 3D–F ) .", "Thus , these data reveal novel , independent roles for SOX2 in regulating epithelial cell survival and generating acini .", "Our previous study indicated that KRT5+ progenitors are maintained in an unspecified state by parasympathetic nerves via acetylcholine/muscarinic activation ( Knox et al . , 2010 ) .", "As a subpopulation of KRT5+ cells of the developing SMG co-express SOX2 ( Lombaert et al . , 2011 ) , we next asked if nerves also maintain SOX2+ progenitor cells by comparing SG where the ganglia had been mechanically or genetically ablated with nerve-containing controls .", "SMG+SLG in which the epithelium and mesenchyme was recombined without the ganglia and cultured for 48 hr ( Knox et al . , 2010 ) exhibited a severe reduction in the number of acini , SOX2 protein and SOX2+ cells .", "However , not only were SOX2+ progenitors adversely affected , the acinar lineage was also greatly depleted in the absence of innervation .", "In the ganglia-free SG , we found a significant reduction in SOX10-positive acinar progenitors and differentiated AQP5-positive acinar cells ( Figure 4A–C ) compared to ganglia-containing controls .", "Similarly , acini , SOX10+ cells , Sox2 and acinar gene expression were reduced in SMG+SLG derived from Phox2b mutant embryos that are deficient in the submandibular parasympathetic ganglion and other craniofacial ganglia ( Pattyn et al . , 1999 ) ( Figure 4D–F ) .", "However , absence of innervation did not impair the expression of ductal marker KRT19 or ductal gene transcripts ( Figure 4A and F ) .", "Thus , peripheral innervation not only preserves the progenitor cell pool , as previously found , but also selectively preserves those progenitors that contribute specifically to the acinar cell lineage . 10 . 7554/eLife . 26620 . 017Figure 4 . The acinar cell lineage and SOX2 are selectively depleted in the absence of parasympathetic nerves .", "( A–C )", "E13 murine SMG+SLG cultured for 48 hr ± parasympathetic ganglion ( nerves ) and subjected to immunofluorescent analysis ( A ) .", "Glands were immunostained for markers of duct cells ( KRT19 , KRT7 ) , SOX2 , SOX10+ acinar progenitors , AQP5+ acinar cells and epithelial cells ( E-cadherin; ECAD ) .", "The number of acini ( B ) and AQP5+ and SOX10+ cells ( C ) were quantified .", "( D–F )", "E11 . 5 murine SMG+SLG deficient in Phox2b were cultured for 60 hr .", "Glands were were immunostained for markers of nerves ( TUBB3 ) , acinar progenitors ( SOX10 ) , basal epithelial progenitors ( KRT5 ) and epithelial cells ( E-cadherin; ECAD; D ) , the number of acini were quantified ( E ) or qPCR was performed .", "( F; n = 3 embryos per genotype ) .", "Scale bars = 200 µm in ( A ) , left panels and ( D ) , left panels; 50 µm in ( A ) , right panels and ( D ) , middle and right panels; 20 µm in ( A ) , middle panels .", "Data in B , C and E are means ± s . d of three biological replicates and three experiments and were subjected a Student’s t-test .", "*p<0 . 05 **p<0 . 01 .", "Data in F are means+s . d . and were analyzed using a one-way analysis of variance with post-hoc Dunnett’s test .", "*p<0 . 05 , **p<0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 26620 . 01710 . 7554/eLife . 26620 . 018Figure 4—source data 1 . Source data relating to Figure 4B . E13 murine SMG+SLG cultured for 48 hr ± parasympathetic ganglion ( nerves ) .", "The number of acini were quantified .", "Data are means of three biological replicates and three experiments .", "s . d . = standard deviation . DOI: http://dx . doi . org/10 . 7554/eLife . 26620 . 01810 . 7554/eLife . 26620 . 019Figure 4—source data 2 . Source data relating to Figure 4C . E13 murine SMG+SLG cultured for 48 hr ± parasympathetic ganglion ( nerves ) and subjected to immunofluorescent analysis .", "The number of AQP5+ and SOX10+ cells were quantified .", "Data are means of three biological replicates and three experiments .", "s . d . = standard deviation . DOI: http://dx . doi . org/10 . 7554/eLife . 26620 . 01910 . 7554/eLife . 26620 . 020Figure 4—source data 3 . Source data relating to Figure 4E . E11 . 5 murine SMG+SLG deficient in Phox2b were cultured for 60 hr .", "The number of acini were quantified .", "Data are means of three biological replicates and three experiments .", "s . d . = standard deviation . DOI: http://dx . doi . org/10 . 7554/eLife . 26620 . 02010 . 7554/eLife . 26620 . 021Figure 4—source data 4 . Source data relating to Figure 4F . E11 . 5 murine SMG+SLG deficient in Phox2b were cultured for 60 hr and qPCR performed .", "Data were normalized to Rsp29 and the WT .", "Data are means of three biological replicates and three experiments .", "s . d . = standard deviation . DOI: http://dx . doi . org/10 . 7554/eLife . 26620 . 021 Next , we determined if acetylcholine/muscarinic signaling regulates SOX2 and production of acinar cells by specifically targeting muscarinic receptors .", "FACS analysis showed that 94% of the SOX2-positive epithelial cells in the SLG at E15 . 0 express the muscarinic receptor CHRM1 ( Figure 5—figure supplement 1A ) , indicating that CHRM1 activation is a potential regulator of SOX2 cells .", "CHRM1 is also expressed throughout the epithelium including ~42% of SOX2-negative epithelial cells ( Figure 5—figure supplement 1A and B ) , indicating that this receptor is not exclusive to SOX2+ cells .", "In support of a direct role for muscarinic receptors in expanding SOX2+ cells , treatment of isolated E14 . 0 SLG epithelial rudiments devoid of nerves and mesenchyme with the muscarinic receptor agonist carbachol ( CCh ) for 48 hr increased the number of SOX2+ cells as well as proliferating SOX2+EdU+ cells compared to the control ( Figure 5A–B ) .", "Furthermore , culture of mouse E14 . 0 SLG with the muscarinic antagonist 4-DAMP for 4 or 24 hr reduced SOX2 expression ( gene and protein ) , the number of SOX2+ cells and SOX2+ cell proliferation ( Figure 5C–D and Figure 5—figure supplement 1C .", "Similar to the outcomes for SMG+SLG in which Sox2 was ablated or nerves removed , inhibition of CHRM1 decreased end bud cell proliferation , differentiated AQP5-positive acinar cells and SOX10-positive acinar progenitors ( Figure 5E and Figure 5—figure supplement 1D and E ) , whereas KRT19-positive cells ( Figure 5E and Figure 5—figure supplement 1E ) or ductal lineage transcript levels were unchanged ( Krt7 ) or increased ( Krt19 and Egfr; Figure 5—figure supplement 1C ) .", "In addition , there was decreased expression of acinar differentiation markers ( Aqp5 , Mist1 and Chrm3; Figure 5—figure supplement 1C ) .", "Finally , to determine if muscarinic activation was sufficient to restore the acinar lineage after denervation , we treated nerve-free SMG+SLG explants ( containing mesenchyme ) with CCh for 48 hr and compared to untreated ganglia-containing and ganglia-free controls .", "As shown in Figure 5F–H , CCh was sufficient to return Sox2 expression in the SLG , and restore AQP5+ cells , and transcript levels of acinar-specific genes including Sox10 , Mist1 and Aqp5 to control levels in the SMG and SLG . 10 . 7554/eLife . 26620 . 022Figure 5 . Muscarinic signaling regulates the acinar lineage and SOX2+ cells .", "( A–B )", "E14 mouse SLG epithelia cultured with FGF10 ±CCh for 24 hr .", "White dashed line outlines acini .", "Arrows indicate double positive SOX2+EdU+ cells .", "Scale bar is 50 µm .", "The number of SOX2+ , EdU+ and SOX2+EdU+ cells was quantified in B . ( C–E ) E14 mouse SLG cultured for 24 hr with DMSO or 4-DAMP ( 10 µM ) .", "The number of SOX2+ and SOX2+Ki67+ cells were counted via FACS , normalized to control and expressed as percentage of total ECAD+ cells ( C ) .", "In D and E , cultured glands were immunostained for SOX2 , nerves ( TUBB3 ) , AQP5 , KRT19 and Ecadherin ( ECAD ) .", "Images are 50 µm projections of 5 µm confocal sections .", "Scale bars are 100 µm .", "( F–H )", "E13 SMG+SLG were cultured ± ganglia and ± CCh ( 100 nM ) for 48 hr and immunostained for AQP5 ( F ) and the number of AQP5+ and KRT19+ cells counted ( G ) .", "SMG+SLG were also subjected to qPCR analysis ( H ) .", "Data in B , C , G and H are means ±s . d of three biological replicates and three experiments .", "Data in B , C and G were subjected a Student’s t-test .", "*p<0 . 05 **p<0 . 01 .", "Data in H were analyzed using a one-way analysis of variance with a post-hoc Dunnett’s test .", "*p<0 . 05 , **p<0 . 01 .", "Additional data for this figure in Figure 5—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 26620 . 02210 . 7554/eLife . 26620 . 023Figure 5—source data 1 . Source data relating to Figure 5B . E14 mouse SLG epithelia cultured with FGF10 ±CCh for 24 hr .", "The number of SOX2+ , EdU+ and SOX2+EdU+ cells were quantified .", "Data are means of three biological replicates and three experiments .", "s . d . = standard deviation . DOI: http://dx . doi . org/10 . 7554/eLife . 26620 . 02310 . 7554/eLife . 26620 . 024Figure 5—source data 2 . Source data relating to Figure 5C . E14 mouse SLG cultured for 24 hr with DMSO or 4-DAMP ( 10 µM ) .", "The number of SOX2+ and SOX2+Ki67+ cells were counted via FACS , normalized to control and expressed as percentage of total ECAD+ cells .", "s . d . = standard deviation . DOI: http://dx . doi . org/10 . 7554/eLife . 26620 . 02410 . 7554/eLife . 26620 . 025Figure 5—source data 3 . Source data relating to Figure 5G . E13 SMG+SLG were cultured ± ganglia and ± CCh ( 100 nM ) for 48 hr and the number of AQP5+ and KRT19+ cells counted .", "Counts were normalized to the control ( nerves ) .", "Data are means of three biological replicates and three experiments .", "s . d . = standard deviation . DOI: http://dx . doi . org/10 . 7554/eLife . 26620 . 02510 . 7554/eLife . 26620 . 026Figure 5—source data 4 . Source data relating to Figure 5H . E13 SMG+SLG were cultured ± ganglia and ± CCh ( 100 nM ) for 48 hr and subjected to qPCR analysis .", "Data were normalized to Rsp29 and control ( nerves ) .", "Data are means of three biological replicates and three experiments .", "s . d . = standard deviation . DOI: http://dx . doi . org/10 . 7554/eLife . 26620 . 02610 . 7554/eLife . 26620 . 027Figure 5—figure supplement 1 . Muscarinic signaling regulates the acinar lineage and SOX2+ cells .", "( A ) Flow cytometry plots show gating strategy to analyze SOX2+ cells versus SOX2- cells ( left ) and gating strategy to analyze percentage of SOX2+/SOX2- cells that express the muscarinic receptor CHRM1 ( right ) .", "SSC-H = side scatter height , 100 , 000 events were analyzed per sample .", "( B ) E13 SLG cultured for 24 hr and stained for CHRM1 , lectin peanut agglutinin ( PNA; basement membrane and epithelial marker ) and nerves ( TUBB3 ) .", "Scale bar is 20 µm .", "( C–E )", "E14 mouse SLGs cultured for 4 or 24 hr with DMSO or the muscarinic inhibitor 4-DAMP ( 10 µM ) .", "In C , glands were cultured for 4 hr and subjected to gene profiling by qPCR .", "Data were normalized to Rsp29 and control values ( DMSO; dashed line ) , and analyzed using a one-way analysis of variance with post-hoc Dunnett’s test .", "*p<0 . 05 , **p<0 . 01 .", "( D ) Representative image of SLG cultured for 24 hr immunostained for SOX10 and Ecadherin ( ECAD ) .", "Image is a 28 µm projections of 2 µm sections .", "Scale bar is 50 µm .", "The number of AQP5+ , KRT19+ , SOX10+ , Ki67+ remaining in SLG cultured with or without 4-DAMP for 24 hr were quantified in E ( n = 2 SLG per treatment and cells were counted in 3–4 end acini per gland ) .", "Data in C and E are means+s . d of four to five biological replicates and three experiments .", "Data in E were subjected a Student’s t-test .", "*p<0 . 05 , **p<0 . 01 .", "( F ) E14 mouse SLGs cultured for four with PD 168393 ( 20 μM ) , KN-93 ( 15 μM ) or vehicle ( water ) .", "Data are means±s . d . of three to four SGs per treatment/genotype normalized to Rsp29 and control values ( vehicle or WT control; dashed line ) .", "Data in C and F were analyzed using a one-way analysis of variance with post-hoc Dunnett’s test .", "*p<0 . 05 , **p<0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 26620 . 02710 . 7554/eLife . 26620 . 028Figure 5—figure supplement 1—source data 1 . Source data relating to Figure 5—figure supplement 1C . E14 mouse SLGs cultured for 4 hr with DMSO or the muscarinic inhibitor 4-DAMP ( 10 µM ) and subjected to gene profiling by qPCR .", "Data were normalized to Rsp29 and control values ( DMSO ) .", "s . d . = standard deviation . DOI: http://dx . doi . org/10 . 7554/eLife . 26620 . 02810 . 7554/eLife . 26620 . 029Figure 5—figure supplement 1—source data 2 . Source data relating to Figure 5—figure supplement 1E . E14 mouse SLGs cultured for 24 hr with DMSO or the muscarinic inhibitor 4-DAMP ( 10 µM ) .", "The number of AQP5+ , KRT19+ , SOX10+ , Ki67+ remaining in SLG cultured with or without 4-DAMP for 24 hr were quantified .", "n = 2 SLG per treatment and cells were counted in 3–4 end acini per gland .", "s . d . = standard deviation . DOI: http://dx . doi . org/10 . 7554/eLife . 26620 . 02910 . 7554/eLife . 26620 . 030Figure 5—figure supplement 1—source data 3 . Source data relating to Figure 5—figure supplement 1F . E14 mouse SLGs cultured for 4 hr with PD 168393 ( 20 μM ) , KN-93 ( 15 μM ) or vehicle ( water; Control ) were subjected to gene profiling by qPCR .", "Data were normalized to Rsp29 and control values .", "Data are means of three to four SGs per treatment/genotype .", "s . d . = standard deviation . DOI: http://dx . doi . org/10 . 7554/eLife . 26620 . 030 As muscarinic receptors in the salivary gland have been reported to signal via intracellular calcium and EGFR ( Jiménez et al . , 2001; Knox et al . , 2010 ) , we determined if either of these downstream targets was required for SOX2 and the acinar lineage .", "Culture of E14 . 0 SLG for 4 hr with the CaMK-II inhibitor KN-93 but not the EGFR inhibitor PD168393 decreased expression levels of Sox2 and Sox10 ( Figure 5—figure supplement 1F ) .", "In comparison , inhibition of EGFR or calcium signaling reduced expression of more differentiated acinar markers ( Mist1 , Aqp5 ) suggesting that early and late markers of salivary gland development are differentially regulated .", "In contrast , ductal genes were expressed at similar or higher levels than controls upon KN-93 treatment ( Figure 5—figure supplement 1F ) .", "Thus , CHRM1 and calcium signaling regulate epithelial SOX2 in the developing SLG and are required for generation of the acinar lineage .", "Finally , we investigated whether neuronal signaling was an evolutionarily conserved mechanism for generating acini and preserving SOX2+ progenitors by analyzing the impact of nerves and muscarinic activation on SOX2 and the acinar lineage in human tissue .", "Although the murine and human glands differ in gross morphology , they undergo similar stages of morphogenesis ( Teshima et al . , 2011 ) and are highly innervated ( Figure 6A and Figure 6—figure supplement 1A ) .", "Due to the paucity of expression data on human fetal salivary gland , we first characterized expression of acinar and duct markers as well as the location of CHRM1 , SOX10 and SOX2 .", "As shown in Figure 6A and Figure 6—figure supplement 1A , CHRM1 protein and proliferating SOX2+ and SOX10+cells are enriched in the acinar compartment of all major human fetal salivary glands , including the parotid gland ( PG ) .", "We confirmed that human SMG/SLG/PG acinar cells are also enriched in SOX10 , CHRM1 and MIST1 as well as CD44 and AQP3 protein ( acini do not express AQP5 protein at these stages ) and that the duct cells express KRT7 in addition to EGFR , KRT5 , KIT and KRT14 ( Figure 6A and Figure 6—figure supplement 1A ) .", "However , KRT19 was expressed in both acini and ducts , indicating that it is not a bona fide ductal marker in fetal human tissue ( Figure 6—figure supplement 1A ) .", "To determine if parasympathetic nerves promote acinar cell formation in developing human gland , we developed a co-culture assay in which human fetal SLG explants were mixed with murine ganglia or mesenchyme ( Figure 6B ) .", "Unlike mice , the innervating human ganglion resides outside the organ and the tissue is thus denervated upon dissection .", "Co-culture of human SLG ( 20–22 w ) with E13 . 0 parasympathetic ganglia for 7 days resulted in extensive remodeling and innervation of the epithelium ( Figure 6B and C ) .", "As for the murine SLG , innervation increased expression of SOX2 protein ( Figure 6D ) as well as of acinar markers MIST1 ( ~5–7 fold ) , AQP3 ( ~1 . 6–1 . 9 fold ) , CHRM1 ( ~2–10 fold ) and CHRM3 ( ~2 . 5–3 . 4 fold ) compared to mesenchyme alone ( Figure 6E; n = 2 fetuses ) .", "Consistent with the hypothesis that parasympathetic nerves preferentially regulate the acinar lineage , levels of ductal gene transcripts KRT5 , KRT7 , KRT14 , KIT and EGFR were similar to human SLG co-cultured with mesenchyme only ( Figure 6E ) . 10 . 7554/eLife . 26620 . 031Figure 6 . Parasympathetic regulation of SOX2 and the acinar lineage is conserved from mice to humans .", "( A ) Immunofluorescent analysis of TUBB3+ nerves as well as SOX2- , SOX10- , CHRM1- and EGFR-expressing cells in fetal human submandibular ( SMG ) or sublingual ( SLG ) at 16–24 w .", "E-cadherin ( ECAD ) marks epithelial cells .", "Image of the 16 w SLG is a 20 µm stack of 1 µm confocal sections .", "All other images are 1–2 µm confocal sections .", "( B ) Brightfield images of SLG explants cultured with E13 murine parasympathetic ganglion ( PSG ) or mesenchyme alone ( MES ) at day 0 ( upper panels ) and day 7 ( lower panels ) .", "( C–E )", "Explants cultured with murine E13 PSG or mesenchyme for 7 days were subjected to immunofluorescent analysis for SOX10 , ECAD and TUBB3+ nerves and SOX2 ( C , D ) or gene profiling by qPCR ( E ) .", "Data in E ( six biological replicates , two individual experiments ) are means+s . d . Scale bar is 50 µm ( A , C ( right panel ) ) , 500 µm ( B , C ( left panel ) , 20 µm ( D ) .", "( F–H )", "Analysis of fetal human SLG ( 22–23 w ) dissociated cells ( F and H ) or explants ( G ) cultured ± CCh for 48–72 hr . ( H ) The number of ECAD+SOX2+ ( red markers ) and ECAD+SOX2+Ki67+ ( black markers ) cells were measured by FACS as a percentage of cells of total ECAD+ cells .", "Each line represents an independent experiment .", "In F and G fold changes in gene expression in dissociated cells or explants were determined via qPCR with expression normalised to GAPDH and control values ( dashed line ) .", "Data in H were analyzed using a Wilcoxon signed-rank test .", "Data in F and G were analyzed using a one-way analysis of variance with post-hoc Dunnett’s test .", "*p<0 . 05 , **p<0 . 01 .", "Additional data for this figure in Figure 6—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 26620 . 03110 . 7554/eLife . 26620 . 032Figure 6—source data 1 . Source data relating to Figure 6E . Human fetal SLG explants cultured with murine E13 PSG or mesenchyme for 7 days were subjected to gene profiling by qPCR .", "Data were normalized to GAPDH and control ( + mesenchyme ) .", "Data are means of six biological replicates , two individual experiments .", "s . d . = standard deviation . DOI: http://dx . doi . org/10 . 7554/eLife . 26620 . 03210 . 7554/eLife . 26620 . 033Figure 6—source data 2 . Source data relating to Figure 6F . qPCR analysis of fetal human SLG ( 22–23 w ) dissociated cells cultured ± CCh for 48 hr .", "Data were normalized to GAPDH and control ( -CCh ) .", "Data are means of six biological replicates , two individual experiments .", "s . d . = standard deviation . DOI: http://dx . doi . org/10 . 7554/eLife . 26620 . 03310 . 7554/eLife . 26620 . 034Figure 6—source data 3 . Source data relating to Figure 6G . qPCR analysis of fetal human SLG ( 22–23 w ) explants cultured ± CCh for 72 hr .", "Data were normalized to GAPDH and control ( -CCh ) .", "Data are means of six biological replicates , two individual experiments .", "s . d . = standard deviation . DOI: http://dx . doi . org/10 . 7554/eLife . 26620 . 03410 . 7554/eLife . 26620 . 035Figure 6—source data 4 . Source data relating to Figure 6H . Analysis of fetal human SLG ( 22–23 w ) dissociated cells cultured ± CCh for 48 hr .", "The number of ECAD+SOX2+ and ECAD+SOX2+Ki67+ cells were measured by FACS as a percentage of total ECAD+ cells .", "Each # represents an independent experiment .", "s . d . = standard deviation . DOI: http://dx . doi . org/10 . 7554/eLife . 26620 . 03510 . 7554/eLife . 26620 . 036Figure 6—figure supplement 1 . SOX2 and CHRM1 are enriched in acinar cells of human fetal SG , consistent with CCh increasing proliferation of human SG SOX2+ cells .", "( A ) Immunofluorescent analysis of nerves ( TUBB3 ) , acinar ( SOX2 , SOX10 , CD44 , and AQP3 ) and ductal ( KRT7 , KRT5 , KIT , KRT14 ) cells in fetal human submandibular ( SMG ) , sublingual ( SLG ) and parotid ( PG ) at 22–24 w .", "Some SGs were immunostained for E-cadherin ( ECAD ) and the proliferation marker Ki67 .", "Images are 1–2 µm confocal sections .", "Scale bars are 50 µm .", "White dashed line outlines acini .", "( B ) Flow cytometry plots show gating strategy to analyze the percentage of SOX2+ and Ki67+ cells of total ECAD+ cells of fetal human SLG ( 22–23 weeks gestation ) cultured ± carbachol ( CCh; 100 nM ) for 48 hr ( data presented in Figure 6H ) .", "#1–3 represent three individual human fetuses .", "Red boxes indicate SOX2+Ki67+ cells .", "100 , 000 events were analyzed per sample . DOI: http://dx . doi . org/10 . 7554/eLife . 26620 . 036 To determine the impact of CHRM1 stimulation on human SOX2+ cells and the acinar lineage , we cultured human fetal SLG explants or dissociated salivary cells with CCh for 48–72 hr .", "As for the murine SLG , CCh treatment was sufficient to increase the expression of SOX2 and acinar cell markers in human explants as well as dissociated cells ( consisting of epithelium and mesenchyme; 20–22 weeks ( w ) of gestation ) ( Figure 6F–G ) .", "Furthermore , CCh treatment promoted SOX2-positive cell proliferation ( Figure 6H and Figure 6—figure supplement 1B ) .", "CCh also increased expression of KRT5 which is enriched in basal duct cells of human tissue and myoepithelial cells , suggesting these cell types are regulated by CHRM1 signaling ( Figure 6F–G ) .", "Thus , muscarinic stimulation is sufficient to rescue the acinar cell lineage and SOX2 expression and to promote the proliferation of SOX2-positive cells in the developing human SLG .", "Combined , these data support a conserved role for cholinergic innervation in the generation of the acinar lineage from mice to humans ." ], [ "Our study demonstrates a critical role for SOX2 in the generation of the acinar lineage during SG development .", "SOX2 acts as a master regulator of the acinar lineage by modulating the expression of lineage-specific genes and controlling both cell proliferation and survival .", "However , SOX2 function in acinar cell production is dependent on parasympathetic nerves that maintain its expression via acetylcholine-muscarinic-calcium signaling .", "The observation of similar outcomes in the human SG-nerve co-culture system provides strong evidence that the regulation of SOX2 and acini by innervation is an evolutionarily conserved mechanism that regulates the generation of acinar cells .", "SOX2 also regulates cell lineage commitment in other organs such as the pituitary and skin ( Andoniadou et al . , 2013; Driskell et al . , 2009; Goldsmith et al . , 2016; Lesko et al . , 2013 ) .", "In the pituitary , loss of SOX2 expression in progenitors leads to a switch in cell fate between the two closely related cell types , melanotrophs and corticotrophs , in the intermediate lobe of the pituitary ( Goldsmith et al . , 2016 ) .", "In the adult skin , SOX2 is expressed in dermal papilla cells that give rise to the three hair follicle subtypes and ablation of Sox2 alters the proportion of these subtypes ( Driskell et al . , 2009; Lesko et al . , 2013 ) .", "Despite SOX2+ progenitors contributing to acini and ducts in the SMG+SLG , we unexpectedly found that Sox2 is only required for the specification of the SOX10-positive acinar lineage .", "However , in contrast to the cell fate switch observed in the pituitary and skin , Sox2-deficient cells in the end buds do not divert to ductal cell fates .", "This suggests that acquisition of a duct cell fate requires active re-specification and that ductal identity is not the default cell fate in the absence of SOX2 or innervation .", "This contrasts with a previous proposal that progenitor cells , in the absence of induction , simply differentiate into duct cells ( Knox et al . , 2010 ) .", "This conclusion is corroborated by recent genetic lineage-tracing studies in the adult SG showing that acinar cells give rise to more acinar cells but not duct cells ( Aure et al . , 2015 ) .", "Thus , despite SOX2 being expressed by some duct cells ( Lombaert et al . , 2011 ) and SOX2-positive cells being able to contribute to ducts during development , SOX2 functions in cell specification are limited to the acinar lineage .", "How salivary acinar cells are established in the SG is not known .", "However , acinar cell expansion in the developing SG has previously been shown to be regulated by FGF10/FGFR2b signaling ( Lombaert et al . , 2013; Matsumoto et al . , 2016 ) .", "This indicates that FGF10/FGFR2b signaling is either adversely affected by loss of SOX2 or that SOX2 acts downstream of this pathway .", "As we did not observe a loss of FGF signaling in Sox2-deficient SMG+SLG and FGF10/FGFR2b signaling reduces SOX2 expression in isolated SMG epithelia ( Lombaert et al . , 2011 ) , we conclude that SOX2 acts downstream of FGF signaling .", "As such , FGF signaling most likely regulates the differentiation of SOX2-positive acinar progenitors and/or their proliferative expansion .", "SOX2 has been shown to promote the survival of cancer cells in vivo and has been linked to lens cell survival in Astyanax surface fish ( Boumahdi et al . , 2014; Chou et al . , 2013; Herreros-Villanueva et al . , 2013; Ma et al . , 2014 ) , but a role for SOX2 in cell survival during mammalian organogenesis has not been reported .", "In this study , we reveal that genetic ablation of Sox2 but not denervation or muscarinic inhibition , which reduces but not eliminate SOX2 , is sufficient to trigger cell death in the SMG+SLG .", "This is likely due to the levels of SOX2 remaining in epithelial cells from denervated or muscarinic receptor antagonized salivary glands being conducive to cell survival , although single-cell expression analysis is required to confirm such a mechanism .", "Levels of SOX2 may also influence lineage outcomes .", "It is possible that SOX2 may be activated in formerly SOX2-negative cells in response to cholinergic stimulation thereby specifying the acinar lineage .", "However , as both acinar and duct cells express SOX2 , there must be other regulators at play besides SOX2 that specify this lineage .", "Furthermore , despite both duct and acinar cells expressing SOX2 during early gland development , apoptosis was restricted to cells in the end buds , which points to an essential and selective role of SOX2 in promoting survival of the acinar but not ductal lineage .", "A direct role for SOX2 in cell survival is supported by chromatin precipitation studies in skin tumor cells where a number of survival genes were directly regulated by SOX2 ( Boumahdi et al . , 2014 ) .", "We , and others , have previously shown that peripheral nerves maintain progenitors in an undifferentiated state , providing a pool of unspecified cells for developmental or regenerative processes ( Knox et al . , 2010; Xiao et al . , 2015 ) .", "We have also shown that nerves , through release of vasoactive intestinal peptide , regulate duct formation ( Nedvetsky et al . , 2014 ) .", "Our current study demonstrates an unexpected role for peripheral innervation in furnishing the ductal system with acini to form a functional organ .", "Multiple signaling pathways such as those of the WNT and EGFR families have been reported to control epithelial differentiation and consequently tissue structure in a diverse range of organs .", "However , compared to neuronal signals that continuously operate during all stages of salivary gland ( and organism ) development , these cell/tissue intrinsic pathways tend to be transient in appearance , for example , FGF10 regulates acinar cell proliferation but is only expressed during early branching morphogenesis ( Steinberg et al . , 2005 ) .", "As such , peripheral nerves offer a way of shaping tissue architecture throughout the development of the organism , thereby providing continuity of signals during tissue morphogenesis .", "In summary , our findings support a critical role for SOX2 and nerves in establishing the secretory end units of the salivary gland , thereby selectively generating the tissue architecture necessary for organ function .", "Given the majority of mammalian organs receive innervation from parasympathetic ganglia , and loss of functional innervation alters epithelia and/or epithelial stem cells in multiple organs including those that express SOX2 such as taste buds , prostate and seminar vesicles , stomach and cornea ( Suzuki , 2008; Tatsuta et al . , 1985; Ueno et al . , 2012; Wanigasekara et al . , 2004; Zhao et al . , 2014 ) , our results may have significant implications for gaining insight into the molecular mechanisms underlying lineage specification and architectural outcomes in diverse systems ." ], [ "All procedures were approved by the UCSF Institutional Animal Care and Use Committee ( IACUC ) .", "Mouse alleles used in this study were provided by The Jackson Laboratory and include: Phox2bLacZ/LacZ ( RRID:MGI:2172761 ) ( Pattyn et al . , 1999 ) , Krt14CreERT2 ( RRID:MGI:5435569 ) ( Vasioukhin et al . , 1999 ) , Sox2CreERT2 ( RRID:MIG:5512893 ) ( Andoniadou et al . , 2013 ) , Sox2fl/fl ( RRID:MIG:4366456 ) ( Smith et al . , 2009 ) and Rosa26mTmG ( RRID:MIG:3623345 ) ( Muzumdar et al . , 2007 ) .", "Sample size was calculated using the Resource Calculator ( Mead , 1988 ) .", "Epithelia and mesenchyme were separated using dispase treatment and mechanical dissection and cultured in a drop of laminin on a nucleopore filter over serum-free DMEM/F12 containing holotransferrin and ascorbic acid ( complete media ) as described for the E13 submandibular gland ( Steinberg et al . , 2005 ) .", "Epithelia were cultured with 500 ng/ml FGF10 ( R and D Systems ) and 0 . 5 µl/ml heparin sulfate ( Sigma Aldrich ) in the presence or absence of 100 nM carbachol ( Sigma Aldrich ) or vehicle ( H2O ) , treated for 1 hr with EdU reagent ( Click-iT EdU Alexa-Fluor 488 kit ) and were fixed for immunostaining after 24–48 hr .", "SGs dissected from CD1 timed pregnant females were dissected into epithelia , mesenchyme and PS and epithelia recombined with mesenchyme with or without the PS , as previously described ( Knox et al . , 2010 ) .", "Explants were cultured for 48 hr before being lysed for RNA isolation or fixed for immunofluorescent analysis ( 4% PFA or 1:1 ice cold acetone: methanol ( AcMeOH ) ) .", "Human fetal salivary glands were harvested from post-mortem fetuses between 15 and 24 weeks gestation with patient consent and permission from the ethical committee of the University of California San Francisco .", "Tissue was identified by location and glandular appearance and following dissection was immediately placed in 4% PFA ( Electron Microscopy Sciences ) , RNAlater ( Qiagen ) or DMEM ( ThermoFisher ) for live tissue explant/cell culture .", "We utilized two separate assays for defining the impact of CCh on SOX2 and the acinar cell lineage: human explants and dissociated cell cultures .", "The explant cultures maintain tissue structure and are similar to the murine studies .", "Dissociated cells enabled us to define changes in gene expression of SOX2 and the epithelial lineages , as well as to perform FACS analysis for quantifying SOX2+ and SOX2+Ki67+ cells .", "The impact of CCh on SOX2 and acinar and ductal lineages was analyzed in both assay types .", "For SG explant culture , tissue was dissected into <1 mm pieces and cultured in serum-free media ( as for the embryonic mouse cultures ) .", "Tissue was incubated with 100 nM CCh for 72 hr before being lysed for RNA .", "For culture of dissociated cells , tissue was first processed into single cells ( see Flow Cytometry protocol below ) before being cultured in DMEM/F12 ( ThermoFisher ) containing holotransferrin , ascorbic acid and 0 . 5 µg/mL heparan sulfate ( Sigma Aldrich ) at a density of 2 × 106 cells/mL in 12 well plates .", "Cells were treated with FGF10 ( R and D systems ) and 100 nM Carbachol ( Sigma Aldrich ) or vehicle ( H2O ) .", "Cells were cultured for 72 hr and centrifuged and lysed for RNA .", "For SG explant-PSG , co-cultures tissue was dissected into <1 mm pieces and cultured on a floating filter above serum-free media .", "E13 mouse PSGs were isolated as described above ( PS recombination assay ) .", "One PSG per explant was placed next to the human SG and cultured for 7 days and either fixed for immunofluorescent analysis or lysed for RNA .", "After fixation , postnatal and adult SGs ( human and mouse ) were either processed for OCT or paraffin embedding .", "For generation of frozen sections , tissue was incubated in increasing concentrations of sucrose ( 25–75% ) and embedded in OCT . 12 µm sections were cut using a cryostat ( Leica ) and stored at −20°C .", "Tissue for paraffin was dehydrated by incubating in increasing concentrations of ethanol and subsequently Histo-Clear ( National Diagnostics ) before embedding in paraffin wax ( Sigma Aldrich ) .", "7 µm sections were cut using a microtome ( Leica ) and stored at room temperature .", "Whole-mount SG and tissue section immunofluorescence analysis has been previously described ( Knox et al . , 2010 ) .", "In brief , tissue was fixed with either ice cold acetone/methanol ( 1:1 ) for 1 min or 4% PFA for 20–30 min followed by permeabilizing with 0 . 1–0 . 3% Triton-X .", "Tissue was blocked overnight at 4°C with 10% Donkey Serum ( Jackson Laboratories , ME ) , 1% BSA ( Sigma Aldrich ) , and MOM IgG-blocking reagent ( Vector Laboratories , CA ) in 0 . 01% PBS-Tween-20 .", "SGs were incubated with primary antibodies overnight at 4°C: goat anti-SOX2 ( 1:200 , Neuromics , GT15098; AB_2195800 ) ; goat anti-SOX10 ( 1:500 , Santa Cruz Biotechnology , sc-17342; AB_2195374 ) ; mouse anti-TUBB3 ( clone TUJ1 at 1:400 , R and D Systems , MAB1195; AB_357520 ) ; rat anti-E-cadherin ( 1:300 , Life Technologies , 13–1900; AB_2533005 ) ; rabbit anti-EGFR ( 1:200 , Abcam , ab52894; AB_869579 ) ; goat anti-KIT ( 1:200 , Santa Cruz , sc-1494; AB_631032 ) ; rabbit anti-KRT5 ( 1:1000 , Covance , PRB-160P; AB_291581 ) ; rat anti-KRT19 ( 1:300 , troma III , DSHB; AB_2133570 ) ; rabbit anti-KRT14 ( 1:1000 , Covance , PRB-155P; AB_292096 ) ; mouse anti-KRT7 ( 1:50 , Covance , MMS-148S; AB_10719738 ) ; rat anti-CD44 ( Biolegend , 1:200 , 103001; AB_312952 ) ; rabbit anti-CHRM1 ( 1:300 , Santa Cruz , sc-7470; AB_2079955 ) ; mouse anti-Ki67 ( 1:50 , BD Biosciences , 550609; AB_393778 ) ; rabbit anti-Caspase3 ( 1:300 , Cell Signaling , 9664; AB_2070042 ) ; rabbit anti-AQP3 ( 1:400 , Lifespan Biosciences Inc . , LS-B8185; AB_2661881 ) ; rabbit anti-AQP5 ( 1:100 , Millipore , AB3559; AB_2141915 ) ; chicken anti-GFP ( 1:500 , Aves Labs , GFP-1020; AB_10000240 ) ; peanut agglutinin ( PNA; 1:200 , Vector Laboratories , AS-2074; AB2336189 ) ; and rabbit anti-MIST1 ( 1:500 , gift from Stephen Konieczny , Purdue University ) .", "Antibodies were detected using Cy2- , Cy3- or Cy5-conjugated secondary Fab fragment antibodies ( Jackson Laboratories ) and nuclei stained using Hoescht 33342 ( 1:1000 , Sigma Aldrich ) .", "EdU staining was performed using the Click-iT EdU Alexa-Fluor 488 kit .", "Fluorescence was analyzed using a Leica Sp5 confocal microscope and NIH ImageJ software .", "Branching morphogenesis was measured by counting the number of acini ( NIH ImageJ; Images were assessed by blinded researchers ) .", "All data were obtained using 4–5 SGs/group and each experiment repeated three times .", "For immunofluorescent analysis ( e . g . , Figure 4D ) , cells positively stained for markers were counted using ImageJ .", "Acinar cell size was measured using ImageJ .", "All data were obtained using 3–5 fields of view/group and each experiment repeated three times .", "RNA was isolated from whole tissue using RNAqueous Micro Kit ( Ambion ) .", "Total RNA samples were DNase-treated ( Ambion ) , prior to cDNA synthesis using SuperScript reagents ( Invitrogen , CA ) .", "SYBRgreen qPCR was performed using 1 ng of cDNA and primers designed using Primer3 and Beacon Designer software or found using PrimerBank ( http://pga . mgh . harvard . edu/primerbank/ ) .", "Primer sequences are listed in Tables 1 and 2 .", "Melt-curves and primer efficiency were determined as previously described ( Hoffman et al . , 2002 ) .", "Gene expression was normalized to the housekeeping gene S29 ( Rps29 ) for mouse and GAPDH for human and to the corresponding experimental control .", "Reactions were run in triplicate and experiments performed two to three times . 10 . 7554/eLife . 26620 . 037Table 1 . Sequences for mouse primers used for qPCR . DOI: http://dx . doi . org/10 . 7554/eLife . 26620 . 037Gene targForward primer sequenceReverse primer sequenceAqp3CTGCCCGTGACTTTGGACCTCCGAAGACACCAGCGATGGAACCAqp5TCTACTTCTACTTGCTTTTCCCCTCCTCCGATGGTCTTCTTCCGCTCCTCTCAscl3GACAGGCTCTCGGTCTTCGCATCTGTGTAAGAGGCCGGTAAtoh1GAGTGGGCTGAGGTAAAAGAGTGGTCGGTGCTATCCAGGAGBaxTGAAGACAGGGGCCTTTTTGAATTCGCCGGAGACACTCGBbc3AGCAGCACTTAGAGTCGCCCCTGGGTAAGGGGAGGAGTCalm1TGGGAATGGTTACATCAGTGCCGCCATCAATATCTGCTTCTCTCalrGAAGCTGTTTCCGAGTGGTTTGCACAATCAGTGTGTATAGGTGTCnn1AAACAAGAGCGGAGATTTGAGCTGTCGCAGTGTTCCATGCCCcnd1CATCCATGCGGAAAATCGTGGAAGACCTCCTCTTCGCACTTCCdh1GACTGGAGTGCCACCACCAAAGACCGCCTGTGTACCCTCACCATCGGCdkn1aCCCCCAATCGCAAGGATTCTTCTTGGTTCGGTGGGTCTGTCChrm1TCCCAAGGCTCACCCAGATGTCGCTCTGTGTGCTTTATTCTGTTGTTTCCChrm3CATAGCACCATCCTCAACTCTACCAAGGGGCATTTCTCTCTACATCCATAGTCCDcpp1TGGTGGGGTATTATGTGGGCAGGGATCGTTAGGGAAGCTAGADcpp2ATGGGCCAATGTAGATGCTCCCCAAGAGGCAACAGTAGGAdNp63TTGTACCTGGAAAACAATGGCATCGTTTCACAACCTCGEtv4GGTCCTGTGTTCTTGGTGCTGTGGGTCCTGTGTTCTTGGTGCTGTGEtv5AAGCCCTTCAAAGTGATAGCGGAGACGTGTCCACAAACTTTCCTCTTTCTGTCAACTEgfrACACTACGCCGCCTGCTTCAAGAGACTGTGCCAAATGCTCCCGAACCCFgf1GCACCGTGGATGGGACAAGGGACAGGAGCACTTCGCCCGCACTTTCCGCACTGAGFgf7CTCTACAGGTCATGCTTCCACCACAGAACAGTCTTCTCACCCTFgf10TCTTCCTCCTCCTCGTCCTTCTCCTCTCCTTCCCCGCTGACCTTGCCGTTCTTCTCAATCGFgfr2bTGGCTCTGTTCAATGTGACGGAGATGGATGAGGCGCTTGCTGTTTGGGCAGGACKitTGGTTGTGGTTGTTGTTGTTGTTGGAAGGCTTGTTCCGAAGTGTAGACKrt5TCCTGTTGAACGCCGCTGACCGGAAGGACACACTGGACTGGKrt7CGCCGCTGAGTGTGGACATCGCTGGCTGCTCTTGGCTGACTTCTGKrt8GGAGGAGAGCAGGCTGGAGTCTGGTGCGGCTGAAAGTGTTGGKrt14CCTCATCCTCTCAATTCTCCTCTGGCTCTCCTTGGTGCGGATCTGGCGGTTGGKrt15GCTGCTACATGCTGCTCAGGCTTAGGCCAGGAAGGACAAGGGTCAAGTAAAGAGAGTGKrt19GCCACCTACCTTGCTCGGATTGGTCTCTGCCAGCGTGCCTTCMist1GCTGACCGCCACCATACTTACTGTGTAGAGTAGCGTTGCAGGMuc19CTGGGTCTGGAAGTAGAAGTATCTAAGCCACAGAAGGAGATNrtnCGCTACCACACGCTGCAAGAGTCCCACACTTATGTGAAGTCAGTTCTCPipGGGTCTCTCATTCACATTCAGTGTGATCTCCTGATTTTCCTGTGCTPmaip1GCAGAGCTACCACCTGAGTTCCTTTTGCGACTTCCCAGGCAPtch1CACCCAGAAAGCAGACTACCCGAATATCTCTCCTCCAGCATGACATACTTCACATTGProl1CACCTAAGCCTAGCACCTCTAACTTCCAAAACACTTCCGCAAATRab3dTACTATCGCGGAGCTATGGGTTTTGATCTGCGTAGCCCAGTCRps29GGAGTCACCCACGGAAGTTCGGGGAAGCACTGGCGGCACATGSmgcTGGCTCTGCAACACAACAGTGGCGAAAAGCTCCCAGGTAASox2CAGCATGTCCTACTCGCAGCAGTGGAGTGGGAGGAAGAGGTAACCSox10ATCAGCCACGAGGTAATGTCCAACACTGCCCAGCCCGTAGCCSpdefAAGGCAGCATCAGGAGCAATGCTGTCAATGACGGGACACTGSyn2TAGACTGCTGTGGAGGTGAAGCTCTGAAAGGTAAAGGTAACTGTrp53CTCTCCCCCGCAAAAGAAAAACGGAACATCTCGAAGCGTTTATubb3CCAGAGCCATCTAGCTACTGACACTGAGAGCCAAGTGGACTCACATGGAGVachtGAGTGGGAGATGGGCATGGTTTGGGCAGGCAGGTACGACGCAAGAGVipTCCAGTGATAGGTACTCCATCTCCATCCATAGCACACGCAGAAZeb1GCTGGCAAGACAACGTGAAAGGCCTCAGGATAAATGACGGCZeb2ATTGCACATCAGACTTTGAGGAAATAATGGCCGTGTCGCTTCG10 . 7554/eLife . 26620 . 038Table 2 . Sequences for human primers used for qPCR . DOI: http://dx . doi . org/10 . 7554/eLife . 26620 . 038GeneForward primer sequenceReverse primer sequenceAQP3GTTTCTGTGTATGTGTATGTCTGCCTTTCGTCCCACTGCTCCTACTTATGTCDH1AGGTGACAGAGCCTCTGGATAGATGGATGACACAGCGTGAG AGACD44CTGCCGCTTTGCAGGTGTACATTGTGGGCAAGGTGCTATTCHRM1ACCTCTATACCACGTACCTGTGAGCAGCAGATTCATGACGCHRM3ATCGGTCTGGCTTGGGTCCCCGGAGGCACAGTTCTCEGFRTGGCAGGTACAGTAGGATAACAAGTCAGTCTAACGCTCATGAPDHCAGCCTCAAGATCATCAGCATGTGGTCATGAGTCCTTCCAKITGCAGAGGAAGTGGAAGGCATCAGTCAGTGAGACAGTAGCATTATGGAAGGTKRT5CGTGCCGCAGTTCTATATTCTACTTTGGGTTCTCGTGTCAGKRT7TCCGCGAGGTCACCATTAACGCTCTGTCAACTCCGTCTCATKRT8AAGGATGCCAACGCCAAGTTCCGCTGGTGGTCTTCGTATGKRT14ATCCAGAGATGTGACCTCCTCCTCAGTTCTTGGTGCGAAGGKRT19GTCTGCCTCCAAGGTCCTCTGATCTACCCAGAAGACACCCTCCAAAMIST1CGGATGCACAAGCTAAATAACGGCCGTCAGCGATTTGATGTAGSOX2TGGCGAACCATCTCTGTGGTGGAAAGTTGGGATCGAACAAAAGCSOX10TCATCCCTTCAATGCCCCCTTGCGTCTCAAGGTCATGGAGG RNA was isolated from whole tissue using RNAqueous Micro Kit ( Ambion ) and total RNA samples were DNase-treated ( Ambion ) .", "Samples were processed for sequencing using the TruSeq Stranded mRNA sample kit ( Illumina ) using 0 . 5 ug of total RNA as starting material .", "First strand synthesis was performed using SuperScript II Reverse Transcriptase ( Thermo Fisher ) .", "Sample yield and integrity was analyzed using a Qubit ( Thermo Fisher ) and samples run on a 2% agarose gel .", "5 nM RNA was submitted for sequencing on the Illumina platform .", "Reads of between 32 , 000 and 47 , 000 were obtained ( n = 2 individual embryos for each genotype ) .", "Embryonic mouse SGs ( CD1; E15 . 5-E16 . 5 ) or human fetal SG tissue ( 22–23 w ) was dissected and washed in PBS containing gentamycin .", "Cell isolation and flow cytometry was performed as previously described ( Muench et al . , 2002 ) .", "Briefly , a single-cell suspension was created by mincing tissue with a scalpel blade and incubating in a PBS solution containing Liberase TM ( Roche ) and DNaseI ( Roche ) at 37°C for 30–60 min .", "The enzyme reaction was quenched by the addition of FCS or BSA and the solution filtered through a 40 µm strainer ( BD Falcon ) and centrifuged at 1500 rpm for 5 min .", "The resulting cell pellet was washed with sterile PBS , centrifuged and resuspended in blocking buffer ( 5% serum and 0 . 01% NaN3 , Biolegend ) .", "Cell surface staining was achieved by incubating cell suspensions with antibodies against CD324 ( ECAD; eBioscience , 46-3249-80; AB_1834418 ) , CD326 ( EpCAM; Miltenyi , 130-098-113; AB_2660298 ) and CHRM1 ( Santa Cruz , sc-7470; AB_2079955 ) .", "Subsequently , intracellular staining was achieved following fixation and permeabilization using an intracellular staining kit ( eBioscience ) and antibodies against SOX2 ( mouse: BD Pharmingen , 562195; AB_10895118; human: R and D Systems , IC2018P; AB_357273 ) and Ki67 ( mouse: Biolegend , 652405; AB_2561929; human: Biolegend , 350513; AB_10959326 ) .", "Flow cytometry was performed on a LSRII ( BD ) using the appropriate single stained controls and data collected using FACSDiva ( BD ) and analyzed using FlowJo .", "100 , 000 events were collected for each sample .", "Embryonic SGs were collected at E17 from two female CD1-ICR mice and pooled in DMEM on ice .", "Tissue was dissociated into a single-cell suspension as for flow cytometric analysis .", "ChIP was performed as previously described ( Lizama et al . , 2015 ) .", "Briefly , cells were crosslinked with 1% formaldehyde , quenched with 0 . 125 M glycine and resuspended in lysis buffer .", "The chromatin was sonicated using a Biorupter sonicator ( Diaganode ) .", "Rabbit anti-SOX2 antibody ( Cell Signaling , #2748 ) or rabbit IgG control ( Cell Signaling , #2729 ) was incubated with Pierce Protein A/G magnetic beads ( Thermo Scientific ) overnight at 4°C .", "The precipitates were washed and chromatin complexes eluted .", "The cross-linking was reversed ( 65°C for 4 hr ) , and the DNA was purified ( QIAquick PCR Purification kit , Qiagen ) .", "100 pg of DNA was used per PCR reaction .", "Primers used in PCR for quantitative ChIP are listed in Table 3 . 10 . 7554/eLife . 26620 . 039Table 3 . Sequences for primers used for SOX10 ChIP-qPCR . DOI: http://dx . doi . org/10 . 7554/eLife . 26620 . 039PrimerForward primer sequenceReverse primer sequenceAGTGGAGGTTTGTTGATGGATTTGCGATGGGAGAGTCTGABACAGTCAGAACCTGTTGCCTTGATACCTACTGCAGGCTGCCGCAGCCTGCAGTAGGTATCACTTCTTGAAGAGTAGGGCDAAAAGACAGGAACTGCCCTGAAGGGTGCCTTCACTGAGAAEGATAGTGGGGACACAAAGAGTCCTAATTCACTGGGCTCTGFTCTTGTTCGGGGCCTTGAAAATGCTTGCTGCTCCGTCCCTGAGACATCAATGAGCAGCAGGCGCACACACACACTTTCCTA Data were analyzed for statistical significance using Student’s t-test ( two groups ) , one-way ANOVA ( multiple groups ) with post-hoc testing performed using Dunnett or Tukey tests or Wilcoxon signed-rank test ( GraphPad Prism or SPSS ) .", "For multiple testing , we used a false discovery rate of 0 . 05 .", "All graphs show the mean +standard deviation ( s . d ) or mean +standard error of the mean ( s . e . m ) ." ] ]

[ "Acinar cells play an essential role in the secretory function of exocrine organs .", "Despite this requirement , how acinar cells are generated during organogenesis is unclear .", "Using the acini-ductal network of the developing human and murine salivary gland , we demonstrate an unexpected role for SOX2 and parasympathetic nerves in generating the acinar lineage that has broad implications for epithelial morphogenesis .", "Despite SOX2 being expressed by progenitors that give rise to both acinar and duct cells , genetic ablation of SOX2 results in a failure to establish acini but not ducts .", "Furthermore , we show that SOX2 targets acinar-specific genes and is essential for the survival of acinar but not ductal cells .", "Finally , we illustrate an unexpected and novel role for peripheral nerves in the creation of acini throughout development via regulation of SOX2 .", "Thus , SOX2 is a master regulator of the acinar cell lineage essential to the establishment of a functional organ ." ]

[ "The salivary glands produce fluid that contains enzymes to help us to digest our food .", "These glands contain a tree-like network of cells – known as acinar cells – that produce the fluid , and cells that form ducts to transport the fluid out of the glands .", "Both types of cells form from stem cells as animal embryos develop .", "Like all developing organs , the salivary glands receive many different signals that guide how they grow .", "However , the identity of the cues that instruct a stem cell to produce a new acinar cell or duct cell are not known .", "Emmerson et al . studied how the salivary glands develop in mouse embryos .", "The experiments show that a protein called SOX2 – which is an essential regulator of stem cells in embryos – is required for acinar cells to form .", "Loss of SOX2 inhibited the production of acinar but not duct cells .", "Furthermore , nerves that surround the gland provide support to cells that produce SOX2 and promote the formation of acinar cells .", "Further experiments suggest that the nerves also play the same role in humans .", "Adult organs often use developmental signals to repair or regenerate tissue .", "As such , understanding how an organ develops may lead to new therapies that can stimulate salivary glands and other organs to regenerate after they have been damaged in adults ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "cell biology", "structural biology and molecular biophysics" ]

[ [ "Epithelial barriers are essential for the survival of multicellular organisms and allow compartmentalization and controlled interactions between distinct environments ( Marchiando et al . , 2010 , Turner , 2009 ) .", "While transcellular transport is mediated by proteins that span the plasma membrane , molecular details of ions , water , and solute transport across the tight junction , i . e . the paracellular path , are less well-defined .", "This , in part , reflects the absence of tools able to detect single channel events at the tight junction and , therefore , a reliance on methods that measure paracellular flux over large multicellular surfaces ( Shen et al . , 2011 ) .", "The limited spatial and temporal resolution of these techniques has contributed to the widely held view of tight junction channels as constitutively open and has limited biophysical characterization of these paracellular channels .", "Nevertheless , it is clear that paracellular transport is critical to the function of transporting epithelia in many organs ( Bagnat et al . , 2007 , Simon et al . , 1999 , Wada et al . , 2013 ) and can be regulated by physiological and pathophysiological stimuli ( Heller et al . , 2005 , Marchiando et al . , 2010 , Suzuki et al . , 2011 , Turner et al . , 1997 , Weber et al . , 2010 ) .", "Intestinal epithelial expression of the tight junction protein claudin-2 , which increases paracellular Na+ conductance ( Amasheh et al . , 2002 , Wada et al . , 2013 , Weber et al . , 2010 ) , is downregulated after the neonatal period ( Holmes et al . , 2006 ) but markedly upregulated in inflammatory and infectious enterocolitis and by several cytokines , including IL-13 ( Heller et al . , 2005 ) .", "This is essential for IL-13-induced increases in paracellular Na+ permeability , as conductance changes are prevented by siRNA-mediated inhibition of claudin-2 upregulation ( Weber et al . , 2010 ) .", "Thus , claudin-2 expression is regulated during development and disease .", "Detailed functional analysis of claudin-2-based channels , in their own right and as models for all paracellular claudin channels , is therefore critical to understanding fundamental mechanisms of development and disease .", "Claudin-2 expression induces large increases in paracellular flux of small cations ( Amasheh et al . , 2002 , Furuse et al . , 2001 , Weber et al . , 2010 ) .", "Inducible claudin-2 expression in MDCKI monolayers , which lack endogenous claudin-2 expression , is therefore an ideal experimental model in which to define paracellular , trans-tight junction channels ( Angelow and Yu , 2009 ) .", "We analyzed claudin-2 channel function using a novel , trans-tight junction patch clamp technique .", "Here , we show that this approach can detect discreet conductance events , define these in biophysical terms , perform extensive characterization that demonstrates that the currents detected reflect the activity of trans-tight junction channels , and excludes the possibility that these events are due to apical membrane conductances or other artifacts .", "The data show that these tight junction channels are gated and behave in a manner reminiscent of traditional transmembrane ion channels despite radical differences in orientation and function .", "Nevertheless , these similarities , the efficacy of pharmacological effectors of transmembrane ion channel function , and the frequency of epithelial barrier defects in disease suggest that it may be possible to develop agents that positively- or negatively-regulate tight junction channel gating for therapeutic benefit ." ], [ "Measurements of paracellular permeability , such as those above , typically assess relatively large epithelial surfaces and , therefore , reflect global averages rather than local , site-specific conductances .", "Scanning and impedance approaches have been used in an effort to overcome these limitations ( Chen et al . , 2013 , Gitter et al . , 1997 , Krug et al . , 2009 ) , but these lack the spatial and temporal resolution needed for identification of single channel events .", "Overall , the greatest obstacle to single channel analyses of tight junction channels has been the orientation of trans-tight junction channels between lateral surfaces of two adjacent cells , i . e . parallel to plasma membranes ( Figure 2A ) .", "This orientation is orthogonal to traditional ion channels and gap junctions , which cross plasma membranes , and renders most patch clamp techniques unsuitable for measuring trans-tight junction ion flux . 10 . 7554/eLife . 09906 . 004Figure 2 . Claudin-2 expression correlates with the frequency of local tight junction channel openings in MDCKI monolayers .", "( A ) Tight junctions are distinct from plasma membrane ion channels and differ from gap junctions in their ability to define conductance between two extracellular compartments .", "( B ) Trans-tight junction patch clamp placement .", "Yellow arrowheads show intercellular junction ( Bar = 10 μm ) .", "( C ) Conductance events detected at −100 mV when claudin-2 was expressed ( +Cldn-2 ) .", "( D ) In the absence of induced claudin-2 expression ( –Cldn-2 ) , the frequency of similar sized conductance events was dramatically reduced .", "( E ) Small claudin-2 independent events were present in parental MDCKI monolayers ( F ) Conductance events were present at +100 mV when claudin-2 was expressed ( +Cldn-2 ) .", "( G ) Events were infrequent in the absence of induced claudin-2 expression ( –Cldn-2 ) .", "( H ) NPo was reduced by 87% ± 4% ( at –100 mV ) and 88% ± 6% ( at +100 mV ) after suppression of claudin-2 expression .", "Events were rare in recordings from parental tight junctions .", "( I ) Representative recording of voltage ramp in claudin-2-expresing MDCKI monolayers showing linear current voltage relationship and reversal potential close to 0 mV .", "( J ) Average current voltage relationships ( n = 8 to 32 per condition ) reveals that average channel conductance was ~90 pS regardless of whether claudin-2 expression was induced ( green line ) or not ( black line ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 09906 . 004 To overcome these challenges , we developed an approach to seal an apical patch pipette across a region of the bicellular tight junction .", "This required several technical advances , including development of low profile chambers that allowed apical tight junction access using a 50°–60° approach angle while simultaneously viewing cell profiles from below the monolayer .", "Successful patching of tight junctions also required optimization of cell growth to afford clear morphological delineation of tight junctions while minimizing accumulation of cellular debris that could interfere with pipette sealing .", "Electrode configuration was also modified so that the pipette was just large enough to span the tight junction , while not so small that it would slip off of the tight junction .", "This allowed us to achieve a gigaseal with an ~5% success rate .", "Once a high resistance gigaseal was achieved , it was then possible to measure current through the paracellular pathway in response to an externally applied voltage relative to a basal reference electrode ( Figure 2B ) .", "The approach above allowed detection of bursts of sub-millisecond duration , flicker-like openings and closings when using holding potentials of −100 or +100 mV ( Vapical− Vbasal ) in monolayers with claudin-2 expression ( Figure 2C , F , H ) .", "Such events were infrequent in monolayers without induction of claudin-2 expression , i . e . with low level claudin-2 expression ( Figure 2D , G , H ) , and were rare in parental MDCKI monolayers that completely lacked claudin-2 expression ( Figure 2E , H ) .", "All-points histograms , fitted to Gaussian distributions , show the claudin-2 dependent conductance centered at ~9 pA , and ranged from ~5 to >10 pA at –100 mV .", "A separate class of smaller conductance values centered at ~4 . 3 pA was present in all lines , regardless of claudin-2 expression .", "To focus on claudin-2-dependent channels , thresholding was used to exclude the small , claudin-2-independent conductances .", "These analyses showed that opening probability ( NPo ) of claudin-2-dependent channels was similar at +100 or –100 mV in claudin-2 expressing monolayers , but was reduced by 87 ± 4% ( at -100 mV ) and 88% ± 6% ( at +100 mV ) in the absence of claudin-2 induction ( Figure 2H ) .", "In contrast , amplitude was similar at high and low levels of claudin-2 expression .", "Thus , the NPo , but not the amplitude , of these openings with conductances of ~92 pS is a function of claudin-2 expression .", "This further suggests that the number of channels , but not the open probability of individual channels , is a function of claudin-2 expression .", "To further characterize the voltage dependence of claudin-2-dependent conductances we performed voltage ramps beginning at a holding potential of –100 mV .", "Current-voltage ( I-V ) relationship plots ( Figure 2I , J ) showed that the reversal potential ( Vrev ) of these events was close to 0 mV , but slightly negative , and followed a linear function of voltage , consistent with a passive process .", "This result argues strongly that the conductance events cannot be apical cation or anion , e . g . K+ , Na+ , or Cl- channels , since the extracellular:intracellular gradients of these ions would necessitate equilibrium potentials much different than 0 mV .", "Notably , this analysis also shows that , despite there being far fewer events when claudin-2 expression was suppressed , individual conductance events were quantitatively similar , in both amplitude and duration , when claudin-2 expression was low ( Figure 2J ) .", "Therefore , the ~9 pA single channel conductances measured by trans-tight junction patch clamp are non-vectorial , as expected for passive paracellular channels , and is unlikely be due to the activity of apical transmembrane ion channels .", "The observation that claudin-2-dependent conductance events occur in bursts was somewhat surprising , as tight junction conductance has been assumed to be uniform over time .", "This widely-held belief was based on stable measurements of paracellular conductance across large epithelial surfaces .", "However , conductance of individual channels is averaged over space in these measurements , which lack both temporal and spatial resolution of the trans-tight junction patch clamp approach .", "To better characterize the opening and closing behaviors of claudin-2-dependent channels , histograms of all events were generated .", "Opening duration histograms of tight junction patch clamp data from cells with high or low claudin-2 expression at holding potentials of +100 or -100 mV .", "These revealed a single population of openings with τopen< 1 ms ( Figure 3A–D ) .", "In contrast , closed duration histograms under the same conditions revealed two populations of closings , corresponding to closed states between and within event clusters .", "Interburst closings were prolonged with τ closed ( stable ) >1 s while intraburst closings occurred with millisecond kinetics , i . e . τ closed ( transient ) < 2 ms ( Figure 3E–H ) .", "One possibility is that the prolonged state ( closedstable ) could represent channel disassembly , while the shorter closed state ( closedtransient ) results from regulation of assembled channels .", "However , given the absence of a significant vesicular claudin-2 pool and the relatively long ~9 hr half-life of claudin-2 protein in MDCK monolayers ( Van Itallie et al . , 2004 ) , we consider it highly unlikely that vesicular traffic or protein turnover could be responsible for the observed opening and closing events .", "In contrast , although the pool of claudin-2 at the tight junction is largely immobile ( Raleigh et al . , 2011 ) , the limited intramembranous diffusion that does occur ( Shen et al . , 2008 ) has kinetics within the range of the longer closed state ( closedstable ) and we cannot exclude this as a possible regulatory mechanism . 10 . 7554/eLife . 09906 . 005Figure 3 . Patch clamp recordings reveal a single open state and two closed states .", "( A–D )", "A single population of fast openings was observed in the presence ( green ) or absence ( white ) of induced claudin-2 expression at –100 and +100 mV ( a-d total recording times: 40 s , 225 s , 31 s , 52 s . ) .", "( E–H )", "Corresponding closed duration histograms from the same representative recordings reveal two distinct closed states .", "( I ) Opening and closing time constants were voltage independent and were similar with and without claudin-2 induction ( n=7 to 35 recordings for each condition ) .", "( J ) Kinetic analysis demonstrates the presence of both stable ( cstable ) and transient ( ctransient ) closed states and one and open ( o ) state . DOI: http://dx . doi . org/10 . 7554/eLife . 09906 . 005 Similar to amplitude , open and closed state kinetics were similar under high or low claudin-2 expression .", "Claudin-2 channel gating is thus independent of expression level , i . e . is non-cooperative .", "This also indicates that changes in NPo that occur as a function of claudin-2 expression reflect differences in channel number rather than open probability of individual channels .", "Further , because properties were similar at +100 and -100 mV , we can conclude that claudin-2-dependent channels are not gated by voltage , unlike many transmembrane ion channels .", "Overall these data show that claudin-2-dependent channels can exist in a highly dynamic opening state ( o ) as well as stable ( cstable ) and transient ( ctransient ) closed states ( Figure 3J ) .", "Despite the voltage ramp results ( Figure 2I , J ) , we re-considered the possibility that detected events represented transmembrane conductances of apical ion channels within apical membrane captured by the patch pipette .", "The ~9 pA claudin-2-dependent openings were , however , never observed when electrodes were sealed off of the tight junction , i . e . over apical membranes away from the tight junction .", "In place of the ~9 pA openings , small conductances could sometimes be detected when electrodes were sealed over apical membranes , but only when the data were low-pass filtered at 500 Hz ( Figure 4A ) .", "These events differed distinctly from the claudin-2-dependent conducances , as the former were more common at holding potentials of +100 mV , relative to -100 mV , and had amplitudes of less than 2 pA , well below those of claudin-2-dependent events .", "These data provide spatial evidence that the conductances detected by trans-tight junction patch clamp are not traditional , transmembrane apical ion channels . 10 . 7554/eLife . 09906 . 006Figure 4 . Large and small tight junction currents are not due to transmembrane ion channels .", "( A ) Small ( <2 pA ) transmembrane ion channel openings were detectable in off-tight junction recordings after applying a 500 Hz low pass filter .", "( Bar = 10 μm ) .", "( B ) Events detected by trans-tight junction patch clamp were not blocked by three different ion channel inhibitor cocktails ( +Cldn-2; representative of n = 3 to 8 per condition ) .", "( C ) Monolayers were cooled while recording from trans-tight junction patch clamp .", "The number of events detected was reduced at 15°C , relative to 37°C , but event amplitude was unaffected ( +Cldn-2; representative of n = 4 ) .", "( D ) NPo and Na+ permeability measured across a 0 . 33 cm2 monolayer were similarly reduced at 15°C ( +Cldn-2 ) .", "( E ) ~4 pA events remained detectable after chilling monolayers to 10°C ( MDCKI parental monolayers; representative of n = 4 ) .", "( F ) ~4 pA events were not blocked by three different ion channel inhibitor cocktails ( MDCKI parental monolayers; representative of n = 3 to 5 per condition ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 09906 . 006 The data above , including the gigaohm seals achieved , symmetrical behavior , near 0 mV reversal potential , detection only at tight junctions , and claudin-2-dependence , suggest that the ~9 pA events detected by trans-tight junction patch clamp cannot be due to artifacts , such as transmembrane ion channels in apical membrane domains sealed within the patch pipette or pipette leak .", "We nevertheless took a pharmacological approach to further examine the hypothesis that these conductance events represented activity of transmembrane ion channels .", "Three different inhibitor cocktails were added to the patch pipette ( Figure 4B ) .", "The first cocktail contained 10 mM 4-aminopyridine and 10 mM TEA-Cl to inhibit voltage-activated K+ channels .", "The second cocktail contained 100 nM charybdotoxin and 2 μM apamin to inhibit small conductance Ca2+ activated K+ channels .", "The third cocktail contained 100 μM amiloride , 20 μM CFTR inhibitor-172 ( inh-172 ) , and 250 μM 4 , 4'-Diisothiocyano-2 , 2'-stilbenedisulfonic acid ( DIDS ) to block epithelial Na+ channels , CFTR , and anion exchangers respectively .", "None of these affected frequency or amplitude of the claudin-2-dependent ~9 pA events detected by trans-tight junction patch clamp .", "We therefore conclude , on the basis of biophysical , molecular , and pharmacological data , that the ~9 pA events detected represent single-channel conductances across the tight junction .", "Previous studies have shown that overall transepithelial conductance as well as claudin-2-dependent Na+ conductance decrease when temperature is reduced ( Gonzalez-Mariscal et al . , 1984 , Martinez-Palomo et al . , 1980 , Shen et al . , 2008 , Yu et al . , 2009 ) .", "We therefore assessed temperature sensitivity of the single channel events measured by trans-tight junction patch clamp .", "These studies were complicated by the technical challenge of cooling the monolayer without creating excessive electrical noise that prevented analysis and limited cooling to ~20°C while recording from the trans-tight junction patch clamp .", "When monolayers of claudin-2-expressing MDCKI were cooled from 37°C to 15°C while recording , the number of claudin-2-dependent ( ~9 pA ) events fell by 37% ( Figure 4C ) .", "This was closely paralleled by a 41% decrease in Na+ permeability measured across monolayers using traditional approaches ( Figure 4D ) , providing more support for the conclusion that the ~9 pA conductance events reflect activity of paracellular claudin-2 channels .", "We also assessed the claudin-2-independent , ~4 pA events using the parental MDCKI cells that completely lacked claudin-2 expression .", "We did this because these claudin-2-independent currents can be obscured by larger events ( e . g . Figure 2 ) .", "In contrast to the ~9 pA events , the ~4 pA events were resistant to cold ( p = 0 . 35 ) , even when chilled to 10°C ( Figure 4E ) .", "The ~4 pA events were also resistant to all of the ion channel inhibitor cocktails ( Figure 4F ) .", "The MDCK cell line is derived from epithelia of the distal convoluted tubule , which function effectively to absorb Na+ and Cl- in the apical-to-basal direction and secrete K+ ( Gekle et al . , 1994 ) .", "Cell lines derived from epithelia within other parts of the body have distinct specialized functions .", "For example , Caco-2 cells are a human colon epithelial cancer cell line that differentiate as absorptive enterocytes and express brush border enzymes and transporters typical of this cell type ( Peterson and Mooseker , 1992 , Pinto et al . , 1983 , Turner and Black , 2001 , Turner et al . , 1996 , Turner et al . , 1997 ) .", "While both MDCK and Caco-2 cell lines are both commonly used to study polarized epithelial cell function , their distinct phenotypes are reflected by divergence in both function and protein expression .", "Nevertheless , tight junction ultrastructure is similar in MDCK and Caco-2 cells , and both are composed of three to five strands ( Figure 5A ) , which express claudin-2 abundantly ( Figure 5B , C ) .", "We took advantage the availability of a Caco-2BBe line in which claudin-2 expression was stably knocked down ( Raleigh et al . , 2011 ) to assess the effects of claudin-2 depletion , rather than addition , on paracellular channel function .", "This also allowed direct comparison of canine renal epithelia and human intestinal epithelia . 10 . 7554/eLife . 09906 . 007Figure 5 . Global conductance and trans-tight junction patch clamp event frequency correlate with claudin-2 expression in Caco-2BBe intestinal epithelial monolayers .", "( A ) Freeze fracture electron microscopy demonstrating that mature tight junctions in Caco-2BBe monolayers are composed of 3–5 strands ( Shen et al . , 2006 ) , similar to MDCKI .", "( B ) Western blot confirms >99% knockdown of claudin-2 in Caco-2BBe monolayers .", "( C ) Claudin-2 is not detectable by immunofluorescence microscopy of knockdown Caco-2BBe monolayers ( Bar = 10 µm ) .", "( D ) Biionic potential analyses show that claudin-2 knockdown reduces small cation permeability .", "( E ) Trans-tight junction patch clamp recordings of Caco-2BBe cells detected events at −100 mV ( n=5 per condition ) .", "Representative traces of trans-tight junction patch clamp data from control and claudin-2 knockdown Caco-2BBe monolayers .", "( F ) All points histogram analysis of patch clamp data from Caco-2BBe monolayers shows a specific reduction in ~9 pA events with no change in frequency of ~4 pA events after claudin-2 knockdown .", "( G ) Average opening conductances was unaffected by the levels of claudin-2 expression .", "( H ) Channel activity ( NPo ) was reduced by claudin-2 knockdown ( n = 5 to 7 per condition ) .", "( I ) Neither ~9 pA nor ~4 pA events were not detectable when the pipette was sealed away from the tight junction in Caco-2BBe monolayers ( n = 12 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 09906 . 007 Paracellular Na+ conductance across Caco-2BBe monolayers was similar to that of MDCKI monolayers with induced claudin-2 expression ( Figure 5D vs Figure 1F ) .", "When claudin-2 expression was stably suppressed by shRNA-mediated knockdown , Na+ conductance across Caco-2BBe monolayers was greatly diminished ( Figure 5D ) and fell to a level similar to the claudin-2-deficient MDCKI parental line ( Figure 1F ) .", "We next analyzed monolayers of Caco-2BBe human intestinal epithelia by trans-tight junction patch clamp ( Figure 5E ) .", "It was substantially more difficult to achieve a gigaohm seal in these monolayers relative to MDCK , likely due to the well-developed brush border of Caco-2BBe cells .", "As with MDCK monolayers , all points histograms demonstrate conductance values centered around ~8 pA , i . e . ~82 pS , in claudin-2-expressing monolayers ( Figure 5F ) , and there was a specific reduction in this class of events after claudin-2 knockdown ( Figure 5G ) .", "We therefore conclude that claudin-2 depletion in human intestinal epithelia eliminates events similar to those generated by claudin-2 expression in canine renal epithelia .", "While NPo was reduced , conductance of the claudin-2-dependent events was not affected by claudin-2-knockdown ( Figure 5H ) , demonstrating that single channel conductance was not a function of claudin-2 concentration .", "Similar to the data from MDCK monolayers , this indicates that gating of claudin-2-dependent channels is not cooperative .", "A class of smaller conductances that were not affected by claudin-2 knockdown was also detected , similar to the claudin-2-independent events detected in MDCK monolayers .", "Finally , as in MDCK cells , the claudin-2-dependent openings were not detectable when the patch pipette was sealed away from the tight junction in Caco-2BBe monolayers ( Figure 5I ) .", "Thus , these data demonstrate the tight junction opening events are detectable in two very different epithelia derived from different organ systems , and , in both cases , the events were claudin-2 dependent .", "Further , these data exclude the possibility that events could be due to off-target effects induced by the tet transactivator used to drive claudin-2 expression in MDCK cells .", "More importantly , the similar conductance values of these events , despite markedly different NPo values , supports the conclusion that the events are mediated by biophysically similar claudin-2-based paracellular channels , rather than some other paracellular channel that might be expected to differ between canine renal and human intestinal epithelia .", "Paracellular flux across tight junction channels is driven passively by transepithelial electrochemical gradients .", "To determine if flux through the claudin-2 dependent channels detected by trans-tight junction patch clamp behaves similarly , we measured single channel reversal potentials in the presence of large apical:basolateral or basolateral:apical NaCl gradients .", "As predicted for a paracellular conductive pathway , we observed similar shifts in reversal potential but in opposite directions by iso-osmotically replacing 90% of basolateral or apical NaCl with mannitol ( Figure 6A , B ) .", "Importantly , such symmetrical behavior indicates that flux across these channels is electrochemically driven by apical:basolateral gradients .", "In contrast , transmembrane ion channels are driven by extracellular:intracellular gradients .", "As intracellular cation composition does not change as rapidly as the extracellular media , transmembrane ion channels would not be expected to behave symmetrically under these conditions . 10 . 7554/eLife . 09906 . 008Figure 6 . Claudin-2-dependent conductance events measured by trans-tight junction patch clamp display cation- and size-selective properties similar to transepithelial paracellular conductance measured over large areas .", "( A–E )", "Current-voltage ( I-V ) relationships for events detected by trans-tight junction patch clamp in MDCKI monolayers with transgenic claudin-2 expression ( +Cldn-2 ) under the ionic conditions shown .", "Pipette and basolateral buffer composition are indicated in mM . ( F ) Vrev was determined under each of the ionic conditions shown ( n=4 to 7 per condition ) .", "( G ) Cation permeability determined from shifts in trans-tight junction patch clamp Vrev ( green line ) or traditional ( black line ) bi-ionic potential measurements . DOI: http://dx . doi . org/10 . 7554/eLife . 09906 . 008 A second very important feature of the reversal potential measurement approach is that permeation occurs via a passive , yet selective , mechanism defined by the electrochemical gradients of the ions passing through the channel .", "This allowed us to further characterize the charge-selectivity of claudin-2-dependent conductances .", "The magnitude of reversal potential shifts indicates that the PNa+/PCl− , as defined by Nernst equilibrium potentials , of single channel events detected by trans-tight junction patch clamp is 7 . 4 ± 3 . 7 .", "This is similar to the PNa+/PCl− of 9 . 5 ± 0 . 1 measured across intact monolayers using traditional global measurement approaches ( Figure 1D ) .", "In addition to demonstrating that the detected single channel events have PNa+/PCl− that is indistinguishable from that of tight junctions , these data also exclude the possibility that openings represent anion channels , such as CFTR , that would be expected to be Cl- , rather than Na+ , selective .", "When measured across intact monolayers , paracellular tight junction permeability is well recognized to be size-selective .", "This can be assessed by measuring flux of differently-sized uncharged probes .", "However , the temporal averaging required by these approaches precludes detection of transient , single-channel events .", "Fortunately , this obstacle has recently been overcome using the technique of biionic substitution , where Na+ within the basolateral media is replaced with a larger cation ( Angelow and Yu , 2009 , Shen et al . , 2011 ) .", "Use of the trans-tight junction patch clamp in conjunction with biionic substitution showed that the channels were permeable to methylamine ( radius=1 . 9 Å ) and that , like Na+ , the Vrev of methylamine was close to 0 mV ( Figure 6C ) .", "This indicates that conductance of Na+ and methylamine is similar in magnitude .", "In contrast , channels detected by trans-tight junction patch clamp were relatively impermeant to the larger cations tetramethylammonium ( radius=2 . 8 Å ) and N-methyl-D-glucamine ( radius=3 . 6 Å ) .", "Thus , basolateral Na+ replacement with these larger cations induced a large negative shift in channel reversal ( Figure 6D–F ) .", "Global analyses of claudin-2-expressing MDCK by biionic substitution demonstrated tight junction size-selectivity that paralleled that of conductance events detected by trans-tight junction patch clamp ( Figure 6G ) .", "In addition to providing another biophysical measure in which the claudin-2-dependent events detected by trans-tight junction patch clamp are similar to claudin-2-dependent paracellular conductances measured by traditional averaging approaches , these data provide further support for the conclusion that the events detected cannot represent apical K+ channels , as the latter are highly-selective and do not accommodate cations as large as methylamine .", "It is , however , interesting to note that the permeability of methylamine , relative to Na+ , is greater in patch clamp measurements than in global measurements .", "We do not know the reason for this , but could speculate that it may be an artifact of the physical forces created by sealing the patch pipette over the junction .", "However , even if that explanation was correct , the fact that permeabilities of tetramethylamine and N-methyl-D-glucamine , relative to Na+ , are unaffected and that charge selectivity is maintained show that such effects , if present , are limited .", "These data therefore demonstrate that the claudin-2 dependent channel represents a passive paracellular conductance pathway with charge- and size-selectivities similar to those measured across large epithelial sheets .", "La3+ is known to nonspecifically inhibit claudin-2-dependent paracellular flux ( Machen et al . , 1972 , Yu et al . , 2010 ) .", "Consistent with this , addition of La3+ to the basolateral chamber completely blocked claudin-2 dependent opening events ( Figure 7A–C ) .", "These data , along with the ionic substitution experiments above , indicate that the channels being studied are equally accessible from apical and basolateral approaches and again refute hypotheses suggesting that the openings detected represent plasma membrane ion channels . 10 . 7554/eLife . 09906 . 009Figure 7 . Paracellular conductance events are blocked by La3+ or claudin-2 derivatization .", "( A ) LaCl3 ( red bar ) blocked opening events .", "Solution exchange artifact is shown in gray .", "( B ) The ~9 pA events were eliminated from the all-points histogram by La3+ treatment ( before LaCl3 black; after LaCl3 red ) ( C ) La3+ treatment ( red ) reduced NPo to 0 ( closed symbols indicate measurements at -100 mV , open symbols indicate measurements at +100 mV , n = 9 ) .", "( D ) MTSET forms a disulfide bond with Cys66 located within the pore of claudin-2I66C channels ( Angelow and Yu , 2009 ) .", "( E ) Transgenic claudin-2I66C was expressed at levels similar to claudin-2WT .", "( F , G )", "MTSET dramatically reduced the number of detectable events in trans-tight junction patch clamp recordings from MDCKI cells expressing claudin-2I66C within ~20 s , but had no effect on monolayers expressing claudin-2WT .", "Blue bar indicates presence of MTSET ( n = 8 to 16 per condition ) .", "Solution exchange artifacts are shown in gray .", "( H , I )", "NPo of MDCKI cells expressing claudin-2I66C or claudin-2WT before and after ( purple ) MTSET treatment ( closed symbols indicate measurements at -100 mV , open symbols indicate measurements at +100 mV , n = 8 to 16 per condition ) .", "( J ) Derivatization of claudin-2I66C does to affect conductance of residual events ( V = –100 mV ) .", "The histogram depicts frequency of events before and after ( purple ) MTSET treatment . DOI: http://dx . doi . org/10 . 7554/eLife . 09906 . 009 La3+ is , however , a non-selective inhibitor .", "We therefore sought a more specific approach to block claudin-2 channels .", "Recent work has begun to define the structural basis for ion selectivity of paracellular , claudin-dependent , trans-tight junction channels ( Angelow and Yu , 2009 , Li et al . , 2013 , Li et al . , 2014 , Li et al . , 2013 , Yu et al . , 2009 ) .", "Extracellular loop 1 ( ECL1 ) of claudin-15 forms the first four β strands , and charged residues at the end of the fourth β strand are thought to line the claudin channel and serve as critical determinants of charge selectivity ( Angelow and Yu , 2009 , Colegio et al . , 2003 , Suzuki et al . , 2014 ) .", "Within the corresponding region of claudin-2 , Ile66 is thought to be buried within a narrow part of the channel ( Angelow and Yu , 2009 ) .", "Covalent modification of claudin-2I66C using 2- ( trimethylammonium ) ethyl methanethiosulfonate ( MTSET ) reduced global paracellular cation flux in claudin-2I66C , but not claudin-2WT , consistent with obstruction of the claudin-2 pore ( Figure 7D ) .", "We exploited the observation that claudin-2I66C can be inhibited by MTSET to ask whether the single channel conductances can be similarly inhibited using MDCKI monolayers with inducible expression of claudin-2I66C ( Figure 7E ) .", "Trans-tight junction patch clamp recordings demonstrated that MTSET markedly reduced NPo of MDCKI monolayers expressing claudin-2I66C ( Figure 7F , H , J ) with kinetics similar to MTSET inhibition measured by global approaches ( Angelow and Yu , 2009 ) .", "As expected , MTSET had no effect on channel events in monolayers expressing claudin-2WT ( Figure 7G , I ) .", "In most cases , MTSET completely abolished events detected by trans-tight junction patch clamp in monolayers expressing claudin-2I66C ( Figure 7H ) , but a few residual events persisted in some monolayers ( Figure 7H ) .", "These MTSET-resistant events were identical in size to those observed prior to MTSET addition ( Figure 7J ) , suggesting that MTSET inhibits each claudin-2 channel either completely or not at all .", "The small number of MTSET-resistant channels occurred in a subset of monolayers , suggesting that this may reflect the lability of MTSET in aqueous solutions .", "Alternatively , the observation that events detected in MTSET-treated claudin-2I66C-expressing monolayers had conductances of ~92 pS , similar to monolayers expressing claudin-2WT could be interpreted as support for a role of Ile66 in claudin-2 gating .", "In either case , these data show that site-directed mutagenesis and chemical derivatization of Ile66 blocks local conductance events in a specific manner .", "Because claudin-2 is concentrated at the tight junction and has not been demonstrated to create transmembrane channels , this result provides further support for the conclusion that events represent flux across paracellular , i . e . tight junction , channels ." ], [ "Tight junction ultrastructure , as seen using freeze-fracture electron microscopy , is established by an anastomosing arrangement of strands which encircle the cell at the apical intercellular space .", "It has been proposed that these strands establish the paracellular barrier and that the strands are also populated by “channels” which impart charge and size selectivity .", "Whether these channels , commonly referred to as the pore pathway ( Shen et al . , 2011 ) , are open at steady state or can open and close was previously unknown .", "Our data show that tight junctions are populated by highly dynamic , gated channels that transition between at least three different states .", "The means by which these functional state transitions occur and whether they are or can be regulated is one interesting question that follows from the results presented here .", "Further , as investigators in this area seek to model claudin channel function based on the published crystal structure of claudin-15 ( Suzuki et al . , 2014 ) , our data indicate that any model applied to claudin-2 must include an explanation for rapid opening and closing of the channel .", "One could hypothesize that , in contrast to the shorter closed state , the longer closed state may reflect transient disassembly of the claudin-2 channel complex .", "We speculate that some of the properties observed in our single channel recordings are due to the arrangement of channels spanning tight junction strands that are oriented in both series and parallel , implying that multiple simultaneous tight junction channel openings would result in stepwise conductance increases .", "Indeed , we did observe short-lived , step-wise conductance increases , consistent with a parallel opening of a second claudin-2 channel , superimposed on a typical initial opening ( e . g . , Figures 4B , 6B , E and 7G ) .", "Alternatively , the multi-strand tight junction ultrastructure and corresponding conductance model proposed by Claude ( Claude , 1978 ) suggest that channels are also arranged in series .", "If true , this would indicate that the NPo measured here for current across the entire height of the tight junction significantly underestimates the NPo of individual claudin-2 channels .", "Consistent with this , we did observed some variability in claudin-2-dependent current amplitude .", "This could be related to the specific configuration of a claudin-2 channel within a given submicron segment of tight junction .", "For example , heterogeneity may be due to variations in baseline strand conductance , different numbers of tight junction strands , or variations in strand branch points within a particular patch of tight junction .", "Indeed , the anastamosing arrangement of tight junction strands may be a mechanism that limits lateral spread of current from single channel openings within any individual strand .", "To better understand the different types of events detected within the patch clamp recordings presented here , we developed an equivalent circuit diagram ( Figure 8 ) .", "First , we determined resistance of the patch pipette electrode itself .", "When in solution , i . e . free of cells , the resistance was determined to be 2 . 5 MΩ , similar to that of electrode used by others .", "When the pipette was sealed to the monolayer , resistance of the seal leak , i . e . the path resulting from incomplete electrical isolation at the edges of the pipette , was determined to be ~30 GΩ , based on the steady-state current of ( ~3 . 5 pA ) at -100 mV when the pipette was sealed over the junction of claudin-2-deficient monolayers composed of either parental or uninduced , transfected MDCKI cells .", "The resistance of the paracellular shunt pathway ( outside of the pipette ) is simply the measured resistance of the monolayer , which ranged from 100 to 1 , 500 Ω·cm2 , i . e . 0 . 0003 to 0 . 0045 MΩ .", "Because this resistance is so much lower than any other pathway measured , it is essentially 0 for these analyses , as it cannot significantly impact conductance events of the types measured here .", "Thus , after accounting for low pipette seal leak and electrode resistance , the conductance events detected by the patch pipette can only represent transepithelial currents . 10 . 7554/eLife . 09906 . 010Figure 8 . Circuit analysis of current pathways detected by trans-tight junction patch clamp . Resistances of the pipette seal , the electrode , the paracellular pathway of the larger epithelial sheet ( outside of the patch ) , detectable apical membrane channels , and both claudin-2-dependent ( green ) and claudin-2-independent ( blue ) paracellular channels are shown .", "Both paracellular channels were detected only when the patch pipette was sealed over the intercellular junction .", "The apical membrane channels were only detected when the patch pipette was sealed to away from the intercellular junction , but , are likely present within the apical membrane adjacent to the junction as well . DOI: http://dx . doi . org/10 . 7554/eLife . 09906 . 010 To specifically assess non-tight junction conductance pathways that could , for example , be due to apical membrane trapped within the patch pipette , the pipette was sealed over the apical membrane , i . e . away from the junction .", "In this configuration , ~2 pA currents could be detected at 100 mV , which corresponds to a pathway with a resistance of 50 GΩ .", "Two additional pathways , measuring ~9 pA and ~4 pA events were present only when the pipette was sealed over the junction .", "The ~9 pA at 100 mV , or 11 GΩ , events were present in MDCKI or Caco-2BBe cells expressing both high and low levels of claudin-2 , but were absent in cells devoid of claudin-2 , i . e . the parental MDCKI line .", "The frequency of detection correlated with claudin-2 expression indicating that these events are strictly claudin-2 dependent .", "In contrast , the ~4 pA at 100 mV , or 25 GΩ , events were detected in the presence or absence of claudin-2 expression .", "At this point , we cannot define these events as due to a specific protein or channel .", "However , they are only detected at the tight junction and may , therefore represent a paracellular , i . e . tight junction , channel that accounts for the electrical conductance measured across sheets of claudin-2-deficient cells using traditional methods .", "In addition to the claudin-dependent size- and charge-selective pore pathway , a charge non-selective leak pathway that allows paracellular flux of small and large molecules is also present ( Anderson and Van Itallie , 2009 , Turner , 2009 ) .", "We and others have shown that occludin- and ZO-1 are both important to leak pathway regulation ( Buschmann et al . , 2013 , Van Itallie et al . , 2009 ) and that , in the intestine , increased leak pathway permeability is induced by TNF via a myosin light chain kinase-dependent process ( Buschmann et al . , 2013 , Clayburgh et al . , 2005 , Van Itallie et al . , 2010 , Zolotarevsky et al . , 2002 ) .", "Our in vitro studies suggest that this pathway may have an effective radius of 62 . 5 Å ( Buschmann et al . , 2013 ) .", "We did not , however , detect a defined population of very large openings in any of our patch clamp recordings .", "This suggests that the leak pathway has limited or no dynamic gating , are extremely rare , or cannot be distinguished from electrode seal loss .", "Alternatively , some investigators have proposed that the leak pathway opens as one tight junction strand at a time , with materials trapped between strands until the next channel opens ( Sasaki et al . , 2003 ) .", "In this case , it would be very difficult to detect these larger events .", "However , they could impact the effective conductance of claudin-2 channel opening events , and thus potentially explain the variability in amplitude that we observed .", "It has also been suggested that leak pathway flux occurs primarily at tricellular tight junctions .", "We therefore attempted to seal patch pipettes over tricellular contacts .", "Unfortunately , either the geometry or small size of these regions makes it exceedingly difficult to place and seal an electrode .", "We therefore conclude that either a significant technical revision to our trans-tight junction patch clamp or completely different approaches will be required to perform single channel analyses of the leak pathway or tricellular tight junction .", "In summary , our novel trans-tight junction patch clamp recordings reveal sub-millisecond timescale gating of single paracellular channels that define trans-tight junction , paracellular conductance .", "Unlike conventional transmembrane ion channels , which regulate flux between extracellular and intracellular compartments , the channels depicted here bridge two extracellular compartments , lumen and tissue , and , therefore , represent an entirely new class of gated ion channels .", "Together with mutagenesis and structural analyses , the ability to detect individual conductance events , as described here , will be a critical tool in determination of molecular mechanisms that regulate channel assembly and gating .", "Such studies may lead to development of pharmacological means of modulating gating activity for therapeutic purposes ." ], [ "Madin-Darby Canine Kidney ( MDCK ) I cells expressing claudin-2 under control of a tet-off inducible expression system were maintained in media with 50 ng/ml of doxycycline , and claudin-2 expression was induced by culture without doxycycline for 4 days , as previously described ( Yu et al . , 2009 ) .", "Human colonic Caco-2BBe epithelial cells , with or without stable claudin-2 knockdown , were maintained and plated as previously described ( Raleigh et al . , 2011 ) .", "Cells were grown on 0 . 33 cm2 polycarbonate semi-permeable membranes with 0 . 4 µm pores ( Corning Life Sciences , Corning , NY ) and used 4 and 10 days after plating for MDCKI and Caco-2 cells , respectively .", "Bridges prepared using 1% agarose in Hank’s balanced saline solution ( HBSS; 135 mM NaCl , 0 . 3 mM Na2HPO4 , 0 . 4 mM MgSO4 , 0 . 5 mM MgCl2 , 0 . 3 mM KH2PO4 , 1 . 3 mM CaCl2 , 10 mM HEPES , 5 mM KOH , pH 7 . 4 ) were used .", "Liquid junction potentials were negligible ( < 1 mV ) .", "Bridges were connected to calomel and Ag-AgCl electrodes and a current clamp ( Physiologic Instruments , San Diego , CA ) , as previously described , with all experiments performed at 37°C ( Weber et al . , 2010 , Yu et al . , 2009 ) .", "Transepithelial resistance was determined using current clamp pulses and Ohm’s law , as described ( Turner et al . , 1997 ) .", "Reversal potentials ( Vrev ) were measured using current clamp ramps from −10 to +10 µA before and after basolateral or apical replacement of HBSS with media in which 90% or 50% of NaCl was iso-osmotically replaced by mannitol .", "All biionic potential measurements were performed in quadruplicate or greater in at least three independent experiments .", "135 mM NaCl was substituted with 135 mM XCl , where X refers to the monovalent cations methylamine ( MA+ ) , tetramethylammonium ( TMA+ ) , ethylamine ( EA+ ) , or N-methyl-D-glucamine ( NMDG+ ) .", "Relative permeabilities ( PNa+/PCl- ) or ( PX+/PNa+ ) were determined using the Goldmann-Hodgkin-Katz voltage equation , measured Vrev , and known composition of basolateral and apical solutions ( Weber et al . , 2010 , Yu et al . , 2009 ) .", "Osmolarity of all buffers was confirmed using a model 3320 osmometer ( Advanced Instruments , Norwood , MA ) .", "Absolute Na+ permeabilities were determined from transepithelial resistance and PNa+/PCl− by the Kimizuka and Koketsu method ( Kimizuka and Koketsu , 1964 , Yu et al . , 2009 ) using activity coefficients of 0 . 755 , 0 . 812 , and 0 . 882 for NaCl at 135 , 67 . 5 , and 13 . 5 mM NaCl , respectively ( Truesdell , 1968 ) .", "MDCKI and Caco-2BBe cells were used 4 and 10 days respectively after plating on shallow-walled clear polyethylene terephthalate membrane supports ( 0 . 4 µm pore size , Corning , Tewksbury MA ) , mounted in glass-bottom 35 mm Petri dishes .", "Currents were measured using an Axopatch 200B amplifier and pClamp software ( Axon Instruments , Union City , CA ) in voltage clamp mode with 5 kHz analog filtering .", "Borosilicate capillary tubes ( World Precision Instrument , Sarasota , FL ) were pulled to outer diameters of ~1 µm , resulting in access resistances of 2 . 5–3 MΩ when fire-polished .", "Monolayers were continuously perfused with apical and basolateral HBSS solution with 5 mM D-glucose .", "Gigaohm seals were obtained allowing current measurement relative to a reference electrode without interference from apical conductances outside of the patch .", "Negative currents reflect cations moving in the direction towards the recording electrode .", "To facilitate sealing it was helpful to stream pipette solution from the pipette as electrodes approached their points of contact .", "This was particularly true for Caco-2BBe recordings and we speculate that this prevents disordered well-developed microvilli from interfering with seal formation .", "Osmolarity was adjusted to 300 mOsm using mannitol to facilitate seal formation .", "The normal pipette solution was 135 mM NaCl , 5 mM KOH , 1 mM MgCl2 , 1 . 3 mM CaCl2 , 10 mM HEPES , pH 7 . 4 , with 290 mOsm .", "When 4-aminopyridine , TEA , charybdotoxin , or apamin were included in the pipette , [NaCl] was reduced to maintain osmolarity .", "La3+ ( 5 mM ) and MTSET ( 1 mM ) were added to both apical and basolateral chambers , but not included in the apical pipette solution .", "Pipette offsets were zeroed upon access of the recording electrode to the extracellular solution and with the electrode far from the monolayer .", "For local dilution potential measurements , bathing solution or pipette solution was replaced with HBSS in which 90% of the NaCl was isosmotically replaced with mannitol .", "For local biionic potential measurements , bathing solution was replaced isosmotically with methylamine chloride ( MACl ) , tetramethylamine chloride ( TMACl ) , or N-methyl-D-glucamine chloride ( NMDGCl ) as indicated .", "Vrev of opening events was determined during repetitive 1 s voltage ramps from −100 to +100 mV with holding potentials of −100 mV .", "For the purposes of subtracting steady state conductance , ramps in which no openings were detected were subtracted from ramps which contained openings .", "The relatively short duration of these ramps permitted data oversampling at 100 KHz and post hoc data averaging down to 10 KHz .", "This provided a somewhat smaller amount of noise compared to the steady state recordings and allowed more accurate measurement of current reversal potentials .", "Patch clamp data and simulated currents were analyzed using Clampfit ( Molecular Devices , Sunnyvale , CA ) .", "All points histograms were generated using 0 . 1 pA bin size and normalized to record duration ( samples/s ) .", "Data were fit to Gaussian distributions .", "Secondary analyses to determine absolute event counts , open and closed durations , and event conductances were performed using Clampfit event detection software .", "Channel activity was expressed as open probability ( NPo ) which was determined from the equation below ( Huang and Rane , 1993 ) : NPo=∑ ( open time x number of channels open ) / ( total time of record ) Liquid junction potentials ( < 5 mV ) were small relative to dilution potentials and not included in calculations .", "Single event opening and closing duration histograms were fit to single and double exponentials using the maximum likelihood method ( TACFit X4 . 3 . 3 software , Bruxton Corporation , Seattle , WA ) .", "A bin size of 2 per decade was chosen to allow sufficient sampling of prolonged closed durations .", "Cell lysates were separated by SDS-PAGE and transferred to PVDF membranes , as described previously ( Weber et al . , 2010 ) .", "Immunoblots were performed using antibodies to claudin-2 ( Abcam , Cambridge , MA ) , E-cadherin ( Cell Signaling Technology , Danvers , MA ) , or β-actin ( Sigma , St . Louis , MO ) followed by horseradish peroxidase-conjugated secondary antibodies ( Cell Signaling Technology ) .", "Proteins were detected by enhanced chemiluminescence .", "Cultured monolayers were fixed with −20°C methanol and bis ( sulfosuccinimidyl ) suberate , as previously described ( Shen and Turner , 2005 ) .", "Tight junction proteins , ZO-1 and claudin-2 were immunostained using mouse anti-ZO-1 and rabbit anti-claudin-2 primary antibodies and Alexa Fluor 488 and 594 conjugated secondary antibodies ( Life Technologies ) .", "Imaging was performed using an epifluorescence microscope ( DM4000; Leica Microsystems , Bannockburn , IL ) equipped with a 63× NA 1 . 32 PL APO oil immersion objective , DAPI , EGFP , and Texas Red laser aligned filter cubes ( Chroma Technology , Rockingham , VT ) , and a Retiga EXi camera ( QImaging , Surrey , BC , Canada ) controlled by MetaMorph 7 . 5 , as previously described ( Su et al . , 2009 ) .", "Student’s t-test was used to compare means .", "Statistical significance was designated as *p < 0 . 05 , **p <0 . 01 , and ***p <0 . 001 .", "The Holm–Bonferroni method was used to correct for multiple comparisons .", "Data are shown as mean ± SEM ." ] ]

[ "Intercellular tight junctions form selectively permeable barriers that seal the paracellular space .", "Trans-tight junction flux has been measured across large epithelial surfaces , but conductance across individual channels has never been measured .", "We report a novel trans-tight junction patch clamp technique that detects flux across individual claudin-2 channels within the tight junction of cultured canine renal tubule or human intestinal epithelial monolayers .", "In both cells , claudin-2 channels display conductances of ~90 pS .", "The channels are gated , strictly dependent on claudin-2 expression , and display size- and charge-selectivity typical of claudin-2 .", "Kinetic analyses indicate one open and two distinct closed states .", "Conductance is symmetrical and reversible , characteristic of a passive , paracellular process , and blocked by reduced temperature or site-directed mutagenesis and chemical derivatization of the claudin-2 pore .", "We conclude that claudin-2 forms gated paracellular channels and speculate that modulation of tight junction channel gating kinetics may be an unappreciated mechanism of barrier regulation ." ]

[ "Epithelial cells form layers that line the inner surface of the gut , lungs and other organs .", "They act as barriers to control the movement of water , ions and small molecules between internal compartments within the body and the external environment .", "Some substances are transported across these barriers by passing through individual epithelial cells , but others pass through the spaces between adjacent cells .", "These spaces are sealed by tight junctions .", "If the tight junctions do not work properly , it can cause problems with regulating the movement of molecules across epithelial-lined surfaces .", "This in turn can contribute to diseases in humans , including inflammatory bowel disease and chronic kidney disease .", "Proteins called claudins form channels that only allow certain molecules to pass through tight junctions .", "One member of this family , called claudin-2 , allows sodium ions and other small positively charged ions to cross between adjacent cells .", "However , it is not clear how these channels work , largely due to the absence of appropriate tools to study this process .", "Here , Weber et al . adapted a technique called patch clamping to study the behavior of individual claudin-2 channels in the tight junctions between mammalian epithelial cells .", "Weber et al . found that claudin-2 allows positively charged ions to move across a tight junction in short bursts rather than in a steady stream as had been suggested by previous work .", "These bursts typically begin and end in less than a millisecond .", "Further experiments revealed that claudin-2 channels have several states; in one state the channel is fully open , in another the channel is firmly closed , and in the third state the channel is temporarily closed but primed to open .", "Further experiments show that mutations in the gene that encodes claudin-2 or drugs that inhibit claudin-2's function alter the open and closed behaviors of these trans-tight junction channels .", "The technique developed by Weber et al . will enable researchers to understand how channel proteins at tight junctions assemble and operate .", "Such studies may lead to the development of drugs that can alter the activity of these channels to treat particular diseases ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "cancer biology" ]

[ [ "TP53 is a tumor suppressor gene that functions to prevent cancer by allowing cells to recover from various stress insults such as DNA damage or by triggering their elimination when the extent of the damage is beyond repair .", "In its normal state , the p53 transcription factor acts in response to oncogenic or other stress signals to induce or repress a variety of target genes involved in cell cycle control , apoptosis , DNA repair , and cellular senescence ( Vogelstein et al . , 2000; Harris and Levine , 2005 ) .", "In normal cells , the levels of p53 protein are tightly regulated by the E3 ubiquitin ligase HDM2 that targets p53 for ubiquitin-dependent proteasome degradation ( Haupt et al . , 1997; Kubbutat et al . , 1997; Marine and Lozano , 2010 ) .", "In addition , HDM2 binding to p53 blocks its transactivation domain preventing p53 transcriptional activation of its target genes ( Momand et al . , 1992 ) .", "HDM2 is itself a p53 target gene and hence acts as part of a negative feedback loop which maintains low cellular concentrations of both partners under non-stressed conditions ( Picksley and Lane , 1993; Wu et al . , 1993; Freedman et al . , 1999; Michael and Oren , 2003; Bond et al . , 2005 ) .", "Approximately , 50% of all tumors display inactivating mutations in p53 ( Hainaut and Hollstein , 2000 ) leading to its partial or complete loss of function ( Vogelstein et al . , 2000; Levine and Oren , 2009 ) .", "In many cancers where TP53 is not mutated , the function of the p53 pathway is often compromised through other mechanisms , including HDM2 gain of function by amplification and/or overexpression ( Bond et al . , 2005; Vousden and Lane , 2007; Brown et al . , 2009; Wade et al . , 2010 ) .", "In these instances blocking the interaction between p53 and HDM2 is hypothesized to stabilize p53 leading to pathway activation and growth arrest and/or apoptosis in cancer .", "Based on this hypothesis and the structural elucidation of the p53–HDM2 interaction , several HDM2 small molecule inhibitors have been developed and are now in clinical trials .", "Indeed , prior work has shown that in human cancer cell lines or xenografts such inhibitors can elicit potent anti-tumor effects as a result of induction of cell cycle growth arrest and an apoptotic response ( Poyurovsky and Prives , 2006; Brown et al . , 2009; Cheok et al . , 2011 ) .", "Here , we describe a novel and highly specific p53–HDM2 inhibitor , NVP-CGM097 , currently in phase I clinical testing ( NCT01760525 ) and a close analog NVP-CFC218 that are both based on an isoquinolinone scaffold .", "In order to identify patients most likely to respond to inhibitors of HDM2 , we sought to develop patient selection biomarkers based on large-scale cancer cell line profiling .", "The analysis of sensitivity profiles across 356 cell lines led to the confirmation that p53 mutant cancer cells fail to respond to HDM2 inhibitors .", "However , among wild-type p53 cancer cells sensitivity was heterogeneous and not solely associated with HDM2 gene amplification .", "Using an unbiased discovery approach , the expression of 13 genes ( including HDM2 ) was found to have robust and superior predictive value for response compared to p53 wild-type status alone .", "This novel 13-gene signature was validated both in vitro and in vivo , and has the potential to improve the selection strategy of patients bearing p53 wild-type tumors who are most likely to respond to treatment with NVP-CGM097 .", "Surprisingly , all 13 genes were p53 target genes suggesting that cells harboring at least partially activated p53 are those that are most susceptible to further p53 activation upon disruption of the p53–HDM2 interaction ." ], [ "NVP-CGM097 and NVP-CFC218 are substituted 1 , 2-dihydroisoquinolinone derivatives that were designed to mimic three key hydrophobic interactions made by p53 residues Phe19 , Trp23 , and Leu26 in the HDM2 pocket ( Kussie et al . , 1996; García-Echeverría et al . , 2000; Furet et al . , 2012 ) ( Figure 1A ) .", "The dihydroisoquinolinone core occupies the center of the cleft and allows for the positioning of appropriate substituents in this sub-pocket ( manuscript in preparation ) . 10 . 7554/eLife . 06498 . 003Figure 1 . TP53 wild-type status is necessary but not sufficient to predict sensitivity to NVP-CGM097 and NVP-CFC218 . ( A ) Chemical structure of NVP-CGM097 and NVP-CFC218 .", "( B ) In vitro activity of NVP-CFC218 and NVP-CGM097 in TR-FRET binding assay", "( a ) and cellular proliferation assay in human cancer cell lines", "( b ) .", "Data are expressed as concentration causing 50% inhibition and shown as mean ± SD from multiple ( n ≥ 8 ) independent experiments .", "( c ) Selectivity is determined by the ratio of GI50 obtained using the HCT-116 p53-null and the HCT-116 p53WT isogenic pair of cell lines .", "( d ) Selectivity is determined by the ratio of GI50 obtained using SAOS-2 ( p53-null ) and SJSA-1 ( p53WT and HDM2-amplified ) osteosarcoma pair of cell lines .", "( C and D )", "Scatter plot showing IC50 values expressed in μM of NVP-CFC218 in cell viability assays of p53 wild-type cell lines ( C ) and p53 mutated cell lines ( D ) , colored by their response to NVP-CFC218 .", "The data used to generate these plots , as well as cell line identity is available in Figure 1—source data 1 .", "( E ) Contingency table indicating the total number of sensitive and insensitive cell lines to NVP-CFC218 .", "The p-value of 5 . 1 × 10−23 shows a significant association between sensitivity to NVP-CFC218 and TP53 wild-type status .", "( F ) Main enriched compound target p-values from the Global Compound Selectivity Analysis .", "p-values are minus log10 transformed .", "The red color refers to compounds that are more selective in the wild-type p53 ( p53WT ) strata than in the mutated p53 ( p53MUT ) strata .", "The blue color indicates the reverse profile .", "Brighter colors indicate which target classes pass the 0 . 25 FDR cut-off .", "The length of each red/blue segment corresponds to the proportion of p53WT selective/p53MUT selective compounds in each target class . DOI: http://dx . doi . org/10 . 7554/eLife . 06498 . 00310 . 7554/eLife . 06498 . 004Figure 1—source data 1 . List of cell lines tested for their sensitivity to NVP-CFC218 ( n = 356 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06498 . 004 The ability of NVP-CFC218 and NVP-CGM097 to disrupt the p53–HDM2 and p53–HDMX complexes was assessed in purified biochemical assays using time-resolved fluorescence resonance energy transfer ( TR-FRET ) to detect interactions between the N-terminal portion of HDM2 ( amino acid 2 to 188 ) or HDMX ( amino acid 2 to 185 ) and the human p53-derived peptide ( amino acid 18 to 26 ) .", "Both NVP-CFC218 and NVP-CGM097 displaced the p53 peptide from the surface of HDM2 with IC50 values of 1 . 6 ± 0 . 2 nM and 1 . 7 ± 0 . 1 nM , respectively .", "In contrast , both compounds were substantially less active against HDMX with IC50 values of 1300 ± 100 nM and 2000 ± 300 nM .", "These data show that NVP-CFC218 and NVP-CGM097 are specific inhibitors of the p53–HDM2 interaction ( Figure 1B ) .", "In measures of cellular viability , NVP-CFC218 and NVP-CGM097 elicited strong anti-proliferative effects in the HCT116 p53WT cell line and showed 34- and 35-fold selectivity , respectively , compared to an isogenic HCT116 cell line in which the TP53 gene was deleted by homozygous recombination .", "Similarly , both compounds blocked proliferation of the osteosarcoma HDM2-amplified SJSA-1 cell line with 56- and 58-fold selectivity , respectively , compared to a control p53-null osteosarcoma SAOS-2 cell line ( Figure 1B ) .", "These results indicate that NVP-CGM097 and NVP-CFC218 inhibit cell proliferation in a p53-dependent manner with a comparable potency and selectivity in vitro .", "In order to investigate the activity of HDM2 inhibitors in preclinical models of cancer and to ultimately identify biomarkers predictive of response , we tested the anti-proliferative activity of NVP-CFC218 in a panel of 477 cell lines from the Cancer Cell Line Encyclopedia ( CCLE ) ( Barretina et al . , 2012 ) .", "After quality control and manual curation of the dose response curves , a total of 356 cell lines met the cell viability quality criteria for NVP-CFC218 and were used for subsequent analyses .", "Cell lines were partitioned into sensitive and insensitive groups based on compound potency ( IC50 ) .", "A rank-order plot of IC50s for NVP-CFC218 showed a natural cut-off of 4 μM , which was used to categorize cell lines .", "Forty seven cell lines were categorized as sensitive and 309 cell lines as insensitive .", "For the sensitive cell lines , the maximal compound effect level ( Amax ) was ≤ −50% ( Figure 1C , D , Figure 1—source data 1 ) .", "As expected , based on the mechanism of action of NVP-CFC218 , most of the sensitive cell lines harbored wild-type p53 ( n = 43 ) and this association was statistically significant by Fisher's exact test ( p-value = 5 . 1 × 10−23 ) ( Figure 1E ) .", "Moreover , repeat cell proliferation assays with the four sensitive cell lines bearing mutations in TP53 showed them to be insensitive ( P12-ICHIKAWA , KBMC-2 , and Hs 294T with IC50 > 8 µM ) or the reported mutation did not lead to complete p53 loss of function ( NCI-H2122 carries both Q16L and C176F p53 mutations which are categorized as neutral and partially deleterious mutations by SIFT , respectively ) .", "A parallel , unbiased orthogonal analysis of cell line sensitivities to over 2000 compounds , grouped by their mechanism of action ( or main target ) , revealed that p53–HDM2 inhibitors including NVP-CFC218 were the most selectively enriched compound class in the p53 wild-type cell line set as compared to the p53 mutant set , with both a false discovery rate ( FDR ) and a p-value below the cut-offs of 0 . 25 and 0 . 05 , respectively ( Figure 1F ) .", "Interestingly , among all p53 wild-type cell lines for which compound data were available , 57% scored insensitive to NVP-CFC218 ( Figure 1C ) .", "These results suggest that a patient selection strategy based only on the p53 status of the tumor will not optimally enrich for patients with a high likelihood of responding to this targeted therapy .", "Hence , there is a need for more sensitive and predictive biomarkers for p53–HDM2 inhibitors .", "We utilized a similar approach as described previously ( Barretina et al . , 2012 ) to identify molecular correlates of compound sensitivity to NVP-CFC218 .", "We first applied a variance filter to remove half of the genes with the lowest variance , yielding 9053 genes .", "The group of sensitive cell lines ( n = 47 ) was then compared to the group of most insensitive ones for which IC50 and Amax of NVP-CFC218 was ≥8 μM and ≤ −50% , respectively ( n = 204 ) .", "To build a predictive model , we used bootstrapping , whereby the cell lines were randomly split into training and testing sets .", "Within each bootstrapped data split where 2/3 of the data was used for training and 1/3 for testing , we used the Wilcoxon uni-variate test to select features that were significantly differentially expressed between sensitive and insensitive lines .", "Using the selected features , a naïve Bayes classifier was trained to predict sensitivity status .", "The number of features selected varied from 5 to 100 and for each , 20 bootstrapped data splits were conducted .", "We then assessed the classifier performance based on accuracy , sensitivity , and specificity of the predictions on the test data .", "The classification accuracy , averaged over twenty bootstraps , was generally higher than 80% with a maximum of ∼93% when 17 genes were selected ( Figure 2A , red dot ) .", "The average classification accuracy with the fewest features within 1 SEM of the maximum was when 13 genes were selected .", "Class-level performance metrics , such as sensitivity and specificity , also showed that the 13-gene solution performed similarly to the optimum or optimally ( Figure 2—figure supplement 1; Figure 2—source data 1 ) .", "Sensitivity was the highest for the 13-gene solution , while the highest specificity was observed with 17 genes .", "However , the 13-gene solution was within 1 SEM of the best specificity .", "Thus , the 13-gene classifier was found to be the optimal solution , yielding the following performance statistics: 93% accuracy ( ±2 . 3% ) , 87% sensitivity ( ±10% ) , and 94% specificity ( ±2 . 8% ) . 10 . 7554/eLife . 06498 . 005Figure 2 . Predictive modeling of NVP-CFC218 chemical sensitivity from CCLE expression data .", "( A ) Prediction accuracy estimation by bootstrapping analysis upon increasing feature set size .", "Estimates are averaged over 20 bootstrapping repeats .", "The maximum accuracy is observed for 17 features ( red dot ) .", "Sensitivity and specificity are shown in Figure 2—figure supplement 1 .", "The averaged data used to generate the accuracy , sensitivity and specificity plots is available in Figure 2—source data 1 .", "( B ) Feature occurrence frequencies of 13 feature selections in 100 bootstrapping repeats .", "Fifty five features were selected at least once .", "The 13 most frequently occurring features ( red dots ) were selected more than 30 times .", "( C ) ROC curves for the three predictive models under comparison , i . e . , the p53 mutation status , the 215-feature and the 13-gene signature models .", "( D ) Precision-Recall plot for the 13-gene signature .", "Five curves are typically shown since cross-validation was repeated five times .", "( E ) Performance estimates of the three compared predictive models: AUC ( Area Under the Curve , from the ROC curve shown in C ) , Sensitivity ( fraction of correctly predicted sensitive cell lines ) , Specificity ( fraction of correctly predicted insensitive cell lines ) , PPV ( positive predicted value , fraction of sensitive cell lines predicted as such ) , and NPV ( negative predictive value , fraction of insensitive cell lines predicted as such ) .", "Measures are averaged over the 5 iterations of 5-fold cross-validations . DOI: http://dx . doi . org/10 . 7554/eLife . 06498 . 00510 . 7554/eLife . 06498 . 006Figure 2—source data 1 . Classifier performance with increasing feature set size . DOI: http://dx . doi . org/10 . 7554/eLife . 06498 . 00610 . 7554/eLife . 06498 . 007Figure 2—figure supplement 1 . Prediction performance estimation by bootstrapping analysis upon increasing feature set size . Estimates are averaged over 20 bootstrapping repeats .", "Maximum sensitivity is observed for 13 features ( sensitivity plot , red dot ) .", "Maximum specificity is observed for 17 features ( specificity plot , red dot ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06498 . 00710 . 7554/eLife . 06498 . 008Figure 2—figure supplement 2 . Prediction performance of NVP-CFC218 in p53WT CCLE cell lines . The measures of prediction accuracy are calculated restricted to the wild-type TP53 subset of CCLE cell lines ( n=68 ) .", "( A ) ROC curves for the 215-feature set and the 13-gene signature predictive models .", "( B ) Precision-Recall plot for the 13-gene signature .", "Five curves are typically shown since cross-validation was repeated 5 times .", "( C ) Performance estimates of the 13-gene signature and TP53 mutation status as predictive models . DOI: http://dx . doi . org/10 . 7554/eLife . 06498 . 008 After determining the number of genes to include in the model and assessing the prediction accuracy , we next wanted to select the 13 genes to include in our final model .", "To this end , we ran 100 bootstraps , selecting 13 genes based on the training set within each bootstrap , and tabulated the number of times each gene was selected .", "Fifty five different features were selected at least once and the top 13 features were selected more than 30 times .", "The 11 most frequent features were selected in more than 70 of the 100 bootstrapped selections , and the 9 most frequent ones appeared in more than 90 selections ( Figure 2B ) .", "The 13 top-ranking genes were strongly enriched in known p53 transcriptional target genes ( Table 1 ) .", "Specifically , 12 out of the 13 expression features were genes whose expression has been shown to be regulated by p53 ( Barak et al . , 1993; el-Deiry et al . , 1993; Miyashita et al . , 1994; Okamoto and Beach , 1994; Varmeh-Ziaie et al . , 1997; Tanaka et al . , 2000; Adimoolam and Ford , 2002; Liu and Chen , 2002; Tan and Chu , 2002; Budanov et al . , 2004; He and Sun , 2007; Kawase et al . , 2008; Xiong et al . , 2011 ) .", "Since no annotation for the 13th signature member was available at the time of the signature development , and to be consistent with the bootstrapping results , we replaced the 13th signature member by the 14th most frequently selected feature , TNFRSF10B , another p53 transcriptional target ( Wu et al . , 1997 ) .", "Interestingly , all genes of the final gene signature were up-regulated in the sensitive cell lines ( Table 1 ) , implying that prior to compound treatment , a subset of p53 target genes is transcriptionally activated in the NVP-CFC218-sensitive cell lines . 10 . 7554/eLife . 06498 . 009Table 1 . 13-gene signature selected for the sensitivity prediction to p53–HDM2 inhibitorsDOI: http://dx . doi . org/10 . 7554/eLife . 06498 . 009FeaturesFold changeWilcoxon p-valueProposed function in p53 pathwayExpr .", "HDM22 . 183 . 09 × 10−14Negative feedback loopExpr .", "CDKN1A3 . 883 . 76 × 10−11Cell cycle/senescenceExpr .", "ZMAT32 . 811 . 20 × 10−10Positive feedback loopExpr .", "DDB22 . 551 . 22 × 10−10DNA repairExpr .", "FDXR2 . 421 . 08 × 10−09ApoptosisExpr .", "RPS27L1 . 971 . 23 × 10−09Positive feedback loopExpr .", "BAX2 . 121 . 84 × 10−09ApoptosisExpr .", "RRM2B2 . 064 . 25 × 10−09DNA repairExpr .", "SESN12 . 271 . 04 × 10−08Oxidative stressExpr .", "CCNG11 . 694 . 81 × 10−08Cell cycleExpr .", "XPC1 . 621 . 56 × 10−07DNA repairExpr .", "TNFRSF10B1 . 913 . 30 × 10−06ApoptosisExpr .", "AEN1 . 463 . 30 × 10−06ApoptosisExpr: gene-level expression values generated by the Affymetrix GeneChip technology with the HG-U133 plus 2 arrays , summarized according to the RMA normalization method .", "After having initially restricted the analysis to gene expression , we evaluated all feature types , including copy number and mutation status in addition to gene expression .", "The same two-class comparison as before yielded a total of 215 significant features from multiple types .", "The TP53 mutation status was the feature most associated with response to NVP-CFC218 ( p-value = 2 . 46 × 10−26 ) , with an odds-ratio of 0 . 013 .", "To further evaluate the 13-gene signature model , we compared its prediction performance to that of both ‘TP53 mutation status’ and a naïve Bayes classifier trained on the ‘215-feature set’ .", "We compared ROC ( receiver operating characteristic ) curves based on the predictions made on the test sets across 5 cross-validation runs for the models based on the 215-feature set , the 13-gene signature and the predictions given by TP53 mutation status ( i . e . the presence of a TP53 mutation predicts insensitivity , while a wild-type genotype predicts sensitivity ) .", "The AUC for the 13-gene signature was higher than for 215-feature set model: 0 . 947 vs 0 . 855 ( Figures 2C and E ) .", "Furthermore , the precision–recall curves for the 13-gene signature showed that an 80% true sensitive prediction rate could be achieved while recalling more than 90% of the truly NVP-CFC218-sensitive cell lines ( Figures 2D and E ) .", "The class-level performance measures showed that the 13-gene signature provided a substantial improvement in predicting response to NVP-CFC218 in particular when performance was evaluated by positive predictive value ( PPV ) ( Figure 2E ) : 76% for the ‘13-gene signature’ vs 59% for the ‘215-feature set’ vs 63% for ‘TP53 mutation status’ .", "The enrichment of response when using the 13-gene signature and associated predictive model as a sample stratification strategy was also strongly significant when compared to the baseline response rate without prior stratification , estimated from the NVP-CFC218 sensitivity data as 19% .", "Additionally in a p53 wild-type background ( n=68 ) , the 13-gene signature was compared to the p53 mutation model ( Figure 2—figure supplement 2 ) .", "Since in this comparison , the p53 model does not predict any insensitive cell lines , no AUC or NPV can be calculated .", "The table in Figure 2—figure supplement 2C shows a sensitivity of 94 . 9% , a specificity of 79 . 2% , a PPV of 88 . 7% , and a NPV of 90% for the 13-gene signature .", "In comparison , p53 mutation model has a sensitivity of 100% , a specificity of 0% , and a PPV of 63 . 2% .", "As before , the 13-gene model outperforms the p53 mutation model in terms of PPV ( Figure 2—figure supplement 2C ) .", "Thus , these results show that the 13-gene signature provides an improvement in response prediction to drug treatment over both the TP53 mutation status and the larger signature consisting of 215 significant features .", "Moreover , these results suggest that the 13-gene signature has utility in TP53 wild-type genetic backgrounds .", "The predictive value of the 13-gene signature was subsequently tested in a second , independent data set of cell lines representative of multiple cancer types representative of the lineages included in the first proliferation screen .", "Cells were assayed for their sensitivity to both p53–HDM2 inhibitors NVP-CGM097 ( n = 52 ) and NVP-CFC218 ( n = 38 ) in in vitro proliferation assays .", "Because of the manual assay format differing slightly from the high-throughput assay , the cut-off for sensitivity was fixed at IC50 ≤ 3 µM for both p53–HDM2 inhibitors and predictions of sensitivity for every cell line were derived using the 13-gene signature .", "As expected , cells that were indeed sensitive or insensitive to NVP-CGM097 were similarly sensitive or insensitive to NVP-CFC218 , except for one cell line ( COLO-829 ) ( Figure 3—source data 1 ) .", "Overall , 36/52 and 26/38 cell lines were predicted to be sensitive to NVP-CGM097 and NVP-CFC218 , respectively , while 27/52 and 23/38 were truly sensitive ( Figure 3A , B ) .", "While we observed a slight decrease in specificity relative to the bootstrap estimate , sensitivity measures were 89% and 91% ( Figure 3C ) , respectively , which is comparable to the sensitivity estimate obtained by bootstrapping the predictive model training data ( 87 . 3% ± 10 . 4% , see Figure 2—source data 1 ) .", "Noticeably , PPV values for both drugs were higher than basal response rate ( Figure 3C ) .", "Taken together these data validate the 13-gene signature as a robust predictor for sensitivity to both NVP-CGM097 and NVP-CFC218 p53–HDM2 inhibitors in an external sample set .", "We also determined the performance of the 13-gene signature in pre-selected cells based upon the presence of wild-type p53 .", "As shown in Figure 3—figure supplement 1 , 35/42 and 25/30 wild-type p53 cell lines were predicted to be sensitive to NVP-CGM097 and NVP-CFC218 , respectively , while 25/35 and 21/25 were truly sensitive .", "PPV values were moderately higher than the basal response rate by 5 to 7% , and the relatively small number of insensitive p53WT cell lines in this external set greatly impaired the specificity and NPV values ( Figure 3—figure supplement 1C ) .", "Taken together , these results indicate the 13-gene signature to be a better predictive feature than TP53 mutation status in unselected cell lines .", "In addition , our results in both the discovery ( Figure", "2 ) and the validation sets ( Figure", "3 ) suggest the 13-gene signature may improve the response rate in wild-type p53 pre-selected cell lines . 10 . 7554/eLife . 06498 . 010Figure 3 . Validation of the predictive 13-gene signature in an external set of cell lines . A total of 52 or 38 cell lines were tested against NVP-CGM097 ( A ) or NVP-CFC218 ( B ) , respectively , in a 3-day proliferation assay .", "Sensitivity call for each compound was applied according to a cut-off of 3 µM .", "Cells that were predicted as sensitive are shown in pink , while the ones predicted as insensitive are shown in blue .", "The data used to generate the waterfall plots and cell line details are available in Figure 3—source data 1 .", "Performance values of the 13-gene signature were derived from this experiment , and are shown in ( C ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06498 . 01010 . 7554/eLife . 06498 . 011Figure 3—source data 1 . Sensitivity prediction and sensitivity to NVP-CFC218 and NVP-CGM097 of an external set of cell lines ( n = 52 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06498 . 01110 . 7554/eLife . 06498 . 012Figure 3—figure supplement 1 . Validation of the predictive 13-gene signature in p53WT cell lines . The same visualization as in Figure 3 is shown restricted to the wild-type p53 cell lines .", "A total of 42 or 30 p53WT cell lines were tested against NVP-CGM097 ( A ) or NVP-CFC218 ( B ) , respectively , in a 3-day proliferation assay .", "The data used to generate the waterfall plots and cell line details are available in Figure 3—source data 1 .", "Performance values of the 13-gene signature were derived from this experiment , and are shown in ( C ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06498 . 012 Given the role of p53 in responding to cellular stress , an obvious concern would be that this signature might simply be reflective of cells undergoing cellular stress during in vitro culture .", "In order to exclude this possibility , we assessed the presence of the signature in primary human tumor samples .", "For this purpose , we used the 13-gene signature , associated with the naïve Bayes predictive model trained on cell line data , to interrogate a collection of ∼21 , 300 human tumor samples for which the whole genome expression profiles are available .", "Here , we reasoned that the proportion of samples of any given lineage that scored positively for the signature would be a measure of the concordance between cell line expression data and primary human tumor data .", "The specific proportion of any given lineage among primary human tumor samples scoring positive for the presence of the 13-gene signature ( Figure 4A ) was found to be very similar to the lineage proportions of signature positive , sensitive cell lines in the CCLE ( Figure 4B ) , as probed by the NVP-CFC218 chemical sensitivity experiment described above .", "These data suggest that the 13-gene signature is not likely an artifact of the in vitro culture .", "In keeping with this observation , the 13-gene signature identified a fraction of predicted sensitive human primary tumor samples within our patient-derived tumor xenograft ( PDX ) collection ( n = 503 ) ( Figure 4C ) , thus allowing the selection of models for further in vivo validation of its predictive power .", "In summary , these analyses provide an estimation of the prevalence of 13-gene signature positivity across various cancer indications and underscore additional cancer types with high signature positivity , such as hepatocellular carcinoma and renal cell carcinoma that may have been underrepresented in the in vitro cell line profiling approach . 10 . 7554/eLife . 06498 . 013Figure 4 . Prevalence of the 13-gene signature in human primary tumors and patient-derived tumor xenograft models .", "( A ) Fraction of human primary tumors predicted sensitive by the 13-gene signature .", "Samples are grouped by primary site .", "Expression data from 21 , 300 human primary tumors were compiled and only primary sites consisting of more than 50 samples were included in the analysis .", "The primary site nomenclature is based on the COSMIC Classification System .", "( B ) Correlation between the fraction of human primary tumor samples predicted sensitive and the observed sensitivity ratio in the CCLE cell line data .", "( C ) Correlation between the sensitivity predictions from the human primary tumor sample collection and the patient-derived tumor xenograft model collection .", "Human primary tumor samples , patient-derived tumor xenografts , and CCLE cell lines are organized by lineage .", "The dashed line corresponds to the identity line . DOI: http://dx . doi . org/10 . 7554/eLife . 06498 . 013 To further assess the predictive performance of the 13-gene signature , tumor response in vivo was tested across a set of PDX models from indications identified from the predictions described above ( Figure 4C ) .", "These in vivo experiments were performed with NVP-CGM097 , owing to its superior pharmacokinetic properties and oral bioavailability as compared to NVP-CFC218 ( Table 2 ) .", "First , the pharmacodynamic effects and optimal daily dose of NVP-CGM097 were determined in the cell line-derived SJSA-1 osteosarcoma xenograft model that harbors an amplification of the HDM2 gene .", "A single oral dose of NVP-CGM097 , 100 mg/kg , led to stabilization of p53 and elevation of p53 target genes such as CDKN1A ( p21 ) at the mRNA level ( Figure 5A , left ) and at the protein level ( Figure 5A , right ) .", "Treatment of SJSA-1 xenografted tumors with NVP-CGM097 led to dose-dependent tumor growth inhibition with 65% tumor regression at 100 mg/kg daily ( Figure 5B , left ) and was well tolerated as measured by body weight ( Figure 5B , right ) .", "The anti-tumor activity of NVP-CGM097 correlated well with a dose-dependent induction in tumors of p21 and HDM2 at the mRNA level and/or protein levels ( Figure 5C , D ) .", "Thus , based on these results , the dose and schedule chosen for the follow up in vivo validation of the 13-gene signature was established at 100 mg/kg NVP-CGM097 given orally , once daily . 10 . 7554/eLife . 06498 . 014Table 2 . PK parameters of NVP-CFC218 and NVP-CGM097 in preclinical speciesDOI: http://dx . doi . org/10 . 7554/eLife . 06498 . 014PK parametersMouseRatDogMonkeyNVP-CFC218 CL ( mL/min . kg ) 912 ± 13 ± 111 ± 2 Vss ( L/kg ) 3 . 18 . 0 ± 1 . 83 . 6 ± 0 . 52 . 1 ± 0 . 3 t1/2app .", "( h ) 3 . 69 . 3 ± 1 . 914 . 4 ± 0 . 72 . 6 ± 0 . 3 AUCinf ( µM . h ) i . v . *2 . 802 . 21 ± 0 . 218 . 08 ± 1 . 682 . 37 ± 0 . 31 AUCinf ( µM . h ) p . o . *1 . 201 . 07 ± 0 . 345 . 74 ± 0 . 680 . 34 ± 0 . 05 Cmax ( µM ) p . o . *0 . 0940 . 075 ± 0 . 0230 . 240 ± 0 . 0430 . 077 ± 0 . 014 Tmax p . o . ( h ) 2 . 04 . 0 ± 1 . 43 . 3 ± 1 . 21 . 0 ± 0 . 1 Oral BA ( %F ) 4349 ± 1571 ± 814 ± 2NVP-CGM097 CL ( mL/min . kg ) 57 ± 13 ± 14 ± 1 Vss ( L/kg ) 2 . 66 . 4 ± 0 . 43 . 8 ± 0 . 42 . 0 ± 0 . 4 t1/2app .", "( h ) 6 . 412 . 1 ± 1 . 114 . 2 ± 1 . 88 . 3 ± 0 . 9 AUCinf ( µM . h ) i . v . *4 . 703 . 66 ± 0 . 279 . 44 ± 1 . 436 . 59 ± 0 . 74 AUCinf ( µM . h ) p . o . *3 . 342 . 97 ± 0 . 407 . 08 ± 1 . 053 . 73 ± 0 . 78 Cmax ( µM ) p . o . *0 . 2070 . 134 ± 0 . 0160 . 250 ± 0 . 0300 . 363 ± 0 . 020 Tmax p . o . ( h ) 4 . 04 . 5 ± 1 . 92 . 7 ± 1 . 21 . 3 ± 0 . 6 Oral BA ( %F ) 7181 ± 1175 ± 1157 ± 12Mouse ( n = 1 per time point ) and rat ( n = 3 per time point ) were treated either with a single dose of the indicated compound at 1 mg/kg i . v . or 3 mg/kg p . o . Dog and monkey ( n = 3 per time-point ) were treated either with a single dose of the indicated compound at 0 . 1 mg/kg i . v . or 0 . 3 mg/kg p . o . *AUC and Cmax values are shown dose-normalized to 1 mg/kg . 10 . 7554/eLife . 06498 . 015Figure 5 . Activity of NVP-CGM097 in SJSA-1 tumor-bearing mice .", "( A ) PK/PD relationship .", "Mice ( n = 3/time-point ) bearing established subcutaneous SJSA-1 xenografts received vehicle or a single oral dose of 100 mg/kg NVP-CGM097 .", "Levels of NVP-CGM097 were measured in plasma and in tumor at 7 different time-points within 48 hr following the dose .", "As a pharmacodynamic readout , the p53 target gene p21 mRNA ( left panel ) and protein ( right panel ) levels were assessed in the tumor samples by qRT-PCR and reverse phase protein array at 7 time-points within 48 hr following NVP-CGM097 single dose .", "Data are plotted as mean ± SEM .", "( B ) In vivo anti-tumor activity of NVP-CGM097 .", "Mice ( n = 6/dosing group ) bearing established subcutaneous SJSA-1 xenografts received vehicle or 25 , 50 , or 100 mg/kg of an oral suspension of NVP-CGM097 daily .", "Tumor volumes were calipered throughout the study , and data are plotted as mean ± SEM ( left panel ) .", "The body-weight ( BW ) is expressed as the percentage change in BW relative to day 0 ( initiation of treatment ) .", "Data are plotted as mean ± SEM ( right panel ) .", "* , p < 0 . 05 vs vehicle control .", "( C and D )", "Tumor pharmacodynamics effects of NVP-CGM097 .", "Tumor samples were retrieved 3 hr post-last dose at the end of the efficacy study described in ( B ) .", "Tumor p21 mRNA levels were assessed by qRT-PCR and data are plotted as mean ± SEM ( C ) .", "Tumor samples were retrieved and paraffin-embedded 3 hr post-last dose at the end of the efficacy experiment described in ( B ) .", "p21 and HDM2 protein levels were assessed by immunohistochemistry ( D ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06498 . 015 A total of 55 PDX models were used for these studies .", "NVP-CGM097 sensitivity was predicted for this sample set using the 13-gene signature ( see ‘Materials and Methods’ section for the specificities of these predictions and Figure 6—source data 2 ) .", "As shown in Figure 6A and Figure 6—source data 1 , 27/55 human primary xenograft tumor models were predicted to be sensitive to NVP-CGM097 , and of these 27 models , 19 were truly sensitive , resulting in a PPV of 70% , improving the basal response rate of 49% ( Figure 6C ) .", "Moreover , 28/55 in vivo models were predicted to be insensitive , and 20/28 were found to indeed be truly insensitive , leading to a significant NPV of 71% ( Figure 6C ) .", "Also , Sensitivity and Specificity features were comparable to the predictive model performance estimated on cell lines by the bootstrapping protocol ( Figure 6C; Figure 2—source data 1 ) .", "We also determined the performance of the 13-gene signature in tumors pre-selected based upon the presence of wild-type p53 .", "As shown in Figure 6B , 21/33 wild-type p53 human PDX models were predicted to be sensitive to NVP-CGM097 and of these , 18 were truly sensitive , resulting in a PPV of 86% , again significantly improving the basal response rate of 70% ( Figure 6C ) .", "In addition , 12/33 models were predicted to be insensitive to the drug , and 7/12 were found to be truly insensitive , leading to a significant NPV of 58% ( Figure 6C ) . 10 . 7554/eLife . 06498 . 016Figure 6 . Validation of the predictive 13-gene signature in patient-derived tumor xenografts ( PDX ) .", "PDX models were established by subcutaneous implantation in nude mice of surgical tumor tissues from treatment-naive cancer patients .", "55 PDX models of multiple cancer types were used for the study: melanoma ( n = 19 ) , colorectal cancer ( n = 17 ) , liposarcoma ( n = 3 ) , renal cell carcinoma ( n = 3 ) , hepatocellular carcinoma ( n = 7 ) , breast cancer ( n = 1 ) , pancreatic cancer ( n = 2 ) , and lung cancer ( n = 3 ) .", "Tumor-bearing mice ( n = 4/dosing group/model ) received vehicle or 100 mg/kg of NVP-CGM097 daily for 4 weeks .", "The response is reported as percentage change in tumor volume at a given day of treatment relative to day 0 ( start of treatment ) .", "The cut-off used for sensitivity to NVP-CGM097 was based on RECIST adapted for full tumor volume measurement .", "The sensitivity call was made at the maximum effect time-point during the treatment period .", "Sensitivity predictions for each PDX model were generated using the 13-gene signature ( Figure 6—source data 2 ) .", "( A ) Waterfall plot showing the tumor response to NVP-CGM097 for all the PDX models in the study ( n = 55 ) , color-coded by the prediction output .", "( B ) The same visualization as in ( A ) is shown restricted to the wild-type p53 PDX models ( n = 33 ) .", "The data used to generate the waterfall plots and PDX model details are available in Figure 6—source data 1 .", "Performance values of the 13-gene signature were derived from this experiment and are shown in ( C ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06498 . 01610 . 7554/eLife . 06498 . 017Figure 6—source data 1 . Sensitivity prediction and sensitivity to NVP-CGM097 of a set of in vivo PDX models ( n = 55 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06498 . 01710 . 7554/eLife . 06498 . 018Figure 6—source data 2 . Expression data of each of the 13 genes included in the gene signature in the set of in vivo PDX models ( n = 55 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06498 . 018 In conclusion , the in vitro predictive model performance shown in Figure 2 and Figure 2—source data 1 was confirmed in vivo using PDX models .", "The results further validate the 13-gene signature as a better predictor for response to p53–HDM2 inhibitors as compared to a selection strategy solely based on p53 mutation status .", "Notably , the findings described here support the use of p53–HDM2 inhibitor sensitivity predictive model in either non-selected tumors or wild-type p53 pre-selected tumors ." ], [ "In this study , we have identified a novel 13-gene signature that robustly predicts sensitivity to p53–HDM2 inhibitors , such as NVP-CGM097 .", "This novel 13-gene signature has a superior predictive value as compared to p53 wild-type status alone and importantly , when applied to p53 wild-type pre-selected tumors , it provides a further enrichment of the drug response rate .", "The newly discovered close analogs , NVP-CGM097 and NVP-CFC218 ( Figure 1A ) , are both members of a novel chemical class ( dihydroisoquinolinones ) which differs from Nutlin analogs ( e . g . , Nutlin 3a and RO5045337/RG7112 ) or other p53–HDM2 inhibitors currently being tested in the clinic ( e . g . , RO5503781/RG7388 , SAR405838/MI-773 , AMG 232 , DS3032b ) ( Masuya et al . , 2014 , manuscript in preparation ) .", "As to compare with Nutlin 3a , NVP-CGM097 and NVP-CFC218 show differences in binding mode within the p53 binding site of HDM2 ( Jeay et al . , 2014; Valat et al . , 2014 , manuscript in preparation ) , which allows better in vitro and in vivo on-target potency , more favorable drug-like properties , and improved in vivo behavior of this compound family ( Ferretti et al . , 2014; Jeay et al . , 2014 ) .", "NVP-CGM097 and NVP-CFC218 were first tested in cell-free assays , where both were shown to selectively inhibit p53–HDM2 binding .", "In cells , consistent with their mechanism of action ( Cheok et al . , 2011 ) and in line with their highly selective nature , both compounds showed comparable anti-proliferation effects in HCT-116 and SJSA-1 wild-type p53-expressing cell lines .", "The isogenic HCT-116 p53-null cells and the osteosarcoma SAOS-2 p53 null cells remained insensitive to NVP-CGM097 and NVP-CFC218 with IC50s ≥ 10 µM .", "Furthermore , in cell viability assays , using 356 cancer cell lines from the CCLE representative of various tumor types ( Barretina et al . , 2012 ) , NVP-CFC218 was shown to inhibit proliferation of about 13 . 2% of the cancer cell lines tested at relevant concentrations ( Figure 1B ) .", "As expected , 91 . 5% ( 43/47 ) of NVP-CFC218-sensitive cells expressed wild-type p53 , consistent with its mechanism of action ( Figure 1C , E ) .", "Importantly , the sensitivity of 3/4 of these cells bearing mutations in TP53 couldn't be reproduced ( P12-ICHIKAWA , KBMC-2 and Hs 294T with IC50 > 8 µM ) , while the sensitivity of the NCI-H2122 TP53 mutant cell line was confirmed in repeat cell proliferation assays .", "Interestingly , the reported p53 mutations in NCI-H2122 cells ( i . e . , Q16L and C176F p53 ) are categorized as being only partially deleterious , suggesting the p53 pathway remains partially functional in these cells .", "In line with the strong association of p53 mutation with lack of sensitivity to NVP-CFC218 , sensitivity response comparison to over 2000 compounds , grouped by their mechanism of action , showed that wild-type p53 cell lines in the CCLE were most sensitive to p53–HDM2 inhibitors , including NVP-CFC218 ( Figure 1F ) .", "Interestingly , 57% ( 57/100 ) of the p53 wild-type cell lines for which compound sensitivity data were available , scored insensitive to NVP-CFC218 ( Figure 1C and E ) .", "These results indicate that the selection of tumors based only on their p53 mutation status may not be optimal in order to tailor patient selection towards maximal response rate .", "With the aim to discover more sensitive biomarkers for patient selection , an in vitro predictive model was developed based on the integrative analysis of the genomic features of this panel of 356 cancer cell lines and their association with sensitivity to NVP-CFC218 ( Barretina et al . , 2012 ) .", "A 13-gene signature was found to be the optimal solution at predicting p53–HDM2 inhibition from gene expression ( Figure 2 ) .", "A further outcome of these studies identified p53 mutation status as being a strong predictor for insensitivity to NVP-CFC218 among about 40 , 000 input features containing genomic , lineage , and gene expression features , confirming in an unbiased manner the underlying hypothesis for this specific mechanism of action .", "In addition , a broader set of 215 newly discovered significant features were identified as being correlated to NVP-CFC218 sensitivity .", "Interestingly , HDM2 expression was found as the second ranked significant feature ( after p53 mutation ) confirming its correlation with sensitivity to p53–HDM2 inhibitors in an unbiased manner .", "However , HDM2 amplification was not revealed by the modeling , most likely because of the low representation of HDM2-amplified cell lines in CCLE .", "Surprisingly , 13/14 of the top most significant expression features corresponded to well-known p53 target genes and their up-regulated expression was found associated with increased sensitivity to NVP-CFC218 ( Table 1 ) .", "The 14th feature , unannotated at the time of this work , was found to be RPL22L1 , previously described as having a central role in αβ T lymphocytes development , together with its highly homologous paralog RPL22 , by up-regulating p53 in a tissue-specific manner ( Anderson et al . , 2007; Zhang et al . , 2013 ) .", "The positive predictive value of this 13-gene signature was found to outperform both the p53 mutation feature alone and the 215 significant feature-set ( 76% vs 63% and 59% , respectively ) ( Figure 2E ) .", "Furthermore , comparable performances were found in an additional set of independent cell lines tested for their sensitivity to NVP-CGM097 and NVP-CFC218 , validating the predictive power of the 13-gene signature in vitro ( Figure 3 ) .", "This cell line-derived 13-gene signature was demonstrated to be suitable for the identification of human primary tumors predicted to be sensitive to a p53–HDM2 inhibitor across a set of about 21 , 300 human tumor samples and across a collection of 503 patient-derived tumor xenograft models , with available genome-wide expression data .", "Interestingly , the prevalence of the 13-gene signature was found to be quite variable within the multiple lineages tested ( Figure 4A ) .", "Importantly , however , the lineage-specific proportion of samples scoring positive for the 13-gene signature was strikingly comparable to the proportion of sensitive cell lines to NVP-CFC218 , in a given lineage .", "Furthermore , the application of the signature across a large set of human tumor samples revealed new indications of potential interest for the development of p53–HDM2 inhibitors , such as renal cell carcinoma and hepatocellular carcinoma ( Figure 4C ) , that had been previously missed due to their under-representation in the set of CCLE cell lines tested .", "The ability of the 13-gene signature to differentiate signature-positive from signature-negative patient-derived tumor xenografts , allowed testing its in vivo predictive value across a set of these models .", "In line with the in vitro findings , the 13-gene signature was found to greatly outperform the random response rate to NVP-CGM097 in vivo , independently upon whether the xenograft models were pre-selected or not for their p53 mutation status ( Figure 6; Figure 6—source data 1 ) .", "Many research efforts have been devoted to the characterization of molecular mechanisms conferring gene-specific regulation within the p53 network ( Vousden and Prives , 2009 ) .", "It has been observed that in proliferating cells , minimal activity of p53 can lead to basal expression of some of its target genes , such as p21 ( Tang et al . , 1998 ) , through mechanisms involving basal promoter-specific recruitment of transcription initiation components ( Espinosa and Emerson , 2001; Espinosa et al . , 2003 ) .", "In a more recent study from Allen et al . ( 2014 ) , using Global Run-On sequencing , gene-specific regulatory mechanisms affecting a number of key survival and apoptotic genes within the p53 network were uncovered .", "The authors imply that some p53 target genes can be ‘primed’ to be switched on even before the p53 protein is activated , leading to a rapid transcription of these genes following p53 activation ( Allen et al . , 2014 ) .", "Some of these p53 target genes , such as CDKN1A , HDM2 , CCNG1 , DDB2 , FDXR , BAX , TNFRSF10B , and AEN , were found to be within the top most transcribed genes following Nutlin-3 treatment ( Allen et al . , 2014 ) and coincidently are all included in the 13-gene signature described in Table 1 .", "Taking together , these observations strongly suggest that the p53 target gene set contained within the predictive 13-gene signature is representative of an underlying activated p53 pathway that renders cancer cell lines and patient-derived tumor xenografts sensitive to p53–HDM2 inhibitors .", "Thus , it is reasonable to think that the 13-gene signature discovered here is best at predicting sensitivity to p53–HDM2 inhibitors , since it not only discriminates mutant p53 from wild-type p53 tumors , but also tumors that have an intact p53 gene but a silent p53 pathway from those that are p53 wild-type and bear an activated pathway .", "We postulate that the latter may be more prone to quickly induce a robust p53-dependent transcriptional response upon exposure to p53–HDM2 inhibitors , thus being more sensitive to this specific anti-cancer therapy .", "Altogether , this work highlights the power of a biomarker discovery approach based on large-scale data generation and unbiased data analysis , followed by robust validation using various independent samples sets .", "Furthermore , the finding that the 13-gene signature corresponds to a set of p53 target genes and that its positivity reflects activity of the p53 pathway , demonstrates the intimate relationship between the biomarker set and the mechanism of action of the targeted therapy , hence its potentially broad applicability to any selective p53–HDM2 inhibitor for optimal patient selection .", "On these bases , we are currently evaluating the 13-gene signature in a Phase I clinical trial ( NCT01760525 ) , in cancer patients bearing p53 wild-type tumors treated with NVP-CGM097 ." ], [ "NVP-CFC218 and NVP-CGM097 were synthesized by Global Discovery Chemistry ( NIBR , Novartis , Basel , Switzerland ) .", "For in vitro studies , 10 mM stock solutions were prepared in 100% dimethyl sulfoxide ( DMSO ) .", "For in vivo experiments , NVP-CGM097 was formulated immediately before each oral administration in a suspension of 0 . 5% hydroxy-propyl-methyl-cellulose ( HPMC ) .", "NVP-CFC218 and NVP-CGM097 biochemical activity against the p53–HDM2 and p53–HDMX interactions was assessed in a TR-FRET assay .", "Standard assay conditions consisted of 60 μl total in PBS buffer containing 125 mM NaCl , 0 . 001% Novexin , 0 . 01% Gelatin , 0 . 2% Pluronic F-127 , 1 mM DTT , and 1 . 7% final DMSO .", "Both NVP-CFC218 and NVP-CGM097 were added at different concentrations to 0 . 1 nM biotinylated HDM2 ( amino acids 2-188 ) or 0 . 1 nM HDMX ( amino acids 2-185 ) , 0 . 1 nM Europium-labeled streptavidin and 10 nM Cy5-p53 peptide ( Cy5-p53 amino acids 18–26 ) .", "After incubation at room temperature for 30 min , samples were measured on a GeniosPro reader ( Tecan ) .", "FRET assay readout was calculated from the raw data of the two distinct fluorescence signals measured in time resolved mode ( fluorescence 665 nm/fluorescence 620 nm × 1000 ) .", "IC50 values were calculated by curve fitting using XLfit ( Fit Model #205 ) .", "The isogenic HCT-116 p53-null cell line was obtained from Horizon ( Cambridge , UK ) .", "All other cell lines were obtained from ATCC ( American Type Culture Collection ) , DSMZ ( Deutsche Sammlung von Mikroorganismen und Zellkulturen ) , and HSRRB ( Health Science Research Resources Bank ) and cultured in RPMI or Dulbecco's modified Eagle's medium plus 10% FBS ( Invitrogen , Carlsbad , CA ) at 37°C , 5% CO2 .", "Cell line identities were confirmed using a 48-variant SNP panel .", "Effects of NVP-CFC218 and NVP-CGM097 on cellular growth and loss of viability shown in Figure 1B were measured using the YOPRO assay ( Invitrogen ) and IC50 values were calculated by curve fitting using XLfit ( Fit Model #201 ) .", "A detailed description of the high-throughput cell viability assays can be found in the report of Barretina et al . ( 2012 ) .", "To identify compounds to which wild-type p53 cell lines were selectively responsive as compared to non-wild-type p53 cell lines across the CCLE , we used the high-throughput cell line profiling described above and reported in Barretina et al . ( 2012 ) .", "First , a selected compound sensitivity metric was log2 transformed and a selectivity score was computed by multiplying the metric Z-score by the absolute value of the metric .", "We then performed a Wilcoxon test opposing the two groups of cell lines and corrected for multiple testing using Benjamini and Hochberg FDR , followed by an enrichment analysis by the compound's main target .", "To this end , compounds were grouped into target classes , and Fisher's exact tests were performed with compounds considered as significantly differentially responsive when the FDR was below 0 . 25 .", "The cut-off for the FDR is lenient as this is used for enrichment purposes .", "Finally , we corrected the p-values obtained from enrichment analysis for multiple testing .", "The genomic and genetic characterization of a panel of cancer-relevant cell lines was undertaken mostly as described ( Barretina et al . , 2012 ) .", "Briefly , gene-level expression values were generated with the HG-U133 plus 2 array ( Affymetrix GeneChip technology ) and summarized according to the RMA normalization method; gene-level chromosome copy number values were obtained with the Affymetrix SNP6 . 0 technology and processed using the Affymetrix apt software and expressed as log2 transformed ratios to a collection of HapMap reference normal samples; gene-level genetic alterations ( point-mutations , insertions , deletions , and complex alterations ) were compiled from the Sanger center COSMIC data and internal sources including Exome Capture Sequencing of 1600 cancer-related genes; pathway-level expression values were generated as referenced in the GeneGo Metacore knowledge base; cell line lineage ( cell line tissue of origin ) ; gene-level ‘tumor suppressor status’ was generated by integrating the genetic alteration , copy number , and expression information .", "Such genomic and genetic data were assembled in a matrix that covers a total of about 40 , 000 genomic features and were used to test their association with cell line chemical sensitivity to NVP-CFC218 .", "All Affymetrix GeneChip Human Genome U133 Plus 2 . 0 arrays were pre-processed with a custom chip definition file ( Entrez Gene-centric custom CDF version 16 from the University of Michigan ) using the RMA ( robust multi-array average ) summarization/normalization algorithm ( Irizarry et al . , 2003; Dai et al . , 2005 ) .", "A set of pre-computed reference quantiles and probe effects was input to the algorithm for normalization of Affymetrix arrays in an approach known as the Extrapolation Strategy or refRMA ( Goldstein , 2006; Katz et al . , 2007 ) .", "The reference quantiles and probe effects were defined from a compendium of publically available arrays representing a broad diversity of oncology indications ( through the expO data set but not exclusively ) and human body normal tissues ( GEO series accessions GSE5764 , GSE6338 , GSE2109 , GSE28504 , GSE6764 , GSE7307 , GSE32317 , GSE7753 , GSE8507 , GSE7904 , GSE8671 , GSE8762 , GSE4107 , GSE4183 , GSE8977 , GSE20238 , GSE20596 , GSE13911 , GSE10282 , GSE9829 , GSE9843 , GSE7553 , GSE9891 , GSE9899 , GSE6004 and GSE4237 ) .", "The RefPlus R package was used to that aim ( Harbron et al . , 2007 ) .", "To define the most accurate and parsimonious predictive model from CCLE expression data , a bootstrapping protocol was used first which is described in the ‘Results’ section .", "All calculations were done with R-3 . 0 . 2 using principally e1071 and caret R libraries .", "The broader , 215-feature set , that discriminates NVP-CFC218 sensitive from insensitive cell lines , was obtained as follows: Wilcoxon signed-rank tests or Fisher's exact tests were used to compare the genomic features of the sensitive to insensitive groups .", "Features having continuous values ( gene expression and copy number , pathway expression features ) were subjected to Wilcoxon sum rank test , while those having discrete values ( genetic alteration , tumor suppressor status , and lineage features ) to Fisher's exact test .", "Significant features passed a local ( by feature type ) FDR of 0 . 25 .", "Irrespective of the FDR , a minimum or maximum number of features per feature type were also required .", "To minimize the impact of the high degree of correlation among the features on the feature selection step , the feature data were clustered before statistical testing by Affinity Propagation method ( Frey and Dueck , 2007 ) to only keep features representing the most variability .", "To compare sensitivity prediction accuracies of the 13-gene signature to both , the 215-feature set and TP53 mutation , naïve Bayes probabilistic models were then built from the respective feature sets .", "The performances of classifications were evaluated with 5 repeats of 5-fold cross-validations of the train data .", "To transform the naive Bayes probabilities into a sensitive or insensitive class-level prediction , a threshold was defined as the probability maximizing the sensitivity and specificity .", "The entire and nearly identical procedure is described in more details in Barretina et al . ( 2012 ) .", "The human primary tumor sample collection and the patient-derived tumor xenograft model collection ( PDX ) consist of about 21 , 300 and 500 samples , respectively , for which gene expression profiles , generated with Affymetrix technology ( Human Genome U133 plus 2 . 0 array ) , are available .", "The samples of the collection were internally annotated with controlled vocabulary for pathology , histology , and primary site using the COSMIC Classification System ( Forbes et al . , 2008 ) , and were submitted for sensitivity prediction ( Figure 6—source data 2 ) .", "The associated GeneChip data were gathered from both public ( GEO , ArrayExpress ) and internal sources , and RMA normalized as described above .", "The naïve Bayes model , used to predict NVP-CFC218 sensitivity , was trained on the 251 CCLE expression data restricted to the 13-gene signature features .", "PDX predictions of NVP-CFC218 sensitivity under the 13-gene signature were undertaken almost as described above .", "However , to account for the latest NVP-CFC218 pharmacological characterization on cell lines and to take advantage of a larger training data set , the naïve Bayes classifier was trained on a wider cell line compendium ( 634 cell lines , 77 and 557 of which being NVP-CFC218-sensitive and NVP-CFC218-insensitive , respectively ) .", "Moreover , the naïve Bayes-predictive model probability threshold , above which a xenograft tumor model is predicted as sensitive to p53–HDM2 inhibition , was also further set to 0 . 2 ( optimizing for PPV/Precision at the cost of Sensitivity/Recall ) .", "All animal studies were conducted in accordance to procedures covered by permit number 1975 issued by the Kantonales Veterinäramt Basel-Stadt and strictly adhered to the Eidgenössisches Tierschutzgesetz and the Eidgenössische Tierschutzverordnung .", "All animals were allowed to adapt for 4 days and housed in a pathogen-controlled environment ( 5 mice/Type III cage ) with access to food and water ad libitum and were identified with transponders .", "Immunohistochemistry has been performed on a Ventana Discovery XT automated immunostainer .", "SJSA-1 xenograft tumors were collected and a 3- to 4-mm slice was cut out of the middle of the tumor , fixed in neutral buffered formalin and embedded in paraffin .", "3 μm sections were processed for immunohistochemistry ( IHC ) .", "Primary antibodies used were mouse monoclonal anti-p21 antibody ( #M7202 , Dako , Carpenteria , CA ) and mouse monoclonal anti-HDM2 antibody ( #965 , Santa-Cruz Biotechnology , Santa-Cruz , CA ) .", "Sections were subsequently stained using the labeled polymer system Simple Stain Mouse MAX PO ( M ) from the N-Histofine Mousestain Kit ( Nichirei Bioscience Inc . , Japan ) and DAB substrate from the DABMap Kit omitting the SA-HRP solution ( Ventana/Roche Diagnostics , Mannheim , Germany ) .", "Counterstaining of sections was done using hematoxylin ( Ventana/Roche Diagnostics ) ." ] ]

[ "Biomarkers for patient selection are essential for the successful and rapid development of emerging targeted anti-cancer therapeutics .", "In this study , we report the discovery of a novel patient selection strategy for the p53–HDM2 inhibitor NVP-CGM097 , currently under evaluation in clinical trials .", "By intersecting high-throughput cell line sensitivity data with genomic data , we have identified a gene expression signature consisting of 13 up-regulated genes that predicts for sensitivity to NVP-CGM097 in both cell lines and in patient-derived tumor xenograft models .", "Interestingly , these 13 genes are known p53 downstream target genes , suggesting that the identified gene signature reflects the presence of at least a partially activated p53 pathway in NVP-CGM097-sensitive tumors .", "Together , our findings provide evidence for the use of this newly identified predictive gene signature to refine the selection of patients with wild-type p53 tumors and increase the likelihood of response to treatment with p53–HDM2 inhibitors , such as NVP-CGM097 ." ]

[ "Stress from daily activities and exposure to chemicals or UV radiation can all damage cells .", "Damaged cells may develop into cancerous tumors if unchecked .", "Normally , a protein called p53 helps to repair or eliminate damaged cells and prevent tumors from forming .", "The p53 protein does this by switching on or off genes that control DNA repair , cell division , and cell death .", "But half of all cancerous tumors have mutations that prevent p53 from doing its job .", "Another protein called HDM2 keeps p53 in check by binding to p53 and preventing it from switching on and off genes after the stress passes .", "In cancers that have normal p53 , sometimes HDM2 is overly active and prevents p53 from suppressing tumor formation and growth .", "Scientists are developing anticancer drugs that work by targeting HDM2; this frees p53 and allows it to wipe out cancerous cells .", "However , it is not always clear which patients with cancer are the most likely to benefit from anti-HDM2 therapy .", "Jeay et al . screened hundreds of cancer cells to determine which ones are sensitive to HDM2-targeting drugs .", "As expected , the screen revealed that cancer cells that have mutations in the gene encoding p53 are insensitive to the anti-HDM2 drug because there is no working p53 to free up .", "But about 60% of the cancer cells that have normal p53 proteins also did not respond to the anti-HDM2 therapy .", "This finding indicates that the presence of normal p53 protein is necessary but not sufficient for tumor cells to respond to anti-HDM2 therapy .", "Next , Jeay et al . compared the patterns of gene expression in the cancer cells that responded to an anti-HDM2 drug with those in cells that didn't respond .", "The analysis showed that a group of 13 genes are expressed more in the cells that responded to the drug .", "All 13 genes are unexpectedly direct targets of p53 , suggesting that p53 remains active in these tumor cells , even if it is not working optimally .", "To verify these results , Jeay et al . grew human tumors in mice and found that tumors with high expression of the 13 genes are sensitive to the anti-HDM2 drug ( called NVP-CGM097 ) .", "The experiments strongly suggest that this 13-gene signature can be used to determine if a patient with cancer will respond to anti-HDM2 therapy .", "Following on from this work , researchers have already launched an early clinical trial with the anti-HDM2 drug and will test whether this gene signature is indeed useful in a real clinical setting ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "biochemistry and chemical biology", "cell biology" ]

[ [ "Niemann-Pick C1 ( NPC1 ) , a lysosomal membrane protein , has emerged as the culprit in two fatal diseases .", "First , mutations in NPC1 underlie Niemann-Pick C disease , a devastating lysosomal storage disease in which accumulation of cholesterol in lysosomes causes abnormalities in brain , liver , lung , and other tissues , leading to death in the teenage years ( Pentchev , 2004 ) .", "Second , NPC1 is the receptor that permits cellular entry of the RNA genomes of lethal Ebola virus , Marburg virus , and other filoviruses ( Carette et al . , 2011; Côté et al . , 2011 ) .", "In this paper , we use the term ‘lysosome’ in its inclusive sense to encompass late endosomes where NPC1 may also function .", "NPC1 mediates the egress of cholesterol that has entered lysosomes through the receptor-mediated endocytosis of low density lipoprotein ( LDL ) ( Liscum et al . , 1989 ) .", "When cell membranes are depleted of cholesterol , LDL receptors bind circulating LDL , internalize it , and deliver it to lysosomes where acid lipase hydrolyzes the lipoprotein’s cholesteryl esters ( Brown and Goldstein , 1986 ) .", "The liberated cholesterol binds to NPC2 , a soluble diffusible protein in the lysosome lumen ( Sleat et al . , 2004 ) .", "NPC2 transports the cholesterol to the lysosomal membrane where it transfers its cholesterol to membrane-embedded NPC1 ( Wang et al . , 2010 ) .", "Designated a hydrophobic handoff , this transfer occurs in such a way that hydrophobic cholesterol is shielded from the aqueous environment ( Kwon et al . , 2009 ) .", "NPC1 is an intrinsic membrane protein of 1278 amino acids that contains 13 membrane spanning helices separated by hydrophilic loops and 19 potential N-linked glycosylation sites ( Davies and Ioannou , 2000 ) ( see Figure 1 ) .", "NPC2 transfers its cholesterol to the N-terminal domain ( NTD ) of NPC1 , which is the hydrophilic sequence of 239 amino acids that projects into the lysosomal lumen after the signal peptide is cleaved ( Infante et al . , 2008a; 2008b ) .", "NPC2 binds to the second luminal loop of NPC1 , an event that may precede the cholesterol transfer to the NTD ( Deffieu and Pfeffer , 2011 ) .", "Mutations that abolish the function of either NPC2 or NPC1 lead to cholesterol accumulation in lysosomes and cause the fatal Niemann-Pick C disease ( Pentchev , 2004; Dixit et al . , 2011 ) . 10 . 7554/eLife . 12177 . 003Figure 1 . Domain structure of human Niemann-Pick C1 ( NPC1 ) .", "The predicted topology of the polytopic protein is based on the data of Davies and Ioannou ( 2000 ) .", "Each of the five known functional domains of the protein are shown in a different color .", "The locations of three loss-of-function mutations referred to in the manuscript are indicated .", "aa , amino acid; NTD , N-terminal domain . DOI: http://dx . doi . org/10 . 7554/eLife . 12177 . 003 After its transfer to NPC1 , cholesterol leaves the lysosome by a mechanism that is poorly understood .", "Some of the cholesterol reaches the endoplasmic reticulum ( ER ) where it has two actions .", "First , it binds to Scap , an ER protein that regulates the cleavage and activation of membrane-bound sterol regulatory element-binding proteins ( SREBPs ) .", "Cholesterol binding to Scap prevents the protease-mediated release and nuclear entry of the active fragment of SREBPs , thereby blocking cholesterol synthesis and uptake from LDL ( Horton et al . , 2002 ) .", "When excess cholesterol reaches the ER , it is esterified with a long chain fatty acid through the action of acyl-coenzyme A cholesterol acyltransferase ( ACAT ) , which converts the cholesterol to its storage form as cholesteryl esters ( Brown et al . , 1975 ) .", "Lysosome-derived cholesterol also reaches the plasma membrane where it fills three distinguishable pools , thereby assuring membrane integrity ( Das et al . , 2014 ) .", "The role of NPC1 in Ebola virus infection was recognized in 2011 through groundbreaking work by two groups .", "One group identified NPC1 through a haploid mutagenesis screen to detect genes required for Ebola infection in cultured cells ( Carette et al . , 2011 ) .", "The other group identified small molecules that block Ebola infection and used ultraviolet crosslinking to trace their target to NPC1 ( Côté et al . , 2011 ) .", "Mutant cells that lack NPC1 were found to resist Ebola virus infection , and infectivity was restored by transfection with a plasmid expressing full length NPC1 ( Carette et al . , 2011 ) .", "In vitro binding studies demonstrated that an Ebola virus surface glycoprotein binds to luminal loop 2 of NPC1 ( Miller et al . , 2012 ) , the same loop that binds to NPC2 ( amino acids 373–620 ) .", "Indeed , Ebola sensitivity was restored when NPC1-deficient Chinese Hamster Ovary ( CHO ) cells were transfected with a plasmid encoding only the luminal loop 2 portion of NPC1 ( Miller et al . , 2012 ) .", "The latter data indicate that NPC1 serves only as the passive attachment site for the Ebola glycoprotein and that the presumed cholesterol transport activity of NPC1 is not required .", "A powerful tool for the study of lysosomal cholesterol transport and Ebola virus infectivity is an androstenolone derivative termed U18666A that inhibits three enzymes in the cholesterol biosynthesis pathway ( Cenedella , 2009 ) .", "In 1989 ( Liscum and Faust , 1989 ) demonstrated that U18666A has another action: it inhibits the exit of LDL-derived cholesterol from lysosomes , thereby creating the equivalent of NPC1 deficiency .", "U18666A is a cationic amphiphile owing to a diethylaminoethyl chain attached to the 3-hydroxyl .", "Other cationic amphiphiles with very different structures also inhibit cholesterol egress from lysosomes ( Lange et al . , 2000 ) .", "The structural discrepancies created some confusion as to mechanism of inhibition .", "However , Wojtanik and Liscum ( 2003 ) reported that U18666A is more than 100-fold more potent than another cationic amphiphile , imipramine , raising the possibility that U18666A directly inhibits a protein required for lysosomal egress , whereas the low potency amphiphiles may have a nonspecific effect .", "U18666A and other amphiphiles have also been reported to block the cellular entry of Ebola virus RNA ( Shoemaker et al . , 2013 ) .", "However , the concentrations required are in the micromolar range rather than the nanomolar range at which U18666A blocks cholesterol egress , suggesting that U18666A acts through a nonspecific effect on Ebola virus as opposed to its specific effect on cholesterol transport .", "In the current study , we used an unbiased chemical screen to identify a postulated cholesterol transport protein that is inhibited by low concentrations of U18666A .", "For this purpose , we synthesized U-X , a derivative of U18666A that contains a benzophenone moiety that attaches covalently to proteins when exposed to ultraviolet light ( Gubbens et al . , 2009 ) .", "The U-X compound also contains an alkyne group that allows attachment of fluorophores and other functional elements through click chemistry ( Kolb et al . , 2001; Sapkale et al . , 2014 ) .", "U-X retains the ability to disrupt LDL-mediated cholesterol egress from lysosomes with high affinity .", "We also synthesized a series of U18666A derivatives that either retain or lose the ability to block lysosomal cholesterol export , and we used these as competitors to assure the specificity of the U-X crosslinking reaction .", "We identified one protein whose U18666A-binding properties matched the requirements for inhibition of cholesterol export , and that protein turned out to be NPC1 ." ], [ "Figure 1 shows the predicted topology of NPC1 ( Davies and Ioannou , 2000 ) together with its five known functional domains .", "The locations of the three loss-of-function mutations discussed in this manuscript are indicated .", "Figure 2 shows the structure of U18666A and the various derivatives that were synthesized for the current studies .", "For each compound , we show the inhibitory constant ( Ki ) that represents the concentration producing a 50% inhibition of the transfer of LDL-derived cholesterol from lysosomes to ER as determined with the cholesterol esterification assay ( see legend to Figure 2 ) .", "U18666A ( designated U ) is a derivative of androstenolone wherein the 3-hydroxyl is modified as a diethylaminoethyl ether , which will be charged ( protonated ) at neutral pH ( pKa 9 . 41 ) .", "As a control for specificity of binding , we synthesized compound A in which a dimethylamino group is introduced at the 17-position and the nitrogen in the ether-linked chain is replaced by a carbon .", "Inasmuch as compound A retains a dialkylamine , it will be similarly protonated at neutral pH ( pKa of 10 . 21 as compared with 9 . 41 for U18666A ) . 10 . 7554/eLife . 12177 . 004Figure 2 . Chemical structures of U18666A and derivatives used in this study . Inhibitory constant ( Ki ) values denote the concentration at which each compound inhibited the incorporation of [14C]oleate into cholesteryl [14C]oleate by 50% in monolayers of intact Chinese Hamster Ovary 7 ( CHO-7 ) cells that were incubated with 10% fetal calf serum ( FCS ) ( mean of 3–14 experiments for each compound ) .", "Assays were carried out under conditions identical to those described in Figure 3A . DOI: http://dx . doi . org/10 . 7554/eLife . 12177 . 004 In order to identify the target of U18666A , we synthesized compound U-X that retains the basic dialkylamino group present in U18666A , but includes a benzophenone and an alkyne group ( Figure 2 , bottom panel ) .", "When U-X binds to a protein and the benzophenone is exposed to 306 nM UV light , the compound attaches covalently to nearby backbone atoms of the peptide chain .", "Through the use of click chemistry , the alkyne group can then be attached to any compound that contains an azide group , including the fluorophore Alexa Fluor 532 Azide .", "The combination of UV crosslinking and fluorescent tagging permits visualization of the protein that has bound U-X .", "Figure 2 also shows the structures of four other compounds ( B , C , D , E ) that were used for various control experiments .", "To measure the transfer of LDL-derived cholesterol from lysosomes to ER in cultured CHO cells , we use two assays: cholesterol esterification and proteolytic cleavage of SREBP-2 .", "In both assays , we first deplete cells of cholesterol by incubating them in the absence of lipoproteins ( i . e . , in lipoprotein-deficient serum , LPDS ) and the presence of compactin , an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A ( HMG CoA ) reductase , the rate-limiting enzyme in cholesterol synthesis .", "We add a low concentration of mevalonate , the product of HMG CoA reductase , to allow the cells to synthesize nonsterol products of the enzyme ( Goldstein and Brown , 1990 ) .", "After 16 hr , we switch the cells to a medium containing lipoproteins in the form of human LDL , fetal calf serum ( FCS ) , or rabbit β-migrating very low density lipoprotein ( β-VLDL ) , all of which deliver cholesterol to lysosomes via the LDL receptor ( Goldstein et al . , 1983 ) .", "For the cholesterol esterification assay , we wait 3–5 hr and then add [14C]oleate for 1–2 hr .", "The cells are harvested and the amount of [14C]oleate incorporated into cholesteryl [14C]oleate is measured by thin layer chromatography and scintillation counting ( see Materials and methods ) .", "As a control for nonspecific effects , we also measure the amount of [14C]oleate incorporated into [14C]triglycerides .", "This incorporation does not depend upon lipoprotein-derived cholesterol , and it was not affected by any of the manipulations in the current experiments .", "The values for [14C]triglycerides are given in the figure legends .", "The second assay relies on the ability of cholesterol to block the proteolytic cleavage and nuclear localization of SREBP-2 ( Brown and Goldstein , 1997 ) .", "Synthesized as an intrinsic protein of ER membranes , SREBP-2 binds immediately to Scap , an escort protein .", "When the ER membranes are low in cholesterol , the Scap/SREBP-2 complex is transported to the Golgi where the SREBP-2 is cleaved by two proteases , liberating a soluble fragment that translocates to the nucleus .", "When LDL-derived cholesterol reaches the ER , it binds to Scap and blocks transport of the Scap/SREBP-2 complex to the Golgi .", "As a result , the amount of nuclear SREBP-2 declines as revealed by sodium dodecyl sulfate polyacrylamide gel electrophoresis ( SDS-PAGE ) and immunoblotting .", "Figure 3A shows a cholesterol esterification assay in which 10% FCS was used as a source of LDL-cholesterol .", "U18666A inhibited the incorporation of [14C]oleate into LDL-derived cholesterol with high affinity ( Figure 3A ) .", "U-X retained similar inhibitory activity .", "On the other hand , compound A was nearly 100-fold less active .", "These experiments were repeated several times , and the calculated Ki values were 0 . 03 , 0 . 06 , and 4 . 5 µM for U18666A , compound U-X , and compound A , respectively .", "These differences in Ki values between U18666A and A indicate the necessity for a correctly positioned dialkylamino group to achieve inhibitory activity .", "In separate experiments , we showed that U18666A did not block cholesterol esterification when it is stimulated by 25-hydroxycholesterol , which enters cells without traversing lysosomes ( Abi-Mosleh et al . , 2009 ) .", "We also found that U18666A does not inhibit cholesteryl [14C]oleate formation when cholesterol is added to ER membranes in vitro . 10 . 7554/eLife . 12177 . 005Figure 3 . Cholesterol esterification , sterol regulatory element-binding protein ( SREBP ) 2 processing , and 125I-LDL ( low density lipoprotein ) degradation in Chinese Hamster Ovary ( CHO ) 7 cells . On day 0 , CHO-7 cells were set up for experiments in medium A with 5% lipoprotein-deficient serum ( LPDS ) at a density of 2 . 5 x 105 cells/60-mm dish .", "On day 2 , the medium was switched to fresh medium containing 5 µM sodium compactin and 50 µM sodium mevalonate .", "On day 3 , the medium was switched to medium B containing either 10% LPDS or 10% fetal calf serum ( FCS ) , 50 µM compactin , 50 µM mevalonate , and various concentrations of the indicated compound and then incubated for either 7 hr ( A ) or 6 hr ( B ) as described below .", "( A ) Cholesterol esterification .", "After a 5 hr incubation with the above medium with 10% FCS and the indicated compounds , each cell monolayer was labeled for 2 hr with 0 . 2 mM sodium [14C]oleate ( 8133 dpm/nmol ) .", "The cells were then harvested for measurement of their content of cholesteryl [14C]oleate and [14C]triglycerides .", "Each value is the average of duplicate incubations .", "Values for [14C]triglyceride content in cells treated with 1 µM of U18666A , compound A , and U-X were 117 , 116 , and 106 nmol/hr/ mg protein , respectively .", "( B ) SREBP-2 processing .", "After a 5 hr incubation with the above medium containing either 10% LPDS ( lane 1 ) or 10% FCS ( lanes 2–11 ) , each monolayer received a direct addition of 20 µg/ml of N-acetyl-leu-leu-norleucinal ( A . G . Scientific , San Diego , CA ) .", "After 1 hr , cells were harvested and fractionated into a nuclear extract and 105g membrane fraction ( Sakai et al . , 1996 ) .", "Aliquots ( 30 and 10 μg protein for SREBP-2 and Niemann-Pick C1 [NPC1] , respectively ) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis ( SDS-PAGE ) .", "Immunoblot analysis and image scanning were carried out with monoclonal antibodies directed against SREBP-2 or NPC1 as described in Materials and methods .", "( C ) 125I-LDL degradation .", "On day 3 , the medium was switched to medium B containing 5% human LPDS , 20 µg protein/ml of either 125I-LDL ( for 125I-LDL degradation ) or unlabeled LDL ( for cholesterol esterification ) , 10 µM compactin , and 50 µM mevalonate in the presence of one of the following compounds: none , 0 . 3 µM U18666A , 0 . 3 µM U-X , and 50 µM chloroquine .", "For the 125I-LDL degradation assay , cells were incubated for 6 hr with 125I-LDL ( 48 cpm/ng protein ) , after which the medium from each monolayer was removed and its content of 125I-monoiodotyrosine was measured as previously described ( Goldstein , 1983 ) .", "The 100% control value for 125I-LDL degradation in the absence of any compound ( none ) was 4 . 1 µg/6 hr/mg of protein .", "The cholesterol esterification assay was carried out as in A except that the cells were pulse-labeled with 0 . 1 mM [14C]oleate ( 6515 dpm/nmol ) .", "The 100% control value for cholesteryl [14C]oleate formed was 5 . 9 nmol/hr/mg protein .", "The content of [14C]triglycerides in cells receiving the various compounds were not significantly different: 50 , 57 , 55 , and 81 nmol/hr/mg protein , respectively , for no addition , U18666A , U-X , and chloroquine .", "All values are the mean of triplicate incubations with individual values shown . DOI: http://dx . doi . org/10 . 7554/eLife . 12177 . 005 In the SREBP-2 cleavage assay , the addition of 10% FCS led to a major reduction in the amount of nuclear SREBP-2 ( Figure 3B ) .", "At the lowest concentration tested ( 0 . 1 µM ) , U18666A prevented this reduction .", "U-X also blocked at low concentrations , whereas compound A at 1 µM had no activity .", "For completeness , we show the immunoblots for the membrane-bound precursor form of SREBP-2 and NPC1 , the latter used as a loading control .", "To confirm that U18666A does not block the receptor-mediated uptake of LDL or the lysosomal degradation of its protein , we incubated CHO cells with 125I-labeled LDL for 6 hr , after which we measured the amount of 125I-monoiodotyrosine released into the medium .", "As shown in Figure 3C , U18666A and U-X did not inhibit degradation of 125I-LDL even though both compounds inhibited the lysosomal exit of cholesterol as determined by inhibition of incorporation of [14C]oleate into cholesteryl [14C]oleate .", "As a positive control , we incubated the cells with chloroquine , a cationic compound that accumulates in lysosomes where it raises the pH above the optimum for lysosomal proteases and lipases ( Goldstein et al . , 1975 ) .", "Chloroquine blocked the degradation of 125I-LDL as well as the release of LDL-derived cholesterol .", "These data indicate that U18666A acts specifically as an inhibitor of cholesterol export and not nonspecifically as a lysosomotropic agent .", "To test the hypothesis that U18666A targets NPC1 , we compared its ability to block the esterification of LDL-derived cholesterol in CHO-7 cells and in TR-4139 cells , a clone of CHO-7 cells that were engineered to stably express excess NPC1 ( Figure 4 ) .", "Overexpression of NPC1 raised the threshold for U18666A inhibition by more than 100-fold ( Ki 0 . 02 µM vs . 2 . 7 µM ) .", "Immunoblots revealed the overexpression in the TR-4139 cells ( Figure 4 , inset ) .", "These quantitative results confirm previous nonquantitative results using Filipin staining to visualize resistance to U18666A in CHO-K1 cells that were engineered to overexpress NPC1 ( Ko et al . , 2001 ) . 10 . 7554/eLife . 12177 . 006Figure 4 . Cholesterol esterification in Chinese Hamster Ovary ( CHO ) 7 and TR-4139 cells that overexpress Niemann-Pick C1 ( NPC1 ) .", "CHO-7 cells and TR-4139 cells were set up for experiments in medium A with 5% lipoprotein-deficient serum ( LPDS ) as described in the legend to Figure 3 .", "On day 2 , the medium was switched to fresh medium containing 5 µM sodium compactin , 50 µM sodium mevalonate , and the indicated concentration of U18666A .", "After incubation for 4 hr , each cell monolayer was labeled for 2 hr with 0 . 2 mM sodium [14C]oleate ( 11 , 662 dpm/nmol ) .", "The cells were then harvested for measurement of their content of cholesteryl [14C]oleate and [14C]triglycerides .", "Each value is the average of duplicate incubations .", "The cellular content of [14C]triglycerides in CHO-7 and TR-4139 cells was 38 and 40 nmol/hr/mg protein , respectively .", "Inset shows immunoblots of sodium dodecyl sulfate polyacrylamide gel electrophoresis ( SDS-PAGE ) gels of whole cell extracts ( 30 µg ) from the indicated cell line incubated with 0 . 5 µg/ml anti-NPC1 or 1 µg/ml anti-calnexin as described in Materials and methods . DOI: http://dx . doi . org/10 . 7554/eLife . 12177 . 006 Inasmuch as U-X retains the ability to block cholesterol exit from lysosomes ( Figure 3 ) , we used this compound to identify the target of U18666A .", "For this purpose , we studied wild-type CHO-K1 cells and 10–3 cells , a mutant CHO-K1 cell line that lacks NPC1 .", "The cells were incubated with U-X alone or in the presence of U18666A or the inactive derivative compound A . As described in Materials and methods , cells were irradiated with UV light after which we prepared homogenates solubilized with sodium dodecyl sulfate ( SDS ) .", "We used click chemistry to attach Alexa Fluor 532 azide to the alkyne on U-X .", "The proteins were then subjected to SDS-PAGE and fluorescently tagged proteins were visualized with a fluorescence imager .", "Several fluorescent proteins were visualized in a UV-dependent manner ( Figure 5A , lanes 4–13 ) .", "Only one of these fluorescent bands disappeared when the cells were incubated with excess U18666A , but not with compound A ( lanes 4–6 ) .", "The apparent molecular mass of this protein was 190 kDa , which matches the apparent molecular mass of NPC1 in the same gel system .", "The 190-kDa band was not seen when the NPC1-deficient mutant cells were subjected to the same procedure ( lanes 7–9 ) . 10 . 7554/eLife . 12177 . 007Figure 5 . Ultraviolet ( UV ) crosslinking of U-X to Niemann-Pick C1 ( NPC1 ) in Chinese Hamster Ovary ( CHO ) -K1 cells , but not in mutant 10–3 cells . On day 0 , CHO-K1 cells and 10–3 cells ( mutant derivative of CHO-K1 cells that lack NPC1 ) were set up in a six-well plate in medium A with 5% lipoprotein-deficient serum ( LPDS ) ( 2 ml/35-mm well ) .", "( A ) In-gel fluorescence of U-X binding proteins in parental CHO-K1 cells and mutant 10–3 cells that lack NPC1 .", "On day 3 , each well of cells received a direct addition of ethanol ( final concentration , 0 . 2% ) containing 0 . 3 µM of U-X crosslinker ( lanes 1–9 ) and one of the following compounds: none ( lanes 1 , 4 , 7 ) ; 6 µM U18666A ( lanes 2 , 5 , 8 ) ; or 6 µM compound A ( lanes 3 , 6 , 9 ) .", "After incubation for 1 hr at 37°C , cells in lanes 4–9 were exposed to UV light as described in Materials and methods .", "Cell extracts were prepared , and the crosslinked U-X was fluorescently tagged using click chemistry .", "Proteins were then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis ( SDS-PAGE ) followed by in-gel fluorescence scanning .", "Arrow denotes a 190-kDa protein crosslinked to U-X and competed by U18666A , but not compound A . ( B ) Fluorescent labeling of 190-kDa protein in wild-type ( WT ) CHO-K1 cells , but not in 10–3 cells lacking NPC1: restoration by expression of NPC1 .", "On day 1 , mutant 10–3 cells were transfected with 1 µg/well of either control plasmid ( pcDNA3 . 1; lane 12 ) or plasmid encoding NPC1 ( pCMV-NPC1-Flag-TEV-StrepTactin; lane 13 ) .", "Nontransfected CHO-K1 and 10–3 cells were set up in parallel ( lanes 10 and 11 , respectively ) .", "On day 3 , all cells were incubated with 0 . 3 µM U-X for 1 hr , after which they were exposed to UV light .", "Cell extracts were prepared , and the cross-linked U-X was fluorescently tagged using click chemistry , followed by SDS-PAGE and in-gel fluorescence as in Panel A . Closed arrow denotes a 190-kDa protein crosslinked to U-X; open arrow shows the appearance of a similar band in transfected 10–3 cells expressing epitope-tagged NPC1 . DOI: http://dx . doi . org/10 . 7554/eLife . 12177 . 007 To confirm the identity of the 190-kDa protein , we repeated the crosslinking experiment with CHO-K1 cells and the mutant 10–3 cells ( Figure 5B ) .", "Again , we observed that the 190-kDa band was absent in the 10–3 cells ( lane 11 ) .", "It was restored when we transfected the cells with a plasmid encoding NPC1 but not a control protein ( lanes 12 and 13 ) .", "The transfected NPC1 protein migrated slightly more slowly than native NPC1 , owing to the presence of epitope tags .", "Figure 6A shows an immunoprecipitation assay to further confirm that U-X crosslinks to NPCl .", "We incubated CHO-7 cells with U-X , irradiated the cells with UV light , and solubilized the proteins in Nonidet P40 ( NP40 ) .", "We incubated the extracts with a monoclonal antibody to NPC1 , and captured the antibody-bound protein on Protein A/G agarose beads .", "The proteins that did not adhere to the beads ( designated Sup . ) and the proteins eluted from the pelleted beads were then reacted with Alexa Fluor 532 using click chemistry .", "The proteins were subjected to SDS-PAGE and visualized with a fluorescence imager .", "When a control antibody was used , we observed a fluorescent doublet of proteins at about 190 kDa in the supernatant fraction .", "In the presence of anti-NPC1 , the labeled doublet was found in the pellet fraction .", "The bottom panel in Figure 6A shows immunoblots of the same fractions with an antibody to NPC1 .", "Figure 6B shows a repetition of this immunoprecipitation experiment , but in this case we incubated the cells with various derivatives of U18666A to test for specificity through competition .", "Crosslinking of U-X to NPC1 was blocked by U18666A and by compounds B and C ( see Figure 2 ) , all of which contain a dialkylamino group similar to U18666A and all of which block cholesterol exit from lysosomes .", "Crosslinking was not inhibited by compounds A , D , and E , which fail to block cholesterol exit ( see Figure 2 ) .", "Again , these data emphasize the role of a correctly positioned dialkylamino moiety appended to an adrostenolone skeleton as a requirement for achieving a specific block of cholesterol exit versus a nonspecific lysosomotropal effect of similarly basic compounds , such as A and E . 10 . 7554/eLife . 12177 . 008Figure 6 . Immunoprecipitation of Niemann-Pick C1 ( NPC1 ) crosslinked with U-X ( A ) and specificity of crosslinking reaction ( B ) .", "On day 0 , Chinese Hamster Ovary ( CHO ) 7 cells were set up at 1 . 6 x 106/150-mm dish in 25 ml medium A with 5% lipoprotein-deficient serum ( LPDS ) per dish as described in Materials and methods .", "( A ) Immunoprecipitation with anti-NPC1 antibody .", "On day 3 , each monolayer received 25 µl of ethanol containing U-X ( final concentration , 0 . 3 µM ) .", "After incubation for 1 hr at 37°C , cells were exposed to ultraviolet ( UV ) light .", "Cell lysates from two dishes were pooled and incubated with 2 µg/ml control rabbit monoclonal antibody ( lanes 1–3 ) or anti-NPC1 antibody ( lanes 4–6 ) .", "The solutions were applied to Protein A/G beads that were washed and eluted as described in Materials and methods .", "Aliquots of the input , supernatant ( Sup . ) , and pellet fractions were obtained , and the crosslinked U-X was fluorescently tagged using click chemistry .", "The proteins were then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis ( SDS-PAGE ) and in-gel fluorescence scanning ( upper lanes ) or immunoblot analysis with 0 . 5 µg/ml anti-NPC1 ( lower lanes ) as described in Materials and methods .", "( B ) Specificity of crosslinking of U-X to NPC1 .", "On day 3 , each monolayer received a direct addition of 25 µl of ethanol containing U-X crosslinker ( final concentration , 0 . 3 µM ) in the absence ( lane 7 ) or presence ( lanes 8–13 ) of 6 µM of one of the indicated compounds whose structures are shown in Figure 2 .", "After incubation for 1 hr at 37°C , cells and homogenates were processed as described in Panel A . Proteins eluted from the Protein A/G beads were subjected to fluorescent labeling using click chemistry , followed by SDS-PAGE and in-gel fluorescence scanning ( upper lanes ) or immunoblot analysis with 0 . 5 µg/ml anti-NPC1 ( lower lanes ) as described in Materials and methods .", "The Ki values for the competing compounds denote the concentration at which each compound inhibited the incorporation of [14C]oleate into cholesteryl [14C]oleate by 50% in monolayers of CHO-7 cells that were incubated with 10% FCS ( see Figure 2 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 12177 . 008 An amino acid substitution in the membrane region of NPC1 ( P691S and P692S in the human and mouse proteins , respectively ) allows the protein to reach its normal site in lysosomes , but prevents it from transporting cholesterol out of the organelle ( Watari et al . , 1999; Ko et al . , 2001; Ohgami et al . , 2004 ) .", "This mutation abolishes the binding and crosslinking of [3H]azocholesterol to NPC1 ( Ohgami et al . , 2004 ) .", "To determine whether the P691S mutation affects the binding of U18666A , we transfected NPC1-deficient 10–3 cells with plasmids encoding epitope-tagged wild-type NPC1 or the P691S mutant ( Figure 7A ) .", "The cells were incubated with U-X and exposed to ultraviolet ( UV ) light , after which the bound U-X was tagged with Alexa Fluor 532 .", "After SDS-PAGE , fluorescent NPC1 was detected in cells expressing wild-type NPC1 ( lane 3 ) , which was competed by U18666A ( lane 4 ) .", "Fluorescent NPC1 was not detected in cells expressing the P691S mutant ( lanes 5 and 6 ) . 10 . 7554/eLife . 12177 . 009Figure 7 . Ultraviolet ( UV ) crosslinking of U-X to transfected wild-type Niemann-Pick C1 ( NPC1 ) , mutant versions of NPC1 , and wild-type NPC1L1 in 10–3 cells that lack NPC1 . ( A and B ) Crosslinking , fluorescent labeling , and in-gel fluorescence .", "On day 0 , 10–3 cells were set up in a six-well plate with 2 ml medium A with 5% lipoprotein-deficient serum ( LPDS ) per 35-mm well as described in Materials and methods .", "On day 1 , cells were transfected by direct addition of 1 µg DNA per dish ( FuGENE HD reagent ) with one of the following plasmids: pcDNA3 . 1 control plasmid ( lanes 1 , 2 , 9 , 10 ) ; pCMV-NPC1-Flag-TEV-StrepTactin ( lanes 3 , 4 , 11 , 12 ) ; pCMV-NPC1 ( P691S ) -Flag-TEV-StrepTactin ( lanes 5 , 6 ) ; pCMV-NPC1 ( P202A/F203A ) -Flag-TEV-StrepTactin ( lanes 7 , 8 ) ; pCMV-NPC1L1-Flag-TEV-StrepTactin ( lanes 13 , 14 ) .", "On day 3 , all cells were incubated ( without change of media ) for 1 hr with 0 . 3 µM U-X crosslinker in the absence ( – ) or presence ( + ) of 6 µM U18666A , after which they were exposed to UV light .", "Cell extracts were prepared , and the crosslinked U-X was fluorescently tagged using click chemistry , followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis ( SDS-PAGE ) and in-gel fluorescence as in Figure 5 .", "( C ) Cholesterol esterification .", "On day 0 , 10–3 cells were set up in medium A with 5% fetal calf serum ( FCS ) at 2 . 5 x 105 cells/60-mm dish .", "On day 1 , monolayers were washed once with Dulbecco’s phosphate-buffered saline ( PBS ) , switched to fresh medium A with 5% LPDS ( devoid of penicillin and streptomycin sulfate ) , and then transfected with 2 µg DNA per dish with the indicated plasmids as described above .", "After incubation for 24 hr , cells were washed once with PBS and switched to medium A with 5% LPDS containing 10 µM sodium compactin and 50 µM sodium mevalonate .", "On day 3 , the cells received fresh medium B containing compactin and mevalonate in the presence of either 10% LPDS or 10% FCS as indicated .", "After incubation for 3 hr at 37°C , each cell monolayer was pulse-labeled for 1 hr with 0 . 1 mM sodium [14C]oleate ( 5436 dpm/nmol ) .", "The cells were then harvested for measurement of their content of cholesteryl [14C]oleate and [14C]triglycerides .", "Each value is the mean of triplicate incubations with individual values shown .", "The cellular content of [14C]triglycerides in all transfected cell lines did not differ significantly in cells treated with LPDS ( 81–91 nmol/hr/mg ) or FCS ( 88–105 nmol/hr/mg ) .", "Inset shows immunoblot analysis of whole cell extracts ( 6 µg ) from the indicated transfection using a 1:1000 dilution of anti-Flag and anti-β-actin . DOI: http://dx . doi . org/10 . 7554/eLife . 12177 . 009 Another mutation that abolishes the cholesterol export function is the conversion of two adjacent amino acids in the NTD to alanine ( P202A/F203A ) .", "This mutation abolishes the binding of [3H]cholesterol ( Kwon et al . , 2009 ) , and it also reproduces the phenotype of complete NPC1 deficiency when knocked into the mouse npc1 gene by homologous recombination ( Xie et al . , 2011 ) .", "However , this mutation did not prevent crosslinking of U-X ( Figure 7A , lanes 7 and 8 ) .", "NPC1L1 is a close relative of NPC1 that is expressed on the surface of intestinal epithelial cells where it mediates cholesterol uptake ( Altmann et al . , 2004 ) .", "Figure 7B shows that U-X did not crosslink to NPC1L1 when the latter was expressed in NPC1-deficient 10–3 cells ( lanes 13 and", "14 ) as compared to the wild-type NPC1 control ( lanes 11 and 12 ) .", "Figure 7C employs the cholesterol esterification assay to show that expression of wild-type NPC1 restored transport of cholesterol derived from the LDL in FCS when transfected into NPC1-deficient 10–3 cells .", "Transport was not restored by transfection of the P691S mutant , the P202A/F203A mutant , or NPC1L1 .", "Immunoblots confirmed that all four proteins were expressed ( Figure 7C , inset ) ." ], [ "The current data provide further insight into the complex transport functions of NPC1 , a lysosomal membrane protein required for cellular cholesterol homeostasis as well as for susceptibility to Ebola and other filoviruses .", "As shown in Figure 1 , NPC1 is a polytopic membrane protein with 13 membrane-spanning helices separated by hydrophilic luminal or cytoplasmic projections .", "Previous studies had assigned functions to two of the hydrophilic luminal segments .", "The NTD , which projects into the lumen , binds cholesterol that is delivered directly from NPC2 ( Infante et al . , 2008b; Kwon et al . , 2009 ) , and the second luminal loop binds NPC2 ( Deffieu and Pfeffer , 2011 ) and a glycoprotein of Ebola virus that is required for release of viral RNA into the cytoplasm ( Miller et al . , 2012 ) .", "Here , we find evidence for another functional domain of NPC1 , namely , a domain that binds the cationic amphiphile U18666A .", "At concentrations as low as 0 . 03 µM , U18666A inhibits the transport of LDL-derived cholesterol from lysosomes to ER as indicated by its failure to suppress SREBP-2 cleavage and its failure to undergo re-esterification by ACAT ( Figure 3 ) .", "These findings suggest a high affinity binding site for U18666A , and UV crosslinking experiments with the U18666A derivative U-X identified NPC1 as a protein that harbors such a binding site .", "U18666A derivatives that retained the ability to block cholesterol transport inhibited U-X crosslinking to NPC-1 , whereas those that failed to inhibit transport also failed to inhibit U-X crosslinking ( Figure 6B ) .", "Although the precise location of the U18666A binding site is unknown , it does not appear to be the same as the cholesterol-binding site in the luminal NTD .", "We earlier showed that U18666A does not block binding of [3H]25-hydroxycholesterol to the NTD of recombinant NPC1 in vitro ( Infante et al . , 2008a ) .", "Moreover , U-X crosslinked normally to NPC1 bearing a P202A/F203A mutation , which abrogates [3H]cholesterol binding in vitro and cholesterol transport in intact cells ( Kwon et al . , 2009 ) ( see Figure 7A ) .", "Crosslinking of U-X to NPC1 was abolished by the P691S mutation , which lies in the sterol-sensing domain of NPC1 ( Figure 7A ) .", "The sterol-sensing domain is a segment containing five transmembrane helices ( helices 3–7 in NPC1 ) ( Nohturfft et al . , 1998; Davies and Ioannou , 2000 ) .", "This domain was initially identified in Scap , the SREBP escort protein that is regulated by cholesterol ( Hua et al . , 1996 ) .", "A similar domain is found in HMG CoA reductase , another membrane protein that is regulated by sterols ( Hua et al . , 1996 ) .", "Proline 691 is a highly conserved residue that lies in the middle of the predicted third transmembrane helix of the sterol-sensing domain of NPC1 .", "A mutation that changes this proline to serine did not prevent normal localization of NPC1 to lysosomes , but it abolished the cholesterol transport function of NPC1 ( Watari et al . , 1999; Ko et al . , 2001 ) ( also , see Figure 7C ) .", "Moreover , this mutation was also shown to prevent the crosslinking of [3H]azocholesterol to NPC1 in intact cells ( Ohgami et al . , 2004 ) .", "Considered together , the data with the P691S mutant is consistent with the idea that NPC1 contains a second cholesterol-binding site located in the sterol-sensing domain .", "Cholesterol initially binds to the NTD as demonstrated biochemically ( Infante et al . , 2008b ) and confirmed by X-ray crystallography ( Kwon et al . , 2009 ) .", "By comparing the structures of sterols bound to NPC2 and NPC1 , it was possible to postulate a hydrophobic handoff by which cholesterol moves from NPC2 to NPC1 without encountering the water phase .", "Mutations predicted to disrupt the interaction of NPC2 with the NTD of NPC1 also disrupt cholesterol exit from lysosomes ( Kwon et al . , 2009; Wang et al . , 2010 ) .", "The hydrophobic handoff may be facilitated by the binding of NPC2 to the second luminal loop of NPC1 ( Deffieu and Pfeffer , 2011 ) .", "This binding may orient NPC2 so that it can transfer cholesterol to the NTD .", "The current data raise the possibility that cholesterol may move in relay fashion from the NTD to a second binding site in the sterol-sensing domain .", "This second site may play a role in transferring cholesterol across the lysosomal membrane .", "It may lack the exquisite specificity of the NTD , and it may bind U18666A and other cationic amphiphiles that thereby block cholesterol egress from lysosomes .", "The postulated cholesterol-binding site in the sterol-sensing domain may require proper membrane orientation in order to bind .", "Although U-X crosslinks to NPC1 in intact cells , so far we have been unable to crosslink U-X to purified NPC1 in detergent solution .", "Moreover , our previous studies indicated that the NTD contains the only site that binds [3H]cholesterol in detergent .", "Thus , a single point mutation in the NTD ( Q79A ) abolished [3H]cholesterol binding to full length NPC1 when the protein was purified in detergents ( Infante et al . , 2008a ) .", "We are currently attempting to reconstitute purified NPC1 into phospholipid bilayers to find conditions that retain the proper membrane orientation , allowing the binding of U-X as well as [3H]cholesterol .", "It is unlikely that binding to the sterol-sensing domain constitutes the mechanism by which U18666A inhibits Ebola virus infection .", "According to published data ( Shoemaker et al . , 2013; Carette et al . , 2011; Miller et al . , 2012 ) , this inhibition requires concentrations of U18666A ( ~3–12 µM ) that are several orders of magnitude higher than are required to block cholesterol transport ( Figure 3A ) .", "Moreover , Ebola infection does not require the sterol-sensing domain of NPC1 .", "The second luminal loop of NPC1 is sufficient ( Miller et al . , 2012 ) .", "The data raise the possibility that high concentrations of cationic amphiphiles may interact at low affinity with luminal loop 2 of NPC1 , thereby blocking virus infection ." ], [ "We obtained U18666A , Dulbecco’s phosphate-buffered saline ( PBS ) , CuSO4 , Tris-HCl , NaCl , FCS , chloroquine , biotin , thiourea , and Benzonase Nuclease from Sigma-Aldrich , St . Louis , MO; [1-14C]oleic acid ( 55 mCi/mmol ) from Perkin Elmer , Waltham , MA; Zeocin and pcDNA3 . 1/Zeo ( - ) from Life Technologies , Grand Island , NY; and FuGENE HD from Promega , Madison , WI .", "Protein A/G Plus-Agarose Immunoprecipitation Reagent:sc 2003 from Santa Cruz Biotechnology , Dallas , TX; cOmplete , EDTA-free Protease Inhibitor Cocktail and Nonidet P40 ( NP-40 ) from Roche Diagnostics , Indianapolis , IN; Alexa Fluor 532 Azide from Joseph M . Ready ( U . T . Southwestern Medical Center ) ; TBTA ( tris[ ( 1-benzyl-1H-1 , 2 , 3-triazol-4-yl ) methyl]amine ) , and Diazo Biotin-Azide from Click Chemistry Tools Bioconjugate Technology Co . , Scottsdale , AZ; and TCEP ( tris ( 2-carboxyethyl ) phosphine ) SDS , and urea from Thermo Fisher , Rockford , IL .", "All tissue culture supplies and stock solutions of sodium compactin and sodium mevalonate were obtained from sources and prepared as previously described ( Das et al . , 2013 ) .", "Human and newborn calf LPDS ( d<1 . 215 g/ml ) , human LDL ( d1 . 019–1 . 063 g/ml ) , and rabbit β-VLDL ( <1 . 006 g/ml ) were prepared by ultracentrifugation as described ( Goldstein et al . , 1983 ) .", "Iodination of purified LDL ( 4 mg protein/reaction ) was performed using IODO-GEN precoated tubes ( Thermo Fisher Scientific Co . , Grand Island , NY ) ( Das et al . , 2013 ) .", "More than 90% of the 125I-radioactivity in 125I-LDL was precipitable after incubation with 10% ( vol/vol ) trichloroacetic acid .", "U18666A and its derivatives ( Figure 2 ) were each dissolved in ethanol and stored as a 10 mM stock solution in multiple aliquots at –20°C .", "Medium A is a 1:1 mixture of Ham’s F-12 medium and Dulbecco’s modified Eagle’s medium containing 2 . 5 mM L-glutamine ( DMEM ) .", "Medium B is L-glutamine-free DMEM .", "All media contained 100 units/ml penicillin and 100 µg/ml streptomycin sulfate unless otherwise noted in figure legends .", "Stock cultures of hamster CHO-K1 cells; CHO-7 cells ( a clone of CHO-K1 cells selected for growth in LPDS ) ( Dahl et al . , 1992; Metherall et al . , 1989 ) ; 10–3 cells ( mutant CHO-K1 cells that lack detectable NPC1 mRNA ) ( Wojtanik and Liscum , 2003 ) ; and TR-4139 cells ( a clone of CHO-7 cells stably transfected with a plasmid encoding human recombinant pCMV-NPC1-Flag-TEV-StrepTactin; see below ) were maintained in monolayer culture at 37°C in a 8 . 8% CO2 incubator .", "pCMV-NPC1-Flag-TEV-StrepTactin encodes wild-type human NPC1 ( amino acids 1–1278 ) followed sequentially by three tandem copies of the Flag epitope tag ( DYKDDDK ) , one copy of the TEV cleavage site ( ENLYFQ ) , and two copies of the StrepTactin epitope tag ( WSHPQFEK ) under control of the cytomegalovirus ( CMV ) promoter .", "This plasmid was constructed by ligating the component DNA sequences into the 5′-XbaI and 3′-HindIII sites of pcDNA3 . 1/Zeo ( - ) ( Infante et al . , 2008a ) .", "The original construct encoding human NPC1 was obtained from Origene Technologies , Rockville , MD .", "Point mutations ( P202A/F203A and P691S ) were introduced into the coding region of the NPC1 plasmid using QuikChange II XL Site-Directed Mutagenesis Kit ( Agilent Technologies , Santa Clara , CA ) .", "The coding region of each plasmid was sequenced to ensure integrity of the construct .", "CHO-7 cells were set up on day 0 at a density of 4 x 105 cells/100-mm dish in medium A with 5% ( vol/vol ) LPDS .", "On day 2 , cells were transfected with 1 µg pCMV-NPC1-Flag-TEV-StrepTactin using FuGENE HD transfection reagent according to the manufacturer’s instructions .", "Twenty-four hr after transfection , cells were switched to the above medium supplemented with 700 µg/ml Zeocin for selection .", "Fresh medium was added every 2–3 days until colonies formed at ~15 days .", "Individual colonies were isolated with cloning cylinders , and expression of NPC1-Flag-TEV-StrepTactin was assessed by immunoblot analysis with anti-Flag antibody .", "Cells from single colonies were cloned by limiting dilution , maintained in medium A with 5% LPDS containing 500 µg/ml Zeocin , and hereafter referred to as TR-4139 cells .", "The rate of incorporation of [14C]oleate into cholesteryl [14C]esters and [14C]triglycerides by monolayers of CHO-K1 , CHO-7 , 10–3 , and TR-4139 cells was measured as described previously in detail ( Goldstein et al . , 1983 ) .", "In brief , 16 hr prior to experiments , cells were switched to medium containing LPDS supplemented with compactin and mevalonate .", "On the day of the experiment , cells were incubated for 3–5 hr with fresh medium containing one of the following lipoproteins as a source of cholesterol: FCS , LDL , or β-VLDL .", "Cells were then pulse-labeled for 1–2 hr with sodium [14C]oleate-albumin complex as described in figure legends .", "The cells were then washed , and the lipids were extracted in hexane:isopropanol ( 3:2 ) , separated on a silica gel G thin-layer chromatogram ( developed in heptane:ethylether:acetic acid , 90:30:1 ) , and quantified by scintillation counting .", "The amounts of cholesteryl [14C]oleate and [14C]triglycerides formed are expressed as nanomoles formed/hour per milligram cell protein .", "CHO-7 cells were set up for experiments as described in the legend to Figure 3 .", "After incubation with the indicated compounds , nuclear and membrane fractions were prepared as described ( Sakai et al . , 1996 ) and then subjected to SDS-PAGE and immunoblot analysis as described below .", "For fluorescent labeling of U-X binding proteins , CHO-K1 and 10–3 cells were set up on day 0 in 2 ml medium A with 5% LPDS at a density of 1 x 105 cells/35-mm well in a six-well plate .", "On day 1 , the cells were transfected with 1 µg of either empty plasmid or pCMV-NPC1-Flag-TEV-StrepTactin using FuGENE HD transfection reagent .", "On day 3 , each monolayer received a direct addition of ethanol ( final concentration , 0 . 2% ) containing the indicated concentration of U-X crosslinker and various compounds as described in figure legends .", "After incubation for 1 hr at 37°C in the dark and without a change of media , the cells were irradiated for 15 min at room temperature under 306 nM UV light ( Atlanta Light Bulb Co . , Cat . No . G15T8E ) in a UV Stratalinker 2400 apparatus ( Stratagene ) .", "Cells were then washed once with PBS and resuspended in 0 . 1 ml buffer containing 50 mM HEPES at pH 7 . 8 , 1 . 5 mM MgCl2 , 10 mM KCl , 100 U/ml Benzonase Nuclease , Protease Inhibitor Cocktail , and 1% ( w/v ) SDS .", "The protein concentration of each lysate was adjusted to 1 . 5 mg/ml using the bicinchoninic acid ( BCA ) protein assay .", "To attach a fluorophore , we employed click chemistry reactions involving copper-catalyzed azide-alkyne cycloaddition ( Kolb et al . , 2001; Sapkale et al . , 2014 ) .", "The reactions were carried out by mixing an aliquot of each SDS lysate ( 43 µl ) , in a final volume of 50 µl , with 3 µl of 1 . 7 mM Tris ( benzyltriazolylmethyl ) amine ( TBTA ) , 2 µl of 50 mM CuSO4 , 1 µl of 50 mM tris ( 2-carboxyethyl ) phosphine ( TCEP ) , and 1 µl of 1 . 25 mM Alexa Fluor 532 Azide .", "The mixture was incubated at room temperature for 1 hr .", "To visualize the fluorescent-labeled proteins by in-gel fluorescence , 20-µl aliquots of each sample were mixed with 5X loading dye and subjected to 8% SDS-PAGE , after which the gels were scanned with a Typhon Imager ( filter settings: excitation , 533 nM; emission , 555 nm; high sensitivity ) .", "For immunoprecipitation of U-X binding proteins , CHO-7 cells were set up on day 0 in 25 ml medium A with 5% LPDS at 1 . 6 x 106/150-mm dish .", "On day 3 , each monolayer received a direct addition of ethanol ( final concentration , 0 . 2% ) containing the indicated concentration of U-X crosslinker and various compounds as described in figure legends .", "After incubation for 1 hr at 37°C , the cells were subjected to UV crosslinking as described above .", "Cells were scraped from the dish , and the cell suspensions from two dishes were pooled .", "All subsequent operations were carried out at 4°C .", "Each pooled cell suspension was centrifuged at 1 x 103 g for 5 min .", "The pellet was washed once with phosphate-buffered saline ( PBS ) and then solubilized with 1 ml of buffer containing 50 mM Tris-HCl at pH 7 . 5 , 150 mM NaCl , 0 . 5% ( v/v ) NP40 , and Protease Inhibitor Cocktail .", "Each solubilized lysate was passed through a 22 . 5-gauge needle 10 times , rotated for 1 hr , and clarified by centrifugation at 1 . 5 x 106 g for 30 min .", "An aliquot of the supernatant ( 1 mg protein ) was adjusted to a final volume of 1 ml with the above buffer and precleared by rotation for 1 hr with 10 μg/ml of preimmune rabbit immunoglobulin G ( IgG ) and 100 μl of Protein A/G PLUS-Agarose Beads .", "After centrifugation at 1 x 103 g for 5 min , the supernatant ( designated as input ) was transferred to a fresh tube and incubated with various monoclonal antibodies as described in figure legends .", "After rotating for 1 hr , 100 μl of Protein A/G PLUS Agarose Beads were added , followed by rotation overnight , and centrifugation at 1 x 103 g for 5 min .", "The resulting supernatant ( ~1 ml ) was transferred to a fresh tube and designated as supernatant .", "The pelleted beads were washed five times with 1 ml of wash buffer ( 50 mM Tris-HCl at 7 . 5 , 150 mM NaCl , and 0 . 02% NP40 ) and suspended in 1 ml of wash buffer mixed with 1% SDS and boiled for 5 min , after which the mixture was clarified by centrifugation for 5 min at 1 x 103 g , and the resulting fraction was designated as pellet .", "Prior to click reactions , the input and supernatant fractions received a direct addition of SDS ( final concentration 1% ) followed by boiling for 5 min .", "All three fractions were then tagged with Alexa-Fluor using click chemistry as described above , after which an aliquot of each fraction representing 2% of the starting sample was subjected to SDS-PAGE , and fluorescently labeled proteins were visualized by in-gel fluorescence .", "After scanning the gel , the proteins were transferred to nitrocellulose , blotted with rabbit monoclonal anti-NPC1 , and visualized by chemiluminescence using goat anti-rabbit IgG conjugated to horseradish peroxidase ( see below ) .", "Cell fractions were subjected to electrophoresis in 1% SDS on 8% polyacrylamide gels , after which the proteins were transferred to nitrocellulose filters .", "The filters were incubated at room temperature with 10 µg/ml of one of the following primary antibodies as indicated in the figure legends: rabbit monoclonal IgG-22D5 against SREBP-2 ( McFarlane et al . , 2015 ) ; rabbit monoclonal IgG against amino acids 1261–1278 of human NPC1; ( Cat . No . 134113 , Abcam , Cambridge , MA ) ; mouse monoclonal IgG against Flag epitope DDDDK ( Cat . No . M185 , MBL International Corp . , Woburn , MA ) ; rabbit polyclonal IgG against β-actin ( Cat . No . 4970 , Cell Signaling Technology , Boston , MA ) , and rabbit polyclonal IgG against calnexin ( Cat . No . ADI-SPA-860 , Enzo Life Sciences , Farmingdale , NY ) .", "Bound antibodies were visualized by chemiluminescence ( SuperSignal West Pico Chemiluminescent Substrate , Thermo Scientific ) after incubation with a 1:5000 dilution of either donkey anti-mouse or goat anti-rabbit IgG conjugated to horseradish peroxidase ( Jackson ImmunoResearch , West Grove , PA ) .", "The images were scanned using an Odyssey FC Imager ( Dual-Mode Imaging System; 2-min integration time ) and analyzed using Image Studio ver . 5 . 0 ( LI-COR , Lincoln , NE ) .", "Similar results were obtained when each experiment was repeated on multiple occasions .", "Three or more independent studies were done for each experiment except for the experiment in Figure 3C , which was done twice ." ] ]

[ "Niemann-Pick C1 ( NPC1 ) is a lysosomal membrane protein that exports cholesterol derived from receptor-mediated uptake of LDL , and it also mediates cellular entry of Ebola virus .", "Cholesterol export is inhibited by nanomolar concentrations of U18666A , a cationic sterol .", "To identify the target of U18666A , we synthesized U-X , a U18666A derivative with a benzophenone that permits ultraviolet-induced crosslinking .", "When added to CHO cells , U-X crosslinked to NPC1 .", "Crosslinking was blocked by U18666A derivatives that block cholesterol export , but not derivatives lacking blocking activity .", "Crosslinking was prevented by point mutation in the sterol-sensing domain ( SSD ) of NPC1 , but not by point mutation in the N-terminal domain ( NTD ) .", "These data suggest that the SSD contains a U18666A-inhibitable site required for cholesterol export distinct from the cholesterol-binding site in the NTD .", "Inasmuch as inhibition of Ebola requires 100-fold higher concentrations of U18666A , the high affinity U16888A-binding site is likely not required for virus entry ." ]

[ "Cholesterol is a type of fat molecule and is a vital component of animal cell membranes .", "It is taken up into cells within particles called low density lipoproteins ( LDLs ) that are then digested in cell compartments known as lysosomes to release the cholesterol .", "Then , the cholesterol leaves the lysosome with the help of a transport protein called NPC1 .", "Mutations in the gene that encodes NPC1 lead to the accumulation of cholesterol in lysosomes; this can cause a devastating illness that affects the brain , liver and other organs .", "The NPC1 protein also plays a crucial role in allowing Ebola viruses to infect animal cells and multiply .", "U18666A is a drug that blocks the movement of cholesterol out of lysosomes and also inhibits Ebola virus infections , but it was not known what components it targets in cells .", "Lu et al . used a technique called ultraviolet-induced crosslinking to identify the proteins that U18666A can bind to .", "The experiments show that U18666A can directly bind to a site that is within a section of the NPC1 protein called the sterol-sensing domain .", "The binding of U18666A to this site blocks the movement of cholesterol out of lysosomes .", "Lu et al . ’s findings indicate that the sterol-sensing domain of NPC1 plays a crucial role in cholesterol’s export from lysosomes .", "A future challenge is to use structural biology techniques ( such as X-ray crystallography or cryo-electron microscope tomography ) to understand the three-dimensional structure of NPC1 ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "chromosomes and gene expression", "structural biology and molecular biophysics" ]

[ [ "Cellular organisms initiate DNA synthesis during genome duplication by the universal mechanism of RNA priming , the assembly of short RNA molecules on the unwound strands of the DNA helix by a specialized DNA-dependent RNA polymerase known as primase ( Frick and Richardson , 2001; Kuchta and Stengel , 2010; DePamphilis and Bell , 2011 ) .", "The RNA primers are extended in an obligate 5′ to 3′ direction by the replicative DNA polymerases that synthesize the bulk of chromosomal DNA .", "The initiation of DNA synthesis is made more complicated by the concurrent duplication of the antiparallel strands of parental DNA ( Hamdan and van Oijen , 2010 ) .", "The repeated priming events necessary for the discontinuous synthesis of the lagging strand require the constant activity of primase at the replication fork .", "In bacteria and bacteriophages , RNA priming is performed by a single-chain primase , acting in combination with the replicative helicase , to which it is bound by a dynamic interaction or is fused together in the same polypeptide ( Patel et al . , 2011 ) .", "The molecular apparatus responsible for initiation of DNA synthesis in eukaryotic replication is more complex .", "The eukaryotic primase is a heterodimer of catalytic and regulatory subunits that is associated with the catalytic subunit of Pol α and its accessory B subunit in a constitutive heterotetrameric assembly , the Pol α/primase complex ( Loeb et al . , 1986; Kaguni and Lehman , 1988; Foiani et al . , 1997; Lao-Sirieix et al . , 2005b ) .", "Reflecting its critical importance to DNA replication , the Pol α/primase complex is an integral component of the eukaryotic replisome ( Calzada et al . , 2005 ) .", "The oligonucleotides synthesized by bacterial and bacteriophage primases are between 4 and 12 nucleotides long and made exclusively of RNA .", "In contrast , the Pol α/primase complex produces longer , composite RNA-DNA primers that result from the concerted enzymatic activities of primase and Pol α ( Chang et al . , 1984; Hu et al . , 1984; Singh , et al . , 1986 ) .", "The constitutive association of Pol α and primase in the cell reflects presumably their tight functional coordination , demanded by the frequent priming necessary for lagging strand synthesis .", "Detailed knowledge of the mechanism of primer synthesis is lacking , but it must involves initiation , extension and completion of RNA synthesis by primase , intramolecular hand-off of the RNA oligonucleotide to Pol α and limited RNA extension with deoxynucleotides ( dNTPs ) ( Brooks and Dumas , 1989; Kuchta et al . , 1990; Copeland and Wang , 1993; Sheaff and Kuchta , 1993; Sheaff et al . , 1994 ) .", "The large subunit of primase performs a critical function in the primer initiation step via its Fe-S domain ( Klinge et al . , 2007; Weiner et al . , 2007 ) , whereas the B subunit of Pol α lacks enzymatic activity and likely acts as a scaffold to mediate interactions with other components of the replicative apparatus ( Uchiyama and Wang , 2004; Klinge et al . , 2009; Zhou et al . , 2012 ) .", "After completion of primer synthesis by Pol α/primase , the primer is elongated by the processive Pols δ and ε that synthesize the majority of chromosomal DNA on the lagging and leading strand templates , respectively .", "Synchronization of priming with Okazaki fragment synthesis requires a concerted mode of primer transfer from Pol α to Pol δ and several mechanisms of polymerase switch have been put forward ( Yuzhakov et al . , 1999; Maga et al . , 2000 ) .", "Before ligation of the completed Okazaki fragments , the RNA portion of the primer is excised by specific nucleases such as Fen1 and Dna2 ( Burgers , 2009 ) .", "The composite nature of the RNA-DNA primers poses a special challenge to the eukaryotic replication apparatus , that must further correct the DNA segment synthesized by Pol α , which lacks proofreading activity; current evidence indicates that the DNA portion of the primer might be corrected by Pol δ ( Pavlov et al . , 2006 ) .", "Despite its central role in genomic duplication , the molecular mechanism of primer synthesis by the Pol α/primase complex is poorly understood and structural insights remain limited .", "Here we use a multi-disciplinary approach to elucidate the structural basis for the catalytic role of Pol α in the synthesis of the RNA-DNA oligonucleotides that prime DNA synthesis in eukaryotic replication .", "We demonstrate that Pol α recognizes the intrinsic and induced conformation of the A-form RNA primer/DNA template helix and that the resulting synthesis of B-form DNA forms the basis for a feedback mechanism of primer termination .", "Our findings provide a new paradigm for a critical step in the complex choreography of events that ultimately lead to replication of the lagging DNA strand ." ], [ "We have determined crystal structures of the catalytic core ( 349–1258; 910 amino acids ) of yeast Pol α in unliganded form ( apo ) , bound to an RNA primer/DNA template duplex ( binary complex ) and in a productive complex with RNA primer/DNA template and incoming dGTP ( ternary complex ) .", "For structural studies of the ternary complex , we used a polymerase mutant ( D998N ) with attenuated catalytic activity .", "During crystallization of the ternary complex , the polymerase extended the 3′-end of the RNA primer by addition of two deoxyguanosine nucleotides .", "Thus , our crystal structure captures the actively copying Pol α in the act of extending the RNA primer with deoxynucleotides ( Figure 1A and Figure 1—figure supplement 1–4 , Table 1 ) . 10 . 7554/eLife . 00482 . 003Figure 1 . Overall structure of actively copying Pol α .", "( A ) The polymerase domain of yeast Pol α is shown in complex with an RNA primer/DNA template and dGTP bound in the active site .", "The polymerase is represented as ribbon , colored blue to red from the N-terminus .", "The RNA primer and DNA template are shown in dark and light gray , respectively .", "The two deoxyguanosine nucleotides added to the RNA primer by Pol α during the crystallization experiment as shown in light gray .", "The dGTP nucleotide is shown in spacefill representation .", "The different subdomains of the polymerase structure are marked in the figure .", "( B ) A view of the RNA primer/DNA template double helix bound to Pol α .", "The nucleic acids are shown as stick models; carbon atoms in light brown , nitrogen atoms in blue , oxygen atoms in red and phosphate atoms in orange .", "The 2′-hydroxyl oxygen atoms of the ribose moieties of the RNA primer are shown in magenta .", "The polymerase is shown as a clipped molecular surface , in blue .", "The conformation assigned to each dinucleotide step in the RNA/DNA helix is indicated on the left-hand side of the panel .", "The first five di-nucleotide steps of the hybrid RNA/DNA helix are numbered one to five , from the 3′-terminus of the RNA primer .", "( C ) Scatter plot of zP , the mean z-coordinate of the backbone phosphorus atoms with respect to individual dinucleotide reference frames , against the mean value for the four χ torsion angles at each di-nucleotide step , for the RNA/DNA helix bound to Pol α ( black dots ) .", "The data points of the first five steps in the RNA/DNA helix bound to Pol α are numbered .", "For comparison , the values obtained for the DNA double helix bound to yeast Pol δ ( empty dots; PDB entry 3IAY ) and phage RB69 Pol ( gray dots; PDB entry 1IG9 ) are also shown .", "For reference , the values obtained for A- and B-form DNA models based on fiber diffraction analysis are marked in the plot with a black cross .", "The stereochemical analysis of the RNA/DNA helix was performed with 3DNA ( Lu and Olson , 2003 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 00482 . 00310 . 7554/eLife . 00482 . 004Figure 1—figure supplement 1 . Primer extension assay for the D998N , R508A , N509A Pol α mutant . The assay was performed as described in the 'Materials and methods' .", "The range of protein concentrations tested in the experiment is indicated in the figure .", "Incubation time was 90 s .", "Polymerase activity is readily detectable at micromolar levels of protein concentrations . DOI: http://dx . doi . org/10 . 7554/eLife . 00482 . 00410 . 7554/eLife . 00482 . 005Figure 1—figure supplement 2 . Mass spectrometry analysis of the RNA 10mer used in the crystallisation of the ternary complex . The results of the analysis for the RNA 10mer in solution ( control; top panel ) and recovered from a sample of washed crystals of the ternary complex ( bottom panel ) are shown .", "The analysis shows primer extension by two dGMP nucleotides and partial incorporation of a third dGMP , during the crystallization process .", "Translocation onto the next templating base ( Adenine at position -1 , see Figure 2C ) was prevented from formation of the crystal lattice based on the non-crystallographic dimer of ternary complexes , illustrated in Figure 1—figure supplement 6 . DOI: http://dx . doi . org/10 . 7554/eLife . 00482 . 00510 . 7554/eLife . 00482 . 006Figure 1—figure supplement 3 . Details of the 2Fo-Fc electron density map at 3 . 1 Å resolution , after B-sharpening and contoured at 2 . 5 rmsd . The map was calculated in PHENIX and displayed in Chimera .", "( A ) Overall view of the double-stranded region of the RNA primer-DNA template in the ternary complex .", "The refined crystallographic model is shown in stick representation , superimposed on the electron density .", "( B ) Two views of electron density for the ribophosphate backbone of the DNA template ( left panel ) and RNA primer ( right panel ) , with the refined model superimposed on the density . DOI: http://dx . doi . org/10 . 7554/eLife . 00482 . 00610 . 7554/eLife . 00482 . 007Figure 1—figure supplement 4 . Close-up view of Pol α's active site , showing the dGTP nucleotide and side chains of amino acids important for catalysis and nucleotide binding . For comparison , the active site of yeast Pol δ ( PDB id: 3IAY ) is shown , superimposed on Pol α .", "The polymerase chains are represented as blue ( Pol α ) or light blue ( Pol δ ) ribbons; the nucleic acid , nucleotides and amino acid side chains are drawn as sticks .", "Carbon atoms in Pol α are coloured white , whereas in Pol δ are coloured in light brown .", "Only amino acids belonging to Pol α are labeled , for clarity .", "The 3′-hydroxyl in the templated primer bound to Pol α is labelled .", "Three Ca2+ ions identified in the active site of Pol δ are also shown , as green spheres . DOI: http://dx . doi . org/10 . 7554/eLife . 00482 . 00710 . 7554/eLife . 00482 . 008Figure 1—figure supplement 5 . Interaction of the single-stranded DNA template with Pol α .", "( A ) Trajectory of the ssDNA template on the surface of Pol α .", "The deoxy-nucleotides of the DNA template ahead of the templating base fit in a groove formed by the exonuclease and N-terminal regions of Pol α .", "( B ) In the ternary complex , the aromatic bases project inwards towards the polymerase , whereas the ribophosphate backbone remains largely exposed to solvent .", "Phe685 in the exonuclease domain protrudes into the groove and stacks its phenyl ring against the base of guanidine in position -3 of the DNA template . DOI: http://dx . doi . org/10 . 7554/eLife . 00482 . 00810 . 7554/eLife . 00482 . 009Figure 1—figure supplement 6 . The actively copying Pol α crystallizes with two copies of the ternary complex in the asymmetric unit . The figure shows a view of the asymmetric unit that highlights the non-crystallographic dyad relating the two copies of ternary complex .", "The protein is shown as a ribbon in light blue , and the RNA-DNA duplex as an all-atom stick model , with a thin tube running through the positions of the phosphorus atoms of the ribo-phosphate backbone . DOI: http://dx . doi . org/10 . 7554/eLife . 00482 . 00910 . 7554/eLife . 00482 . 010Figure 1—figure supplement 7 . Interaction with the non-crystallographic copy of Pol α captures the 5′-terminal nucleotide of the RNA primer in a surface pocket of the palm domain , in the crystals of ternary complex .", "( A ) General view of the contact between Pol α and the second , non-crystallographic RNA-DNA molecule in the asymmetric unit .", "The polymerase is shown as molecular surface in light blue and the position of its RNA-DNA substrate is shown by thin gray tubes .", "The non-crystallographic RNA-DNA molecule is shown as an all-atom stick model , with the 5′-terminal nucleotide of the RNA primer highlighted in yellow .", "( B ) Atomic details of the non-crystallographic interface between Pol α and the second copy of the RNA-DNA molecule in the asymmetric unit of the ternary complex crystals .", "A negatively-charged loop in the palm domain of Pol α , including residues D889 , D891 and E892 , unwinds the 5′-terminal nucleotide of the templated RNA primer .", "The 5′-terminal nucleotide of the RNA primer remains trapped by multiple polar and hydrophobic interactions in a pocket on the surface of the palm domain , whilst its templating nucleotide in the DNA molecule is disordered in the electron density map and is not included in the crystallographic model . DOI: http://dx . doi . org/10 . 7554/eLife . 00482 . 01010 . 7554/eLife . 00482 . 011Table 1 . X-ray data processing and crystallographic refinementDOI: http://dx . doi . org/10 . 7554/eLife . 00482 . 011X-ray data processingSeleno-methionineApoBinary complexTernary complexSpace groupP 2 21 21P21P1P 21 21 21Unit cell ( Å ) 74 . 0 127 . 5 144 . 174 . 4 127 . 1 74 . 572 . 1 74 . 8 117 . 0111 . 7 145 . 7 197 . 2Angles ( ° ) 90 . 0 90 . 0 90 . 090 . 0 104 . 8 90 . 082 . 3 72 . 6 82 . 490 . 0 90 . 0 90 . 0Mosaicity0 . 390 . 540 . 770 . 35OverallInnershellOutershellOverallInnershellOutershellOverallInnershellOutershellOverallInnershellOutershellLow resolution limit ( Å ) 73 . 9773 . 972 . 8153 . 5153 . 512 . 4258 . 7758 . 773 . 1649 . 3949 . 393 . 26High resolution limit ( Å ) 2 . 678 . 432 . 672 . 37 . 272 . 339 . 4933 . 19 . 793 . 1Rmerge0 . 1330 . 0520 . 6630 . 0850 . 0390 . 5120 . 0730 . 030 . 9210 . 1050 . 0420 . 974Rmerge in top intensity bin0 . 061--0 . 051--0 . 029--0 . 046--Rmeas ( within I+/I− ) 0 . 140 . 0540 . 7030 . 1040 . 0480 . 6210 . 0850 . 0351 . 0680 . 1130 . 0461 . 05Rmeas ( all I+ & I− ) 0 . 1480 . 0710 . 7020 . 10 . 0450 . 6090 . 0850 . 0351 . 0680 . 1130 . 0461 . 05Rpim ( within I+/I− ) 0 . 0430 . 0170 . 2290 . 0580 . 0270 . 350 . 0430 . 0180 . 5390 . 0420 . 0180 . 386Rpim ( all I+ & I− ) 0 . 0330 . 0160 . 1650 . 040 . 0190 . 2460 . 0430 . 0180 . 5390 . 0420 . 0180 . 386Fractional partial bias−0 . 008−0 . 043−0 . 028−0 . 035−0 . 025−0 . 045−0 . 039−0 . 034−0 . 178−0 . 007−0 . 006−0 . 127Total number of observations781 , 85525 , 82697 , 175355 , 35710 , 42750 , 930175 , 884521725 , 591428 , 39713 , 18161 , 528Total number unique39 , 6261401565658 , 9061764852345 , 0271413654659 , 15419908380Mean ( ( I ) /sd ( I ) ) 17 . 939 . 64 . 611 . 330 . 1313 . 655 . 61 . 510 . 828 . 92 . 1Completeness10099 . 699 . 899 . 391 . 499 . 197 . 896 . 797 . 599 . 797 . 298 . 4Multiplicity19 . 718 . 417 . 265 . 963 . 93 . 73 . 97 . 26 . 67 . 3Anomalous completeness99 . 999 . 799 . 5Anomalous multiplicity10 . 3118 . 8DelAnom correlation between half-sets0 . 6020 . 8230 . 068Mid-slope of Anom normal probability1 . 372--Crystallographic refinementSeleno-methionineApoBinary complexTernary complexR-factor ( overall/outershell ) 0 . 1959 ( 0 . 2405 ) 0 . 2051 ( 0 . 3079 ) 0 . 2551 ( 0 . 3623 ) 0 . 2111 ( 0 . 3570 ) R-free ( overall/ outershell ) 0 . 2341 ( 0 . 3058 ) 0 . 2361 ( 0 . 3453 ) 0 . 2858 ( 0 . 4040 ) 0 . 2479 ( 0 . 3909 ) Number of atoms13 , 49713 , 69027 , 70729 , 108 macromolecules6637668313 , 91414 , 680 ligands62 water11021600Protein residues82782916871754RMS ( bonds ) 0 . 0020 . 0020 . 0030 . 003RMS ( angles ) 0 . 570 . 590 . 770 . 75Ramachandran favored ( % ) 97979694Ramachandran outliers ( % ) 0 . 1200 . 0610 . 24Clashscore3 . 742 . 7510 . 0411 . 55Wilson B-factor45 . 9442 . 3191 . 6497 . 34Average B-factor56 . 562 . 4108111 . 6 macromolecules56 . 762 . 7108111 . 6 solvent45 . 854 . 9 The catalytic region of Pol α adopts the universal ‘right-hand' DNA polymerase fold consisting of a palm domain harboring the active site , a fingers domain that interacts with the incoming nucleotide and a thumb domain that grips the primer-template duplex .", "As the prototypical member of the B-family of DNA polymerases , distinctive structural features that had been identified previously in bacteriophage RB69 Pol ( Wang et al . , 1997 ) , bacterial DNA Pol II ( Wang and Yang , 2009 ) and yeast Pol δ ( Swan et al . , 2009 ) , such as the extended N-terminal region or the antiparallel hairpin fold of the helical fingers domain , are also present in Pol α .", "The majority of the contacts of the polymerase with the primer/template duplex takes place within a region of 7 bp from the 3′-terminus of the templated primer ( Figure 1B ) .", "Pol α's footprint on the primer-template duplex is smaller than normally observed in B-family DNA polymerases and matches the minimal primer size that is efficiently utilized by the polymerase ( Kuchta et al . , 1990 ) .", "Four deoxynucleotides of single-stranded DNA template fit in extended conformation within the groove formed by the exonuclease domain and N-terminal region of Pol α , in agreement with the position of template DNA upstream of the active site previously observed in other B-family DNA polymerases ( Hogg et al . , 2007; Swan , et al . , 2009; Figure 1—figure supplement 5 ) .", "In the crystals , two ternary complexes are related by non-crystallographic dyad symmetry , which brings into contact one end of each RNA/DNA duplex with the palm domain of the other polymerase molecule ( Figure 1—figure supplements 6 and 7 ) .", "The acidic tip of a long loop connecting the palm and finger domains unwinds the first base pair of the RNA/DNA duplex , capturing the 5′-end nucleotide of the RNA primer in a surface pocket on the palm domain .", "Such interaction would potentially begin the unwinding of the templated RNA primer , in preparation for its excision .", "However , the physiological relevance of our structural observation is currently unclear .", "The RNA primer–DNA template helix bound to Pol α adopts overall an A-DNA conformation .", "Clustering analysis for each dinucleotide step of the mean z-coordinates of the phosphorus atoms with the glycosyl torsion angle χ ( Lu et al . , 2000 ) identifies a continuous stretch of six dinucleotide steps as A-DNA and one step as B-DNA flanked by dinucleotides in A-like conformation ( Figure 1C ) .", "As a control , the clustering analysis identifies correctly the double-stranded DNA bound productively by B-family Pol δ and RB69 Pol as B-form DNA and the dinucleotide step at the primer 3′-end as of A-like character .", "Extensive structural analysis has demonstrated that the palm and thumb domains of actively copying DNA polymerases make continuous contact with the minor groove of the primer-template DNA over a full turn of the DNA double helix .", "Interactions of the palm domain with the primer-template help position the 3′-terminus of the primer in optimal arrangement for catalysis , whereas the thumb domain secures the grip of the polymerase onto the DNA duplex .", "Strikingly , the structure of Pol α bound productively to RNA primer/DNA template shows that , although the palm domain makes a canonical set of interactions with the first 3 bp of the primer-template helix , the thumb domain engages almost exclusively with the RNA primer strand ( Figure 2A , B ) . 10 . 7554/eLife . 00482 . 012Figure 2 . Specific recognition of the RNA/DNA helix by Pol α .", "( A ) Identical views of active complexes of yeast Pols α ( left ) and δ ( Swan et al . , 2009; right; PDB entry 3IAY ) , bound to primer/template duplexes and incoming deoxynucleotide .", "The polymerase structures are depicted as ribbons ( Pol α , blue; Pol δ , cyan ) .", "Primer/template duplexes are shown in spacefill representation and colored light brown , with the exception of the phosphate groups that have red oxygen atoms and orange phosphate atoms .", "The 2′-hydroxyl oxygen atoms in the ribose moieties of the RNA are highlighted in magenta .", "( B ) The interface between the thumb domain of Pol α and the RNA primer/DNA template duplex .", "The ribo-phosphate backbone is shown as a thin tube , in salmon and pale yellow color for the RNA and DNA strands , respectively , and the bases are depicted as rungs of a ladder .", "Pol α is shown as a molecular surface , colored gray except for atoms that are within a 5 Å radius of the RNA primer or the DNA template , that are colored as the nucleic acid strand .", "( C ) Schematic diagram of the interactions of Pol α′s thumb domain with the RNA/DNA helix .", "The ribophosphate portion of nucleotides that interact with the thumb domain is shaded gray .", "A small circle at position two of the ribose ring indicates the RNA nucleotides .", "( D ) Hydrophobic contacts of the thumb domain with the exposed minor groove of the RNA/DNA helix .", "The protein is shown as thin blue tube and amino acid side chains as sticks with white carbon atoms , yellow sulfur atoms and red oxygen atoms .", "The RNA primer is shown as sticks , with atoms colored as in panel A . Protein-RNA contacts are shown as dashed green lines . DOI: http://dx . doi . org/10 . 7554/eLife . 00482 . 012 The interactions between the thumb domain and the RNA primer are concentrated in a contiguous region of five nucleotides , from position two to six of the RNA primer and involve solely the ribose and phosphate moieties of the RNA backbone ( Figure 2C ) .", "Two sequence motifs , residues 1074–1077 in the long segment of the L-shaped thumb , and residues 1130–1150 in the tip of the thumb , form a continuous protein interface that tracks the position of the phosphodiester backbone of the RNA primer .", "Pol α exploits the wide , shallow nature of the minor groove of the RNA/DNA helix with a series of hydrophobic contacts involving the side chains of Met1131 , Leu1133 , Tyr1140 , Pro1141 , Met1146 , which recognize the C3′-endo conformation of the ribose moieties of RNA nucleotides in position four to six ( Figure 2D ) .", "The contiguous pair of invariant arginine residues 1075 and 1076 straddles the ribose-phosphate backbone between nucleotides three and four from the 3′-end of the RNA primer ( Figure 3 ) .", "The side chain of Arg1075 extends into the minor groove and packs its guanidinium moiety against the ribose sugar of Ade4 ( Figures 2C and 3A ) , thus engaging in an interaction that matches closely the contact made by the equivalent Arg839 of yeast Pol δ with the DNA primer ( Figure 3B ) .", "However , in the case of Pol α the close contact with Arg1075 induces a local rearrangement of the RNA conformation , which includes a B-DNA C2′-endo ribose pucker for Ade4 and unstacking between the aromatic bases of nucleotides four and five ( Figure 3A , C ) .", "The local conformation of the RNA primer is stabilized by insertion of the Arg1076 side chain between the phosphate groups of nucleotides three and four ( Figures 2C and 3C , D ) .", "The ‘snap-clip' interaction of arginines 1075 and 1076 with the RNA backbone anchors the RNA primer to the thumb domain of Pol α , by facilitating the formation of three hydrogen bonds between the phosphate groups of nucleotides four , five and six and the mainchain nitrogens of residues Arg1076 , Lys1132 and Ser1134 , respectively ( Figure 3D ) . 10 . 7554/eLife . 00482 . 013Figure 3 . Arginines 1075 and 1076 anchor Pol α to the RNA primer .", "( A ) Close-up view of the interaction of Arg1075 with the RNA primer .", "A hydrogen bond interaction between the carboxylate group of Glu1077 and Arg1075 is also shown , as a yellow dashed line .", "The protein is shown as thin blue tube and amino acid side chains as sticks with white carbon atoms , blue nitrogen atoms and red oxygen atoms .", "The RNA primer is shown as sticks , with atoms colored as in Figure 2A .", "A black arrow highlights the position of the 2′-hydroxyl moiety of the ribose ring .", "For clarity , the DNA template has been omitted .", "( B ) Close-up view of the interaction of Pol δ Arg839 with the DNA primer , in the same orientation as Pol α in panel A . The interaction of Asp841 with Arg839 is also shown .", "( C ) Conformation of the RNA primer bound to Pol α .", "The polymerase is shown as a clipped molecular surface , in light blue .", "The position of Arg1075 and Arg1076 is indicated in dark blue .", "Color scheme is as in Figure 2A .", "The DNA template strand is omitted for clarity .", "( D ) Polar contacts at the interface between Pol α's thumb domain and the RNA primer .", "Hydrogen bonds between the phosphate groups of the RNA primer and main chain nitrogen atoms of the thumb domain are depicted as dashed yellow lines .", "Color scheme is as in Figure 2A .", "The DNA template strand is omitted for clarity . DOI: http://dx . doi . org/10 . 7554/eLife . 00482 . 013 The hydrogen-bond network between thumb domain and RNA is augmented by polar interactions contributed by the side chains of Lys1132 , Ser1134 , Lys1135 and Tyr1140 ( Figure 3D ) .", "The G1116E mutation in fission yeast Pol α at the equivalent position to Ser1134 causes a defect in mating-type switching ( Singh and Klar , 1993 ) : the close interaction of Ser1134 with the RNA primer suggests that the defect might be caused by weakened affinity of Pol α for its primer-template substrate .", "The crystallographic analysis pointed to a specific mechanism for the intrinsic and induced recognition of the templated RNA primer by Pol α .", "The result of the structural study prompted us to investigate whether the polymerase activity of Pol α reflected such specificity .", "In order to test this hypothesis , we examined the ability of the polymerase domain of yeast Pol α to extend with deoxynucleotides a templated primer made of either RNA or DNA .", "Whereas Pol α displayed only weak activity and limited processivity when extending a DNA primer , extension of an RNA primer resulted in much higher levels of polymerization ( Figure 4A ) .", "Strikingly , the profile of RNA-dependent extension products showed a pronounced peak at between 10 to 12 nucleotides , indicating strong processivity limited to a full-turn of double helix ( Figure 4A , B ) .", "Such characteristics of the catalytic activity of Pol α were observed for the isolated polymerase core as well as the Pol α /primase complex and are independent of primer size ( Figure 4C ) . 10 . 7554/eLife . 00482 . 014Figure 4 . DNA- and RNA-primed dNTP polymerization by Pol α .", "( A ) Primer extension assay , analyzed by denaturing acrylamide gel electrophoresis and [α-32P]dATP phosphorimaging .", "The wild-type polymerase domain of yeast Pol α was incubated at 37°C with poly ( dT ) 70mer template , dATP and either an oligo ( A ) or oligo ( dA ) 15mer primer ( see ‘Materials and methods' for details ) .", "The reaction products were analyzed at the indicated time points .", "( B ) Quantitative product size-distribution analysis of the primer extension assay in panel A . For each time point , the intensities of the RNA and DNA primer extension products were measured and normalized relative to the measured intensity of the 12mer product in the RNA primer lane .", "The normalized values of the RNA and DNA-primer extension products are plotted against the size of the extension product , as number of bases , in the top and bottom panels , respectively .", "The normalized values are the average of three independent measurements .", "( C ) Primer extension assay .", "The catalytic domain of yeast Pol α and a recombinant version of the yeast Pol α/primase complex were incubated at 37°C and for 60 s with a poly ( dT ) 70mer template , dATP and either an oligo ( A ) or oligo ( dA ) of different length , as indicated in the panel .", "( D ) Primer extension assay .", "The catalytic domain of yeast Pol α was incubated at 37°C and for 60 s with dATP and one of the following 5′-to-3′ primer/template pairs: A12 , A12/ ( dT ) 50; dA12 , ( dA ) 12/ ( dT ) 50; 2 G , A5GGA5/T43CCT5; 4 G , AGGA2GGA5/T43CCT2CCT ; 6 G , AGGA2GGAGGA2/T40CCTCCT2CCT; 7 G , AGGAGGGAGGA2/T40CCTCCCTCCT . DOI: http://dx . doi . org/10 . 7554/eLife . 00482 . 014 The marked difference in catalytic behavior of Pol α in the presence of either an RNA or a DNA primer is consistent with the crystallographic evidence that Pol α recognizes structural features of the primer-template helix .", "Elongation of the RNA primer with deoxynucleotides would cause translocation of the polymerase beyond the RNA/DNA duplex region and onto B-DNA , with loss of the RNA-specific contacts , eventually causing Pol α to stall and terminate primer synthesis .", "We sought to test this mechanism of primer termination by design of DNA primer sequences with altered conformational properties .", "It is known that , although double-stranded DNA normally adopts a B-DNA conformation , its structure can display pronounced conformational variation depending on the local sequence of bases .", "Short runs of consecutive deoxyguanosine nucleotides can induce A-like DNA conformation in double-stranded DNA ( Svozil et al . , 2008; Marathe et al . , 2009 ) .", "We examined the ability of Pol α to extend a templated oligo ( dA ) primer with increasing deoxyguanylate content , introduced incrementally as di- and tri-nucleotides .", "Indeed , as the deoxyguanosine content of the DNA primer increased , the size profile of the extension products synthesized by Pol α changed from that expected of a DNA primer to that of an RNA primer ( Figure 4D ) .", "The observation that extension of the RNA primer is limited to a single turn of double helix indicates that termination of primer synthesis might be triggered by the loss of specific interactions of Pol α with the RNA/DNA duplex .", "Further insight as to the possible structural basis for a termination mechanism can be obtained by comparing the structures of isolated and actively copying Pol α , which reveals a striking difference in the position of the thumb domain .", "In order to adopt the conformation observed in ternary complex , the thumb domain of Pol α must rotate inwards by about 20° , a large conformational rearrangement compared for instance with the limited movement of the thumb domain observed in structures of isolated and copying RB69 Pol ( Figure 5A ) .", "The large difference in the position of the thumb domain in the ternary complex and in the isolated polymerase suggests a molecular basis for the release of the completed primer-template by Pol α: loss of the interactions with the primer-template duplex would allow the thumb domain to rotate away from the DNA , terminating the productive engagement of the polymerase with its substrate . 10 . 7554/eLife . 00482 . 015Figure 5 . Conformational changes of Pol α during DNA priming .", "( A ) Structural superposition of the isolated and active conformations of the polymerase domain of Pol α ( left ) and RB69 Pol ( Wang et al . , 1997; Franklin et al . , 2001; right; PDB entries 1IH7 and 1IG9 ) .", "The protein is shown as ribbons and the nucleic acid duplex as sticks .", "The polymerase domain is colored light blue ( apo ) and dark blue ( active conformation ) .", "In order to help visualize the difference between apo and active conformations of the polymerase , the major inertia axis of the thumb domain for each structure is also shown ( Pettersen et al . , 2004 ) .", "( B ) Change in root mean square deviation ( RMSD ) of the Cα positions over the time of simulation for the HOLO ( 100 ns; left panel ) and APO trajectories ( 150 ns; right panel ) , calculated relative to the starting structure of the trajectory ( see ‘Materials and methods' for details ) .", "( C ) Time-dependent projection on the first two principal components of the trajectory of the apo polymerase structure ( 150 ns , light blue trace ) , the polymerase structure in the ternary complex ( 100 ns , dark blue trace ) and the polymerase structure starting from the conformation adopted in the ternary complex , but in absence of the RNA/DNA duplex and deoxynucleotide ( 100 ns; green trace ) .", "A black arrow marks the starting position of the green trace .", "The position of the crystallographic models of the polymerase in the apo form and in the ternary complex is marked by an orange cross . DOI: http://dx . doi . org/10 . 7554/eLife . 00482 . 01510 . 7554/eLife . 00482 . 016Figure 5—figure supplement 1 . Difference between the root mean square fluctuation ( RMSF ) values of the APO and HOLO trajectories at each Cα position of the polymerase structure . The mean RMSF values of the two HOLO trajectories were subtracted from the mean RMSF value of the two APO trajectories .", "The approximate boundaries of the polymerase domains are indicated below the diagram . DOI: http://dx . doi . org/10 . 7554/eLife . 00482 . 016 We explored this model by performing nanosecond ( ns ) molecular dynamics simulations of the catalytic domain of Pol α , based on the crystallographic models of isolated and actively copying polymerase ( Figure 5B and Figure 5—figure supplement 1 ) .", "Principal component analysis of the trajectories shows that the actively copying polymerase maintains a stable conformation over the 100 ns duration of the simulation .", "In comparison , the structure of the isolated polymerase displays considerable conformational fluctuations , indicative of large-scale conformational changes and local loss of secondary structure that affect in particular the N-amino terminal region and the thumb domain .", "Importantly , the conformational flexibility of the isolated polymerase does not seem to be sufficient to sample conformations adopted by the polymerase in the ternary complex .", "In order to determine whether the conformation of the actively copying polymerase can be maintained in the absence of the RNA/DNA duplex and deoxynucleotide , we analyzed the behavior of the polymerase taking the conformation adopted in the ternary complex as the starting point for the simulation .", "The trajectory showed an increased conformational instability , accompanied by a tendency to relax towards the apo conformation of the polymerase ( Figure 5C and Video 1 ) .", "Taken together , the results of the molecular dynamics simulations indicate that Pol α reaches the active conformation observed in the ternary complex via an ‘induced fit' mechanism that is dependent on its interactions with the RNA/DNA duplex and deoxynucleotide .", "Loss of the optimal set of protein-RNA contacts would weaken the grip of the polymerase on its primer/template substrate and promote primer release . 10 . 7554/eLife . 00482 . 017Video 1 . Principal motion of the Pol α structure in the simulated trajectory of the polymerase , starting from the conformation adopted in the ternary complex but in the absence of RNA/DNA and dGTP ( APOFORCED trajectory; see ‘Materials and methods' for details ) .", "The movie was prepared with the MD movie tool in Chimera , by interpolation of 30 Pol α structures representative of the APOFORCED trajectory .", "The Pol α structure is shown as a ribbon , coloured blue to red from the N- to the C-terminal end of the polypeptide . DOI: http://dx . doi . org/10 . 7554/eLife . 00482 . 017 It is well established that adoption of a conformation competent for catalysis by DNA polymerases requires the concerted movements of the fingers domain , that rotates upwards to form the nucleotide binding site , and of the thumb domain , that embraces the primer-template duplex .", "Comparison of Pol α structures in the apo , binary and ternary complex reveals a further and unexpected rearrangement: as the polymerase morphs from the apo to the active conformation , the palm domain tilts away from the rising fingers , by rotating on the short helix containing the highly conserved 864-DFNSLYPS-871 motif , known as region II of B-family DNA polymerases ( Wang et al . , 1989; Delarue et al . , 1990; Hubscher et al . , 2002; Figure 6A , Figure 6—figure supplement 1 , Video 2 ) . 10 . 7554/eLife . 00482 . 018Figure 6 . Palm domain movement controls nucleotide access to active site .", "( A ) Structural superposition of the polymerase domain of Pol α in the apo form ( light blue ) , in the binary ( blue ) and ternary ( dark blue ) complex .", "Only the palm and fingers domains of the polymerase are shown .", "The position of the region II sequence , conserved in B-family DNA polymerases , is indicated .", "( B ) Polypeptide conformation in the region II sequence of Pol α .", "From left to right , the panel shows the conformation of the polymerase in the apo form ( light blue ) , in the two polymerase molecules in the asymmetric unit of the binary complex crystals ( blue ) and in the ternary complex form ( dark blue ) .", "The polypeptide is shown as a thin tube .", "The 866-NS-867 di-peptide and the dGTP nucleotide are shown as sticks . DOI: http://dx . doi . org/10 . 7554/eLife . 00482 . 01810 . 7554/eLife . 00482 . 019Figure 6—figure supplement 1 . Structures of Pol α in the apo form , in a binary complex with RNA/DNA and in a ternary complex with RNA/DNA and dGTP . Both structures present in the asymmetric unit of the binary complex crystals are shown , as they differ markedly in conformation of the thumb and palm domains ( see also text for details ) .", "The polymerase is shown as ribbon , coloured blue to red from the N-terminus .", "The RNA/DNA molecule is shown as all-atom stick model . DOI: http://dx . doi . org/10 . 7554/eLife . 00482 . 01910 . 7554/eLife . 00482 . 020Video 2 . Animation that shows morphing between the Pol α structures in the ternary complex and in the apo form .", "Pol α is shown in ribbon representation , coloured light blue .", "The animation was prepared with the MD movie tool in Chimera . DOI: http://dx . doi . org/10 . 7554/eLife . 00482 . 020 Rotation of the palm domain is accompanied by a conformational rearrangement of region II that is necessary in order to accommodate the phosphoribose moiety of the incoming nucleotide ( Figure 6B ) .", "The two copies of binary complex present in the asymmetric unit of the crystals capture intermediate states of the transition , as in one binary complex the conformation of region II is apo-like , whereas in the second copy region II adopts a conformation akin to that observed in the ternary complex .", "The range of positions adopted by the palm domain in the apo , binary and ternary complex structures suggests that adoption of an active conformation by Pol α might require a wider set of conformational rearrangements than predicted by current models of DNA polymerase activity .", "The observed movement of the palm domain could participate in a ‘ratchet' mechanism for unidirectional translocation of Pol α , as return of the palm domain to the apo conformation after catalysis would propel the polymerase forward onto the next templating base .", "Interestingly , site-specific mutations of Ser863 in region II of human Pol α ( Ser867 of yeast Pol α ) , which acts as the pivot during rotation of the palm domain , cause a drastic reduction in the specific activity of the polymerase without changing its kinetic parameters , in agreement with a potential structural role of this serine residue in DNA polymerisation ( Dong et al . , 1993 ) ." ], [ "Here we have presented structural , biochemical and computational experiments that address the essential role of Pol α in the initiation of DNA synthesis in eukaryotic replication .", "Our findings provide evidence for a specific mechanism of RNA primer recognition , limited extension and termination by Pol α , that is encoded in the interaction of the polymerase with the hybrid RNA primer/DNA template helix ( Figure 7 ) .", "Recognition of the intrinsic and induced geometry of the RNA/DNA helix by Pol α would prompt rapid dNTP polymerization , until a full turn of DNA double helix has been synthesized .", "Translocation away from the RNA/DNA helix and synthesis of B-form DNA would cause Pol α to stall , akin to a locomotive running into railtrack of different gauge .", "Loss of the optimal interface contacts between Pol α and the primer-template helix would favor a conformational change towards the apo conformation of the polymerase , with consequent disengagement of Pol α from the completed RNA-DNA primer . 10 . 7554/eLife . 00482 . 021Figure 7 . Mechanism of DNA primer synthesis by Pol α .", "( A ) Priming eukaryotic replication requires synthesis of an RNA primer by the primase subunit of the Pol α/primase complex , intramolecular primer hand-off to the catalytic subunit of Pol α and limited primer extension with DNA; processive elongation of the completed RNA-DNA primer by Pol δ results in Okazaki fragment synthesis .", "The ribbon model of the Pol α/primase complex was assembled from the crystal structures of apo Pol α ( this manuscript ) , the yeast Pol α CTD–B subunit complex ( Klinge et al . , 2009 ) , and the archaeal primase ( Lao-Sirieix et al . , 2005a ) .", "The location of the Pol α/primase complex components in the model is based on previous electron microscopy and biochemical data ( Nunez-Ramirez et al . , 2011; Kilkenny et al . , 2012 ) and is not meant to give an accurate representation of their reciprocal spatial relationship .", "The crystal structures of isolated and copying Pol α and Pol δ are shown in spacefill representation .", "The model of Pol δ is based on PDB entry 3IAY ( Swan et al . , 2009 ) .", "In the diagram , DNA is shown in yellow and RNA in magenta .", "( B ) The catalytic cycle of Pol α consists of specific recognition of the templated RNA primer , rapid and limited primer extension with dNTPs and termination of synthesis induced by polymerization of B-form DNA .", "Constitutive tethering of primase to Pol α ( not drawn in the panel; Kilkenny et al . , 2012 ) ensures efficient recycling of Pol α onto the 3′-terminus of the next RNA primer . DOI: http://dx . doi . org/10 . 7554/eLife . 00482 . 021 The emerging picture of Pol α as a specialized polymerase that extends processively an RNA primer with deoxynucleotides to the limited size of a helical turn has important implications for our understanding of lagging strand synthesis .", "Uninterrupted DNA synthesis during replication depends on the tight coordination of Pol α function with the upstream activity of primase , that synthesizes the RNA primer , and the downstream activity of Pol δ , that elongates the resulting RNA-DNA primers .", "The efficient mechanism for the rapid extension and controlled termination of RNA primers by Pol α uncovered here seems ideally suited for the frequently repeated , highly integrated priming events taking place on the lagging strand template .", "After termination of synthesis and release of the completed primer , the physical tethering of Pol α to primase would facilitate its recycling onto the 3′-end of the next RNA primer ( Nunez-Ramirez et al . , 2011; Kilkenny et al . , 2012 ) .", "The evidence for a mechanism of primer release which stems from Pol α's ability to discriminate between different shapes of the primer-template helix is relevant to our mechanistic understanding of the coordinated sequence of events that take place during Okazaki fragment synthesis .", "Spontaneous primer release once a helical equivalent of deoxynucleotides has been added to the RNA primer would make the 3′-terminus of the RNA-DNA primer directly available for extension by Pol δ and Pol ε , the DNA polymerases that synthesize the bulk of genomic DNA .", "Such a mechanism provides a straightforward model of primer hand-off in eukaryotic replication , by removing the need for molecular transactions between primer-bound Pol α and the replicative apparatus deputed to leading and lagging strand synthesis .", "We note that , although the molecular details differ , the mechanism of primer release by Pol α described here is conceptually similar to the mechanism of polymerase switching proposed for Pol η after lesion by-pass ( Biertumpfel et al . , 2010; Silverstein et al . , 2010 ) : for both polymerases , termination of DNA synthesis appears to be a direct consequence of the specific way in which they interact with their nucleic acid substrate .", "The ability of primases to synthesise RNA primers of discrete size has been well characterized ( Kuchta and Stengel , 2010 ) .", "Our work has highlighted a previously unappreciated functional similarity between primase and Pol α , as extension of an RNA primer by Pol α leads to controlled termination of synthesis , in a functionally analogous manner to the known counting ability of primase .", "This functional symmetry in the behavior of the two polymerases responsible for priming DNA synthesis seems logical , as the lack of proof-reading ability by Pol α poses a potential threat to genomic stability .", "Thus , in addition to excising the RNA portion of the primer , eukaryotic replication faces the additional challenge of correcting possible mismatches introduced by Pol α in the DNA segment of the primer .", "How mistakes made by Pol α are corrected by the replication apparatus has not been fully elucidated yet but current evidence points to a proofreading role of Pol δ ( Pavlov et al . , 2006 ) .", "The mechanism of primer termination indicated by our findings would constitute a simple and elegant way to limit the extent of inaccurate DNA that needs to be corrected from the 5′-terminus of each Okazaki fragment ." ], [ "A gene segment corresponding to amino acids 349–1258 of the yeast Pol α ( 910 residues; hereinafter referred to as Pol α ) was PCR amplified from Saccharomyces cerevisiae genomic DNA and inserted by enzymatic restriction into the pRSFDuet-1 vector ( Merck KGaA , Darmstadt , Germany ) for bacterial over-expression , as a polypeptide fused at its N-terminus to a dual histidine and streptavidin TEV-cleavable tag .", "The pRSF-Pol α construct was over-expressed with IPTG in Escherichia coli BL21 ( DE3 ) Rosetta2 strain ( Invitrogen Life Technologies Ltd , Paisley , UK ) growing at 20°C in Turbo Broth™ ( Molecular Dimensions Ltd , Newmarket , UK ) .", "The Pol α protein was purified by successive steps of Co-NTA affinity chromatography ( Qiagen Ltd , Manchester , UK ) , Heparin Sepharose chromatography ( GE Healthcare Life Sciences , Little Chalfont , UK ) and Strep-Tactin chromatography ( IBA GmbH , Göttingen , Germany ) , followed by tag removal and a final step of size exclusion chromatography on a Superdex 200 16/60 ( GE Healthcare ) .", "The eluted Pol α sample was concentrated to 100 μM in 20 mM Hepes pH 6 . 8 , 300 mM NaCl , 10% glycerol buffer and divided in small aliquots that were flash frozen in liquid nitrogen for crystallography and functional studies .", "A version of pRSFDuet-1-Pol α carrying mutations R508A , N509A , D998N construct was prepared with QuikChange site-directed mutagenesis kit ( Agilent Technologies UK Ltd , Stockport , UK ) , according to the manufacturer's instructions .", "Catalytic Asp998 was mutated to asparagine in order to produce an inactive polymerase; unexpectedly , the D998N mutation greatly reduced but did not abolished polymerisation by Pol α ( Figure 1—figure supplement 1 ) .", "The double mutation R508A , N509A was introduced in order to promote crystallization of the ternary complex .", "As expression levels of the Pol α mutant were lower than observed for the wild-type protein , over-expression was carried out in a 30 l BIOSTAT® ( Sartorius Stedim UK Ltd , Epsom , UK ) bioreactor .", "Purification of the Pol α mutant was carried out in the same way as for the wild-type protein .", "For preparation of recombinant Pol α/primase complex , the pRSFDuet-1 vector was adapted for polycistronic expression of Pol α , the B subunit and the heterodimeric primase ( RP and LP , unpublished data ) .", "Expression of the Pol α/primase complex was carried out in 30 l BIOSTAT® ( Sartorius Stedim ) bioreactor , using 20 l of auto induction media .", "Purification of the Pol α/primase complex was carried out according to a similar protocol followed for Pol α purification , including steps of Ni-NTA chromatography ( Qiagen ) , Heparin sepharose chromatography ( GE Healthcare ) , Strep-Tactin chromatography ( IBA ) , tag removal by TEV cleavage and gel filtration chromatography .", "All steps of this purification were carried out at 4°C to avoid degradation of the complex .", "The purified Pol α/primase complex was concentrated to 20 μM in 20 mM Hepes pH 7 . 0 , 300 mM KCl , 10% glycerol buffer and divided in small aliquots that were flash frozen in liquid nitrogen for functional studies .", "Crystals of Pol α ( apo ) were grown by mixing equal volumes of protein at 50 μM and 0 . 1 M Bicine pH 9 . 4 , 6–10% PEG 8000 and improved by streak seeding .", "The X-ray crystal structure of Pol α was determined by single-wavelength anomalous scattering using selenomethionine-labelled ( SeMet ) protein crystals .", "Pol α crystallized in the monoclinic space group P21 , with cell dimensions: a = 74 . 4 Å b = 127 . 1 Å c = 74 . 5 Å , β = 104 . 8° .", "X-ray diffraction data for native and SeMet crystals were collected at European Synchrotron Research Facility ( ESRF ) on beam line ID23-1 at the peak wavelength of the Selenium K edge .", "Data were indexed , integrated , scaled and merged using MOSFLM ( Battye et al . , 2011 ) and SCALA ( Evans , 2011 ) of the CCP4 program suite ( Collaborative Computational Project , 1994 ) .", "Phasing and initial automatic model building of SeMet Pol α was carried out in PHENIX ( Adams et al . , 2010 ) and the crystallographic model was completed manually and refined at 2 . 67 Å resolution in REFMAC 5 . 5 ( Murshudov et al . , 1997 ) , BUSTER ( Bricogne et al . , 2011 ) and PHENIX , to R-factor/R-free ( % ) of 19 . 6/23 . 4 .", "As SeMet Pol α crystallized in a different space group ( orthorhombic space group P22121 , cell dimensions: 74 . 0 Å 127 . 4 Å 144 . 1 Å ) determination of the native Pol α structure was achieved by molecular replacement with PHASER ( McCoy et al . , 2007 ) , using the crystal structure of SeMet Pol α as search model .", "The crystal structure of native Pol α structure was refined at 2 . 3 Å resolution using BUSTER and PHENIX , to R-factor/R-free ( % ) of 20 . 5/23 . 6 .", "For both SeMet and native Pol α structures , amino acids 677–679 ( 3 residues ) , 816–847 ( 32 residues ) , 1056–1062 ( 7 residues ) , 1176–1185 ( 10 residues ) and 1129–1258 ( 30 residues ) were not visible or poorly ordered in the electron density maps during refinement and were therefore excluded from the final refined model .", "Details of data processing and crystallographic refinement are provided in Table 1 .", "Co-crystals of Pol α bound to an RNA/DNA duplex ( binary complex ) were grown by vapour diffusion in hanging drop at 18°C , by mixing Pol α with 5′-AGGCGGGCAG-3′ RNA 10mer annealed to 5′-TTTTCGCTGCCCGCCT-3′ DNA 16mer and 1 mM di-deoxycytidine triphosphate with 0 . 1 M Bicine pH 8 . 0 , 12% PEG 3350 and 10 mM MgCl2 .", "The binary complex crystallized in the triclinic space group P1 , with cell dimensions: a = 72 . 1 Å b = 74 . 8 Å c = 117 . 0 Å α = 82 . 3° β = 72 . 6° γ = 82 . 4° , and two copies of the binary complex in the asymmetric unit .", "X-ray diffraction data were collected at ESRF beam line ID14-1 , and processed as for the native Pol α crystal structure .", "The structure was solved by molecular replacement in PHASER , using the crystallographic model of the apo Pol α as search model , and refined to 3 . 0 Å resolution with PHENIX , to R-factor/R-free ( % ) of 25 . 5/28 . 6 .", "Amino acids 349–350 ( 2 residues ) , 816–847 ( 32 residues ) , 1176–1186 ( 11 residues ) , 1243–1258 ( 16 residues ) in both polymerase chains were not visible or poorly ordered in the electron density maps during refinement and were therefore excluded from the final refined model .", "In addition , amino acids 1056–1062 ( 7 residues ) and 1133–1166 ( 34 residues ) in polymerase chain B were disordered and excluded from the final model .", "Of the DNA 16mer/RNA 10mer substrate , only a duplex region spanning 8 bp from the 3′-terminus of the RNA primer was visible in the electron density map and was included in the final model .", "Three RNA nucleotides at the 5′-end of chains D , F and three DNA nucleotides at the 5′-end of chain E are poorly ordered in the map and their position must be considered tentative .", "Despite the presence of nucleotide in the crystallization buffer , no electron density for ddCTP was visible in the map .", "Details of data processing and the crystallographic refinement are provided in Table 1 .", "For co-crystallization of Pol α with RNA/DNA and deoxynucleotide ( ternary complex ) , the R508A , N509A , D998N Pol α mutant was used .", "The ternary complex was reconstituted prior to crystallization by incubating Pol α with a twofold excess of 5′-CGGCGGGCAG-3′ RNA 10mer annealed to 5′-TGAGCGTGTGTACCCCTGCCCGCCG-3′ DNA 25mer and 5 mM deoxyguanosine triphosphate .", "Crystals of ternary complex were grown under oil in micro-batch setup , by mixing the sample with 200 mM MgAc2 , 10% PEG 8000 crystallization buffer , at a protein to buffer ratio of 1 . 1:1 . 8 ( v/v ) .", "Crystals were optimised by addition of 3% glycerol and by batch seeding .", "The ternary complex crystallized in the orthorhombic space group P212121 , with cell dimensions: a = 111 . 7 Å b = 145 . 7 Å c = 197 . 2 Å , and two copies of the ternary complex in the asymmetric unit .", "X-ray diffraction data were collected at beamline I02 of the Diamond Light Source , and processed as for the native Pol α crystal structure .", "The structure was solved by molecular replacement in PHASER , using the crystallographic model of the native Pol α as search model , and refined to 3 . 1 Å resolution with BUSTER and PHENIX , to R-factor/R-free ( % ) of 21 . 1/24 . 8 .", "Amino acids 507–510 ( 4 residues ) , 677–680 ( 4 residues ) , 816–839 ( 24 residues ) , 1175–1187 ( 13 residues ) and 1243–1258 ( 16 residues ) were not visible or poorly ordered in the electron density maps during refinement and were therefore excluded from the final refined model .", "Details of data processing and the crystallographic refinement are provided in Table 1 .", "The assay was performed in a 20 μl reaction containing 10 nM wild-type Pol α ( amino acid 349–1258 ) , 0 . 75 μM template poly ( dT ) DNA 70mer ( Sigma-Genosys ) , 0 . 5 μM oligo ( dA ) DNA or oligo ( A ) RNA 15mer ( Sigma-Genosys ) , 100 μM dATP in 20 mM Tris-HCl pH 8 . 0 , 50 mM NaCl , 10 mM MgCl2 , 0 . 2 mg/ml BSA , 2 mM DTT buffer .", "Reactions were initiated by the addition of 1 μl of radio-labeled 2 mM dATP ( 1:30 v/v dilution of 0 . 7 μCi [α32P] dATP with cold dATP ) , incubated at 37°C for the appropriate time and quenched by addition of 12 μl of formamide loading buffer ( 95% formamide , 0 . 025% bromophenol blue , 0 . 025% xylene cyanol , 5 mM EDTA and 0 . 025% SDS ) and heating at 70°C for 5 min .", "The reaction products were separated by electrophoresis in denaturing conditions , on a pre-run 7 M urea 12 . 5% polyacrylamide gel run for 2 hr at 2000 V . Gels were fixed in 10% acetic acid , 10% ethanol for 5 min , dried under vacuum and imaged by storage phosphor autoradiography in a Typhoon™ FLA 9000 biomolecular imager ( GE Healthcare ) .", "Quantitative gel analysis was performed with ImageQuant TL ( GE Healthcare ) .", "The RNA and DNA sequences used in the experiment of Figure 4D are shown in Table 2 . 10 . 7554/eLife . 00482 . 022Table 2 . Oligonucleotides used in the primer extension assay of Figure 4DDOI: http://dx . doi . org/10 . 7554/eLife . 00482 . 022Primer nameTypePrimer5–3′Template5–3′A12RNAA12T50dA12DNAdA12T50G2DNAAAAAAGGAAAAAT43CCT5G4DNAAGGAAGGAAAAAT43CCTTCCTG6DNAAGGAAGGAGGAAT40CCTCCTTCCTG7DNAAGGAGGGAGGAAT40CCTCCCTCCT Residual activity of the D998N R508A N509A Pol α mutant was demonstrated by the primer extension assay , as described; the protein concentration in the assay was varied over a range of concentrations up to 10 μM ( Figure 1—figure supplement 1 ) .", "In preparation for the simulation , three loop regions of Pol α corresponding to amino acids 677–679 ( 3 residues ) , 1056–1062 ( 7 ) and 1176–1185 ( 10 ) , that are disordered in the crystal structures of Pol α , were modeled using MODELLER9v8 ( Sali and Blundell , 1993; Fiser et al . , 2000 ) .", "A longer , disordered loop region spanning amino acids 816–840 ( 25 residues ) was too long for modeling without template; the equivalent region in the crystal structure of Pol δ ( residues 578–585; PDB ID 3IAY ) was used instead .", "The C-terminal helix of the thumb domain ( residues 1229–1242 ) is disordered in the apo structure and was modeled based on its conformation in the structure of the ternary complex .", "After modelling of the missing loops , the Pol α polypeptide comprises 877 residues .", "In addition to the Pol α polypeptide , the ternary complex contains one template DNA molecule of 16 deoxyribonucleotides , one RNA primer of nine ribonucleotides extended at the 3′-end with two deoxynucleotides and one deoxyguanosine triphosphate ( dGTP ) .", "The 5′-terminal ribonucleotide of the RNA primer was excluded from the model of ternary complex , as it makes extensive contacts with the other Pol α chain in the asymmetric unit and does not form a Watson-Crick base pair .", "The dynamic behaviour of Pol α was explored by simulating the trajectory of the apo structure ( APO trajectory ) , of the ternary complex ( HOLO trajectory ) and of the polymerase structure in the conformation adopted in the ternary complex but in the absence of RNA/DNA and dGTP ( APOFORCED trajectory ) .", "A total of six trajectories were simulated: two APO trajectories of 150 ns , two HOLO trajectories of 100 ns and two APOFORCED trajectories of 100 ns .", "Molecular dynamics simulations were performed with the AMBER11 package , using the AMBER FF99SB force field ( Wang et al . , 2004 ) .", "A dodecahedral box of water molecules , treated as in the TIP3P model ( Jorgensen et al . , 1983 ) , was built around the complexes and a physiological concentration of 0 . 15 M NaCl ( 72 counterions each ) was added to the box .", "The dGTP molecule , present in the HOLO structure , has been parameterized using the GAFF code , as implemented in AMBER .", "The following protocol was used for all the simulations: ( 1 ) in vacuo minimization ( 1000 steps ) ; ( 2 ) minimization , keeping the complexes fixed , allowing water molecules and ions to equilibrate ( 1000 steps of steepest descent plus 1000 steps of conjugate gradient ) ; ( 3 ) minimization of all the system , without restrictions ( 1000 steps of steepest descent plus 1000 steps of conjugate gradient ) ; ( 4 ) NVT equilibration , 1 ns; ( 5 ) production phase .", "All calculations were performed with the CUDA-enabled version of PMEMD ( Goetz , et al . , 2012 ) , using TESLA GPUs at the High Performance Computing ( HPC ) cluster of the University of Cambridge .", "Two TESLA-GPUs perform approximately 4 ns/day , when computing a system of approximately 115000 atoms .", "Analysis of the trajectories was performed with the AMBERTOOLS 1 . 5 and GROMACS packages ( Hess et al . , 2008 ) .", "Principal component analysis was used to compare the principal modes of motion of Pol α in the APO , HOLO and APOFORCED trajectories .", "The covariance matrix of the APOFORCED trajectories was constructed , based on the three-dimensional positional fluctuations of C-α atoms from their ensemble average position , and diagonalized , creating a set of eigenvectors and eigenvalues that represent the direction and the amplitude of the motion , respectively .", "All the trajectories were projected on the first two eigenvectors and eigenvalues of the APOFORCED trajectories , in order to compare the regions sampled along common eigenvectors and to analyze the distribution of the motion .", "Construction and diagonalization of the covariance matrix was performed using the g_covar command of GROMACS .", "Projection of structures onto eigenvectors was performed using the g_anaeig command of GROMACS .", "All the other analyses have been performed with GROMACS tools , such as g_rms , g_rmsf , g_cluster .", "AmberTools's ptraj was used for the DSSP calculations and for all the transformation of the trajectories .", "Artwork figures were prepared with PyMOL ( Version 1 . 5 . 0 . 4 , Schrödinger LLC , http://www . pymol . org/ ) , Chimera ( Pettersen et al . , 2004 ) and QuteMol ( Tarini et al . , 2006 ) .", "Movies were prepared with Chimera ." ] ]

[ "The DNA Polymerase α ( Pol α ) /primase complex initiates DNA synthesis in eukaryotic replication .", "In the complex , Pol α and primase cooperate in the production of RNA-DNA oligonucleotides that prime synthesis of new DNA .", "Here we report crystal structures of the catalytic core of yeast Pol α in unliganded form , bound to an RNA primer/DNA template and extending an RNA primer with deoxynucleotides .", "We combine the structural analysis with biochemical and computational data to demonstrate that Pol α specifically recognizes the A-form RNA/DNA helix and that the ensuing synthesis of B-form DNA terminates primer synthesis .", "The spontaneous release of the completed RNA-DNA primer by the Pol α/primase complex simplifies current models of primer transfer to leading- and lagging strand polymerases .", "The proposed mechanism of nucleotide polymerization by Pol α might contribute to genomic stability by limiting the amount of inaccurate DNA to be corrected at the start of each Okazaki fragment ." ]

[ "During mitosis , a cell duplicates its DNA and then divides , ultimately generating two genetically identical daughter cells .", "In eukaryotes , the process of DNA duplication occurs at multiple sites throughout the genome: at each site , the antiparallel strands of the parental DNA separate and provide a template for DNA polymerase ( Pol ) , the enzyme that synthesizes the two new DNA strands .", "Duplication of the DNA proceeds in both directions from each site through the polymerization of nucleotides to form new strands of DNA that are complementary to the template strands .", "However , since DNA polymerases can only polymerize nucleotides in one direction , the 5′ to 3′ direction , synthesis of the so-called leading strand proceeds continuously , whereas the other , lagging strand is synthesized in fragments .", "The task of duplicating the bulk of the DNA is shared between Pol δ , which is primarily responsible for synthesis of the lagging strand , and Pol ε , which fulfils the same role for the leading strand .", "However , Pols δ and ε cannot initiate DNA synthesis by themselves; short RNA-DNA chains called primers must also be paired to each template strand .", "Production of the primers requires the concerted action of two more enzymes: an RNA polymerase known as primase , and another DNA polymerase called Pol α .", "It is known that completion of the RNA-DNA primer requires Pol α to increase the length of the RNA segment by adding extra nucleotides , but the details of this process are poorly understood .", "Perera et al . combined crystallographic , biochemical and computational evidence to describe how Pol α first recognizes and then extends the RNA strand in the primer .", "They found that Pol α recognizes the particular shape of double helix—an A-form helix—that is formed by the DNA template and the RNA primer .", "The geometry of this helix prompts the Pol α enzyme to start adding nucleotides to the RNA in the primer .", "Perera et al . determined that once a full turn of double-helix DNA has been synthesized , Pol α is no longer in direct contact with the A-form helix , which causes the enzyme to disengage and terminate polymerization , leaving behind the now complete RNA-DNA primer .", "Perera et al . offer a new paradigm for understanding the initiation of DNA synthesis in eukaryotic replication .", "Their work suggests that Pol α has the ability to discriminate between different shapes of the primer-template helix , thus providing a mechanistic understanding of primer release .", "The spontaneous release of the primer offers a simple and elegant way to limit DNA synthesis by Pol α , a polymerase that is prone to error , and to make the RNA-DNA primer directly available for extension by Pol δ and Pol ε ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "biochemistry and chemical biology", "cell biology" ]

[ [ "The Wnt/β-catenin signaling cascade is an ancient cell communication pathway that operates context-dependent transcriptional switches to control animal development and tissue homeostasis ( Cadigan and Nusse , 1997 ) .", "Deregulation of the pathway in adult tissues can lead to many different cancers , most notably colorectal cancer ( Clevers and Nusse , 2012 ) .", "Wnt-induced transcription is mediated by T cell factors ( TCF1/3/4 , LEF1 ) bound to Wnt-responsive enhancers , but their activity depends on the co-activator β-catenin ( Armadillo in Drosophila ) , which is rapidly degraded in unstimulated cells .", "Absence of β-catenin thus defines the OFF state of these enhancers , which are silenced by Groucho/TLE co-repressors bound to TCF via their Q domain .", "This domain tetramerizes to promote transcriptional repression ( Chodaparambil et al . , 2014 ) , which leads to chromatin compaction ( Sekiya and Zaret , 2007 ) apparently assisted by the interaction between Groucho/TLE and histone deacetylases ( HDACs ) ( Jennings et al . , 2008; Turki-Judeh and Courey , 2012 ) .", "Wnt signaling relieves this repression by blocking the degradation of β-catenin , which thus accumulates and binds to TCF , converting the Wnt-responsive enhancers into the ON state .", "This involves the binding of β-catenin to various transcriptional co-activators via its C-terminus , most notably to the CREB-binding protein ( CBP ) histone acetyltransferase or its p300 paralog ( Mosimann et al . , 2009 ) , resulting in the transcription of the linked Wnt target genes .", "Subsequent reversion to the OFF state ( for example , by negative feedback from high Wnt signaling levels near Wnt-producing cells , or upon cessation of signaling ) involves Groucho/TLE-dependent silencing , but also requires the Osa/ARID1 subunit of the BAF ( also known as SWI/SNF ) chromatin remodeling complex ( Collins and Treisman , 2000 ) which binds to β-catenin through its BRG/BRM subunit ( Barker et al . , 2001 ) .", "Cancer genome sequencing has uncovered a widespread tumor suppressor role of the BAF complex , which guards against numerous cancers including colorectal cancer , with >20% of all cancers exhibiting at least one inactivating mutation in one of its subunits , most notably in ARID1A ( Kadoch and Crabtree , 2015 ) .", "Thus , it appears that failure of Wnt-inducible enhancers to respond to negative feedback imposed by the BAF complex strongly predisposes to cancer .", "How β-catenin overcomes Groucho/TLE-dependent repression remains unclear , especially since β-catenin and TLE bind to TCF simultaneously ( Chodaparambil et al . , 2014 ) .", "Therefore , the simplest model envisaging competition between β-catenin and TLE cannot explain this switch , which implies that co-factors are required .", "One of these is Pygo , a chromatin reader binding to histone H3 tail methylated at lysine 4 ( H3K4m ) via its C-terminal PHD finger ( Fiedler et al . , 2008 ) .", "In Drosophila where Pygo was discovered as an essential co-factor for activated Armadillo , its main function appears to be to assist Armadillo in overcoming Groucho-dependent repression ( Mieszczanek et al . , 2008 ) .", "We recently discovered that Pygo associates with TCF enhancers via its highly conserved N-terminal NPF motif that binds directly to the ChiLS complex , composed of a dimer of Chip/LDB ( LIM domain-binding protein ) and a tetramer of SSDP ( single-stranded DNA-binding protein , also known as SSBP ) .", "Notably , ChiLS also binds to other enhancer-bound NPF factors such as Osa/ARID1 and RUNX , and to the C-terminal WD40 domain of Groucho/TLE , and thus forms the core module of a multiprotein complex termed ‘Wnt enhanceosome’ ( Fiedler et al . , 2015 ) .", "We proposed that Pygo renders this complex Wnt-responsive by capturing Armadillo/β-catenin through the Legless adaptor ( whose orthologs in humans are BCL9 and B9L , also known as BCL9-2 ) ( Kramps et al . , 2002; Städeli and Basler , 2005 ) .", "The salient feature of our model is that the Wnt enhanceosome keeps TCF target genes repressed prior to Wnt signaling while at the same time priming them for subsequent Wnt induction , and for timely shut-down via negative feedback depending on Osa/ARID1 ( Fiedler et al . , 2015 ) .", "Here , we assess the function of Legless and BCL9/B9L within the Wnt enhanceosome .", "Using a proximity-labeling proteomics approach ( called BioID; Roux et al . , 2012 ) in human embryonic kidney ( HEK293 ) cells , we uncovered a compelling association between BCL9/B9L and the core Wnt enhanceosome components , regardless of Wnt signaling .", "Co-immunoprecipitation ( coIP ) and in vitro binding assays based on Nuclear Magnetic Resonance ( NMR ) revealed that BCL9 and B9L associate with TLE3 through their C-termini , and that they bind directly to ChiLS via their evolutionary conserved homology domain 3 ( HD3 ) .", "These elements are outside the sequences mediating the adaptor function between Pygo and Armadillo/β-catenin , but they are similarly important for Wnt responses during Drosophila development and in human cells , as we show by CRISPR/Cas9-based genome editing .", "Our results consolidate and refine the Wnt enhanceosome model , indicating a constitutive scaffolding function of BCL9/B9L within this complex .", "Our evidence further suggests that BCL9/B9L but not Pygo undergoes a β-catenin-dependent rearrangement within the enhanceosome upon Wnt signaling , gaining proximity to TCF , which might trigger enhanceosome switching ." ], [ "Legless and BCL9/B9L paralogs ( collectively referred to as Legless/BCL9 below ) bind to Pygo and Armadillo/β-catenin via their conserved N-terminal homology domains 1 and 2 ( HD1 , HD2 ) , respectively ( Figure 1A ) .", "Previous evidence suggested that the sole function of Legless during Drosophila development is to link Pygo to Armadillo ( Kramps et al . , 2002 ) .", "However , C-terminal truncations of BCL9/B9L behave as dominant-negatives regarding Wnt responses in mammalian cell lines ( Adachi et al . , 2004; de la Roche et al . , 2008 ) , which suggested that their C-termini harbor functionally relevant elements .", "Indeed , these elements are missing in a Legless truncation encoded by a known lgs mutation ( lgs7I ) , owing to a stop codon downstream of HD2 ( Kramps et al . , 2002 ) , yet this truncation should be fully competent to link Pygo to Armadillo .", "Consistent with the notion of functional elements downstream of HD2 , the C-termini of Legless/BCL9 exhibit several conserved sequence blocks , in particular HD3 ( Figure 1A ) which is found in all orthologs from the most primitive animals to humans .", "No ligand has been identified for HD3 , nor for the C-terminus of Legless/BCL9 . 10 . 7554/eLife . 20882 . 003Figure 1 . The C-terminus of Legless is required for Wg-dependent patterns in flies .", "( A ) Cartoon of lgs mutants , with domain boundaries indicated ( grey , deleted sequences ) .", "( B ) Western blot of lysates from lgs mutant embryos ( genotypes indicated above panels ) , probed with antibodies as indicated , confirming stability of the lgs2-8 truncation product ( ~65 kDa , arrowhead; an unspecific cross-reactivity of this α-Lgs antiserum is marked by asterisk ) .", "( C ) Developmental rates and survival of wt and lgs homozygous mutant larvae as indicated; first-instar larvae were picked ( n = 25 ) , and % pupation ( left ) or hatching of flies ( right ) was scored daily; error bars , SEM of four independent experiments .", "( D ) Posterior leg and abdominal phenotypes of wt and lgs mutant flies , as indicated; representative examples are shown; arrow , missing sternite . DOI: http://dx . doi . org/10 . 7554/eLife . 20882 . 00310 . 7554/eLife . 20882 . 004Figure 1—figure supplement 1 . CRISPR/Cas9-based gene editing strategies for Drosophila legless . Cartoon of lgs exon boundaries and targeting sites in exon 4; sgRNA target sequences and PAM sites are highlighted in the wt sequence , with corresponding mutant sequences underneath; red , premature stop codons generated in 1-5 and 2-8 alleles . DOI: http://dx . doi . org/10 . 7554/eLife . 20882 . 004 To test the function of these sequences downstream of HD2 , we deleted HD3 from endogenous Drosophila lgs ( lgsΔHD3 ) using the CRISPR/Cas9 system ( Port et al . , 2014 ) .", "We also generated a truncation allele ( lgs2-8 ) that deletes HD3 plus the entire C-terminus ( Figure 1—figure supplement 1 ) .", "We isolated fly lines bearing these alleles , and confirmed that they express mutant proteins of the predicted sizes , and at normal levels ( Figure 1B ) .", "Notably , the lgs2-8 truncation allele causes pupal lethality if combined with a strong lgs allele ( lgs20F; Kramps et al . , 2002 ) .", "Furthermore , homozygous lgs2-8 animals ( derived from homozygous mothers ) show severely delayed development , and most die in their pupal cases , with <25% of them hatching as flies ( Figure 1C ) .", "A high fraction of these escapers exhibit patterning defects indicative of attenuated signaling by Wingless ( Wg , Drosophila Wnt ) , as previously observed in flies bearing weak alleles of lgs , pygo , and dTCF ( Brunner et al . , 1997; Kramps et al . , 2002; Thompson et al . , 2002 ) —namely proximal leg duplications and dorso-ventral polarity leg defects ( in ~1/2 mutant flies ) as well as loss of sternites in the ventral abdomen ( in ~1/6 mutant flies; Figure 1D ) .", "Identical phenotypes were observed in homozygotes bearing an equivalent truncation allele isolated independently ( lgs1-5; Figure 1—figure supplement 1 ) , ruling them out as off-target effects of the CRISPR/Cas9 engineering process .", "Thus , the sequences downstream of HD2 are essential for Drosophila development , and appear to participate in Wg signaling responses .", "To confirm this , we examined a Wg-responsive transcriptional enhancer from dpp ( dpp . lacZ; Blackman et al . , 1991 ) , which is repressed by high levels of Wg signaling emanating from the Wg source in ventral compartments of leg discs via a homeodomain protein called Brinker ( Theisen et al . , 2007 ) .", "Immunofluorescence revealed a striking derepression of dpp . lacZ in the ventral compartment of >1/3 of the leg discs from lgs2-8 homozygotes ( 28/78 discs ) ( Figure 2A , B; Figure 2—figure supplement 1 ) , demonstrating that the function of the mutant Legless in transducing the Wg signal is severely compromised in these discs .", "This was also true for wing discs ( dissected from of pupating larvae , to ensure matched developmental timing between mutants and wild-type , wt ) : these discs exhibit much reduced expression of Senseless ( Sens ) ( n = 50 discs; Figure 2C , D ) , a Wg target gene that specifies bristles along the wing margin and whose expression is abolished in pygo mutant clones ( Parker et al . , 2002; Fiedler et al . , 2008 ) .", "As expected , this attenuation of Sens results in fewer margin bristles in the homozygous mutant escaper flies ( 61 versus 80 stout margin bristles , and 15 . 6 versus 18 . 8 chemosensory bristles , per wt versus mutant wing , respectively ) , and larger gaps between individual stout bristles ( 22 . 7 versus 17 . 3 μm , per mutant versus wt wing , respectively ) ( Figure 2E , F; mean values from 10 wings dissected from different homozygous lgs2-8 females; the sizes of wt and mutant wings were the same ) . 10 . 7554/eLife . 20882 . 005Figure 2 . The C-terminus of Legless is required for nuclear Wg responses in imaginal discs .", "( A , B )", "Third leg discs from third instar ( A ) wt or ( B ) lgs2-8/lgs2-8 larvae , fixed and stained with antibodies as indicated in panels ( merges on the right ) , showing derepression of dpp . lacZ in the ventral compartment ( arrow ) ; space bar , 50 µm .", "( C , D )", "Corresponding wing discs , showing attenuated Sens expression along the prospective wing margin ( see also insets ) ; space bar , 100 µm .", "( E , F )", "Anterior margin segments of escaper flies , focused on stout margin bristles; yellow arrows , chemosensory bristles; red arrows , missing stout bristles causing gaps that are occupied by ectopic chemosensory bristles .", "( G ) RT-qPCR assays of wing discs dissected from climbing wt and lgs2-8/lgs2-8 third instar larvae , as indicated; values were normalized relative to RpL32 ( internal control ) , and are shown as mean ± SEM relative to wt ( set to 1 ) ; * = p<0 . 05 , ** = p<0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 20882 . 00510 . 7554/eLife . 20882 . 006Figure 2—figure supplement 1 . Additional analysis of lgs2-8 during fly development .", "( A , B )", "Leg discs as in main Figure 2 but giving rise to first or second leg , showing similar derepression of dpp .", "LacZ in the ventral compartments ( arrow ) of lgs2-8/lgs2-8 homozygous larvae as seen in their third leg discs; space bar , 50 mm . ( C ) Stereo images of eyes from wt or heterozygous mutant females also expressing activated Armadillo ( F76 ) ( Freeman and Bienz , 2001 ) , as indicated in the panels , showing comparable suppression of the rough eye phenotype by lgs2-8/+ as by heterozygosity of the strongest known lgs allele ( lgs20F; Kramps et al . , 2002 ) , or by a pygo null allele ( pygoS123; Thompson et al . , 2002 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 20882 . 006 We also used RT-qPCR to examine RNA expression levels of the endogenous Wg target genes engrailed , Distalless and H15 ( Cadigan and Nusse , 1997; Estella et al . , 2008; Wilder and Perrimon , 1995 ) in lysates from wing discs dissected from climbing ( that is , fully grown ) homozygous lgs2-8 larvae .", "We found significant reductions in the expression levels of all three target genes compared to controls ( Figure 2G; note that neither dpp nor dpp . lacZ expression is controlled by Wg in wings discs; see Figure 2C , D ) .", "Finally , we also found that heterozygosity of lgs2-8 suppresses the rough eye phenotype caused by activated Armadillo ( Freeman and Bienz , 2001 ) , indistinguishably from heterozygosity of pygoS123 or lgs20F ( Thompson et al . , 2002; Kramps et al . , 2002 ) ( Figure 2—figure supplement 1 ) .", "These results demonstrate that the C-terminal Legless truncation encoded by our lgs2-8 allele is severely compromised in its ability to transduce the Wg signal in multiple developmental contexts , despite retaining normal adaptor function in linking Pygo and Armadillo .", "Evidently , this adaptor function is not sufficient to sustain normal development if Legless is expressed at endogenous levels , suggesting that the previously reported rescue activities of equivalent Legless fragments ( Kramps et al . , 2002 ) may have stemmed from overexpression .", "We also examined homozygous lgsΔHD3 animals , which hatched as flies without showing any developmental delay ( Figure 1C ) .", "However , ~5% of these flies exhibited leg abnormalities , similarly to those exhibited by lgs2-8 ( Figure 1D ) although the penetrance of this phenotype was much higher in the latter ( see above ) .", "Nevertheless , these leg defects in the lgsΔHD3 homozygotes suggest that HD3 contributes to the function of Legless in transducing Wg responses .", "Given these results from flies , we decided to also assess the function of the BCL9/B9L C-terminus and HD3 in the human Wnt response .", "We thus applied the CRISPR/Cas9 system to HEK293T cells ( Ran et al . , 2013 ) , to generate single knock-out ( KO ) cells lacking BCL9 or its nuclear paralog B9L , or double knock-out ( DKO ) cells lacking both ( Figure 3—figure supplement 1 ) .", "Using a TCF-dependent reporter assay ( called SuperTOP; Veeman et al . , 2003 ) , we found the Wnt-induced transcriptional activity of these DKO cells to be substantially reduced ( to <20% of their parental control cells; Figure 3A ) .", "Loss of BCL9 reduces the transcriptional activity nearly as much as loss of both paralogs , suggesting that BCL9 may be the physiologically limiting paralog in these cells , at least for transient Wnt responses .", "Likewise , the endogenous Wnt target genes AXIN2 ( Lustig et al . , 2002 ) and SP5 ( Hoverter et al . , 2012; Weidinger et al . , 2005 ) are no longer Wnt-inducible in the DKO cells ( Figure 3B ) , reconfirming the critical role of BCL9/B9L in the Wnt response of these cells . 10 . 7554/eLife . 20882 . 007Figure 3 . The C-terminus of BCL9/B9L is required for transcriptional Wnt responses in human cells .", "( A ) SuperTOP assays in wt or KO HEK293T cells lacking BCL9 and/or B9L , as indicated , ±6 hr of Wnt stimulation ( WCM , Wnt3a-conditioned-media ) ; mean ± SEM ( n = 3 independent experiments ) .", "( B ) RT-qPCR assays in wt or DKO HEK293T cells , ±6 hr of 20 mM NaCl or LiCl as indicated , revealing relative transcript levels of AXIN2 or SP5; values were normalized to TBP ( internal control ) , and are shown as mean ± SEM relative to unstimulated wt controls ( set to 1 ) ; note that the uninduced levels of SP5 were unusually high in one of the two DKO clones , but neither of the DKO clones showed Wnt-inducible SP5 .", "( C ) SuperTOP assays in wt or DKO HEK293T cells as in ( A ) , 24 hr after transfection with wt and mutant B9L as indicated ( below , corresponding Western blots; α-ABC , active unphosphorylated β-catenin ) ; ev , empty vector control . DOI: http://dx . doi . org/10 . 7554/eLife . 20882 . 00710 . 7554/eLife . 20882 . 008Figure 3—figure supplement 1 . CRISPR/Cas9-based gene editing strategies for BCL9 and B9L in HEK293T cells .", "( A ) Cartoon of BCL9 and B9L exon boundaries ( with non-coding 5’-and-3’-UTR exons omitted ) ; sgRNA targeting sites are marked for both genes .", "( B ) sgRNA sequences , with PAM sites in bold .", "( C ) Western blot of lysates from individual HEK293T clones with BCL9 or B9L single KO , or with BCL9/B9L DKO , probed with antibodies as indicated on the left .", "( D ) Summary of BCL9/B9L KO and DKO lines generated in this study . DOI: http://dx . doi . org/10 . 7554/eLife . 20882 . 008 When we re-expressed FLAG-B9L in the DKO cells , this restored full Wnt-responsiveness ( Figure 3C ) .", "However , the C-terminal truncation mutants of B9L ( △C , △HD3△C ) failed to do so ( despite elevated expression levels in the case of △HD3△C ) , and the HD3 deletion provided only partial rescue activity , similarly to a deletion mutant lacking the HD1 Pygo-binding element ( Figure 3C ) .", "This demonstrates the crucial role of the B9L C-terminus for the Wnt response of these cells , and it indicates a functional contribution of HD3 to this response .", "Restoration of Wnt-responsiveness is also provided by re-expressed BCL9 tagged with green fluorescent protein ( GFP-BCL9 ) albeit not by its C-terminal truncation , but we did not analyze this paralog further since its transcriptional activation potential is not as strong as that of the nuclear B9L ( de la Roche et al . , 2008 ) .", "Given the requirement of the BCL9/B9L C-terminus for the Wnt response , we set out to identify its ligands that confer this function .", "Since previous attempts based on tandem-affinity pull-down experiments were unsuccessful ( M Graeb , PhD thesis ) , we adopted a proximity-labeling approach called BioID , tagging B9L and BCL9 with BirA* ( a promiscuous version of the biotin ligase BirA; Roux et al . , 2012 ) and using these as baits to probe the proteome associated with BCL9/B9L in cells .", "This method is capable of identifying transient low-affinity ligands of the bait as well as indirect bystanders ( that is , indirect interactors and ‘vicinal’ proteins ) , provided these are within range of the BirA* tag: in the case of Lamin A , ~50% of the hits were estimated to be within 20–30 nm of its BirA* tag ( Roux et al . , 2012 ) .", "The upper limit appears to be <100 nm , according to BioID studies with Cep250 , an extended coiled-coil protein that spans ~100 nm within the centrosomal complex ( as determined by super-resolution microscopy; Sonnen et al . , 2012 ) : the C-terminal ligand of Cep250 was only labeled by its C-terminal but not its N-terminal BirA* tag ( Firat-Karalar et al . , 2014 ) .", "Therefore , the BioID approach can provide insight into the position and reach of a bait’s BirA* tag within a protein complex .", "Note that BCL9/B9L is presumed to be an extended protein , with >90% of its sequence predicted to be intrinsically unstructured .", "To keep the expression of our BCL9 and B9L baits near endogenous levels , we chose a tetracycline-controlled transcriptional activation system based on T-REx-293 cells ( Al-Jassar et al . , 2017 ) , isolating clonal T-REx-293 cell lines that express B9L-BirA* and BCL9-BirA* integrated at a specific genomic locus .", "Cells were labeled with biotin for 12 hr , with or without stimulation by Wnt3A-conditioned media or 50 mM LiCl ( to inhibit glycogen synthase kinase 3 , which causes activation of β-catenin , and thus mimics Wnt stimulation ) .", "Lysates were prepared for one-step biotin-avidin affinity purification and subsequent analysis by LC-MS/MS mass spectrometry .", "Recall that the probability of identifying a hit by this approach ( reflected by the total number of unweighted spectral counts derived from this hit ) is primarily determined by its proximity to the bait , but is also affected by the strength and duration of their interaction during the labeling period , and by other factors including size and cellular abundance of interactors and suitably exposed biotin-acceptor sites ( primary amines ) .", "We first conducted several experiments with B9L-BirA* as a bait since the B9L paralog is predominantly nuclear , and thus likely dedicated to the transcriptional Wnt response , while BCL9 is distributed throughout the nucleus and cytoplasm ( de la Roche et al . , 2008 ) ( Figure 4—figure supplement 1 ) , possibly carrying out additional functions in the cytoplasm such as chaperoning β-catenin ( de la Roche et al . , 2012 ) .", "Strikingly , each experiment identified known components of the Wnt enhanceosome , even in the absence of Wnt stimulation: we consistently found ARID1A and ARID1B as the top hits , followed closely by TLE3 , TLE1 , TLE4 , LDB1 and PYGO2 and , further down the list , TCF factors , β-catenin and SSBP3/4 ( initially named SSDP1/2; the peptides obtained do not distinguish between these closely related paralogs ) ( Figure 4A ) .", "We also found nine additional subunits of the BAF complex ( Kadoch and Crabtree , 2015 ) on our list , topped by BRG-1/SMARCA4 , with only BAF155/SMARCC1 , BAF47/SMARCB1 , SS18 and BCL7 missing ( the latter three likely for technical reasons; Figure 4—figure supplement 2 ) .", "This indicates that the fully assembled BAF complex is tethered to the Wnt enhanceosome , presumably through its enhancer-binding Osa/ARID1 subunit which also binds to ChiLS ( see Introduction ) .", "Furthermore , we found the CBP co-activator and its p300 paralog , as well as DNA-binding proteins of the LHX and GATA families known to tether ChiLS to DNA ( Love et al . , 2014 ) .", "Essentially the same set of proteins were found with BCL9-BirA* as a bait , albeit with lower efficiency ( Figure 4B ) , possibly because of its largely cytoplasmic localization .", "Indeed , we found several cytoplasmic proteins specifically associated with BCL9 but not B9L , including α-catenin ( Figure 4—figure supplement 2 ) . 10 . 7554/eLife . 20882 . 009Figure 4 . BCL9/B9L and PYGO2 are constitutively associated with the Wnt enhanceosome , and nuclear co-receptor complexes .", "( A , B )", "List of BioID hits for ( A ) B9L-BirA* and ( B ) BCL9-BirA*±10–12 hr of WCM; names above the dotted line refer to the top hits , while names below this line refer to hits selected on relevance to Wnt ( blue ) or nuclear co-receptors ( green ) ; only specific hits with a > 5 spectral count ratio relative to the BirA* control are shown; numbers represent unweighted spectral counts ( >95% probability ) .", "( C ) RIME hits for FLAG-B9L-BirA*; only specific hits with a >5 spectral count ratio relative to the control are shown .", "( D ) List of BioID hits for PYGO2-BirA* , as in ( A , B ) ; * , identified with lower confidence ( >55% probability ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 20882 . 00910 . 7554/eLife . 20882 . 010Figure 4—figure supplement 1 . Stably transfected BCL9/B9L cell lines for BioID , and summary of wt and mutant BirA* baits .", "( A ) Immunofluorescence of stable BioID T-REx-293 cell lines expressing wt or mutant BCL9-BirA* , B9L- BirA* or BirA* alone , showing subcellular distribution of the various baits; nuclei are labeled by DAPI staining .", "( B ) Cartoons of wt and mutant BCL9-BirA* , B9L-BirA* and PYGO2-BirA* .", "( C ) Western blot probed with a-ABC antibody , to confirm Wnt induction of the stimulated PYGO2-BirA* cell lysates prior to BioID pull-downs and analysis . DOI: http://dx . doi . org/10 . 7554/eLife . 20882 . 01010 . 7554/eLife . 20882 . 011Figure 4—figure supplement 2 . Additional analysis of BioID hits .", "( A ) Venn diagrams , showing the number of hits shared between the BCL9 , B9L and PYGO2 BioID baits with >95% probability ( excluding the BirA*-only control ) ;right , a selection of high-confidence BCL9-specific hits not found with the other two ( nuclear ) baits .", "( B ) Components of the SWI/SNF complex found by all three baits . DOI: http://dx . doi . org/10 . 7554/eLife . 20882 . 01110 . 7554/eLife . 20882 . 012Figure 4—figure supplement 3 . Constitutive association between B9L and TCF prior to Wnt stimulation . CoIP assays in transiently transfected HEK293T cell , with Western blots showing ( A ) Wnt-independent association between GFP-TCF and FLAG-B9L , or ( B ) Wnt-dependent association between GFP-BCL9 with endogenous b-catenin or ABC ( and Wnt-independent association with endogenous Pygo , as internal control ) .", "Transfected cells were exposed to LiCl , or NaCl as control , as described in the main Materials and methods . DOI: http://dx . doi . org/10 . 7554/eLife . 20882 . 012 To determine whether any of these hits might be direct ligands of B9L , we applied the RIME ( rapid IP mass spectrometry of endogenous proteins ) technique ( Mohammed et al . , 2016 ) to our cell line stably expressing B9L-BirA* at levels comparable to endogenous B9L , using FLAG resin to capture its N-terminal FLAG tag ( Figure 4—figure supplement 1 ) .", "This method relies on limited crosslinking of proteins in live cells followed by mass spectrometry of peptides cross-linked to the affinity-purified bait , and is thus capable of identifying direct ligands of the bait .", "It has been extensively validated for hit lists from immunoprecipitation-based approaches obtained for nuclear hormone receptors ( for example , estrogen receptor ) and other DNA-binding proteins ( Mohammed et al . , 2016 ) .", "These RIME experiments identified only five specific hits; these proteins associated with B9L-BirA* ( Figure 4C ) include PYGO2 , its only known direct ligand in the absence of Wnt .", "This short list also contained LDB1 , which we shall identify below as another direct B9L ligand .", "It was topped by TLE3 , suggesting that TLE3 may also be a direct ligand of B9L .", "Importantly , the majority of the hits identified by B9L-BirA* or BCL9-BirA* hardly change after Wnt stimulation ( Figure 4A , B ) .", "This implies that BCL9/B9L is associated with the Wnt enhanceosome prior to Wnt stimulation and independently of β-catenin .", "We note however that the spectral counts of many hits tend to be slightly increased upon Wnt signaling , a trend that is also apparent for TLE1 and TLE3 whose labeling by B9L-BirA* was increased 2-3x upon Wnt stimulation ( Figure 4A , B ) .", "Similarly , the labeling of GSE1 by B9L-BirA* is increased 3-5x in stimulated cells ( Figure 4A ) : GSE1 is a subunit of the BRAF-HDAC complex ( also known as BHC ) ( Hakimi et al . , 2003 ) , which might interact with Groucho/TLE through its HDAC subunit , potentially explaining the Wnt-dependent increase in the labeling of GSE1 .", "In any case , TLE1/3 behaves like the LDB1 core component of the Wnt enhanceosome in remaining associated with Wnt-responsive enhancers even when these are active , consistent with recent findings that TLE1 and β-catenin can bind simultaneously to TCF1 ( Chodaparambil et al . , 2014 ) .", "Association of β-catenin with BCL9/B9L is undetectable prior to Wnt stimulation , as expected from its low abundance in the absence of signaling .", "This may also explain why the labeling of CBP and p300 by B9L-BirA* is stimulated ~4–6x upon Wnt stimulation , given that these histone acetyltransferases are known ligands of β-catenin .", "However , both proteins exhibit a significant level of labeling even without Wnt signaling , suggesting a constitutive association with the Wnt enhanceosome , consistent with the identification of CBP by RIME as a putative direct ligand of B9L .", "It thus appears that the binding of CBP and p300 to β-catenin upon its docking to the Wnt enhanceosome increases their proximity to the C-terminus of BCL9/B9L , which bears the BirA* tag .", "Three other factors exhibit a striking increase in labeling after Wnt stimulation , namely the TCF factors , APC and SSBP3/4 ( Figure 4A , B ) .", "TCF4 is the only TCF paralog labeled prior to Wnt signaling , albeit at very low levels ( also by the BirA* control ) , but its labeling by B9L-BirA* or BCL9-BirA* is Wnt-inducible by 13x or 2 . 5x , respectively .", "For TCF1/3 and LEF1 , no labeling is detectable in unstimulated cells , whereas each of these factors is labeled efficiently upon Wnt signaling ( >19–30x ) .", "The same is true for the APC tumor suppressor ( >29x ) , a BCL9-specific hit that is recruited to TCF target genes upon Wnt signaling by β-catenin ( Sierra et al . , 2006 ) , owing to direct high-affinity binding ( Choi et al . , 2006 ) .", "Finally , the labeling of SSBP3/4 by B9L-BirA* is moderately increased ( >6x ) in Wnt-stimulated cells; as in the case of CBP/p300 , SSBP3/4 is also detectable prior to Wnt signaling , albeit close to background levels .", "As far as we know , the expression levels of APC , SSBP3/4 and TCF factors do not change after Wnt stimulation , except for LEF1 whose levels increase ~2x in Wnt-stimulated HEK293T cells ( de la Roche et al . , 2014 ) .", "Therefore , a likely explanation for the Wnt-inducible labeling of these factors is that Wnt signaling , or β-catenin , induces their proximity to the C-terminus of BCL9/B9L .", "We note that TCF factors are bound constitutively to their cognate enhancers ( for example , TCF4 in unstimulated HEK293 cells; Frietze et al . , 2012 ) although , in Drosophila , the association of dTCF with Wg-responsive enhancers appears to be strengthened by Wg signaling ( Parker et al . , 2008 ) .", "To obtain independent evidence for the constitutive association between BCL9/B9L and TCF , we conducted coIP assays in HEK293T cells co-expressing FLAG-B9L and GFP-TCF4 .", "As expected , the two proteins coIP with similar efficiency in cells with or without Wnt stimulation ( Figure 4—figure supplement 3 ) .", "Likewise , coIP of GFP-BCL9 with endogenous PYGO2 is also Wnt-independent , whereas its coIP with endogenous β-catenin ( or activated β-catenin , ABC ) is strictly Wnt-inducible , as expected ( Figure 4—figure supplement 3 ) .", "This underscores our notion that the Wnt-inducible labeling of TCF factors by B9L-BirA* and BCL9-BirA* reflects β-catenin-dependent apposition of the C-terminus of BCL9/B9L to TCF , rather than β-catenin-dependent recruitment of Legless/BCL9 to TCF ( as initially envisaged; Kramps et al . , 2002; Städeli and Basler , 2005 ) .", "Strong independent support for this notion came from BioID experiments with PYGO-BirA* , which we expressed in T-REx-293 cells under identical conditions as the BCL9 and B9L baits; Wnt stimulation was confirmed by Western blotting against ABC ( Figure 4—figure supplement 1 ) .", "By and large , PYGO2-BirA* identified the same Wnt enhanceosome components as B9L-BirA* , with ARID1A/B topping the list ( Figure 4D ) .", "Complete lists of specific BioID hits obtained with B9L-BirA* , BCL9-BirA* and PYGO2-BirA* whose total number of unweighted spectral counts are ≥1 , and 5x above background ( that is , counts obtained for that hit with BirA* ) , can be found in Supplementary files 1–3 .", "SSBP3/4 was labeled more efficiently by PYGO2-BirA* than by B9L-BirA* , consistent with our evidence that Pygo binds directly to an interface shared between LDB1 and SSDP ( Fiedler et al . , 2015 ) .", "Importantly , except for p300 and CBP whose labeling by PYGO2-BirA* was moderately Wnt-inducible ( ~3x ) , all other components of the Wnt enhanceosome and BAF complex were labeled with similar efficiency regardless of Wnt signaling , including TCF1/3/4 and LEF1 ( Figure 4D ) .", "Thus , the proximity of PYGO2 to TCF factors does not change as a result of β-catenin docking the Wnt enhanceosome .", "To identify ligands binding to HD3 or the C-terminus of BCL9/B9L , we applied BioID mass spectrometry to T-REx-293 cells stably transfected with mutant BCL9-BirA* and B9L-BirA* baits lacking these sequences ( △HD3 and △C , respectively ) , and also with baits lacking HD1 ( △HD1 ) expected to abrogate Pygo binding ( Fiedler et al . , 2008 ) .", "We confirmed that the subcellular distributions of these mutant BCL9-BirA* and B9L-BirA* baits were not affected by the various deletions ( Figure 4—figure supplement 1 ) .", "The lists of proteins associated with △C baits are similar to those found with the wt , but a few of them show reduced spectral counts , most notably in the case of BCL9ΔC which barely associates with TLE1 nor TLE3 ( Figure 5A ) .", "coIP assays revealed robust binding between HA-tagged TLE3 ( HA-TLE ) and wt GFP-BCL9 but not with two different C-terminal truncations ( Figure 5B ) .", "Indeed , the C-terminal WD40 domain of TLE3 is both necessary and sufficient for this coIP ( Figure 5C ) .", "This domain binds to short motifs within a range of DNA-binding repressors ( Jennings et al . , 2006 ) , but is not involved in binding to TCF ( Chodaparambil et al . , 2014 ) .", "Therefore , BCL9/B9L could bind directly to TCF-associated TLE/Groucho ( as indicated by RIME , see above ) . 10 . 7554/eLife . 20882 . 013Figure 5 . The Legless/BCL9 C-terminus binds to core Wnt enhanceosome components .", "( A ) Top BioID hits showing differential association with wt versus mutant BCL9-BirA* ( unweighted spectral counts > 95% probability ) .", "( B , C )", "Western blots of coIPs of wt or mutant GFP-BCL9 with ( B ) HA-TLE3 or ( C ) HA-tagged truncations , after co-expression in HEK293T cells ( lysed 48 hr after transfection ) , probed with antibodies as indicated on the left .", "( D ) Top differential BioID hits of B9L-BirA* as in ( A ) .", "( E ) CoIP assays between co-expressed wt or mutant GFP-BCL9 , LDB1-FLAG and SSDP-FLAG , as in ( B ) ; the band in the top panel corresponds to LDB1-FLAG . DOI: http://dx . doi . org/10 . 7554/eLife . 20882 . 01310 . 7554/eLife . 20882 . 014Figure 5—figure supplement 1 . HD3-dependent interaction between LDB1 and Legless/B9L . CoIP assays between co-expressed wt or mutant ( A ) GFP-B9L or ( B ) GFP-Lgs , and LDB1-FLAG and SSDP-FLAG , as in main Figure 5E . DOI: http://dx . doi . org/10 . 7554/eLife . 20882 . 01410 . 7554/eLife . 20882 . 015Figure 5—figure supplement 2 . CRISPR/Cas9-based gene editing strategy for PYGO2 in HEK293T cells .", "( A ) Cartoon of PYGO2 exon boundaries and targeting site in exon 3; top , sgRNA sequence , with PAM site in bold; underneath , chromosomal location .", "( B ) Western blot of lysates from individual HEK293T clones with PYGO2 KO , probed with antibodies as indicated on the left .", "( C ) CoIP assays between co-expressed wt or mutant GFP-BCL9 , and LDB1-FLAG plus SSDP-FLAG in wt or PYGO2 KO cells , confirming that the association of this BCL9 paralog with the ChiLS core complex is partly mediated by its binding to Pygo via HD1 . DOI: http://dx . doi . org/10 . 7554/eLife . 20882 . 015 Likewise , a small number of proteins show reduced association with BCL9 or B9L baits lacking HD3 , with the top hit being LDB1 whose association with B9L-BirA* is reduced by half compared to the wt control ( Figure 5D ) .", "Indeed , binding between LDB1-FLAG and GFP-B9L , GFP-BCL9 or GFP-Lgs is readily detectable in coIP assays , but is significantly reduced if HD3 is deleted ( Figure 5E; Figure 5—figure supplement 1 ) .", "Binding is similarly reduced by an alanine substitution of the most conserved residue in HD3 ( W472 in BCL9 , W520 in B9L; see also below ) , and eliminated if △HD1 is tested ( Figure 5E; Figure 5—figure supplement 1 ) .", "This indicates the importance of HD3 and HD1 for the interaction between ChiLS and BCL9/B9L , whereby HD1 likely contributes indirectly to this interaction via its binding to PYGO2 ( Figure 5A ) ( Fiedler et al . , 2008 ) .", "We generated PYGO2 KO cells by CRISPR/Cas9 ( noting that HEK293T cells do not express PYGO1 ) , to confirm that ChiLS coIPs less efficiently with BCL9 in the absence of Pygo ( Figure 5—figure supplement 2 ) .", "This supports the notion that Pygo promotes the association between BCL9/B9L and ChiLS .", "Next , we asked whether HD3 is a direct binding site for ChiLS .", "This short conserved element is predicted to form a single α-helix ( Peng and Xu , 2011 ) , with an invariant tryptophan and a phenylalanine/tyrosine ( F/Y ) doublet further downstream whose hydrophobic side chains extend in the same direction ( Figure 6A , B ) .", "Together , they form a hydrophobic surface to which ChiLS may bind . 10 . 7554/eLife . 20882 . 016Figure 6 . HD3 binds directly to ChiLS .", "( A ) Top , sequence alignments of HD3; yellow , conserved residues; grey , semi-conserved residues .", "Bottom , position-specific alignment ( HMMER; Finn et al . , 2011 ) for B9L HD3; yellow , conserved tryptophan and phenylalanine/tyrosine doublet .", "( B ) Predicted structure of HD3 ( by I-TASSER ) , with heat-map indicating relative line broadenings upon incubation of 15N-HD3 with ChiLS ( see D ) , ranging from 80% ( red ) to 40% ( yellow ) ; grey , proline ( not detectable ) .", "( C ) TALOS+ predictions of α-helicity of HD3 , based on backbone secondary chemical shifts in Figure 6—figure supplement 1; for each position , the rigidity index ( RCI-S2 ) and helical probability ( p ) are indicated ( pink , N-terminal linker residues ) .", "( D–F )", "Overlays of HSQC spectra of 100 μM 15N-labeled ( D , E ) wt HD3 or ( F ) W520R mutant alone ( red ) , and probed with 300 μM ( D , F ) MBP-Chip205-436-Lip-SSDP1-92 or ( E ) Lip-SSDP1-92 ( blue ) ; line broadening owing to ChiLS binding to wt HD3 ( D ) results in the disappearance of selected resonances in the blue ( HD3 + ChiLS ) spectrum , revealing the corresponding resonances from the red ( HD3-only ) spectrum . DOI: http://dx . doi . org/10 . 7554/eLife . 20882 . 01610 . 7554/eLife . 20882 . 017Figure 6—figure supplement 1 . Assignment of the [1H-15N]-HSQC spectrum of HD3 . Assignments of 1H-15N correlations for the backbone amide resonances of [15N-13C]-Lip-HD3 residues , after overlay with [15N-13C]-Lip ( to identify Lip residues ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 20882 . 01710 . 7554/eLife . 20882 . 018Figure 6—figure supplement 2 . Modification of the chip phenotype by loss of HD3 . ( A ) Genetic interactions between chipe55/+ heterozygosity ( causing wing margin defects , marked by arrowheads ) and heterozygosity of Wnt pathway components; representative wings are shown .", "Note that this phenotype is exacerbated by lowering the dose of SSDP protein ( as expected , given that Chip and SSDP form an obligatory complex; see Fiedler et al . , 2015 ) , but ameliorated by lowering the dose of associated proteins such as dTCF , Armadillo , Pygo , Legless or Groucho , possibly because normal levels of the latter sequester some of the limiting amounts of Chip ( which is reduced in these heterozygotes ) .", "Note the strong suppression by the HD3 allele , possibly indicative of the direct binding of Chip to this domain .", "( B ) Restoration of normal wing margins by heterozygosity of genes , as indicated ( see also A ) ; black , partial restoration ( only anterior margin ) ; white , restoration of both margins . DOI: http://dx . doi . org/10 . 7554/eLife . 20882 . 018 To test this , we purified 15N-labelled 6xHis-Lipoyl ( Lip ) -tagged B9L509-537 ( Lip-HD3 ) after bacterial expression , for binding assays with purified ChiLS complex , as previously described ( Fiedler et al . , 2015 ) .", "15N-Lip-HD3 proved soluble and monomeric ( as judged by size exclusion chromatography ) , and produces well-dispersed heteronuclear single-quantum correlation ( HSQC ) spectra .", "Incubation of 15N-Lip-HD3 with purified ChiLS complex , but not with purified SSDP alone , produces clear line broadening ( ‘bleaching’ ) of selective peaks ( Figure 6D , E ) – strong evidence for a direct interaction .", "Assignments of these peaks ( obtained with a double-labeled 13C15N-Lip-HD3 sample; Figure 6—figure supplement 1 ) provided experimental evidence for the helical nature of HD3 ( as ascertained by TALOS+; Shen et al . , 2009 ) ( Figure 6C ) .", "It allowed us to generate a heat-map that implicates most residues of HD3 in the interaction with ChiLS ( Figure 6B ) .", "Importantly , no spectral changes are observed if a W520R-mutant version of 15N-Lip-HD3 is incubated with ChiLS ( Figure 6F ) , showing that this point mutation abrogates the binding of HD3 to ChiLS .", "Thus , ChiLS binds directly and specifically to HD3 .", "Given this physical interaction between HD3 and ChiLS , we asked whether we could also detect genetic interactions between lgs and chip in Drosophila .", "Flies heterozygous for chip exhibit multiple wing notches ( Shoresh et al . , 1998 ) that are strongly exacerbated by heterozygosity of ssdp; however , this phenotype is ameliorated by heterozygosity of several Wnt signaling components including pygo , groucho and lgs ( Figure 6—figure supplement 2 ) .", "Interestingly , the strongest interaction is seen with lgsΔHD3 whose heterozygosity restores normal margins in >25% of the flies .", "The same is also seen with lgs2-8 heterozygosity although full suppression is less penetrant ( Figure 6—figure supplement 2 ) .", "These strong genetic interactions between chip and HD3-defective lgs alleles further underscore the close functional link between these two proteins .", "Unexpectedly , we consistently found multiple components of nuclear co-receptors complexes amongst the top hits for all three BirA* baits , including several NCOA co-activators and NCOR co-repressors ( Figure 4 ) .", "In the case of B9L-BirA* , we also found the DNA-binding components of the resident complex , namely the arylhydrocarbon receptor ( AHR ) and its partner ARNT ( Figure 4A ) .", "Clearly , there is close proximity between BCL9/B9L-PYGO2 and nuclear co-receptor complexes .", "This suggests that a substantial fraction of Wnt-responsive enhancers also contain binding sites for nuclear receptors ( for example , AHR in HEK293T cells ) , and that these sites are near TCF-binding sites ." ], [ "Our study has uncovered genetic and physical interactions between two constitutive core components of the Wnt enhanceosome and the C-terminus of Legless/BCL9 .", "The first of these is ChiLS , the core module of the Wnt enhanceosome ( Fiedler et al . , 2015 ) ( Figure 7 ) : we have shown that ChiLS is a direct and specific ligand of the α-helical HD3 element of B9L and , likely , of other Legless/BCL9 orthologs , given the strong sequence conservation of this α-helix ( Figure 6 ) .", "The physiological relevance of this interaction with ChiLS is underscored by genetic analysis in flies .", "Our evidence thus implicates HD3 as an evolutionary conserved contact point between Legless/BCL9 and ChiLS , although the primary link between these two proteins appears to be provided by Pygo . 10 . 7554/eLife . 20882 . 019Figure 7 . Refined model of the Wnt enhanceosome . The Wnt enhanceosome complex associated with a Wnt-responsive enhancer in its OFF or ON state ( for initial model , see Fiedler et al . [2015] , illustrating multivalent constitutive interactions of Legless/BCL9 with Pygo ( through HD1 ) , ChiLS ( through HD3 ) and Groucho/TLE ( through its C-terminus ) .", "OFF , HD2 is poised to interact with Armadillo/β-catenin upon Wnt-induced stabilization; ON , rearrangement of Legless/BCL9 upon recruitment of Armadillo/β-catenin , resulting in the apposition of its C-terminus to TCF .", "CBP/p300 is associated with both states ( possibly through direct binding to Legless/BCL9 ) ; its activity may be directed towards histones upon Armadillo/β-catenin binding , which antagonizes Groucho/TLE-dependent silencing and promotes the transcription of linked target genes .", "Likewise , the BAF complex is associated with both states ( through the NPF motif of its subunit Osa/ARID1 ) , earmarking the complex for feedback inhibition ( see Figure 7—figure supplement 1 ) .", "S , SSDP; Q , Q domain of TLE ( tetramerizing , and binding to TCF ) ; W , WD40 domain of TLE ( binding to ChiLS ) ; arrows , NPF-mediated interactions; green , positively-acting components; red , negatively-acting components; black circles , nucleosomes bearing H3K4me marks of poised or active enhancers ( Kharchenko et al . , 2011 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 20882 . 01910 . 7554/eLife . 20882 . 020Figure 7—figure supplement 1 . Reinstalling silencing on a Wnt-responsive enhancer . Revised model for feedback inhibition of the Wnt enhanceosome in response to high signaling levels at the Wnt signaling source , which depends on the BAF complex and its Osa/ARID1 subunit ( for initial model , see Fiedler et al . , 2015 ) .", "The homeodomain protein Brinker has been implicated in this process by studies of the Wg-responsive Ubx midgut enhancer in flies , in which the Osa response element maps to the primary Brinker binding site ( Collins and Treisman , 2000 ) .", "Brinker confers repression of this enhancer in response to high Wg signaling levels by binding to the WD40 domain of Groucho , but its repressive activity also requires recruitment of its co-repressor Teashirt , whose expression is locally induced by high levels of signaling emanating from the Wg source ( Saller et al . , 2002 ) .", "Repression of the dpp leg enhancer near the Wg source in the ventral leg disc also depends on Brinker ( Theisen et al . , 2007 ) .", "Notably , Teashirt binds to Armadillo ( Gallet et al . , 1999 ) and could thus compete with its binding to dCBP , suggesting a mechanism by which the Brinker-Teashirt complex may reinstall silencing on a Wnt-responsive enhancer .", "Note that the BAF complex might also be responsible for silencing the enhanceosome after cessation of Wnt signaling . DOI: http://dx . doi . org/10 . 7554/eLife . 20882 . 020 A second link between the Legless/BCL9 C-terminus and the Wnt enhanceosome is mediated by the WD40 domain of TLE/Groucho .", "Given our evidence from RIME , this link is also likely to be direct although , for technical reasons , we have not been able to prove this .", "The function of the C-terminus of Legless/BCL9 for transducing Wnt signals was revealed by the wg-like phenotypes in Drosophila larvae and flies and by their defective transcriptional Wg responses ( Figure 1 and 2 ) , and by the loss of transcriptional Wnt responses in BCL9/B9L-deleted human cells ( Figure 3 ) .", "Our evidence indicates that Legless/BCL9 undergoes three separate functionally relevant interactions with distinct components of the Wnt enhanceosome—with Pygo , ChiLS and Groucho/TLE ( Figure 7 ) .", "Importantly , BioID revealed that these interactions are constitutive , preceding Wnt signaling , and that they hardly change upon Wnt stimulation ( Figure 4 ) .", "Taken together with its multivalent interactions with the Wnt enhanceosome , this is consistent with Legless/BCL9 being a core component of this complex , providing a scaffolding function that facilitates its assembly and/or maintains its cohesion .", "Following Wnt stimulation , Legless/BCL9 undergoes an additional physiologically relevant interaction , by binding to ( stabilized ) Armadillo/β-catenin via HD2 ( Kramps et al . , 2002 ) .", "Legless/BCL9 thus confers Wnt-responsiveness on the Wnt enhanceosome through its ability to capture Armadillo/β-catenin .", "In other words , in addition to scaffolding the enhanceosome , Legless/BCL9 also earmarks this complex for Wnt responses .", "Intriguingly , our BioID data indicated that the capture of β-catenin by Legless/BCL9 triggers its rearrangement within the complex , apposing its C-terminus to TCF ( Figure 7 ) .", "This apparent β-catenin-dependent apposition is consistent with structural data showing that BCL9/B9L HD2 is closely apposed to TCF when in a ternary complex with β-catenin ( Sampietro et al . , 2006 ) .", "Our evidence support the notion of Legless/BCL9 acting as an ‘Armadillo loading factor’ , facilitating access of Armadillo/β-catenin to TCF ( de la Roche and Bienz , 2007; Townsley et al . , 2004 ) , but argues against the original co-activator hypothesis which posited that Legless/BCL9 is recruited to TCF by Armadillo/β-catenin exclusively in Wnt-stimulated cells ( Kramps et al . , 2002; Städeli and Basler , 2005 ) .", "Whatever the case , the β-catenin-dependent apposition of the Legless/BCL9 C-terminus to TCF is likely to trigger Wnt enhanceosome switching from OFF to ON , resulting in the relief of Groucho/TLE-dependent repression and culminating in the Wnt-dependent transcriptional activation of linked target genes ( Figure 7 ) .", "This transition of the Wnt enhanceosome from OFF to ON is accompanied by a proximity gain between Legless/BCL9 and CBP/p300 ( Figure 4 ) , likely to reflect at least in part its de novo binding to Armadillo/β-catenin .", "However , our evidence indicates that CBP/p300 is associated with the Wnt enhanceosome prior to Wnt signaling , possibly via direct binding to B9L as suggested by RIME ( Figure 4C ) , and that the docking of Armadillo/β-catenin to the Wnt enhanceosome strengthens its association with CBP/p300 , and/or directs the histone acetyltransferase activity of CBP/p300 towards its substrates , primarily the histone tails .", "By acetylating these tails , CBP/p300 appears to promote Wnt-dependent transcription in flies and human cells ( Li et al . , 2007 ) .", "Indeed , CBP-dependent histone acetylation has been observed at Wg target enhancers in Drosophila although , interestingly , this preceded transcriptional activation ( Parker et al . , 2008 ) .", "This is consistent with our BioID data , indicating constitutive association of CBP/p300 with the Wnt enhanceosome .", "It seems plausible that histone acetylation at Wnt target enhancers is instrumental in antagonizing the compaction of their chromatin imposed by Groucho/TLE , which depends on its tetramerization via its Q domain ( Chodaparambil et al . , 2014 ) as well as its binding to HDACs ( Jennings et al . , 2008; Turki-Judeh and Courey , 2012 ) .", "Indeed , we found HDACs near the bottom of our BioID lists , and one of the top hits identified by B9L was GSE1 , a subunit of the BRAF-HDAC complex ( Hakimi et al . , 2003 ) .", "However , CBP/p300 also has non-histone substrates within the Wnt enhanceosome , including dTCF in Drosophila whose Armadillo-binding site can be acetylated by dCBP , which thus blocks the binding between the two proteins ( Waltzer and Bienz , 1998 ) and antagonizes Wg responses ( Li et al . , 2007 ) .", "It thus regulates Wnt-dependent transcription positively as well as negatively , similarly to Groucho/TLE which not only silences Wnt target genes but also earmarks them for Wnt inducibility , as a core component of the Wnt enhanceosome .", "It is intriguing that both bimodal regulators are associated constitutively with this complex .", "A corollary is that the docking of Armadillo/β-catenin to the Wnt enhanceosome changes their substrate specificities and/or activities .", "An important refinement of our initial enhanceosome model is with regard to the BAF complex , which appears to be a constitutive component of the Wnt enhanceosome ( Figure 7 ) , as indicated by our BioID data .", "This complex is highly conserved from yeast to humans , and it contains 15 subunits in human cells ( Kadoch and Crabtree , 2015 ) , including the DNA-binding Osa/ARID1 subunit .", "A wealth of evidence from studies in flies and mammals indicates that this complex primarily antagonizes Polycomb-mediated silencing of genes , most notably of the INK4A locus which encodes an anti-proliferative factor , which could explain why the BAF complex functions as a tumor suppressor in many tissues .", "However , recall that this complex also specifically antagonizes Armadillo/β-catenin-mediated transcription ( Collins and Treisman , 2000 ) , likely via its BRG/BRM subunit which directly binds to β-catenin ( Barker et al . , 2001 ) .", "Evidence from studies in Drosophila of Wg-responsive enhancers suggests that this complex mediates a negative feedback from high Wg signaling levels near Wg-producing cells which results in re-repression ( Collins and Treisman , 2000 ) , imposed by the Brinker homeodomain repressor ( Saller et al . , 2002; Waltzer et al . , 2001; Theisen et al . , 2007 ) and its Armadillo-binding Teashirt co-repressor ( Gallet et al . , 1999 ) ( Figure 7—figure supplement 1 ) .", "The same factors may also instal silencing on Wnt-responsive enhancers upon cessation of Wnt signaling .", "Notably , mammals do not have a Brinker ortholog , which could explain some of the apparent functional differences between flies and mammals with regard to the BAF complex ( Kadoch and Crabtree , 2015 ) .", "However , the closest mammalian relatives of Teashirt are the Homothorax/MEIS proteins , a family of homeodomain proteins whose expression can be Wnt-inducible ( for example , Wernet et al . , 2014 ) .", "They are thus candidates for Wnt-induced repressors that confer BAF-dependent feedback inhibition .", "Notably , none of our BioID lists contained RUNX proteins .", "Based on our functional evidence from Drosophila midgut enhancers , we proposed that these proteins ( which bind to both enhancers and Groucho/TLE ) are pivotal for initial assembly of the Wnt enhanceosome at Wnt-responsive enhancers during early embryonic development , or in uncommitted progenitor cells of specific cell lineages ( Fiedler et al . , 2015 ) .", "However , HEK293 cells are epithelial cells and may thus not express any RUNX factors .", "In any case , our negative BioID results suggest that RUNX factors function in a hit-and-run fashion .", "Evidently , the Wnt enhanceosome complex , once assembled at Wnt-responsive enhancers , can switch between ON and OFF states without RUNX .", "In summary , we have uncovered a fundamental role to Legless/BCL9 as a scaffold of the Wnt enhanceosome , far beyond its role in linking Armadillo/β-catenin to Pygo .", "Indeed , the function of Legless/BCL9 may extend beyond transcriptional Wnt responses , as indicated by the unexpected discovery of its strong association with nuclear co-receptor complexes .", "Potentially , these associations underlie the observed cross-talk between Wnt/β-catenin and nuclear hormone receptor signaling , documented extensively in the literature ( for example , Beildeck et al . , 2010; Mulholland et al . , 2005; Schneider et al . , 2014 ) , including evidence for direct activation of the androgen receptor by β-catenin ( Kypta and Waxman , 2012 ) .", "Furthermore , a strong association between TLE1 and the estrogen receptor has been discovered in breast cancer cells , where TLE1 assists the estrogen receptor in its interaction with chromatin and its proliferation-promoting function ( Holmes et al . , 2012 ) .", "This is reminiscent of the role of Groucho/TLE as a cornerstone of the Wnt enhanceosome , proposed to earmark TCF enhancers for subsequent β-catenin docking and transcriptional Wnt responses ( Fiedler et al . , 2015 ) .", "It will be interesting to test experimentally the putative roles of BCL9/B9L and Pygo in enabling cross-talk between β-catenin and nuclear hormone receptor signaling , both during normal development and in cancer ." ], [ "The following plasmids have been described: LDB1-FLAG , SSDP-FLAG ( Fiedler et al . , 2015 ) ; murine FLAG-B9L , FLAG-ΔC ( called FLAG-ΔCter ) , GFP-B9L ( Adachi et al . , 2004 ) ; GFP-Lgs , GFP-TCF4 ( Townsley et al . , 2004 ) .", "Human GFP-BCL9 was generated by exchanging the FLAG tag of FLAG-BCL9 ( de la Roche et al . , 2008 ) .", "Mutants were made from parental plasmids using standard site-directed mutagenesis procedures , and confirmed by sequencing .", "TLE3 was amplified by PCR from 6xMyc-TLE3 ( Hanson et al . , 2012 ) and subcloned in pcDNA3 . 1-N-HA , and mutants ( WD40 , ΔWD40 ) were generated from this subclone .", "The following antibodies and resins were used: α-GFP RRID:AB_439690 , α-FLAG RRID:AB_262044 , α-HA RRID:AB_390918 , α-β-actin ( for human lysates ) RRID:AB_476744 ( Sigma-Aldrich , St . Louis MO , USA ) ; α-BCL9 RRID:AB_2063609 , α-BCL9-2 RRID:AB_2063747 ( Abingdon , UK ) ; α-β-actin ( for fly lysates ) RRID:AB_2305186 , α-BirA RRID:AB_300830 , α-Pygo2 RRID:AB_10863482 ( Abcam , Cambridge , UK ) ; α-ABC RRID:AB_11127203 ( Cell Signaling Technology ) .", "Rat α-Lgs antiserum ( Eurogentec , Liège , Belgium ) was obtained from pre-bleed immunizations with gluthathione S-transferase-purified Lgs232-555 .", "GFP-Trap resin RRID:AB_2631357 ( Chromotek , Planegg , Germany ) was used for coIP assays , and α-FLAG M2 affinity gel RRID:AB_10063035 ( Sigma-Aldrich ) was used for RIME .", "The CRISPR design tool at crispr . mit . edu was used to design single-stranded oligomers for sgRNA targeting vectors .", "pCFD3-1S and pCFD3-2S were generated by hybridizing single-stranded oligomers with their complementary strands in 2 mM Tris-HCl ( pH 7 . 4 ) , 10 mM NaCl , 200 μM EDTA at 95°C for 5 min , and by subsequently ligating the double-stranded oligomers into a BbsI restriction site of pCFD3 ( kindly provided by Simon Bullock , MRC LMB , Cambridge , UK ) .", "pCFD4-HD3 was generated by inserting a double-stranded oligomer coding for sgRNAs targeting upstream and downstream of HD3 ( Figure 1—figure supplement", "1 ) into pCFD4 ( from Simon Bullock ) using Gibson assembly ( Gibson et al . , 2009 ) .", "Prior to injections into fly embryos ( from a vermilion strain ) , pCFD3-1S , pCFD3-2S and pCFD4-HD3 were purified using plasmid midi kit columns ( Qiagen , Venlo , Netherlands ) , and dissolved in injection buffer at 200 ng μl−1 containing 100 μM phosphate buffer and 5 mM KCl .", "Dechorionated embryos were injected by standard procedures , and sgRNA-expressing transgenic lines were identified on the basis of their vermilion+ eye color , and subsequently crossed to a transgenic fly strain bearing act5c . cas9 ( from Simon Bullock ) , essentially as described ( Port et al . , 2014 , 2015 ) .", "For genotyping , DNA was extracted from individual flies by standard methods , and lgs sequences were determined after PCR amplification using the following primers: 5’-CATCGGGAAGAACAGTTGGC-3’ and 5’-GGACTGGATGCAGCAAATCG-3’ ( for PCR amplification ) , and 5’-TGAATCAATTTCTTTTTCCTG-3’ ( for sequencing; see also below ) .", "HEK293T cells were purchased from the European Collection of Cell Cultures ( authenticated by STR DNA profiling ) .", "Upon receipt , cells were frozen , and individual aliquots were taken into culture , typically for analysis within <10 passages .", "Single KO ( of BCL9 , B9L , or PYGO2 ) and BCL9/B9L DKO cells were generated essentially as described ( Ran et al . , 2013 ) , using guide RNA-encoding plasmid derivatives of pSpCas9 ( BB ) −2A-GFP ( PX458 ) obtained as described above for their fly equivalents ( Figure 3—figure supplement 1; Figure 5—figure supplement 2 ) .", "Cells were sorted 48 hr post-transfection at a density of 1 cell/well in a 96-well plate and grown in Dulbecco’s modified Eagles medium ( supplemented with 10% fetal bovine serum , 100 U ml−1 penicillin , 100 µg ml−1 streptomycin ) for 20–25 days to isolate individual clones .", "These were screened by genotyping ( see below ) and Western Blot analysis ( for lack of BCL9 , B9L or PYGO2 expression ) .", "For genotyping , 2–5 × 104 HEK293T cells were homogenized in 15 μl MicroLYSIS-Plus ( Microzone , Haywards Heath , UK ) and thermocycled as specified by the manufacturers .", "2 μl supernatant was used for PCR amplification , and the resulting PCR products were purified using the QIAquick purification kit ( Qiagen , Hilden , Germany ) and sequenced .", "Sequence chromatograms were analyzed using MacVector software ( MacVector Inc . , Cary NC , USA ) .", "The following primers were used: 5’- CGAGATTTTCCTCTGGCAGC-3’ and 5’-AAGGAGTCGGCGGAAATACT-3’ ( amplification of BCL9 ) ; 5’-GGATCCTGGCTAACAAGACAAG-3’ and 5’-AGAAGTCCGACCACTCTGTG-3’ ( amplification of B9L ) ; 5’-AGTCCAGAAAAGAAGCGAAGG-3’ and 5’-CAGAAGCTTCAGTGGTCAGC-3’ ( amplification of PYGO2 ) ; 5’-ACCCACTTCCACAGCAGAG-3’ ( sequencing of BCL9 ) ; 5’- TGTCTGAGGAAGCCATGGAG-3’ ( sequencing of B9L ) ; 5’-CTCGATCTCCTGACCTCGTG-3’ ( sequencing of PYGO2 ) .", "The following mutant Drosophila strains were used: lgs20F ( Kramps et al . , 2002 ) ; chipe55 ( Morcillo et al . , 1997 ) ; ssdpL7 ( van Meyel et al . , 2003 ) ; dTcf3 ( van de Wetering et al . , 1997 ) ; armXM19 ( Peifer and Wieschaus , 1990 ) ; groMB5 ( Jennings et al . , 2008 ) ; pygoS123 ( Thompson et al . , 2002 ) .", "The strains for CRISPR-mediated genome engineering ( act5c . cas9 , vermilion bearing an attP2 landing site ) were provided by Simon Bullock ( see also Port et al . [2015] ) .", "Larvae were raised at 22°C for scoring of pupation , survival and mutant phenotypes; homozygous flies ( Figure", "1 ) were generated from homozygous mothers where possible ( to eliminate maternal contributions ) .", "Preparation of wings , legs and abdomens were done according to standard protocols , and bright-field images were acquired with a Zeiss Axiophot microscope .", "Wing and leg discs dissected from late third instar larvae ( or pupating larvae if specified ) were fixed with paraformaldehyde , and stained with α-Sens ( Nolo et al . , 2000 ) , α-Wg RRID:AB_528512 ( Developmental Studies Hybridoma Bank ) or chicken α-β-galactosidase RRID:AB_2313752 ( Immune Systems , Paignton , UK ) as described ( Fiedler et al . , 2015; Miller et al . , 2013 ) .", "All discs were counter-stained with DAPI , to control for the focal plane ( though DAPI images are not shown in triple antibody stainings ) , and single confocal images were acquired at identical settings with a Zeiss Confocal Microscope .", "RNA was isolated from dissected wing discs with the RNeasy kit ( Qiagen , Hilden , Germany ) and converted to cDNA with Super RT ( HTBiotechnology Ltd , Cambridge , UK ) .", "RT-qPCR reactions were subsequently run in Fast-96-well format on a Vii7 Real-Time PCR System ( Applied Biosystems , Foster City CA , USA ) .", "The following TaqMan probes were used: Dm01792952_m1 , Dm01820389_m1 , Dm01804677_m1 , Dm01842959_m1 , Dm01803388_m1 , Dm01843776_s1 and Dm02151827_g1 ( Applied Biosystems , Foster City CA , USA ) .", "Statistical significance was assessed with two-tailed Student’s t-tests .", "To generate BioID plasmids , BirA* ( R118G ) was amplified from pcDNA3 . 1 mycBioID ( Roux et al . , 2012 ) by PCR and subcloned into pcDNA5/FRT/TO using megaprimer PCR .", "Coding sequences for PYGO2 , BCL9 and B9L ( and mutants thereof ) were amplified likewise and inserted directly upstream of BirA* in pcDNA5/FRT/TO using Gibson assembly ( Gibson et al . , 2009 ) .", "For each stably transfected BioID cell line , 1 . 4–2 . 1 × 108 cells were grown adherent to full confluence , washed once with phosphate-buffered saline ( PBS ) , flash-frozen in liquid nitrogen , and stored at −80°C for 1–20 days ( for further details , see Al-Jassar et al . , 2017 ) .", "BioID pull-downs were then essentially done as described ( Roux et al . , 2013 ) , and protein was eluted from the beads by boiling for 15 min in LDS sample buffer ( ThermoScientific , San Jose CA , USA ) .", "RIME pull-downs were essentially done as described ( Mohammed et al . , 2016 ) , except that 3–5 × 107 cells were washed in PBS and incubated for 6 min in 4% methanol-free formaldehyde ( Polysciences , Warrington , USA ) before lysis and boiling in sample buffer .", "All samples were resolved on 4–12% Bis-Tris polyacrylamide gels ( Life Technologies , Carlsbad CA , USA ) , and gels were stained with Imperial Protein Stain ( ThermoScientific , San Jose CA , USA ) .", "Gel slices ( 2–3 mm ) were prepared for mass spectrometric analysis by manual in situ digestion with trypsin , and digests were analyzed by nano-scale capillary LC-MS/MS using an Ultimate U3000 HPLC ( ThermoScientific Dionex , San Jose , USA ) .", "The analytical column outlet was directly interfaced via a nano-flow electrospray ionization source , with a hybrid dual pressure linear ion trap mass spectrometer ( Orbitrap Velos , ThermoScientific , San Jose , USA ) .", "LC-MS/MS data were searched against a protein database ( UniProt KB ) using the Mascot search engine program ( Matrix Science , London , UK ) .", "MS/MS data were validated using the Scaffold program ( RRID:SCR_014345; Proteome Software Inc . , Portland OR , USA ) , and processed with R ( RRID:SCR_001905 ) .", "HEK293T cells were cultured and transfected essentially as described ( Metcalfe et al . , 2010 ) .", "For coIP assays , cells were lysed 48 hr post-transfection in lysis buffer ( 20 mM Tris–HCl pH 7 . 4 , 10% v/v glycerol , 100 mM NaCl , 1 mM EDTA , 5 mM NaF , 2 mM Na3VO4 , 0 . 2% v/v Triton-X-100 ) supplemented with protease inhibitors ( Roche , Basel , Switzerland ) , and sonicated twice for 10 s with an amplitude of 15 μm using a Soniprep 150 plus ( MSE , London , UK ) .", "Samples were cleared by centrifugation at 16 , 100 x g for 10 min , and supernatants were incubated with resin for 2 hr at 4°C .", "All inputs shown are equivalent to 10% of IPs .", "For luciferase reporter assays , SuperTOP ( Veeman et al . , 2003 ) was co-transfected with CMV-Renilla as internal control , and assays were performed initially 48 hr post-transfection , but subsequently 24 hr post-transfection ( for all figures shown in this study ) .", "Wnt inductions were for 6 hr ( unless specified otherwise ) , either with Wnt3a-conditioned-media ( WCM ) or 20 mM LiCl ( or 20 mM NaCl as control ) , and values of uninduced cells were set to 1 .", "RNA extractions , cDNA synthesis and RT-qPCR reactions were conducted as described above for fly wing discs .", "The following TaqMan probes were used: Hs00610344_m1 , Hs01370227_mH and Hs00427620_m1 ( Applied Biosystems , Foster City CA , USA ) .", "The expression and purification of 6xHis-MBP-LDB156-285 , 6xHis-Lip-SSDP1-92 and 6xHis-Lip-HD3509-537 ( from human B9L ) as well as the acquisition and analysis of [1H-15N]fast-HSQC spectra were done as described ( Fiedler et al . , 2015 , 2008; Miller et al . , 2013 ) .", "Spectra were acquired on a Bruker Avance-3 spectrometer operating at 600 MHz 1H frequency and with a sample temperature of 298 °K .", "All samples were prepared in aqueous phosphate buffer of physiological ionic strength ( 25 mM phosphate pH 6 . 7 , 150 mM sodium chloride ) .", "Backbone resonance assignments were obtained for [13C-15N]-double-labelled Lip-HD3 using standard procedures ( Fiedler et al . , 2015 , 2008; Miller et al . , 2013 ) ." ] ]

[ "Wnt/β-catenin signaling elicits context-dependent transcription switches that determine normal development and oncogenesis .", "These are mediated by the Wnt enhanceosome , a multiprotein complex binding to the Pygo chromatin reader and acting through TCF/LEF-responsive enhancers .", "Pygo renders this complex Wnt-responsive , by capturing β-catenin via the Legless/BCL9 adaptor .", "We used CRISPR/Cas9 genome engineering of Drosophila legless ( lgs ) and human BCL9 and B9L to show that the C-terminus downstream of their adaptor elements is crucial for Wnt responses .", "BioID proximity labeling revealed that BCL9 and B9L , like PYGO2 , are constitutive components of the Wnt enhanceosome .", "Wnt-dependent docking of β-catenin to the enhanceosome apparently causes a rearrangement that apposes the BCL9/B9L C-terminus to TCF .", "This C-terminus binds to the Groucho/TLE co-repressor , and also to the Chip/LDB1-SSDP enhanceosome core complex via an evolutionary conserved element .", "An unexpected link between BCL9/B9L , PYGO2 and nuclear co-receptor complexes suggests that these β-catenin co-factors may coordinate Wnt and nuclear hormone responses ." ]

[ "In every animal , different cells must be able to communicate with each other to make sure that the body is correctly formed and maintained .", "Animal cells have many ways of communicating , but one important and well-studied mechanism involves a signaling molecule called Wnt that is released by some cells and received by others .", "The Wnt molecule and its effects are similar in all animals , and over-active Wnt signaling in humans contributes to a number of diseases including various cancers .", "The Wnt signal is carried from the surface of the receiving cell to the DNA in its nucleus via a protein called β-catenin .", "The β-catenin protein then helps to switch on a large number of genes .", "However , to do this β-catenin must interact with an assembly of other proteins collectively called the Wnt enhanceosome .", "There are still many unknowns about how exactly β-catenin cooperates with the enhanceosome .", "Now , van Tienen et al . investigated one component of the Wnt/β-catenin pathway called BCL9/B9L: a large protein that contains a number of flexible regions .", "First , a biochemical technique called BioID was used with human embryonic kidney cells to determine the proteins that BCL9/B9L encounters during a 12-hour period .", "This technique can detect when two proteins come close together , even if the interaction is weak or does not last very long .", "The BioID experiments showed that BCL9/B9L is close to two proteins in the Wnt enhanceosome in addition to β-catenin , and other techniques were used to confirm that one of these proteins contacts BCL9/B9L directly .", "The experiments also showed that BCL9/B9L acted as a tether to bring β-catenin close to the protein within the enhanceosome that binds to the DNA .", "Importantly , BCL9/B9L interacted with the enhanceosome both in the presence and absence of Wnt , indicating that the assembly is ready to switch genes on as soon as β-catenin reaches the DNA .", "Next , van Tienen et al . confirmed that the parts of BCL9/B9L that bind to the enhanceosome are important for its activity by using a gene-editing technology called CRISPR/Cas9 to essentially delete them both in the human cells and in fruit flies .", "Unexpectedly , the BioID experiments also revealed that BCL9/B9L binds to proteins that transmit signals from molecules other than Wnt , in particular from hormones such as estrogen and androgen .", "Future experiments could explore if , and how , BCL9/B9L integrates these signals from hormones with the signal from Wnt .", "A better understanding of this process might have important implications for the treatment of certain cancers , such as breast and prostate cancers that can be driven by over-active hormone signals ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "microbiology and infectious disease" ]

[ [ "A vaccine that induces high-level ( >90% ) sterile protection by inducing immunity that attacks the non-pathologic , asymptomatic pre-erythrocytic stages of Plasmodium falciparum ( Pf ) will prevent infection , disease , and transmission and could be a powerful instrument to eliminate Pf malaria from geographically defined areas ( Plowe et al . , 2009; malERA Consultative Group on Vaccines , 2011 ) .", "In rodent models , sterile protection can be induced by immunization with live Plasmodium sporozoites attenuated by either irradiation , genetic modification ( GAP ) , or by concomitant anti-parasitic drug treatment ( For reviews see Hoffman et al . , 2010; Butler et al . , 2012; Khan et al . , 2012; Nganou-Makamdop and Sauerwein , 2013 ) .", "In humans , induction of complete sustained protective immunity against a challenge infection has been achieved by previous exposure to the bites of mosquitoes infected with", "i ) live radiation-attenuated Plasmodium sporozoites that invade but then completely arrest in the liver ( Clyde et al . , 1973; Hoffman et al . , 2002 ) and", "ii ) live sporozoites in volunteers taking chloroquine chemoprophylaxis ( CPS ) with full parasite liver-stage development; once released into the circulation asexual blood stages are killed by chloroquine ( Roestenberg et al . , 2009 , 2011 ) .", "More recently it has been demonstrated for the first time that sterile immunity can be achieved by intravenous immunization with radiation-attenuated aseptic , purified , cryopreserved Pf sporozoites ( SPZ ) called PfSPZ Vaccine ( Seder et al . , 2013 ) .", "From a product manufacturing perspective , GAPs have the clear advantage of representing a homogeneous parasite population with a defined genetic identity .", "The genetic attenuation is an irreversible , intrinsic characteristic of the parasite that does not require additional manufacturing steps like irradiation .", "Furthermore , in the manufacturing process of GAP-infected mosquitoes , operators are never exposed to Pf parasites that can cause disease .", "However , clinical development of GAPs has suffered from safety problems related to breakthrough infections during immunization leading to pathological blood stage infections responsible for clinical symptoms and complications .", "Strains of mice showed differential susceptibility to breakthrough infections after injection of sporozoites of rodent malaria GAPs , demonstrating the need for extensive preclinical rodent screening ( Annoura et al . , 2012 ) .", "The P . falciparum GAP PfΔp52Δp36 is the only GAP so far that has been assessed in humans but the trial in which the Pf sporozoites were administered by mosquito bite had to be terminated , because of breakthrough infections in one volunteer during immunization ( Spring et al . , 2013 ) .", "Our in vitro experiments with PfΔp52Δp36 confirm that this double gene deletion GAP ( i . e . two genes removed from the genome ) is not fully attenuated similar to the equivalent rodent GAP PbΔp52Δp36 in the Plasmodium berghei/C57BL/6 model ( Annoura et al . , 2012 ) .", "Therefore , identification of additional genes critical and uniquely selective for liver-stage development has become a major challenge for GAP vaccine development ( Annoura et al . , 2012; Khan et al . , 2012; Ploemen et al . , 2012 ) .", "Furthermore , single gene deletion GAPs will most likely not be adequate ( Ploemen et al . , 2012 ) .", "This prompted us to generate and test a GAP with deletions of two independent genes critical for liver-stage development .", "We recently identified a novel P . berghei ( Pb ) gene deletion mutant , PbΔb9 , lacking the expression of the B9 protein ( Pf ortholog: PFC_0750w; PF3D7_0317100 ) ( Annoura et al . , 2014 ) .", "This protein is a newly identified member of the Plasmodium 6-Cys family of proteins .", "Initial safety evaluation in rodents demonstrated that PbΔb9 mutants have a stronger attenuation phenotype than mutants lacking the 6-Cys proteins P52 and P36 ( van Dijk et al . , 2005; van Schaijk et al . , 2008; VanBuskirk et al . , 2009; Annoura et al . , 2014 ) .", "As second target gene for liver-stage attenuation , we selected the slarp and sap1 orthologs reported in Pb and Plasmodium yoelii ( Py ) , respectively ( Pf ortholog: PF11_0480; PF3D7_1147000; hereafter termed slarp ) .", "These slarp mutants show an excellent safety profile by full arrest in the liver in mice ( Aly et al . , 2008; Silvie et al . , 2008 ) .", "The SLARP protein is expressed in sporozoites and in early liver-stages and is involved in the regulation of transcription ( Silvie et al . , 2008; Aly et al . , 2011 ) .", "In this study , we report the generation and evaluation of a rodent GAP lacking the genes encoding for B9 and SLARP ( PbΔb9Δslarp ) and the generation and evaluation of the equivalent human Pf GAP lacking the Pf ortholog genes .", "PfΔb9Δslarp was generated using constructs that allowed for the removal of the drug selectable marker from the genome by FRT/FLPe recombinase methodology ( van Schaijk et al . , 2010 ) .", "The safety and efficacy of PbΔb9Δslarp and the lack of development of PfΔb9Δslarp in human hepatocytes , in vitro , and , in vivo , in chimeric mice provide strong support for clinical development of a PfΔb9Δslarp PfSPZ vaccine ." ], [ "Previously , we generated a Pb mutant with disruption of the b9 locus ( PbΔb9 ) by standard genetic modification using a double cross-over integration event , followed by removal of the drug-selectable marker cassette by negative selection ( Lin et al . , 2011 ) .", "Characterization of the PbΔb9 phenotype showed that liver-stage development was fully abrogated in BALB/c mice and severely compromised in the more stringent C57BL/6 murine model for P . berghei ( Annoura et al . , 2014 ) .", "Immunization of a single dose of 10k ( i . e . 10 , 000 sporozoites ) or 5k PbΔb9 protected BALB/c mice against a 10k WT-sporozoite challenge , while 80% of mice were still protected after a single 1k immunizing dose ( Table 1 ) .", "In C57BL/6 mice , immunization with 50K/20K/20K of PbΔb9 resulted in complete protection lasting up to 180 days , reducing to 45% protection when challenged at 1 year post-immunization .", "However , sporozoite administration occasionally resulted in blood stage infections after administration of high doses , thereby compromising the safety profile ( Annoura et al . , 2014 ) . 10 . 7554/eLife . 03582 . 003Table 1 . Protection of mice after immunization with P . berghei PbΔb9 or PbΔb9Δslarp sporozoitesDOI: http://dx . doi . org/10 . 7554/eLife . 03582 . 003Mouse strainPb mutantDay of challenge*Immunization regimes no .", "protected/no challengedBALB/c10k†5k1kPbΔb91010/10‡18/208/10PbΔb9Δslarp1020/2010/1020/20C57Bl650/20/20k§10/10/10k1/1/1kPbΔb9104/4ndnd905/51809/9#3655/11PbΔb9Δslarp10Nd10/106/101806/6ndnd*Number of days post last immunization; 104 wild-type sporozoites were injected by IV route .", "†Immunization dose: number of sporozoites x1000 . ‡Protected/total # of immunized mice ( % ) ; protection was 0/15 in naive control BALB/c and 0/10 in C57BL/6 mice .", "§Immunization dose with 7 day intervals between immunizations .", "#Immunization dose 50/10/20k with 7 day intervals between immunizations .", "nd = not done .", "Previously , it has been shown by others that PbΔslarp parasites are completely arrested in liver-stage development with a complete lack of breakthrough blood-stage infections ( Aly et al . , 2008; Silvie et al . , 2008 ) .", "Therefore , we generated a new single gene deletion mutant PbΔslarp in a parasite line that constitutively expressed a fusion of the reporter proteins GFP and luciferase , using a slarp-targeting DNA-construct for deletion by double cross-over homologous integration ( Figure 1—figure supplement 1 ) .", "The PbΔslarp mutant showed blood stage growth and mosquito infections with functional sporozoites similar to wild-type ( Supplementary file 1 ) .", "However , intravenous injection of up to 500k PbΔslarp sporozoites never led to full development of parasites in the liver as assayed by in vivo imaging ( Figure 1—figure supplement 1 ) or analysis of blood stage infection ( Table 2 ) .", "PbΔslarp sporozoites arrested very soon after invasion of cultured Huh7 hepatocytes corroborating the excellent safety findings by Silvie et al . ( 2008 ) . 10 . 7554/eLife . 03582 . 004Table 2 . Breakthrough blood-stage infections after intravenous injection of PbΔslarp and PbΔb9Δslarp sporozoitesDOI: http://dx . doi . org/10 . 7554/eLife . 03582 . 004Mouse strainMutantInfection* Spz x 103Breakthrough blood infection/total # micePre-patent period‡ ( days ) BALB/cWT†105/54–5PbΔslarp500/5PbΔslarp250/10PbΔb9Δslarp250/10C57BL/6WT†105/54–5PbΔslarp5000/5PbΔslarp2000/10PbΔb9Δslarp2000/10PbΔb9Δslarp1500/5*Inoculation dose of sporozoites administered IV .", "†P .", "berghei ANKA strain: line cl15cy1 . ‡Day with parasitemia of 0 . 5–2% .", "Therefore , in order to create a completely attenuated and safe rodent GAP , we additionally disrupted the slarp gene in the PbΔb9 genome by double cross-over integration ( Figure 1 ) .", "Asexual growth and sporogonic development/function equaled wild-type ( Supplementary file 1 ) .", "However , PbΔb9Δslarp sporozoites arrested soon after invasion of cultured Huh7 hepatocytes ( Figure 1 ) and intravenous injection of 150–200K PbΔb9Δslarp sporozoites never resulted in breakthrough blood-stage infections in mice ( Table 2 ) .", "Finally , protective efficacy induced by PbΔb9Δslarp was studied in both BALB/c and C57BL/6 mice .", "A single immunization dose of 10K , 5K , or even 1K of PbΔb9Δslarp sporozoites in BALB/c mice induced full protection against a 10K wild-type sporozoite challenge ( Table 1 ) .", "C57BL/6 mice were 100% protected after 3 × 10K immunization with PbΔb9Δslarp sporozoites , and the protective efficacy reduced to 60% after a 3 × 1K immunization dose .", "A challenge at day 180 post-immunization of a 50/20/20K dose still resulted in complete protection .", "The combined data showed that PbΔb9Δslarp completely arrest during liver-stage development and induce a highly efficient protective immunity in two different strains of mice . 10 . 7554/eLife . 03582 . 005Figure 1 . Generation and genotype analyses of P . berghei mutant PbΔb9Δslarp .", "( A ) Generation of mutant PbΔb9Δslarp .", "For PbΔb9Δslarp , the DNA-construct pL1740 was generated containing the positive/negative selectable marker cassette hdhfr/yfcy .", "This construct was subsequently used to generate the mutant PbΔb9Δslarp in the PbΔb9Δsm mutant .", "See Supplementary file 2A for the sequence of the primers .", "( B ) Diagnostic PCR and Southern analysis of Pulse Field Gel ( PFG ) -separated chromosomes of mutant PbΔb9Δslarp confirming correct disruption of the slarp and the b9 locus .", "See Supplementary file 2A for the sequence of the primers used for the selectable marker gene ( SM ) ; 5′-integration event ( 5′ ) ; 3′-integration event ( 3′ ) ; and the slarp and the b9 ORF .", "For Southern analysis , PFG-separated chromosomes were hybridized using a 3′UTR pbdhfr probe that recognizes the construct integrated into P . berghei slarp locus on chromosome 9 , the endogenous locus of dhfr/ts on chromosome 7 , and a 3′UTR pbdhfr probe that recognizes the construct integrated into P . berghei b9 locus on chromosome", "8 . In addition , the chromosomes were hybridized with the hdhfr probe recognizing the integrated construct into the slarp locus on chromosome", "9 . ( C ) Development of liver-stages in cultured hepatocytes as visualized by staining with antibodies recognizing the parasitophorous vacuole membrane ( anti-EXP1; green ) and the parasite cytoplasm ( anti-HSP70; red ) .", "Nuclei are stained with Hoechst-33342 .", "Hpi: hours post-infection .", "Scale bar represents 10 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 03582 . 00510 . 7554/eLife . 03582 . 006Figure 1—figure supplement 1 . Generation and genotype analyses of P . berghei mutant PbΔslarp-a .", "( A ) Generation of mutant PbΔslarp-a .", "For PbΔslarp-a , the DNA-construct pL1740 was generated containing the positive/negative selectable marker cassette hdhfr/yfcy .", "This construct was subsequently used to generate the mutant PbΔslarp-a ( 1839cl3 ) in the PbGFP-Luccon reference line .", "See Supplementary file 2A for the sequence of the primers .", "( B ) Diagnostic PCR and Southern analysis of Pulse Field Gel ( PFG ) -separated chromosomes of mutant Δslarp-a confirming correct disruption of the slarp-locus .", "See Supplementary file 2A for the sequence of the primers used for the selectable marker gene ( SM ) ; 5′-integration event ( 5′ ) ; 3′-integration event ( 3′ ) ; and the slarp ORF .", "Mutant PbΔslarp-a has been generated in the reference P . berghei ANKA line PbGFP-Luccon which has a gfp-luciferase gene integrated into the silent 230p locus ( PBANKA_030600 ) on chromosome 3 .", "For Southern analysis , PFG-separated chromosomes were hybridized using a 3′UTR pbdhfr probe that recognizes the construct integrated into P . berghei slarp locus on chromosome 9 , the endogenous locus of dhfr/ts on chromosome 7 , and the gfp-luciferase gene integrated into chromosome 3 .", "In addition , the chromosomes were hybridized with the hdhfr probe recognizing the integrated construct into the slarp locus on chromosome", "9 . ( C ) Real time in vivo imaging of Δslarp luciferase-expressing liver-stage parasites in C57BL/6 mice at 24 , 35 , and 45 hr post-infection .", "C57BL/6 mice were IV injected with either 5 × 104 Pb-GFPLuccon sporozoites ( n = 5 ) , resulting in a full liver infection ( upper panel: representative image of WT infected mice ) , or with 5 × 105 PbΔslarp-a sporozoites ( n = 5 ) ( lower panel: representative image of PbΔslarp-luc infected mice ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 03582 . 006 Considering the desired phenotype as observed in P . berghei , we generated a Pf mutant lacking expression of both B9 ( PF3D7_0317100 ) and SLARP ( PF3D7_1147000; sporozoite asparagine-rich protein ) .", "These genes are conserved between rodent and human species , both at the level of syntenic location in their respective genomes on chromosomes 3 and 11 respectively , and at the sequence level .", "Pfb9 shows 37% amino acid sequence identity and 54% sequence similarity with Pbb9 ( ( Annoura et al . , 2014 ) ) ; Pfslarp shows 28% amino acid sequence identity and 46% sequence similarity with Pbslarp .", "First , we generated two independent Pf mutants lacking slarp by standard double cross-over integration of a DNA construct and analyzed their phenotype throughout the parasite life cycle ( Figure 2—figure supplement 1 , 2 ) .", "Blood-stage development of two independently derived PfΔslarp ( i . e . PfΔslarp-a and–b ) parasites was comparable to WT parasites .", "PfΔslarp mutants produced WT numbers of gametocytes , oocysts and sporozoites ( Figure 2 ) .", "The intra-cellular PfΔslarp-a and -b parasite development in primary human hepatocytes was not significantly different in number and morphologically identical to WT parasites at 3 and 24 hours post-infection ( hpi ) ( Figure 3 ) .", "However , their number was more than 10-fold reduced at 48 hpi and not detectable from day 3 onwards to day 7 post-infection .", "Parasites lacking b9 in P . falciparum arrested before day 2 post-infection of primary human hepatocytes with the exception of one observed liver schizont at a later timepoint ( Annoura et al . , 2014 ) .", "PfΔslarp-a and -b parasites still showed positive HSP70 staining and morphologically normal parasites at 48 hpi in primary human hepatocytes , indicating time point of arrest later compared to PfΔb9 parasites . 10 . 7554/eLife . 03582 . 007Figure 2 . Phenotypes of P . falciparum PfΔslarp and PfΔb9Δslarp parasites .", "( A ) Gametocyte , oocyst , and sporozoite production .", "Gametocyte numbers ( stage II and IV–V ) per 1000 erythrocytes at day 8 and day 14 after the start of gametocyte cultures .", "Exflagellation ( Exfl ) of male gametocytes in stimulated samples from day 14 cultures ( ++ score = >10 exflagellation centers per microscope field at 400× magnification ) .", "Median number of oocysts at day 7 , IQR is the inter quartile range and sporozoite ( day 21 ) production ( ×1000 ) in A . stephensi mosquitoes .", "( B ) Gliding motility of P . falciparum WT ( cytochalasin D treated and untreated ) , PfΔslarp-b , PfΔb9Δslarp-F7 , and PfΔb9Δslarp-G9 parasites .", "Gliding motility was quantified by determining the mean percentage ± standard deviation of parasites that exhibited gliding motility by producing characteristic CSP trails ( ≥1 circles ) or parasites that did not produce CSP trails ( 0 circles ) .", "( C ) Cell traversal ability of P . falciparum NF54 , PfΔslarp-b and PfΔb9Δslarp-F7 sporozoites as determined by FACS counting of Dextran positive Huh7 cells .", "Shown is the mean percentage ±standard deviation of FITC positive cells .", "Dextran control ( control ) : hepatocytes cultured in the presence of Dextran but without the addition of sporozoites . DOI: http://dx . doi . org/10 . 7554/eLife . 03582 . 00710 . 7554/eLife . 03582 . 008Figure 2—figure supplement 1 . Consecutive gene deletion of slarp and b9 in P . falciparum . Schematic representation of the genomic loci of ( A ) slarp ( PF11_0480; PF3D7_1147000 ) on chromosome 11 ( Chr . 11 ) and ( B ) b9 ( PFC_0750w; PF3D7_0317100 ) on chromosome 3 ( Chr . 3 ) of wild-type ( wt; NF54wcb ) , PfΔslarp and PfΔb9Δslarp gene deletion mutants before ( PfΔslarp a and PfΔb9Δslarp ) and after the FLPe mediated removal of the hdhfr::gfp resistance marker ( PfΔslarp b and PfΔb9Δslarp clones F7/G9 ) , respectively .", "The constructs for the targeted deletion of slarp ( pHHT-FRT-GFP slarp ) and b9 ( pHHT-FRT-GFP-B9 ) contain two FRT sequences ( red triangles ) that are recognized by FLPe .", "P1 , P2 and P3 , P4 primer pairs for LR-PCR analysis of slarp and b9 loci respectively; T ( TaqI ) and R ( RcaI ) : restriction sites used for Southern blot analysis and sizes of restriction fragment are indicated; cam: calmodulin; hrp: histidine rich protein; hsp: heatshock protein; fcu: cytosine deaminase/uracil phosphoribosyltransferase; hdhfr::gfp: human dihydrofolate reductase fusion with green fluorescent protein; pbdt: P . berghei dhfr terminator . DOI: http://dx . doi . org/10 . 7554/eLife . 03582 . 00810 . 7554/eLife . 03582 . 009Figure 2—figure supplement 2 . Genotype analysis of the generated PfΔslarp and PfΔb9Δslarp parasites .", "( A ) Long range PCR analysis of genomic DNA from WT , PfΔslarp and PfΔb9Δslarp asexual parasites confirms the slarp gene deletion and consecutive gene deletions of both slarp and b9 respectively and subsequent removal of the hdhfr::gfp resistance marker .", "The PCR products are generated using primers P1 , P2 for slarp and P3 , P4 for b9 ( see A and B respectively; for primer sequences see primer table in Supplementary file 2B ) and PCR products are also digested with restriction enzymes x ( XmaI ) and kx ( KpnI/XcmI ) respectively for confirmation ( i . e . slarp LR-PCR product sizes: WT , 12 kb , is undigested; Δslarp-a , 5 . 4 kb is digested into 1 . 3 kb and 4 . 0 kb fragments , Δslarp-b , 2 . 4 kb is digested into 1 . 3 kb and 1 . 1 kb fragments . b9 LR-PCR product sizes: WT , 5 . 5 kb , is digested into 756 bp , 793 bp , and 4 . 0 kb fragments; Δb9-b , 2 . 6 kb is digested into 756 bp , 793 bp , and 1 . 1 kb fragments ) .", "( B ) Southern analysis of restricted genomic DNA from WT , PfΔslarp-a , PfΔslarp-b , PfΔb9Δslarp-F7 , and PfΔb9Δslarp-G9 asexual parasites .", "DNA was digested with restriction enzyme ( E: TaqI ) and probed with the 5′ slarp targeting region ( P: 5′ slarp-T; see A ) on the left side of the slarp Southern or probed with the 3′slarp targeting region ( P: 3′ slarp-T; see A ) on the right side of the slarp panel .", "For analysis of the b9 , integration DNA was digested with restriction enzymes ( E: RcaI ) and probed with the 5′ b9 targeting region ( P: 5′ b9-T; see A ) on the right panel .", "The expected fragment sizes are indicated in panel ( A ) .", "( C ) RT-PCR analysis showing the absence of b9 and slarp transcripts in P . falciparum PfΔslarp-a , PfΔslarp-b , PfΔb9Δslarp-F7 , and PfΔb9Δslarp-G9 mutant sporozoites .", "PCR amplification using purified sporozoite RNA was performed either in the presence or absence of reverse transcriptase ( RT+ or RT− , respectively ) and generated the expected 506 bp and 580 bp fragments for slarp and b9 respectively , the positive control was performed by PCR of 18S rRNA using primers 18Sf/18Sr ( for primer sequences see Supplementary file 2B ) and generated the expected 130 bp fragment . DOI: http://dx . doi . org/10 . 7554/eLife . 03582 . 00910 . 7554/eLife . 03582 . 010Figure 3 . Development of P . falciparum PfΔslarp and PfΔb9Δslarp parasites in human primary hepatocytes .", "( A ) In vitro invasion of P . falciparum wt , PfΔslarp-a , PfΔslarp-b , PfΔb9Δslarp-F7 , and PfΔb9Δslarp-G9 sporozoites in primary human hepatocytes .", "Invasion is represented as the mean ratio ± standard deviation of extra- and intra-cellular sporozoites by double staining at 3 and 24 hr post-infection , determined after three wash steps to remove sporozoites in suspension .", "( B ) Immunofluorescence assay of PfΔslarp-b parasites in human primary hepatocytes at 3 and 24 hr post-infection .", "Parasites are visualized by staining with anti-PfCSP antibodies ( green; Alexa-488 ) and parasite , and hepatocyte nuclei are stained with DAPI ( blue ) .", "Images were photographed on an Olympus FV1000 confocal microscope .", "Scale bar represents 5 µm .", "( C ) Development of P . falciparum wt , PfΔslarp-a , PfΔslarp-b ( top panel ) , PfΔb9Δslarp-F7 , and PfΔb9Δslarp-G9 ( bottom panel ) liver-stages in primary human hepatocytes following inoculation with 40 , 000 sporozoites .", "From day 2 to 7 , the mean number ± standard deviation of parasites per 96-well was determined by counting parasites stained with anti-P .", "falciparum HSP70 antibodies .", "The bottom panel represents experiments performed in primary human hepatocytes from 2 different donors .", "No parasites present ( NP ) .", "( D ) Development of liver-stages of PfΔb9Δslarp GAP in chimeric mice engrafted with human hepatocytes .", "Mice were infected with 106 wt or PfΔb9Δslarp-G9 sporozoites by intravenous inoculation .", "At 24 hr or at 5 days after sporozoite infection , livers were collected from the mice and the presence of parasites determined by qPCR of the parasite-specific 18S DNA .", "uPA HuHEP; chimeric homozygous uPA+/+-SCID mice engrafted with human hepatocytes .", "As controls , uPA mice; heterozygous uPA+/−-SCID mice not engrafted with human hepatocytes were used . DOI: http://dx . doi . org/10 . 7554/eLife . 03582 . 010 Next , we generated double gene-deletion PfΔb9Δslarp mutants using the FRT/FLPe recombinase methodology ( van Schaijk et al . , 2010 ) .", "This methodology employs FLPe recombinase to remove a FRT-site flanked drug resistance marker cassette introduced into the Pf genome when the target gene has been removed by double cross-over homologous recombination as shown for PfΔslarp-b parasites in Figure 2—figure supplement 1 , 2 .", "After cloning , this ‘marker-free’ line was subsequently transfected with the Pfb9 gene-targeting construct pHHT-FRT-GFP-b9 ( Annoura et al . , 2014 ) to delete the b9 locus from the PfΔslarp-b genome ( Figure 2—figure supplement 1 , 2 ) .", "Subsequently two ‘marker-free’ clones , PfΔb9Δslarp-F7 and PfΔb9Δslarp-G9 , were obtained containing the correct genotype that is removal of the slarp and b9 gene loci as well as both respective drug selection cassettes ( Figure 2—figure supplement 2 ) .", "In addition , we confirmed the loss of expression of both slarp and b9 by RT-PCR analysis by demonstrating the absence of transcripts in mRNA collected from PfΔb9Δslarp-F7 and PfΔb9Δslarp-G9 salivary gland sporozoites ( Figure 2—figure supplement 2 ) .", "We then examined the phenotype of PfΔb9Δslarp-F7 and PfΔb9Δslarp-G9 mutants during blood stage and mosquito development .", "Asexual blood stage growth of PfΔb9Δslarp parasites was normal as both clones reached an asexual parasitemia between 0 . 5 and 5% during cloning within 21 days and PfΔb9Δslarp clones produced WT-like numbers of gametocytes , oocysts , and sporozoites ( Figure 2 ) .", "We next analyzed the development of PfΔb9Δslarp in human hepatocytes using cultured primary hepatocytes and uPA+/+-SCID mice engrafted with human hepatocytes ( human liver-uPA-SCID mice ) ( Meuleman et al . , 2005 ) .", "PfΔb9Δslarp sporozoites showed normal gliding motility , hepatic cell traversal ( Figure 2 ) , as well as invasion of primary human hepatocytes , but parasites were completely absent in two independent experiments at day 2 up to day 7 post-infection , following inoculation of primary human hepatocytes with 40 , 000 PfΔb9Δslarp F7 or G9 sporozoites ( Figure 3 ) .", "Detailed analyses of 80 individual wells at day 4 post-infection did not result in identification of a single developing parasite .", "The combined day 2 and day 4 data of PfΔb9Δslarp indicated that the timing of arrest is similar to PfΔb9 ( Annoura et al . , 2014 ) and there had been complete arrest of liver-stage development , similar to PfΔslarp parasites .", "In addition , human liver-uPA-SCID mice were intravenously inoculated with 1 × 106 WT or PfΔb9Δslarp sporozoites .", "Two heterozygous uPA+/−-SCID mice , not engrafted with human hepatocytes , served as controls and were also challenged with P . falciparum sporozoites .", "Livers were collected either at 24 hpi or 5 days post-infection for detection of P . falciparum 18S DNA by quantitative real-time PCR ( Foquet et al . , 2013 ) .", "Both mice infected with WT Pf and 1 of the 2 mice infected with PfΔb9Δslarp were positive for Pf 18S DNA at 24 hr post-infection , demonstrating successful sporozoite infection in human hepatocytes ( Figure 3 ) .", "A lower signal was observed in PfΔb9Δslarp-infected mice at day 1 after infection compared to WT parasites , likely reflecting the early time point of arrest of this GAP .", "All mice infected with Pf WT ( 3/3 ) showed a strong increase in parasite 18S DNA at day 5 post-infection , representing successful liver-stage development .", "In contrast , none of the human liver-uPA-SCID mice infected with PfΔb9Δslarp sporozoites showed 18S DNA higher than heterozygous uPA+/−-SCID mice , not engrafted with human hepatocytes that had been infected with PfΔb9Δslarp sporozoites ( Figure 3 ) .", "Although these studies were performed with a limited number of mice , these findings indicate that PfΔb9Δslarp parasites can invade but do not develop in livers of humanized mice .", "Our combined results demonstrate abrogation of development of PfΔb9Δslarp inside human hepatocytes ." ], [ "While this manuscript was in preparation an article was published that also describes a multiple-gene deletion P . falciparum parasite that has undergone pre-clinical evaluation ( Mikolajczak et al . , 2014 ) .", "In that study , the authors describe a P . falciparum mutant that , like our work , also lacks the gene slarp ( sap1 ) as well as the paralogous pair of genes , p52 and p36 ." ], [ "The following reference lines of the ANKA strain of P . berghei were used: line cl15cy1 ( Janse et al . , 2006a , 2006b ) and line 676m1cl1 ( PbGFP-Luccon; see RMgm-29 in www . pberghei . eu ) .", "PbGFP-Luccon expresses a fusion protein of GFP and luciferase from the eef1a promoter ( Franke-Fayard et al . , 2004; Janse et al . , 2006a ) .", "For transfections , the parasite used was directly from a characterized good manufacturing process ( GMP ) and produced working cell bank ( WCB ) of the P . falciparum NF54 wild-type strain ( Ponnudurai et al . , 1981 ) , produced by Sanaria Inc , identical to that described previously ( Hoffman et al . , 2010; Epstein et al . , 2011; Roestenberg et al . , 2013 ) .", "Blood stages of wt , PfΔslarp-a , PfΔslarp-b , PfΔb9Δslarp-F7 , and PfΔb9Δslarp-G9 were cultured in a semi-automated culture system using standard in vitro culture conditions for P . falciparum and induction of gametocyte production in these cultures was performed as previously described ( Ifediba and Vanderberg , 1981; Ponnudurai et al . , 1982 , 1989 ) .", "Fresh human red blood cells and serum were obtained from Dutch National blood bank ( Sanquin Nijmegen , NL; permission granted from donors for the use of blood products for malaria research ) .", "Cloning of transgenic parasites was performed by the method of limiting dilution in 96-well plates as described ( Thaithong , 1985 ) .", "Parasites of the positive wells were transferred to the semi-automated culture system and cultured for further phenotype and genotype analyses ( See below ) .", "For P . berghei infections , female C57BL/6J and BALB/c ( 12-week old; Janvier France ) and Swiss OF1 ( 8 weeks old Charles River ) were used .", "All animal experiments with rodent parasites performed at the LUMC ( Netherlands ) were approved by the Animal Experiments Committee of the Leiden University Medical Center ( DEC 07171; DEC 10099 ) and at the RUNMC ( Netherlands ) by the Radboud University Experimental Animal Ethical Committee ( RUDEC 2008-123 , RUDEC 2008-148 , RUDEC 2010-250 , RUDEC 2011-022 , RUDEC 2011-208 ) .", "The Dutch Experiments on Animal Act is established under European guidelines ( EU directive 86/609/CEE ) regarding the Protection of Animals used for Experimental and Other Scientific Purposes .", "Human liver-uPA-SCID mice ( chimeric mice ) were produced as described before ( Meuleman et al . , 2005 ) .", "The study protocol for infecting these mice with P . falciparum sporozoites was approved by the animal ethics committee of the Faculty of Medicine and Health Sciences of the Ghent University .", "To disrupt the P . berghei slarp gene ( PBANKA_090210 ) , a construct was generated using the adapted ‘Anchor-tagging’ PCR-based method as described ( Annoura et al . , 2012 ) ( Figure 1—figure supplement 1 ) .", "The two targeting fragments ( 1195 bp and 823 bp ) of slarp were amplified using genomic DNA ( parasite line cl15cy1 ) as template with the primer pairs 5960/5961 ( 5′target sequence ) and 5962/5963 ( 3′target sequence ) .", "See Supplementary file 2A for the sequence of the primers .", "Using this PCR-based targeting construct ( pL1740 ) , the mutant PbΔslarp-a ( 1839cl3 ) was generated in the PbGFP-Luccon reference line using standard methods of transfection and positive selection with pyrimethamine ( Figure 1—figure supplement 1 ) .", "The generation of the drug-selectable marker-free mutant PbΔb9Δsm ( 1309cl1m0cl2; RMgmDB no . 934 ) has been described by Annoura et al . ( 2014 ) .", "This mutant , which contains a disrupted b9 gene and is drug-selectable marker free , was used for deleting the slarp gene ( PBANKA_090210 ) .", "To delete the slarp gene , the gene-deletion construct pL1740 was used as described above .", "Using this construct the mutant PbΔb9Δslarp ( line 1844cl1 ) was generated in the PbΔb9Δsm line using standard methods of transfection and positive selection with pyrimethamine ( Figure 1 ) .", "Correct integration of the constructs into the genome of mutant parasites was analyzed by diagnostic PCR-analysis and Southern analysis of PFG-separated chromosomes as shown in Figure 1 and Figure 1—figure supplement 1 .", "PFG-separated chromosomes were hybridized with a probe recognizing hdhfr or the 3′-UTR dhfr/ts of P . berghei ( Janse et al . , 2006b ) .", "The slarp gene ( PF3D7_1147000 ) in P . falciparum WT parasites ( NF54wcb ) was deleted using a modified construct based on plasmid pHHT-FRT- ( GFP ) -Pf52 ( van Schaijk et al . , 2010 ) ( Figure 2—figure supplement 1 ) .", "Targeting regions were generated by PCR using primers BVS179 and BVS180 for the 5′ target region and primers BVS182 and BVS184 for the 3′ target region ( see Supplementary file 2B for primer sequences ) .", "The 5′and 3′ target regions were cloned into pHHT-FRT- ( GFP ) -Pf52 digested with BsiWI , BssHII and NcoI , XmaI , respectively , resulting in the plasmid pHHT-FRT-GFP-slarp .", "The b9 gene ( PF3D7_0317100 ) of PfΔslarp-b P . falciparum parasites was deleted using a modified construct based on plasmid pHHT-FRT- ( GFP ) -Pf52 ( van Schaijk et al . , 2010 ) ( Figure 2—figure supplement 1 ) .", "Targeting regions were generated by PCR using primers BVS84 and BVS85 for the 5′ target region and primers BVS88 and BVS89 for the 3′ target region .", "The 5′and 3′ target regions were cloned into pHHT-FRT- ( GFP ) -Pf52 digested with NcoI , XmaI and MluI , BssHII resulting in the plasmid pHHT-FRT-GFP-b9 .", "All DNA fragments were amplified by PCR amplification ( Phusion , Finnzymes ) from genomic P . falciparum DNA ( NF54 strain ) and all PCR fragments were sequenced after TOPO TA ( Invitrogen , Leek , The Netherlands ) sub-cloning .", "Transfection of WT ( NF54wcb ) parasites with the plasmid pHHT-FRT-GFP-slarp and selection of mutant parasites were performed , as described ( van Schaijk et al . , 2010 ) , resulting in the selection of the parasite line PfΔslarp-a .", "The second PfΔslarp parasite line , originating from an independent transfection , was subsequently transfected with pMV-FLPe to remove the drug-selectable marker cassette using FLPe as described ( van Schaijk et al . , 2010 ) and cloned resulting in the parasite clone PfΔslarp-b .", "Subsequent transfection of PfΔslarp-b parasites with the plasmid pHHT-FRT-GFP-b9 and selection were performed , as described above , resulting in the parasite line PfΔb9Δslarp .", "The parasite line PfΔb9Δslarp was subsequently transfected with pMV-FLPe to remove the drug-selectable marker cassette using FLPe and cloned , as described above , resulting in the cloned parasite lines PfΔb9Δslarp-F7 and PfΔb9Δslarp-G9 that are free of drug resistance markers .", "Genotype analysis of PfΔslarp and PfΔb9Δslarp parasites was performed by Expand Long range dNTPack ( Roche ) diagnostic , long-range , PCR ( LR-PCR ) and Southern blot analysis ( Figure 2—figure supplement 2 ) .", "Genomic DNA of blood stages of WT or mutant parasites was isolated and analyzed by LR-PCR using primer pair p1 , p2 ( slarp ) and p3 , p4 ( b9 ) ( See Supplementary file 2B for primer sequences ) for correct integration of the constructs in the respective slarp and b9 loci by double cross-over homologous recombination .", "The LR-PCR program has an annealing step of 48°C for 30 s and an elongation step of 62°C for 10–15 min .", "All other PCR settings were according to manufacturer's instructions .", "PCR products were directly analyzed by standard agarose gel electrophoresis or first digested with restriction enzymes for further confirmation of the genotype and removal of resistance markers was confirmed by sequencing .", "For Southern blot analysis , genomic DNA was digested with TaqI or RcaI restriction enzymes for analysis of integration into the slarp and b9 loci , respectively .", "Southern blot was generated by capillary transfer as described ( Sambrook and Russel , 2001 ) and DNA was hybridized to radioactive probes specific for the targeting regions used for the generation of the mutants and generated by PCR ( See above ) .", "The presence or absence of slarp and b9 transcripts in WT and mutant sporozoites was analyzed by reverse transcriptase-PCR ( Figure 2—figure supplement 2 ) .", "Total RNA was isolated using the RNeasy mini Kit ( Qiagen ) from 106 salivary gland sporozoites collected by dissection of mosquitoes 16 days after feeding with WT , PfΔslarp-a , PfΔslarp-b , PfΔb9Δslarp-F7 , and PfΔb9Δslarp-G9 parasites .", "Remaining DNA was degraded using DNAseI ( Invitrogen ) .", "cDNA was synthesized using the First Strand cDNA synthesis Kit for RT-PCR AMV ( Roche ) .", "As a negative control for the presence of genomic DNA , reactions were performed without reverse transcriptase ( RT− ) .", "PCR amplification was performed for regions of slarp using primers BVS290 , BVS292 and for regions of b9 using primers BVS286 and BVS288 .", "Positive control was performed by PCR of 18S rRNA using primers 18Sf and 18Sr .", "Asexual multiplication rate and gametocyte production of P . berghei blood stages were determined as described ( Annoura et al . , 2012 ) .", "The P . berghei mutants were maintained in Swiss mice .", "The multiplication rate of blood stages and gametocyte production were determined during the cloning procedure ( Janse et al . , 2006b ) and were not different from parasites of the reference ANKA lines .", "P . falciparum blood stage development and gametocyte production were analyzed as described ( van Schaijk et al . , 2010 ) .", "Feeding of A . stephensi mosquitoes with P . berghei and P . falciparum , determination of oocyst production and sporozoite collection , as well as P . berghei gliding motility were performed as described ( Annoura et al . , 2012 ) .", "P . falciparum gliding motility of sporozoites was determined as described ( Stewart and Vanderberg , 1988; van Schaijk et al . , 2008 ) .", "P . falciparum cell traversal and invasion of hepatocytes were determined in Huh7 cells and primary human hepatocytes respectively as described ( van Schaijk et al . , 2008 ) .", "Infectivity of P . berghei sporozoites and development was determined in cultures of Huh7 cells as described ( van Schaijk et al . , 2008 ) .", "For analysis of liver-stage development by immunofluorescence , parasites were stained with the following primary antibodies: anti-PbEXP1 ( PBANKA_092670; raised in chicken ( Sturm and Heussler , 2007 ) ) and anti-PbHSP70 ( PBANKA_081890; raised in mouse ( Mueller et al . , 2005 ) ) .", "Infectivity of P . falciparum sporozoites and development was analyzed in primary human hepatocytes as described ( van Schaijk et al . , 2008 ) .", "Briefly for analysis of development by immunofluorescence , parasites were stained with the following primary antibodies: anti-HSP70 ( PF3D7_0930300 ( Renia et al . , 1990 ) ) and anti-CSP ( PF3D7_0304600; 3SP2 ) using double labeling .", "Anti-mouse secondary antibodies , conjugated to Alexa-488 or Alexa-594 ( Invitrogen ) , were used for visualization .", "Primary human hepatocytes were isolated from healthy parts of human liver fragments , which were collected during unrelated surgery in agreement with French national ethical regulations ( Gouagna et al . , 2007 ) and after oral informed consent from adult patients undergoing partial hepatectomy as part of their medical treatment ( Service de Chirurgie Digestive , Hépato-Bilio-Pancréatique et Transplantation Hépatique , Hôpital Pitié-Salpêtrière , Paris , France ) .", "The collection and use of this material for the purposes of the study presented here were undertaken in accordance with French national ethical guidelines under Article L . 1121-1 of the ‘Code de la Santé Publique’ .", "Given that the tissue samples are classed as surgical waste , that they were used anonymously ( the patient's identity is inaccessible to the researchers ) , and that they were not in any way genetically manipulated , article L . 1211-2 stipulates that their use for research purposes is allowed provided that the patient does not express any opposition to the surgeon prior to surgery and after being informed of the nature of the research in which they might be potentially employed .", "Within this framework , the collection and use of this material was furthermore approved by the Institutional Review Board ( Comité de Protection des Personnes ) of the Centre Hospitalo-Universitaire Pitié-Salpêtrière , Assistance Publique-Hôpitaux de Paris , France .", "C57BL/6 or BALB/c mice were inoculated with sporozoites by intravenous injection of different sporozoite numbers , ranging from 1 × 104 to 5 × 105 .", "Blood stage infections were monitored by analysis of Giemsa-stained thin smears of tail blood collected on day 4–14 after inoculation of sporozoites .", "The prepatent period ( measured in days post sporozoite infection ) is defined as the day when a blood stage infection with a parasitemia of 0 . 5–2% is observed .", "Liver-stage development in live mice was monitored by real time in vivo imaging of liver-stages as described ( Ploemen et al . , 2012 ) .", "Liver-stages were visualized by measuring luciferase activity of parasites ( expressing luciferase under the eef1a promoter ) in whole bodies of mice ( Ploemen et al . , 2009 ) .", "Prior to immunization , P . berghei sporozoites were collected at day 21–27 after mosquito infection by hand-dissection .", "Salivary glands were collected in DMEM ( Dulbecco's Modified Eagle Medium from GIBCO ) and homogenized in a homemade glass grinder .", "The number of sporozoites was determined by counting in triplicate in a Bürker-Türk counting chamber using phase-contrast microscopy .", "BALB/c and C57BL/6 mice were immunized by intravenous injection using different numbers of mutant sporozoites .", "BALB/c mice received one immunization and C57BL/6 mice received three immunizations with two 7 day intervals .", "Immunized mice were monitored for blood infections by analysis of Giemsa stained films of tail blood at day 4–16 after immunization .", "Immunized mice were challenged at different time points after immunization by intravenous injection of 1 × 104 sporozoites from the P . berghei ANKA reference line cl15cy1 .", "In each experiment , age matched naive mice were included to verify infectivity of the sporozoites used for challenge .", "After challenge , mice were monitored for blood infections by analysis of Giemsa stained films of tail blood at day 4–21 .", "Human liver-uPA-SCID mice were produced as described before ( Meuleman et al . , 2005 ) .", "Briefly , within two weeks after birth homozygous uPA+/+-SCID mice ( Foquet et al . , 2013 ) were transplanted with approximately 106 cryopreserved primary human hepatocytes obtained from a single donor ( BD Biosciences , Erembodegem , Belgium ) .", "To evaluate successful engraftment , human albumin was quantified in mouse plasma with an in-house ELISA ( Bethyl Laboratories Inc . , Montgomery , TX ) .", "The study protocol was approved by the animal ethics committee of the Faculty of Medicine and Health Sciences of the Ghent University .", "Human liver-uPA-SCID mice ( n = 10 ) and non-chimeric heterozygous uPA+/−-SCID mice ( control , n = 2 ) were intravenously injected with 106 fresh isolated PfΔb9Δslarp-G9 or as a control WT sporozoites .", "One and 5 days post-infection livers were removed and each liver was cut into 12 standardized sections and stored in RNAlater ( Sigma ) at 4°C until analysis as described ( Foquet et al . , 2013 ) .", "From each part DNA was extracted to assess the parasite load by Pf18S qPCR and to assess the number of human and mouse hepatocytes by Multiplex qPCR PTGER2 analysis ( Foquet et al . , 2013 ) .", "While this manuscript was in preparation an article was published that also describes a multiple-gene deletion P . falciparum parasite that has undergone pre-clinical evaluation ( Mikolajczak et al , 2014 ) .", "In that study , the authors describe a P . falciparum mutant that , like our work , also lacks the gene slarp ( sap1 ) as well as the paralogous pair of genes , p52 and p36 .", "The materials described in this study must be acquired through a material transfer agreement ." ] ]

[ "A highly efficacious pre-erythrocytic stage vaccine would be an important tool for the control and elimination of malaria but is currently unavailable .", "High-level protection in humans can be achieved by experimental immunization with Plasmodium falciparum sporozoites attenuated by radiation or under anti-malarial drug coverage .", "Immunization with genetically attenuated parasites ( GAP ) would be an attractive alternative approach .", "In this study , we present data on safety and protective efficacy using sporozoites with deletions of two genes , that is the newly identified b9 and slarp , which govern independent and critical processes for successful liver-stage development .", "In the rodent malaria model , PbΔb9ΔslarpGAP was completely attenuated showing no breakthrough infections while efficiently inducing high-level protection .", "The human PfΔb9ΔslarpGAP generated without drug resistance markers were infective to human hepatocytes in vitro and to humanized mice engrafted with human hepatocytes in vivo but completely aborted development after infection .", "These findings support the clinical development of a PfΔb9ΔslarpSPZ vaccine ." ]

[ "Vaccines commonly contain a weakened or dead version of a disease-causing microorganism , or its toxins , or surface proteins .", "These prime the immune system to rapidly recognize , respond to , and eliminate the actual infectious pathogen if later encountered .", "While vaccines are currently available to help prevent a large number of diseases , vaccines for many deadly diseases , including malaria , do not yet exist .", "Malaria is caused by a group of parasites called Plasmodium , which are transferred to humans by mosquitoes .", "While measures to control mosquito populations and prevent mosquito bites have helped to reduce the incidence of malaria in some countries , the number of people—and especially children—that die of malaria every year remains very high .", "When a mosquito carrying Plasmodium in its salivary glands bites a human , the parasite is injected into the human's bloodstream and travels to the liver .", "The parasite reproduces in the liver cells until there are so many of them that the cells rupture , and the parasites are released back into the bloodstream .", "Any mosquito that then feeds on the blood of the infected individual may also suck up the parasite .", "The parasite then goes through a further stage of development in the mosquito , eventually migrating to the salivary glands , from where the parasite can be transmitted into a new human host .", "Recent work in rodents suggests that genetically altered or weakened Plasmodium falciparum sporozoites—the form of the parasite found in mosquito saliva—could be used to vaccinate humans against malaria caused by this parasite species .", "Now , van Schaijk , Ploemen et al . evaluate whether a safe and effective vaccine could be made from sporozoites that lack two genes , called b9 and slarp , which are critical for the parasites to develop inside liver cells .", "When mice were injected with the modified sporozoites , their immune cells were able to detect the parasites and respond against them .", "The mice subsequently did not develop malaria when they were infected with normal , unmodified parasites .", "Furthermore , none of the mice contracted malaria from the modified sporozoites .", "The modified sporozoites behaved similarly in human liver cells: after invading these cells , the parasites were unable to develop .", "Clinical testing and further development are now needed to see if a successful malaria vaccine can be made from these sporozoites ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "ecology", "plant biology" ]

[ [ "More than 70% of all higher plants , including crop plants , form symbiotic associations with arbuscular mycorrhizal fungi ( AMF ) ( Brundrett and Tedersoo , 2018 ) .", "While the fungus facilitates the uptake of mineral nutrients , in particular phosphorous ( P ) and nitrogen , the plant supplies the fungus with carbon ( Helber et al . , 2011; Bravo et al . , 2017; Jiang et al . , 2017; Keymer et al . , 2017; Luginbuehl et al . , 2017 ) .", "The interaction affects plant growth ( Rooney et al . , 2009; Adolfsson et al . , 2015 ) and resistance to various abiotic and biotic stresses ( Pineda et al . , 2010; Vannette et al . , 2013; Chitarra et al . , 2016; Sharma et al . , 2017 ) .", "Although AMF interactions are physically restricted to the roots , they influence whole-plant performance , hence systemic metabolic responses have been anticipated , and searched for , but no general AMF-specific responses have been found ( Bi et al . , 2007; Toussaint , 2007; Schweiger and Müller , 2015 ) .", "While changes in foliar levels of carbohydrates , proteins , and amino acids , as well as secondary metabolites and phytohormones have been shown to respond to AMF inoculation ( Schweiger et al . , 2014; Aliferis et al . , 2015; Adolfsson et al . , 2017 ) , these changes are not specific to AMF interactions and tend to be general responses to various abiotic and biotic stresses .", "Moreover , these metabolic responses also tend to be taxa-specific , and many are likely indirect consequences of AMF-mediated effects on plant growth and development .", "In contrast , large amounts of blumenol-type metabolites accumulate in roots after AMF inoculation .", "These compounds are apocarotenoids , in particular C13 cyclohexenone derivatives , produced by the cleavage of carotenoids .", "After AMF colonization , a C40 carotenoid is cleaved by carotenoid cleavage dioxygenase 7 ( CCD7 ) to produce a C13 cyclohexenone and a C27 apocarotenoid which is further cleaved by CCD1 to yield a second C13 cyclohexenone ( Floss et al . , 2008; Vogel et al . , 2010; Hou et al . , 2016 ) .", "The compounds have been found to accumulate in the roots of AMF-colonized plants in a manner highly correlated with the fungal colonization rate ( Fester et al . , 1999 ) .", "Other stimuli such as pathogen infection and abiotic stresses , do not induce their accumulations ( Maier et al . , 1997 ) .", "The AMF-induced accumulation of these compounds is widespread and has been observed in roots of plant species from different families , including mono- and dicotyledons , ( Hordeum vulgare , Peipp et al . , 1997 ) ; Solanum lycopersicum and Nicotiana tabacum , Maier et al . , 2000; e . g . , Zea mays , Fester et al . , 2002; Lotus japonicus and Medicago truncatula , Fester et al . , 2005; Ornithogalum umbellatum , Schliemann et al . , 2006; Allium porrum , Schliemann et al . , 2008 ) .", "Blumenols are classified into three major types; blumenol A , blumenol B and blumenol C ( Figure 1A ) .", "However , previous studies have reported that only blumenol glycosides containing a blumenol C-based aglycone are positively correlated with mycorrhizal colonization .", "The aglycone can be additionally hydroxylated at the C11 or carboxylated at the C11 or C12 position ( Maier et al . , 1997; Maier et al . , 2000 ) .", "Additionally , 7 , 8-didehydro versions of blumenol C have been reported ( Peipp et al . , 1997 ) .", "The glycosylation usually occurs as an O-glycoside at the C9 position ( Strack and Fester , 2006 ) , but glycosylations at the hydroxylated C11 position have also been observed ( Schliemann et al . , 2008 ) .", "The glycosyl moiety can be a single sugar or combinations of glucose ( Glc ) , rhamnose , apiose , arabinose and/or glucuronic acid , which , in turn can be additionally malonylated or contain a 3-hydroxy-3-methylglutarate decoration ( Strack and Fester , 2006; Schliemann et al . , 2008 ) .", "The connections among sugar components can also vary ( e . g . , glucose- ( 1’’→4’ ) -glucose or glucose- ( 1’’→6’ ) -glucose; Maier et al . , 2000; Fester et al . , 2002 ) .", "The particular type of decorations appears to be highly species-specific and it is likely that additional structural variants remain to be discovered .", "Exemplary structures are shown in Figure 1B .", "Interestingly , blumenols such as blumenol A , blumenol A-9-O-Glc , blumenol B , blumenol C and blumenol C-9-O-Glc , were also reported to occur in the aerial parts of various plant species ( Galbraith and Horn , 1972; Bhakuni et al . , 1974; Takeda et al . , 1997 ) .", "However , most of these studies focused on the identification of natural products using large scale extractions ( up to several kg of plant material ) and were not performed in the context of AMF colonization .", "Furthermore , some blumenol compounds were also found in plant families that are known to have lost their ability to establish AMF interactions ( Brassicaceae: Cutillo et al . , 2005; Urticaceae: Aishan et al . , 2010 ) .", "These reports indicate AMF-independent constitutive levels of particular blumenols in aerial plant parts .", "Adolfsson et al . , 2017 analyzed blumenol accumulations together with other metabolites in leaves of plants with and without AMF colonization .", "None of these studies reported AMF-specific accumulations of blumenols or transcripts specific for their biosynthesis .", "The concentrations of some blumenol derivatives were even reported to be down-regulated in response to AMF colonization ( Adolfsson et al . , 2017 ) .", "The identification of a reliable metabolite marker in aerial plant tissues would be highly useful for AMF research since the characterization of AMF-associations is still laborious and time-consuming , typically requiring destructive root harvesting and microscopic examination or transcript analyses ( Vierheilig et al . , 2005; Parádi et al . , 2010 ) .", "To identify readily accessible AMF-indicative shoot metabolites , we hypothesized that a subset of the AMF-induced root metabolites would accumulate in shoots as a result of transport or systemic signaling ." ], [ "We performed an untargeted metabolomics analysis of root tissues in a transgenic line of Nicotiana attenuata , silenced in the calcium- and calmodulin-dependent protein kinase ( irCCaMK ) and empty vector ( EV ) plants co-cultured with or without Rhizophagus irregularis ( Figure 2A ) .", "By using irCCaMK plants , unable to establish a functional AMF-association ( Groten et al . , 2015 ) , we were able to dissect the AMF association-specific metabolic responses from those changes that result from more general plant-fungus interactions .", "Untargeted metabolome profiling of roots using liquid chromatography ( LC ) coupled to time-of-flight mass spectrometry ( qTOF-MS ) resulted in a concatenated data matrix consisting of 943 mass features ( m/z signals detected at particular retention times ) .", "A co-expression network analysis was conducted in which nodes represent m/z features and edges connect metabolite mass features originating from similar in-source fragmentations and sharing biochemical relationships ( Li et al . , 2015; Li et al . , 2016 ) .", "For example , features representing well-known compounds , like nicotine and phenylalanine , were tightly connected ( Figure 2B ) .", "A STEM clustering pipeline was performed to recognize patterns of metabolite accumulations in the genotype × treatment data matrix [ ( EV/irCCaMK ) × ( -/+AMF inoculation ) ] .", "As a result , 5 of 8 computed distinct expression patterns were mapped onto the covariance network in Figure 2B ( shown in different colors ) .", "A tightly grouped cluster of unknown metabolites , highlighted in red ( Figure 2B ) occupied a distinct metabolic space .", "Metabolites grouped in this cluster were highly elicited upon mycorrhizal colonization in EV , but not in irCCaMK plants and not found in plants without AMF associations ( Figure 2C ) .", "The structures of the compounds of this cluster were annotated based on tandem-MS and NMR data .", "Five metabolites were annotated as blumenols: 11-hydroxyblumenol C-9-O-Glc ( Figure 2C; Compound 1 ) , 11-carboxyblumenol C-9-O-Glc ( Figure 2C; Compound 2 ) , 11-hydroxyblumenol C-9-O-Glc-Glc ( Compound 3 ) , blumenol C-9-O-Glc-Glc ( Compound 4 ) and blumenol C-9-O-Glc ( Compound 5 ) .", "To quantify these compounds throughout the plant , we used a more sensitive and specifically targeted metabolomics approach based on LC-triple-quadrupole-MS .", "The abundance of the five blumenol C-glycosides continually increased with mycorrhizal development ( Figure 2—figure supplement 1A ) and was highly correlated with the mycorrhizal colonization rate as determined by the transcript abundances of classical arbuscular mycorrhizal symbiosis-marker genes ( fungal house-keeping gene , Ri-tub; plant marker genes , Vapyrin , RAM1 , STR1 and PT4; Park et al . , 2015; Figure 2—figure supplement 1B , Data Set 1 ) .", "Compounds 1 and 2 showed a similar AMF-specific accumulation in the leaves , as observed in the roots ( Figure 2D ) .", "The other analyzed blumenols were not detected in leaves ( Compounds 3 and 4; Figure 3—figure supplement 1A ) or showed a less consistent AMF-specific accumulation ( Compound 5; due to its constitutive background level; Figure 3—figure supplement 1A ) .", "The identity of Compounds 1 and 2 in the leaves was verified by high resolution qTOF-MS ( Figure 3—figure supplement 1B–E ) .", "Next , we determined the correlations between the contents of AMF-indicative foliar Compounds 1 and 2 and root colonization rates .", "In a kinetic experiment , the amount of both compounds steadily increased in the leaves of plants inoculated with R . irregularis ( Figure 3A , Figure 3—figure supplement 2 ) .", "At three wpi , the abundance of compounds 1 and 2 in the leaves was sufficient to reflect the colonization level of the roots .", "In contrast , the classical AMF-marker-genes , which are usually analyzed in the roots , did not respond in the leaves ( Figure 4 ) .", "In an inoculum-gradient experiment using increasing inoculum concentrations , proportionally higher Compound 1 and 2 levels were observed ( Figure 3B ) , accurately reflecting the differential colonization of roots across treatments ( Figure 3E ) .", "In addition to inoculation with a single AMF species ( R . irregularis ) , we also tested mycorrhizal inoculum originally collected from the plant’s native habitat , the Great Basin Desert in Utah , USA , which mainly consists of Funneliformis mosseae and R . irregularis .", "EV plants inoculated with this ‘natural inoculum’ also accumulated Compounds 1 and 2 in leaves , while irCCaMK plants did not ( Figure 3C ) .", "The analysis of a second independently transformed irCCaMK line confirmed the result that when the association with R . irregularis was genetically abrogated , Compounds 1 and 2 failed to accumulate in leaves of plants co-cultured with the AMF ( Figure 3—figure supplement 3 ) .", "When planted into the plant’s natural environment in Utah , both EV and irCCaMK plants could be clearly distinguished by their leaf Compound 1 and 2 contents .", "The signature of Compound 2 provided a better quality marker in these field-grown plants ( Figure 3D , Figure 3—figure supplement 4 ) .", "The foliar contents of these two compounds were highly correlated with the percentage of arbuscules in roots , the core structure of AMF interactions ( Figure 3F , Figure 3—figure supplement 2 ) .", "In contrast , other biotic or abiotic stresses , including herbivory , pathogen infection and drought stress , did not elicit the foliar accumulations of Compounds 1 and 2 ( Figure 5 ) .", "Such stimuli also do not induce blumenol accumulation in roots ( Maier et al . , 1997 ) .", "An analysis of various plant tissues , including different leaf positions , stem pieces , flowers and capsules revealed that these AMF-specific signatures accumulated throughout the shoot ( Figure 3G ) .", "Taken together , we conclude that the contents of particular blumenols in aerial plant parts robustly reflect the degree of mycorrhizal colonization in N . attenuata plants .", "Blumenols are apocarotenoids originating from a side branch of the carotenoid pathway ( Hou et al . , 2016 ) .", "Most of the candidate genes for blumenol biosynthesis were upregulated in roots , but not in leaves of N . attenuata plants in response to mycorrhizal colonization ( Figure 6A , Figure 6—figure supplement 1A ) .", "We inferred that these AMF-indicative leaf apocarotenoids are transported from their site of synthesis in colonized roots to other plant parts .", "This is consistent with the occurrence of blumenols in stem sap ( Figure 6—figure supplement 1B ) which was collected by centrifuging small stem pieces .", "To clarify the origins ( local biosynthesis vs . transport ) of these leaf blumenols , we genetically manipulated the carotenoid biosynthesis of N . attenuata plants .", "To minimize the effects of a disturbed carotenoid biosynthesis on the AMF-plant interaction , we used the dexamethasone ( DEX ) -inducible pOp6/LhGR system to silence phytoene desaturase ( PDS ) expression in a single DEX-treated leaf position ( Schäfer et al . , 2013 ) .", "Treated leaves showed clear signs of bleaching , indicating PDS silencing ( Figure 6B , Figure 6—figure supplement 1C ) , but levels of the AMF-indicative Compounds 1 and 2 were not affected , consistent with their transport from other tissues , likely the highly accumulating roots .", "As a control , we analyzed the non-AMF-inducible Compound 6 , showing constitutive levels in aerial tissues ( Figure 6—figure supplement 2 ) .", "In DEX-treated leaves , Compound 6 concentrations were reduced by nearly 40% , consistent with local production ( Figure 6B , Figure 6—figure supplement 1D ) .", "To confirm the within-plant transport potential of blumenols , we dipped roots of seedlings into aqueous solutions of Compounds 1 or 2 .", "After overnight incubation , the blumenol derivatives were clearly detected not only in roots , but also in shoots ( Figure 6C , Figure 6—figure supplement 1E ) .", "To test the potential of the foliar AMF-indicative metabolites as a screening tool , we quantified the concentration of Compounds 1 and 2 in leaves of plants of a population of recombinant inbred lines ( RILs ) of a forward genetics experiment , an experiment which would be challenging with the classical screening tools of root staining or nucleic acid analysis .", "We focused our analysis on Compound 2 due to the superior quality of its signature in the leaves of field-grown plants ( Figure 3—figure supplement 4 ) .", "The experiment consisted of a population of RILs from a cross of two N . attenuata accessions ( Utah , UT and Arizona , AZ ) ( Zhou et al . , 2017 ) which differ in their mycorrhizal colonization ( Figure 7A–B , Figure 7—figure supplement 1 ) and accumulation of foliar Compound 2 in the glasshouse ( Figure 7C ) .", "A QTL analysis of 728 plants grown across a 7200 m2 field plot ( Figure 7D ) revealed that the abundance of Compound 2 mapped to a single locus on linkage group 3 ( Figure 7E ) , which harbored a homologue of NOPE1 ( NIATv7_g02911 ) previously shown to be required for the initiation of AMF symbioses in maize and rice ( Nadal et al . , 2017 ) .", "Transcripts of NaNOPE1 were more abundant in AZ roots after AMF inoculation , but did not differ significantly in leaves ( Figure 7—figure supplement 1B ) .", "While clearly requiring additional follow-up work , these results highlight the value of these signature metabolites for HTP screens , which form the basis of most crop improvement programs .", "The AMF-specific accumulation of blumenol C-derivatives in roots is a widespread phenomenon within higher plants ( Strack and Fester , 2006 ) ; however , how general are the observed blumenol changes in aerial parts across different combinations of plants and AMF species ?", "We analyzed Solanum lycopersicum , Triticum aestivum and Hordeum vulgare plants with and without AMF inoculation and again we found an overlap in the AMF-specific blumenol responses in roots and leaves , consistent with the transport hypothesis .", "Further analyses led to the identification of additional AMF-indicative blumenols in the leaves of Medicago truncatula , Solanum tuberosum and Brachypodium distachyon .", "We identified various types of blumenols that showed an AMF-specific accumulation in the shoot , including blumenol B ( Compound 7 ) , which has not previously been reported in an AMF-dependent context ( Figure 7F; Figure 7—figure supplement 2 ) .", "As reported for roots , the particular blumenol types were species-dependent , but the general pattern was widespread across monocots and dicots in experiments conducted at different research facilities .", "In tests with diverse fungal species ( R . irregularis , F . mosseae and Glomus versiforme ) , the observed effects were not found to be restricted to specific AMF taxa ( Figure 7F; Figure 7—figure supplement 2 ) .", "In short , the method is robust ." ], [ "Metabolites and metabolite responses are often specific to particular parts and tissues of a plant ( Li et al . , 2016; Lee et al . , 2017 ) , but it is also known that local responses can spread to other plant parts .", "Additionally , metabolites do not only accumulate at their place of biosynthesis but can be readily transported throughout the plant ( Baldwin , 1989 ) .", "Therefore , we hypothesized that local changes in the roots might also be reflected in the systemic aerial tissues , either by signaling or transport .", "This allowed us to identify specific AMF-indicative blumenols in the shoot ( Figure 3 ) despite the occurrence of other highly abundant and constitutively produced compounds and blumenols that are not indicative of AMF-associations .", "Interestingly , the confirmation of compound identities in leaf samples with high resolution MS techniques proved to be challenging and required additional sample purification steps .", "Likely , such matrix effects thwarted the detection of these AMF-indicative , systemic blumenol responses in previous investigations .", "Under our conditions , AMF-indicative blumenols began mirroring the colonization rates of roots at around three wpi ( Figure 3—figure supplement 2 ) .", "Although microscopic methods can detect first signs of AMF colonization of the roots already after a few days ( Brundrett et al . , 1985 ) , the sensitivity of our method was found to be sufficient to analyze colonization rates observed in the glasshouse and , more importantly , in nature ( Figures 3 and 7 ) .", "The discovery of these AMF-indicative blumenol compounds in diverse plant species interacting with different AMF species ( Figure 7 , Figure 7—figure supplement 2 ) further indicates that these responses are widespread .", "AMF-induced blumenols in the roots have been shown to be quantifiable by various MS and photodiode array based detector setups .", "However , AMF-induced blumenols occur in many-fold lower amounts in the leaves compared to the roots ( e . g . , Compound 1 in N . attenuata approximately 1/10 ) and , at these low concentrations , their analysis is likely thwarted by complex matrix effects .", "Therefore , their analysis requires detection systems with advanced sensitivity and selectivity as is offered by state-of-the-art triple quadrupole technology and enhanced sample preparations ( e . g . , by solid-phase-extraction based purification and concentration ) which were used in the quantitative detection of leaf blumenols described here .", "In addition to the blumenols , mycorradicin , another biosynthetically related type of apocarotenoids , was reported to accumulate in AMF-colonized roots ( Klingner et al . , 1995 ) and it would be interesting to investigate if mycorradicin accumulates throughout the plant , as well .", "Despite the AMF-induced accumulation of blumenols in the shoot , putative candidate genes of the apocarotenoid biosynthesis pathway were only induced in the roots of AMF-inoculated plants ( Figure 6A , Figure 6—figure supplement 1A ) .", "To exclude other mechanisms ( e . g . , post-transcriptional regulation ) mediating the local production of the blumenol compounds in the leaves , we genetically manipulated the carotenoid pathway in a tissue-specific manner .", "It is challenging to manipulate blumenols without affecting the AMF-colonization of the plant , since other carotenoid-derived compounds , such as strigolactones , are known to play an important role in this process ( Lanfranco et al . , 2018 ) .", "To circumvent these problems , we used the LhGR/pOp6 system for chemically inducible RNAi-mediated gene silencing of PDS ( Schäfer et al . , 2013 ) to impair carotenoid biosynthesis only in a particular leaf of AMF-inoculated plants .", "Interestingly , only the constitutively produced Compound 6 was reduced in the treated leaves , while the AMF-indicative Compounds 1 and 2 were not affected by our treatment ( Figure 6B ) .", "This indicated that instead of being locally produced , Compound 1 and 2 are translocated from the roots , an inference consistent with the occurrence of AMF-indicative blumenols in stem sap and the capacity of seedlings to transport blumenols from the root to the shoot from hydroponic solution ( Figure 6C , Figure 6—figure supplement 1B , D ) .", "It seems likely that the AMF-indicative blumenols are transported in the xylem with the transpiration stream ( Figure 6D ) .", "The blumenol glucosides ( Compounds 1 , 2 and 6 ) are hydrophilic low-molecular weight ( 402 , 388 and 386 Da ) compounds that are unlikely to pass membranes without further support , e . g . , by transporters .", "It was recently demonstrated that ATP-binding cassette ( ABC ) transporters ( G-type ) are involved in the root-to-shoot transport of ABA , a phytohormone with a molecular structure related to blumenols , and these transporters resemble the function of other ABCG-type proteins reported to mediate the long-distance transport of cytokinins and strigolactones ( Borghi et al . , 2015 ) .", "Whether blumenols are transported by similar mechanisms remains an interesting question .", "Blumenols were shown to accumulate in large amounts in the roots of various plants after AMF-inoculation ( Strack and Fester , 2006 ) and our data indicate that they are subsequently distributed throughout the plant ( Figure 3G ) .", "While the conservation of this response in various plants after inoculation with different AMF species ( Figure 7F , Figure 7—figure supplement 2 ) indicates an important functional role in the AMF-plant interaction , this function remains to be explored .", "Unfortunately , the current knowledge of the biological activity of blumenols only vaguely indicates potential systemic functions of AMF-induced blumenols in shoot tissues .", "Activity studies on vomifoliol , the aglycone of the not AMF-indicative Compound 6 , showed that this compound induces stomatal closure similar to the structurally related abscisic acid ( Stuart and Coke , 1975 ) .", "Additionally , blumenols are known to suppress seed germination and plant growth ( Kato-Noguchi et al . , 2012; Kato-Noguchi et al . , 2015 ) .", "Therefore , AMF-induced blumenols could serve as systemic signals that mediate the large-scale adjustments in general physiology that are thought to accompany AMF-interactions .", "For example , AMF-induced blumenols could be involved in the regulation of differential susceptibility of AMF-inoculated plants to stresses , such as drought or pathogen infection .", "Even if classical tools for the quantification of AMF-plant interactions can offer superior sensitivity they are labor intensive and highly destructive which limits their application in studies that require high sample throughput , as well as in experiments that require repeated analysis of plants .", "We propose that the analysis of AMF-indicative blumenols in the shoot provides a convenient , easy-to-use , and minimally destructive tool to interrogate plant-AMF interactions in a HTP manner that allows for forward genetic studies even under field conditions ( Figure 7E ) and empowers plant breeding programs to produce mycorrhiza-responsive and P-efficient high-yielding lines ( van de Wiel et al . , 2016 ) .", "Currently , phosphate fertilizer is derived from phosphate rock , a non-renewable resource , which is predicted to be depleted soon ( Vaccari and Strigul , 2011 ) .", "By enabling breeding programs to select crop varieties which have negotiated AMF symbioses that deliver high yields with minimal P inputs , this discovery could help steer the ‘green revolution’ away from intense agricultural inputs and the collateral environmental damage they cause .", "While some of the ‘green revolution’ crop varieties with gibberellin response defects are potentially more efficient in Pi uptake as a result of their higher root colonization rates by AMF ( Floss et al . , 2013; Foo et al . , 2013 ) this serendipitous breeding event underscores the value of explicitly designing crop breeding programs to produce crops that negotiate more favorable AMF associations ." ], [ "For our experiments with Nicotiana attenuata ( Torr . ex S . Wats . ) , we used plants from the 31st inbred generation of the inbred ‘UT’ line , irCCaMK [A-09-1208-6 and A-09-1212-1 ( Groten et al . , 2015 ) plants that are stably silenced in CCaMK via RNAi , i-irPDS plants ( A-11-92−4 × A-11-325-4; Schäfer et al . , 2013 ) harboring the LhGR/pOp6 system for chemically-inducible RNAi-mediated gene silencing of phytoene desaturase ( PDS ) and the respective empty vector ( EV ) transformed plants ( A-04-266-3; Bubner et al . , 2006 ) as controls .", "Details about the transformation and screening of the irCCaMK plants are described by Groten et al . ( 2015 ) and for the i-irPDS plants by Schäfer et al . ( 2013 ) .", "Seeds were germinated on Gamborg B5 as described by Krügel et al . ( 2002 ) .", "The advance intercross recombinant inbred line ( RIL ) population was developed by crossing two N . attenuata inbred lines originating from accessions collected in Arizona ( AZ ) and Utah ( UT ) , USA ( Glawe et al . , 2003; Zhou et al . , 2017 ) .", "Additionally , we used Solanum lycopersicum ‘Moneymaker‘ , Hordeum vulgare ‘Elbany ‘and Triticum aestivum ‘Chinese Spring ‘plants .", "For glasshouse experiments , plants were treated according to Groten et al . ( 2015 ) .", "In brief , they were transferred into dead ( autoclaved twice at 121°C for 30 min; non-inoculated controls ) or living inoculum ( R . irregularis , Biomyc Vital , inoculated plants ) diluted 1:10 with expanded clay ( size: 2–4 mm ) .", "Pots were covered with a thin layer of sand .", "Plants were watered with distilled water for 7 d and subsequently fertilized every second day either with a full strength hydroponic solution ( for 1 L: 0 . 1292 g CaSO4 × 2H2O , 0 . 1232 g MgSO4 × 7H2O , 0 . 0479 g K2HPO4 , 0 . 0306 g KH2PO4 , 2 mL KNO3 ( 1 M ) , 0 . 5 mL micronutrients , 0 . 5 mL Fe diethylene triamine pentaacetic acid ) or with a low P hydroponics solution containing only 1/10 of the regular P-concentration ( 0 . 05 mM ) .", "Plants were grown separately in 1L pots , if not stated otherwise .", "In the paired design ( Figure 2 ) , irCCaMK plants were grown together with EV plants in 2L pots and the watering regime was changed to ¼ of the regular P-concentration after plants started to elongate .", "Glasshouse experiments with natural inoculum ( Figure 3C ) were conducted in a mesocosm system ( four boxes , each 2 pairs of EV and irCCaMK plants ) .", "Plants were maintained under standard glasshouse conditions ( 16 hr light , 24–28°C , and 8 hr dark , 20–24°C and 45–55% humidity ) with supplemental light supplied by high-pressure sodium lamps ( Son-T-Agro ) .", "The field experiments were conducted as described by Schuman et al . ( 2012 ) .", "Seedlings were transferred to Jiffy pots and planted into a field plot at the Lytle Ranch Preserve in the Great Basin Desert ( Utah , USA: N 37 . 1412 , W 114 . 0275 ) .", "Field season 2016 ( Figure 3D ) : field experiments were conducted under the US Department of Agriculture Animal and Plant Health Inspection Service ( APHIS ) import permission numbers 10-004-105m ( irCCaMK ) and 07-341-101n ( EV ) and the APHIS release permission number 16-013-102r .", "EV and irCCaMK plants were planted in communities of six plants , either of the same genotype or with both genotypes in equal number .", "During harvests , roots were washed and briefly dried with a paper towel .", "Subsequently , they were cut into 1 cm pieces and mixed .", "Plant tissues were shock-frozen in liquid nitrogen immediately after collection , ground to a fine powder and stored at −20°C ( short-term storage ) /−80°C ( long-term storage ) until extraction .", "From the root samples , an aliquot was stored in root storage solution ( 25% ethanol and 15% acetic acid in water ) at 4°C for microscopic analysis .", "For stem sap collection , branches of N . attenuata plants were cut into 1 . 5 cm long pieces and placed into small 0 . 5 mL reaction tubes with a small hole in the tip , which were placed in a larger 1 . 5 mL reaction tube .", "The tubes were centrifuged for 15 min at 10 000 × g .", "The stem sap from the larger reaction tubes was collected and stored at −20°C .", "Medicago truncatula ( Figure 7 and Figure 7—figure supplement", "2 ) and Brachypodium distachyon ( Figure 7 and Figure 7—figure supplement", "2 ) samples were prepared at the laboratory of Prof . Maria Harrison from the Boyce Thompson Institute for Plant Research ( Ithaca , NY , USA ) .", "M . truncatula plants were grown in a growth chamber with a 16 hr light ( 25°C ) /8 hr dark ( 22°C ) cycle .", "B . distachyon plants were grown in growth chamber with a 12 hr light ( 24°C ) /12 hr dark ( 22°C ) cycle .", "All experiments were carried out in surface sterilized containers , autoclaved growth substrates and with surface sterilized spores and seeds as described previously ( Liu et al . , 2004; Hong et al . , 2012; Floss et al . , 2013 ) .", "The growth substrates were mixtures of play sand ( average particle 200–300 μm ) , black sand ( heterogeneous particle size 50–300 μm ) and gravel ( heterogeneous particle size 300 μm −10 mm ) as outlined below .", "For M . truncatula , 2 d-old seedlings were planted into 20 . 5 cm cones ( Cone-tainers ) containing a 1:1 mixture of sterile black sand and gravel with 200 surface-sterilized G . versiforme spores placed on a layer of play sand positioned 4 cm below the top of the cones .", "Five seedlings were planted into each cone .", "Plants were fertilized twice weekly with 20 mL of modified 1/2-strength Hoagland’s solution ( Millner and Kitt , 1992 ) containing 100 µM potassium phosphate .", "Plants were harvested 49 d post planting and tissue frozen in liquid nitrogen and stored at −80 C . One cone containing five seedlings represents one biological replicate .", "The harvest date was 3/3/2015 .", "B . distachyon seedlings were planted into cones ( Cone-tainers ) containing a 2:1:1 mixture of black sand:play sand:gravel with 300 surface-sterilized G . versiforme spores placed on a layer of play sand positioned 4 cm below the top of the cones .", "Plants were fertilized twice weekly with 20 mL of modified 1/4-strength Hoagland’s solution ( Millner and Kitt , 1992 ) containing 20 µM potassium phosphate .", "Plants were harvested 35 d post planting and tissue frozen in liquid nitrogen and stored at −80 C . Each cone contained three plants and each biological replicate consisted of a pool of 4 cones .", "The harvest date was 6/20/2016 .", "S . lycopersicum ‘Moneymaker ‘ ( Figure 7—figure supplement", "2 ) and Solanum tuberosum ‘Wega’ ( Figure 7 ) samples were prepared at the laboratory of Prof . Philipp Franken by Dr . Michael Bitterlich from the Leibniz-Institute of Vegetable and Ornamental Crops ( Großbeeren/Erfurt Germany ) .", "S . lycopersicum were transplanted into 10 L open pots containing a sand/vermiculite mixture ( sand: grain size 0 . 2–1 mm; Euroquarz , Ottendorf-Okrilla , Germany , vermiculite: agra vermiculite , Pullrhenen , Rhenen , The Netherlands; 1:1 v:v ) and grown in the glass house from March to May ( 20-28:17 °C day:night , PAR: 300–2000 µmol m−2 s−1 ) .", "Mycorrhizal plants were inoculated with a commercial inoculum either containing R . irregularis DAOM 197198 ( INOQ , Schnega , Germany ) or F . mosseae BEG12 ( MycAgro Laboratory , Breteniere , France ) with 10% of the substrate volume and were harvested after 11 or 6 weeks , respectively .", "S . tuberosum tubers of similar size were planted into 3 L pots filled with the same substrate and grown in a growth cabinet ( 20:16 °C day:night , 16 hr light , 8 hr dark; PAR: 250–400 µmol m−2 s−1 , 50% rH ) .", "Mycorrhizal plants were inoculated with a commercial inoculum containing F . mosseae BEG12 ( MycAgro Laboratory , Breteniere , France ) with 10% of the substrate volume and were harvested after 6 weeks .", "Non-mycorrhizal counterparts were inoculated with the same amount of autoclaved ( 2 hr , 121°C ) inoculum and a filtrate .", "The filtrate was produced for every pot by filtration of 200 mL deionized water through Whatman filter ( particle retention 4–7 μm; GE Healthcare Europe GmbH , Freiburg , Germany ) containing approx .", "200 mL of inoculum .", "The same amount of deionized water ( 200 mL ) was added to mycorrhizal pots .", "Plants were irrigated every other day with 400–600 mL nutrient solution ( De Kreij et al . , 1997 ) ; 40% of full strength ) with 10% of the standard phosphate to guarantee good colonization ( N: 10 . 32 mM; P: 0 . 07 mM , K: 5 . 5 mM , Mg: 1 . 2 mM , S: 1 . 65 mM , Ca: 2 . 75 mM , Fe: 0 . 02 mM , pH: 6 . 2 , EC: 1 . 6 mS ) .", "For the experiment in the glasshouse , additional irrigation was carried out with deionized water until pot water capacity every other day .", "The pooled bulk leaf sample was dried at 60°C for 48 hr , ground to a fine powder and stored under dry conditions at room temperature until further analyses .", "Herbivory treatments were conducted by placing Manduca sexta neonates , originating from an in-house colony , on the plants .", "After feeding for two weeks , rosette leaves were harvested .", "As controls , we harvested leaves from untreated plants .", "For bacteria and virus infection , plants were inoculated with Agrobacterium tumefaciens carrying the Tobacco Rattle Virus .", "The inoculation was conducted by infiltrating leaves with a bacteria suspension using a syringe .", "The treatment was conducted as described for virus-induced gene silencing described by Ratcliff et al . ( 2001 ) and by Saedler and Baldwin ( 2004 ) .", "After incubation for three weeks , stem leaves of the treated plants and untreated control plants were harvested .", "The fungal infection was done with Botrytis cinerea .", "On each plant , three leaves were treated by applying six droplets , each containing 10 µL of B . cinerea spore suspension ( 106 spores mL−1 in Potato Extract Glucose Broth , Carl Roth GmbH ) , to the leaf surface .", "As control , plants were treated with broth without spores in the same way .", "Samples were collected after four days incubation .", "Drought stress was induced by stopping the watering for four days .", "Subsequently , stem leaves of the drought-stressed plants and the continuously watered control plants were harvested .", "In contrast to the other samples of the stress experiment , leaves were dried before analysis to compensate for weight differences caused by changes in the water content .", "For extraction , samples were aliquoted into reaction tubes , containing two steel balls .", "Weights were recorded for later normalization .", "Per 100 mg plant tissues ( 10 mg in case of dry material ) , approximately 1 mL 80% MeOH was added to the samples before being shaken in a GenoGrinder 2000 ( SPEX SamplePrep ) for 60 s at 1150 strokes min−1 .", "After centrifugation , the supernatant was collected and analyzed .", "For triple-quadrupole MS quantification , the extraction buffer was spiked with stable isotope-labeled abscisic acid ( D6-ABA , HPC Standards GmbH ) as an internal standard .", "Stem sap was diluted 1:1 with MeOH spiked with D6-ABA as an internal standard .", "After centrifugation , the supernatant was collected and analyzed .", "The purification of N . attenuata leaf extracts for high resolution qTOF-MS was conducted by solid-phase-extraction ( SPE ) using Chromabond HR-XC 45 µm benzensulfonic acid cation exchange columns ( Machery-Nagel ) to remove abundant constituents , such as nicotine and phenolamides .", "After purification the samples were evaporated to dryness and reconstituted in 80% methanol .", "Compound identification was conducted by NMR with purified fractions of root and leaf extracts .", "Compounds 1 , 3 and 4 were extracted from root tissues of N . attenuata and purified by HPLC ( Agilent-HPLC 1100 series; Grom-Sil 120 ODS-4 HE , C18 , 250 × 8 mm , 5 μm; equipped with a Gilson 206 Abimed fraction collector ) .", "Compounds 2 and 7 were extracted from a mixture of leaf tissues from different plant species ( M . truncatula , Z . mays , S . lycopersicum and N . attenuata ) .", "The first purification step was conducted by SPE using the Chromabond HR-XC 45 µm benzensulfonic acid cation exchange columns ( Machery-Nagel ) to remove hydrophilic and cationic constituents .", "Additional purification steps were conducted via HPLC ( Agilent-HPLC 1100 series; Phenomenex Luna C18 ( 2 ) , 250 × 10 mm , 5 µm; equipped with a Foxy Jr . sample collector ) and UHPLC ( Dionex UltiMate 3000; Thermo Acclaim RSLC 120 C18 , 150 × 2 . 1 mm , 2 . 2 µm; using the auto-sampler for fraction collection ) .", "For high resolution mass spectrometry ( MS ) , indiscriminant tandem mass spectrometry ( idMS/MS ) , tandem MS ( MS2 ) and pseudo-MS3 were used .", "Ultra-high performance liquid chromatography ( UHPLC ) was performed using a Dionex UltiMate 3000 rapid separation LC system ( Thermo Fisher ) , combined with a Thermo Fisher Acclaim RSLC 120 C18 , 150 × 2 . 1 mm , 2 . 2 µm column .", "The solvent composition changed from a high % A ( water with 0 . 1% acetonitrile and 0 . 05% formic acid ) in a linear gradient to a high % B ( acetonitrile with 0 . 05% formic acid ) followed by column equilibration steps and a return to starting conditions .", "The flow rate was 0 . 3 mL min-1 .", "MS detection was performed using a micrOTOF-Q II MS system ( Bruker Daltonics ) , equipped with an electrospray ionization ( ESI ) source operating in positive ion mode .", "ESI conditions for the micrOTOF-Q II system were end plate offset 500 V , capillary voltage 4500 V , capillary exit 130 V , dry temperature 180°C and a dry gas flow of 10 L min−1 .", "Mass calibration was performed using sodium formiate ( 250 mL isopropanol , 1 mL formic acid , 5 mL 1 M NaOH in 500 mL water ) .", "Data files were calibrated using the Bruker high-precision calibration algorithm .", "Instrument control , data acquisition and reprocessing were performed using HyStar 3 . 1 ( Bruker Daltonics ) .", "idMS/MS was conducted in order to gain structural information on the overall detectable metabolic profile .", "For this , samples were first analyzed by UHPLC-ESI/qTOF-MS using the single MS mode ( producing low levels of fragmentation that resulted from in-source fragmentation ) by scanning from m/z 50 to 1400 at a rate of 5000 scans s-1 .", "MS/MS analyses were conducted using nitrogen as collision gas and involved independent measurements at the following four different collision-induced dissociation ( CID ) voltages: 20 , 30 , 40 and 50 eV .", "The quadrupole was operated throughout the measurement with the largest mass isolation window , from m/z 50 to 1400 .", "Mass fragments were scanned between m/z 50 to 1400 at a rate of 5000 scans s-1 .", "For the idMS/MS assembly , we used a previously designed precursor-to-product assignment pipeline ( Li et al . , 2015; Li et al . , 2016 ) using the output results for processing with the R packages XCMS and CAMERA ( Data Set 2 ) .", "Additional MS/MS experiments were performed on the molecular ion at various CID voltages .", "For the fragmentation of the proposed aglycones via pseudo-MS3 , we applied a 60 eV in-source-CID transfer energy which produced spectra reflecting the loss of all sugar moieties .", "Purified fractions were completely dried with N2 gas and reconstituted with MeOH-d3 prior to analysis by nuclear magnetic resonance spectroscopy ( NMR ) .", "Structure elucidation was accomplished on an Avance III AV700 HD NMR spectrometer ( Bruker-Biospin , Karlsruhe , Germany ) at 298 K using a 1 . 7 mm TCI CryoProbeTM with standard pulse programs as implemented in Bruker TopSpin ( Version 3 . 2 ) .", "Chemical shift values ( δ ) are given relative to the residual solvent peaks at δH 3 . 31 and δC 49 . 05 , respectively .", "Carbon shifts were determined indirectly from 1H-13C HSQC and 1H-13C HMBC spectra .", "The data are shown in Table 1 and compared with previously published reference data ( Matsunami et al . , 2010 ) .", "Blumenol C glucoside and byzantionoside B differ only in the configuration of position C-9; blumenol C glucoside is ( 9S ) -configured whereas byzantionoside B has a ( 9R ) -configuration .", "Characteristic 13C-chemical shift differences can thus be found for the positions C-9 , C-10 and C-1’ .", "In byzantionoside , C-9 and C-10 were reported to have chemical shifts of δC 75 . 7 and δC 19 . 9 , respectively .", "In contrast , the chemical shifts for the same positions in blumenol C glucoside were reported to be lowfield shifted to δC 77 . 7 and δC 22 . 0 , respectively .", "Experimental chemical shifts of C-9 for the compounds identified in this publication were in the range from δC 77 . 2 to δC 78 . 2 , and for C-10 in the range from δC 21 . 6 to δC 21 . 9 , respectively .", "C-1’ of byzantionoside was reported to have a chemical shift of δC 102 . 3 , while for blumenol C glucoside the chemical shift was δC 104 . 1 .", "The experimental chemical shifts for C-1’ of the compounds of this publication are in the range from δC 103 . 8 to δC 104 . 1 .", "Hence the 13C-chemical shift data are completely consistent with the structures being blumenol C glucosides rather than byzantionoside B . More characteristic differences can be found in the 1H chemical shifts .", "The methylene shifts for H-7 of byzantionoside were reported to have chemical shifts of δH 1 . 50 and δH 1 . 98 while for blumenol C glucoside the same position showed chemical shifts of δH 1 . 67 and δH 1 . 81 .", "Experimental 1H chemical shifts for H-7 of the compounds 1–4 of this publication were found in the range of δH 1 . 62 to δH 1 . 69 and δH 1 . 80 to δH 1 . 88 , respectively .", "Consequently , the NMR data clearly establish the structures to be blumenol C derivatives and not byzantionosides .", "For chromatographic separations , a UHPLC ( Dionex UltiMate 3000 ) was used to provide a maximum of separation with short run times .", "This reduced the interference from other extract components ( matrix effects ) , increased the specificity of the method , and met the requirements of a HTP analysis .", "The auto-sampler was cooled to 10°C .", "As a stationary phase , we used a reversed phase column ( Agilent ZORBAX Eclipse XDB C18 , 50 × 3 . 0 mm , 1 . 8 µm ) suitable for the separation of moderately polar compounds .", "Column temperature was set to 42°C .", "As mobile phases , we used: A , 0 . 05% HCOOH , 0 . 1% ACN in H2O and B , MeOH , the composition of which was optimized for an efficient separation of blumenol-type compounds within a short run time .", "We included in the method a cleaning segment at 100% B and an equilibration segment allowing for reproducible results across large samples sets .", "The gradient program was as follows: 0–1 min , 10% B; 1–1 . 2 min , 10–35% B; 1 . 2–5 min , 35–50% B; 5–5 . 5 min , 50–100% B; 5 . 5–6 . 5 min , 100% B; 6 . 5–6 . 6 min , 100–10% B and 6 . 6–7 . 6 min , 10% B . The flow rate was set to 500 µL min−1 .", "Analysis was performed on a Bruker Elite EvoQ triple quadrupole MS equipped with a HESI ( heated electrospray ionization ) ion source .", "Source parameters were as follows: spray voltage ( + ) , 4500V; spray voltage ( - ) , 4500V; cone temperature , 350°C; cone gas flow , 35; heated probe temperature , 300°C; probe gas flow , 55 and nebulizer gas flow , 60 .", "Samples were analyzed in multiple-reaction-monitoring ( MRM ) mode; the settings are described in Table", "2 . The compound list was limited to the AMF-indicative markers in N . attenuata , Compound 1 and 2 , the not AMF-indicative Compound 6 and the internal standard ( D6-ABA ) .", "Accordingly , the gradient program was adjusted as follows: 0–1 min , 10% B; 1–1 . 2 min , 10–35% B; 1 . 2–3 min , 35–42% B; 3–3 . 4 min , 42–100% B; 3 . 4–4 . 4 min , 100% B; 4 . 4–4 . 5 min , 100–10% B and 4 . 5–5 . 5 min , 10% B . The MRM settings are given in Table", "3 . To determine the fungal colonization rates and mycorrhizal structures , root samples were stained and analyzed by microscopy .", "For WGA-Alexa Fluor 488 staining , roots were first washed with distilled water and then soaked in 50% ( v/v ) ethanol overnight .", "Roots were then boiled in a 10% ( w/v ) KOH solution for 10 min .", "After rinsing with water , the roots were boiled in 0 . 1 M HCl solution for 5 min .", "After rinsing with water and subsequently with 1x phosphate-buffered saline solution , roots were stained in 1x phosphate-buffered saline buffer containing 0 . 2 mg mL−1 WGA-Alexa Fluor 488 overnight in the dark .", "Zeiss confocal microscopy ( LSM 510 META ) was used to detect the WGA-Alexa Fluor 488 ( excitation/emission maxima at approximately 495/519 nm ) signal .", "Trypan blue staining was performed as described by Brundrett et al . ( 1984 ) to visualize mycorrhizal structures .", "For the counting of mycorrhizal colonization , 15 root fragments , each about 1 cm long , were stained with either trypan blue or WGA-488 followed by slide mounting .", "More than 150 view fields per slide were surveyed with 20x object magnification and classified into five groups: no colonization , only hyphae ( H ) , hyphae with arbuscules ( H + A ) , hyphae with vesicles ( V + H ) , and hyphae with arbuscules and vesicles ( A + V + H ) .", "The proportions of each group were calculated by numbers of each group divided by total views .", "For the molecular biological analysis of colonization rates , RNA was extracted from the roots using the RNeasy Plant Mini Kit ( Qiagen ) or NucleoSpin RNA Plant ( Macherey-Nagel ) according to the manufacturer’s instructions and cDNA was synthesized by reverse transcription using the PrimeScript RT-qPCR Kit ( TaKaRa ) .", "Quantitative ( q ) PCR was performed on a Stratagene Mx3005P qPCR machine using a SYBR Green containing reaction mix ( Eurogentec , qPCR Core kit for SYBR Green I No ROX ) .", "We analyzed the R . irregularis specific housekeeping gene , Ri-tub ( GenBank: EXX64097 . 1 ) , as well as the transcripts of the AMF-induced plant marker genes RAM1 , Vapyrin , STR1 and PT4 .", "The signal abundance was normalized to NaIF-5a ( NCBI Reference Sequence: XP_019246749 . 1 ) .", "The primer sequences are summarized in Table", "4 . The transcript analysis of the ( apo ) carotenoid pathway was conducted based on RNA-seq ( Data Set 3 ) by using N . attenuata roots with or without R . irregularis inoculations .", "The data analysis methods are based on the previously published pipeline of Ling et al . ( 2015 ) .", "Representative values for transcripts abundances are TPM ( Transcripts per kilobase of exon model per million mapped reads ) .", "To analyze the root-to-shoot transfer potential of blumenols , we placed three N . attenuata seedlings , previously germinated on petri dishes with GB5 Agar for approximately 10 days , into 0 . 5 mL reaction tubes .", "The roots were placed into the tube , while the shoot projected out of the tube .", "The tubes were carefully covered with parafilm , which held the seedlings in place and isolated roots from shoots ( see Figure 6C ) .", "The tubes were filled with tap water supplemented with 0 . 5% v/v plant extracts enriched in Compounds 1 or 2 ( unknown concentration; purified fractions ) , or a commercial available standard of Compound 6 ( 25 ng µL−1 end concentration; Roseoside; Wuhan ChemFaces Biochemical Co . , Ltd . ) .", "Compound 1 or 2 were prepared from a mix of leaf tissues from different plant species ( M . truncatula , Z . mays , S . lycopersicum and N . attenuata ) by methanol extraction followed by purification by SPE ( Chromabond HR-XC column ) and HPLC ( Agilent-HPLC 1100 series; Phenomenex Luna C18 ( 2 ) , 250 × 10 mm , 5 µm; equipped with a Foxy Jr . fraction collector ) .", "As a control , we used tap water supplemented with the respective amounts of MeOH .", "The seedlings were incubated for one day in a Percival climate chamber ( 16 hr of light at 28°C , and 8 hr of dark at 26°C ) .", "During sample collection , roots and shoots were separated and the roots were rinsed in water ( to reduce the surface contamination with the incubation medium ) .", "While the shoots were analyzed separately , the roots of all seedlings from the same treatment were pooled .", "Sample extraction was conducted as described above .", "For the temporal and spatial restriction of PDS gene silencing , we treated the petiole of the second oldest stem leaf of AMF-inoculated and non AMF-inoculated i-irPDS and EV plants with a 100 µM dexamethasone-containing lanolin paste ( 1% v/v DMSO ) .", "The lanolin paste was prepared and applied as described by Schäfer et al . ( 2013 ) .", "The treatment started three weeks after potting and was conducted for three weeks .", "The lanolin paste was refreshed twice per week .", "On each plant the treated leaf and the adjacent , untreated leaves were harvested for analysis .", "The field experiments for QTL analysis were conducted in 2017 .", "Collected leaf samples were extracted as described with 80% MeOH spiked with D6-ABA as internal standard and analyzed with the method described under ‘Method for targeted blumenol analysis in N . attenuata’ .", "The peak areas for Compound 2 were normalized by the amount of extracted tissue and internal standard and log-transformed .", "Samples with missing genotype or phenotype information were removed .", "In total , 728 samples were used for QTL mapping analysis .", "For quantitative trait loci ( QTL ) mapping , we used the AZ-UT RIL population and data analysis described by Zhou et al . ( 2017 ) .", "Statistical analysis of the data was performed with R version 3 . 0 . 3 ( http://www . R-project . org/ ) .", "The statistical methods used and the number of replicates are indicated in the figure legends ." ] ]

[ "High-through-put ( HTP ) screening for functional arbuscular mycorrhizal fungi ( AMF ) -associations is challenging because roots must be excavated and colonization evaluated by transcript analysis or microscopy .", "Here we show that specific leaf-metabolites provide broadly applicable accurate proxies of these associations , suitable for HTP-screens .", "With a combination of untargeted and targeted metabolomics , we show that shoot accumulations of hydroxy- and carboxyblumenol C-glucosides mirror root AMF-colonization in Nicotiana attenuata plants .", "Genetic/pharmacologic manipulations indicate that these AMF-indicative foliar blumenols are synthesized and transported from roots to shoots .", "These blumenol-derived foliar markers , found in many di- and monocotyledonous crop and model plants ( Solanum lycopersicum , Solanum tuberosum , Hordeum vulgare , Triticum aestivum , Medicago truncatula and Brachypodium distachyon ) , are not restricted to particular plant-AMF interactions , and are shown to be applicable for field-based QTL mapping of AMF-related genes ." ]

[ "All plants need a nutrient called phosphorus to grow and thrive .", "Phosphorus is found in soil , but the supply is limited so plants often struggle to acquire enough of it .", "To overcome this problem , many plants form friendly relationships ( or symbioses ) with certain fungi in the soil known as arbuscular mycorrhizal fungi .", "The fungi colonize plant roots and supply phosphorus and other nutrients in return for sugars and various molecules .", "Although many crop plants – including barley and potatoes – are able to form these symbioses , farmers commonly apply fertilizers containing phosphate and other nutrients to their fields to increase the amount of food they produce .", "Breeding new crop varieties that are better at forming symbioses with the fungi could reduce the need for fertilizers .", "However , the methods currently available to study these relationships are laborious and time-consuming , typically requiring samples of plant roots to be examined in a laboratory .", "Wang , Schäfer et al . used an approach called metabolomics to search for molecules in coyote tobacco plants that indicate the plants have formed symbioses with arbuscular mycorrhizal fungi .", "The experiments found that a group of molecules called blumenols accumulate in the roots and also in the shoots and leaves of plants with these symbioses , but not in the tobacco plants that were not able to associate with the fungi .", "Experiments in several other plant species including tomato , potato and barley produced similar findings , suggesting that the blumenols may be a useful and potentially universal indicator of symbioses between many different plants and fungi .", "Measuring the levels of blumenols in plant shoots and leaves is much quicker and easier than current methods of identifying fungal symbioses in plant root samples .", "Therefore , blumenols may be a useful tool for plant breeders who would like to screen large numbers of plants for these symbioses , and breed crops that negotiate better interactions with the beneficial fungi ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "neuroscience" ]

[ [ "Sensory attenuation – also known as self-suppression – refers to the phenomenon that self-generated sensations feel less salient , and evoke a smaller neurophysiological response , than externally-generated sensations which are physically identical ( Hughes et al . , 2013; Cardoso-Leite et al . , 2010 ) .", "Sensory attenuation is believed to result from the action of an internal forward model , or IFM ( Blakemore et al . , 2000a; Wolpert and Miall , 1996 ) .", "According to this account , the sensory consequences of self-generated movements are predicted based on a copy of the outgoing motor command , known as an efference copy .", "These predicted sensations are compared to the actual sensations resulting from the movement , and the difference between predicted and actual sensation ( i . e . , the sensory discrepancy – Wolpert and Miall , 1996 ) is sent higher up the neuronal hierarchy for further processing ( Seth and Friston , 2016 ) .", "In the case of a self-generated movement , the internal prediction is able to account for , and ‘explain away’ , much of the resulting sensation , which is why self-initiated sensations typically feel less salient , and evoke a smaller neurophysiological response , than externally-initiated sensations ( Blakemore et al . , 1998 ) .", "Sensory attenuation has been extensively studied in the context of overt speech production .", "Auditory stimuli elicit an electrophysiological brain response ( the auditory-evoked potential ) with a characteristic N1 component .", "The amplitude of this component is known to be sensitive to sound intensity; i . e . , loud sounds evoke larger N1 amplitudes than soft sounds ( Näätänen and Picton , 1987; Hegerl and Juckel , 1993 ) .", "Numerous electroencephalographic ( EEG ) and magnetoencephalographic ( MEG ) studies have found that self-generated vocalizations elicit an N1 component ( M100 in the MEG ) of smaller amplitude than the N1 component elicited when passively listening to the same sounds ( Ford et al . , 2007a; Curio et al . , 2000; Heinks-Maldonado et al . , 2005; Oestreich et al . , 2015; Houde et al . , 2002 ) .", "This phenomenon has been dubbed N1-suppression , and it suggests that self-generated sounds are processed as though they were physically softer than externally-generated sounds , reflecting the action of an IFM ( Greenlee et al . , 2011; Heinks-Maldonado et al . , 2006 ) .", "The suggestion that an IFM is responsible for sensory attenuation to overt speech is bolstered by the finding that experimentally altering auditory feedback ( e . g . , by pitch-shifting or delaying an individual’s voice , such that auditory sensations do not match the predictions of the IFM ) reduces the amount of N1-suppression ( Heinks-Maldonado et al . , 2006; Behroozmand and Larson , 2011; Behroozmand et al . , 2011; Aliu et al . , 2009 ) .", "The central aim of the present study is to explore whether N1-suppression , which has consistently been observed in response to overt speech , also occurs in response to inner speech , which is a purely mental action .", "Inner speech – also known as covert speech , imagined speech , or verbal thoughts – refers to the silent production of words in one’s mind ( Perrone-Bertolotti et al . , 2014; Alderson-Day and Fernyhough , 2015 ) .", "Inner speech is one of the most pervasive and ubiquitous of human activities; it has been estimated that most people spend at least a quarter of their lives engaged in inner speech ( Heavey and Hurlburt , 2008 ) .", "An influential account of inner speech suggests that it ultimately reflects a special case of overt speech in which the articulator organs ( e . g . , mouth , tongue , larynx ) do not actually move; that is , inner speech is conceptualized as ‘a kind of action’ ( Jones and Fernyhough , 2007 , p . 396 – see also Feinberg , 1978; Pickering and Garrod , 2013; Oppenheim and Dell , 2010 ) .", "Support for this idea has been provided by studies showing that inner speech activates similar brain regions to overt speech , including audition and language-related perceptual areas and supplementary motor areas , but does not typically activate primary motor cortex ( Palmer et al . , 2001; Zatorre et al . , 1996; Aleman et al . , 2005; Shuster and Lemieux , 2005 ) .", "While previous data suggest that inner and overt speech share neural generators , relatively few neurophysiological studies have explored the extent to which these two processes are functionally equivalent .", "If inner speech is indeed a special case of overt speech – ‘a kind of action’ – then it would also be expected to have an associated IFM ( Tian and Poeppel , 2010 – see also Feinberg , 1978; Numminen and Curio , 1999; Whitford et al . , 2012; Ford et al . , 2001a ) .", "A significant challenge in determining the existence ( or otherwise ) of an IFM to inner speech is that inner speech does not elicit a measurable auditory-evoked potential , which means that N1-suppression to inner speech cannot be determined directly using current methodologies .", "However , the existence of an IFM may be inferred based on how the production of inner speech suppresses the brain’s electrophysiological response to overt speech ( Tian and Poeppel , 2010; Tian and Poeppel , 2015; Tian and Poeppel , 2013; Tian and Poeppel , 2012 ) .", "A critical feature of the IFM associated with overt speech is that the efference copy is", "( a ) time-locked to the onset of the action , and", "( b ) contains specific predictions as to the expected sensory consequences of that action ( i . e . , is content-specific – Wolpert et al . , 1995 ) .", "Correspondingly , if inner speech were , in fact , associated with an IFM , then its associated efference copy would be expected to be:", "( a ) time-locked to the onset of the inner speech , and", "( b ) contain information as to the specific content of the inner speech .", "In this case , engaging in inner speech would be expected to result in maximal N1-suppression to overt speech in the case where two conditions were met: ( 1 ) the external sound was presented at precisely the same time as the inner speech was produced , and ( 2 ) the content of the external sound matched the content of the inner speech .", "The present study introduces a new experimental procedure that allowed us to test whether inner speech produces N1-suppression to audible speech in the absence of any overt motor action .", "In this protocol , participants were instructed to produce a single phoneme in inner speech at a specific time , which was designated by means of a precise visual cue .", "At the same time , an audible phoneme was presented in participants’ headphones; the audible phoneme could be either the same as ( Match condition ) or different from ( Mismatch condition ) the inner phoneme .", "In the Passive condition , participants were instructed not to produce an inner phoneme .", "The results indicated that inner speech resulted in N1-suppression , but only if the content of the inner phoneme matched the content of the audible phoneme .", "These results suggest that inner speech production is associated with a time-locked and content-specific internal forward model , similar to the one that operates in the production of overt speech .", "Furthermore , these results suggests that inner speech , by itself , is able to elicit an efference copy and cause sensory attenuation , even in the absence of an overt motor action ." ], [ "On each trial of the experiment , participants watched a short animation of approximately 5 s duration .", "As illustrated in Figure 1a , the animation depicted a red vertical line ( the ‘fixation’ line ) that remained in a fixed location in the middle of the screen .", "This fixation line was overlaid upon a thick green horizontal bar ( the ‘ticker tape’ ) .", "A second , green , vertical line ( the ‘trigger’ line ) , which was embedded in the ticker tape , was initially presented at the far right-hand side of the screen .", "At the start of each trial , participants fixated their eyes on the fixation line .", "After an interval of 1–2 s , the green ticker tape and the green trigger line began to move leftwards across the screen towards , and ultimately beyond , the stationary fixation line .", "At the exact time at which the trigger line intersected the fixation line – the ‘sound-time’ – an audible phoneme was delivered to participants’ headphones ( Figure 1c ) .", "The audible phoneme was a recording of a male speaker producing either the phoneme /BA/ or the phoneme /BI/ .", "( Note: for clarity , audible phonemes are capitalized in text while inner phonemes are written in lower case . Our justification for choosing these two audible phonemes in particular is presented below ) .", "Participants were instructed to generate an inner phoneme at exactly the moment the fixation line intersected the trigger line ( i . e . , at the sound-time ) .", "The experimental manipulation was the content of the inner phoneme that participants were instructed to produce .", "There were three different types of trial blocks .", "In the first type of trial block , participants were asked to produce the inner phoneme /ba/ at the sound-time .", "The second type of trial block was identical , except that participants were asked to produce the inner phoneme /bi/ .", "On each trial , participants were asked to imagine themselves moving their articulator organs ( i . e . , mouth , tongue , larynx , etc . ) and vocalizing the inner phoneme , but not to actually make any movements .", "In the third type of trial block , participants were not instructed to produce an inner phoneme , but were instructed to simply listen to the sounds .", "Following the sound-time , the trigger line continued to move for an additional 1 s before a text-box was displayed and participants were asked to rate how successfully they followed the instructions on the trial .", "The data were parsed into three discrete trial-types , which were analyzed as separate conditions: ( 1 ) Match trials , in which the inner phoneme matched the audible phoneme ( i . e . , inner phoneme: /ba/; audible phoneme: /BA/ OR inner phoneme: /bi/; audible phoneme: /BI/ ) , ( 2 ) Mismatch trials , in which the imagined phoneme did not match the audible phoneme ( i . e . , inner phoneme: /ba/; audible phoneme: /BI/ OR inner phoneme: /bi/; audible phoneme: /BA/ , and ( 3 ) Passive trials , in which the participant was not instructed to imagine a phoneme ( i . e . , inner phoneme: none; audible phoneme: /BA/ OR inner phoneme: none; audible phoneme: /BI/ ) .", "Following pre-processing ( see EEG Processing and Analysis for full details ) , EEG data epochs ( time-locked to the onset of the audible phoneme ) were averaged separately for each of the three conditions ( Match , Mismatch , Passive ) .", "The dependent variable was the amplitude of the N1 component of the auditory-evoked potential elicited by the auditory phoneme .", "The N1 peak was identified on each individual participant’s average waveform for each of the three conditions .", "Figure 2 shows the auditory-evoked potentials averaged across electrodes FCz , Fz , and Cz , as these were the electrodes at which N1 was maximal ( see Figure 2a , voltage maps ) .", "Figure 2b shows a box-and-whiskers plot of raw N1 amplitudes for each condition ( Match , Mismatch , Passive ) .", "Given that this experiment used a repeated measures design , Figure 2c shows a scatterplot of the magnitude of the within-subjects differences between conditions , which constitute the critical contrasts .", "These difference scores were approximately normally distributed with no clear outliers .", "Repeated measures ANOVA revealed a significant main effect of Condition ( F ( 2 , 82 ) = 4 . 21 , p = 0 . 018 , ηp2 = 0 . 09 ) on the amplitude of the N1 peak .", "Analysis of simple effects revealed that N1-amplitude in the Match condition was significantly smaller than both the Mismatch ( t ( 41 ) = 2 . 54 , p = 0 . 015 , dz = 0 . 39 , CI ( 95% ) = [0 . 187 , 1 . 649] ) and Passive ( t ( 41 ) = 2 . 77 , p = 0 . 008 , dz = 0 . 43 , CI ( 95% ) = [0 . 278 , 1 . 776] ) conditions ( Figure 2c ) .", "There was no difference in N1-amplitude between the Mismatch and Passive conditions ( t ( 41 ) = 0 . 26 , p = 0 . 800 , dz = 0 . 04 , CI ( 95% ) = [−0 . 758 , 0 . 977] ) .", "As can be seen in Figure 2 , while the topographies exhibited a fronto-central negativity in all three conditions , centered on electrode FCz , there was a hint of a leftward shift in the scalp distribution in the Match condition .", "Thus , in order to ensure the stability of the results , we performed a supplementary analysis with an expanded set of nine electrodes: specifically , Fz , FCz , Cz , F1 , FC1 , C1 , F2 , FC2 , and C2 .", "The pattern of results was identical to when the analysis was restricted to the three midline electrodes .", "Specifically , the main effect of Condition remained significant ( F ( 2 , 82 ) = 3 . 61 , p = 0 . 031 , ηp2 = 0 . 08 ) , as did the difference between the Match and Mismatch conditions ( t ( 41 ) = 2 . 35 , p = 0 . 024 , dz = 0 . 36 , CI ( 95% ) = [0 . 122 , 1 . 607] ) and the Match and Passive conditions ( t ( 41 ) = 2 . 57 , p = 0 . 014 , dz = 0 . 40 , CI ( 95% ) = [0 . 193 , 1 . 624] ) .", "The difference between the Mismatch and Passive conditions remained non-significant ( t ( 41 ) = 0 . 11 , p = 0 . 916 , dz = 0 . 02 , CI ( 95% ) = [−0 . 889 , 0 . 800] ) .", "The Condition × Electrode interaction was also not significant ( F ( 16 , 656 ) = 1 . 18 , p = 0 . 323 , ηp2 = 0 . 03 ) .", "There was also a main effect of Condition on the latency of the N1 peak ( F ( 2 , 82 ) = 5 . 03 , p = 0 . 016 , ηp2 = 0 . 11 .", "Analysis of the simple effects revealed that the N1-peak occurred significantly earlier in the Match condition compared to the Passive condition ( t ( 41 ) = 3 . 15 , p = 0 . 003 , dz = 0 . 49 , CI ( 95% ) = [−6 . 927 , –1 . 518] ) .", "There was no significant difference in N1-latency between the Mismatch and Passive conditions ( t ( 41 ) = 1 . 76 , p = 0 . 087 , dz = 0 . 27 , CI ( 95% ) = [−6 . 298 , 0 . 441] ) , nor between the Match and Mismatch conditions ( t ( 41 ) = 1 . 29 , p = 0 . 204 , dz = 0 . 20 , CI ( 95% ) = [−3 . 316 , 0 . 729] ) .", "A visual inspection of Figure 2 also suggested between-condition differences in the P2 ( 150–190 ms ) and P3 ( 250–310 ms ) components of the auditory-evoked potential .", "While these components were not directly relevant to our hypotheses , for completeness the data and analyses for these components are presented below .", "The P2 component occurred around 150–190 ms post-sound ( see Figure 3a ) , while the P3 component occurred around 250–310 ms post-sound ( see Figure 4a ) .", "However , not all three conditions generated a distinct peak for the P2 and P3 components .", "Specifically , the Match and Mismatch conditions did not elicit a distinct P2 , whereas the Passive condition did not exhibit a distinct P3 .", "This meant that ( unlike for analysis of the N1 ) it was not possible to use a peak-detection approach for these components .", "Instead , time-windows were identified for the P2 ( 150–190 ms ) and P3 ( 250–310 ms ) components , and the average voltage within these time-windows were analyzed ( see EEG Processing and Analysis for more detail ) .", "Figure 3a shows the average waveforms for the P2 component , averaged across the Cz , FCz , and CPz electrodes , and Figure 3B shows a box-and-whiskers plot of raw P2 amplitudes for each condition .", "One-way ANOVA revealed a main effect of Condition ( F ( 2 , 82 ) = 6 . 60 , p = 0 . 006 , ηp2 = 0 . 14 ) .", "Analysis of the simple effects revealed that amplitude of the P2 component was significantly smaller in the Mismatch condition , relative to both the Match ( t ( 41 ) = 3 . 54 , p = 0 . 001 , dz = 0 . 55 , CI ( 95% ) = [0 . 555 , 2 . 028] ) and Passive ( t ( 41 ) = 3 . 21 , p = 0 . 003 , dz = 0 . 50 , CI ( 95% ) = [−3 . 549 , –0 . 810] ) conditions ( Figure 3C ) .", "The difference between the Match and Passive conditions was not significant ( t ( 41 ) = 1 . 26 , p = 0 . 216 , dz = 0 . 19 , CI ( 95% ) = [−0 . 540 , 2 . 316] ) .", "Figure 4a shows the average waveforms for the P3 component , averaged across the CPz , Cz , and Pz electrodes , and Figure 4B shows a box-and-whiskers plot of raw P3 amplitudes for each condition .", "ANOVA revealed a main effect of Condition ( F ( 2 , 82 ) = 5 . 86 , p = 0 . 004 , ηp2 = 0 . 13 ) .", "Analysis of the simple effects revealed that the amplitude of the P3 component was significantly larger in the Match condition relative to both the Mismatch ( t ( 41 ) = 2 . 23 , p = 0 . 032 , dz = 0 . 34 , CI ( 95% ) = [0 . 117 , 2 . 433] ) and Passive ( t ( 41 ) = 3 . 26 , p = 0 . 002 , dz = 0 . 50 , CI ( 95% ) = [0 . 813 , 3 . 444] ) conditions ( Figure 4c ) .", "There was no significant difference between the Passive and Mismatch conditions ( t ( 41 ) = 1 . 31 , p = 0 . 196 , dz = 0 . 20 , CI ( 95% ) = [−0 . 458 , 2 . 165] ) .", "To provide a point of reference with the inner speech experiment , we also conducted a follow-up ‘overt speech’ experiment .", "The overt speech experiment had an identical experimental procedure to the inner speech experiment , except that participants were instructed to overtly – as opposed to covertly – vocalize the phonemes at the sound-time .", "Just as in the inner speech experiment , an audible phoneme ( i . e . , /BA/ or /BI/ ) was delivered to participants’ headphones at the sound-time .", "Participants were instructed to vocalize the overt phonemes softly , so as to minimize the amount of bone conduction of the sound to the ear .", "An additional ‘Motor-Control’ condition was also included in which participants overtly vocalized the phonemes at the sound-time , but no audible phoneme was delivered .", "The purpose of this condition was to allow us to identify and correct for the electrophysiological activity generated by the motor act of producing the overt phoneme per se .", "This was done by subtracting participants’ activity in the motor-only condition from their waveforms in the active conditions ( i . e . , Match and Mismatch ) , as is common in sensory attenuation studies which compare motor-active and motor-passive conditions ( Ford et al . , 2014 ) .", "The order of the conditions was randomized for each participant , with the caveat that the Motor-Control condition was always run last .", "Thirty individuals participated in the overt speech experiment .", "Participants’ mean age was 25 . 0 years ( SD = 6 . 0 ) and 20 were female .", "Thirty-three participants were originally recruited for the study , however three participants generated ≤60 usable epochs in one or more conditions ( based on the exclusion criteria described for the inner speech experiment ) and were excluded from further analysis .", "Figure 5 shows the auditory-evoked potentials averaged across electrodes FCz , Fz , and Cz for the uncorrected ( Figure 5a ) and motor-corrected data ( Figure 5b ) .", "Voltage-maps are presented for the motor-corrected data .", "Raw N1 amplitudes for each motor-corrected condition are presented as box-and-whiskers plots in Figure 5c , and Figure 5d shows scatterplots of the ( within-subjects ) difference scores between the conditions .", "For the uncorrected data , repeated-measures ANOVA revealed a significant main effect of Condition ( F ( 2 , 58 ) = 10 . 99 , p < 0 . 001 , ηp2 = 0 . 28 ) on the amplitude of the N1 peak .", "Critically , analysis of simple effects revealed that N1-amplitude in the Match condition was significantly smaller than the Mismatch condition ( t ( 29 ) = 2 . 61 , p = 0 . 014 , dz = 0 . 48 , CI ( 95% ) = [0 . 472 , 3 . 897] ) , consistent with the results of the inner speech experiment .", "However , contrary to the results of the inner speech experiment , N1-amplitude in the Passive condition was significantly smaller than both the Match ( t ( 29 ) = 2 . 63 , p = 0 . 013 , dz = 0 . 48 , CI ( 95% ) = [0 . 646 , 5 . 137] ) and Mismatch ( t ( 29 ) = 3 . 97 , p < 0 . 001 , dz = 0 . 72 , CI ( 95% ) = [2 . 461 , 7 . 691] ) conditions in the overt speech experiment ( see Figure 5a ) .", "The pattern of results was identical for the motor-corrected data .", "Repeated-measures ANOVA revealed a significant main effect of Condition ( F ( 2 , 58 ) = 8 . 43 , p = 0 . 001 , ηp2 = 0 . 23 ) .", "Analysis of simple effects revealed that N1-amplitude in the Match condition was significantly smaller than the Mismatch condition ( t ( 29 ) = 2 . 46 , p = 0 . 020 , dz = 0 . 45 , CI ( 95% ) = [0 . 384 , 4 . 190] ) , and that N1-amplitude in the Passive condition was significantly smaller than both the Match ( t ( 29 ) = 2 . 20 , p = 0 . 036 , dz = 0 . 40 , CI ( 95% ) = [0 . 150 , 4 . 243] ) and Mismatch ( t ( 29 ) = 3 . 43 , p = 0 . 002 , dz = 0 . 63 , CI ( 95% ) = [1 . 808 , 7 . 159] ) conditions ( see Figure 5b , c and d ) .", "In order to select which two audible phonemes would be presented to participants in the inner and overt speech experiments , we presented nine phonemes to 10 participants ( age = 18 . 7 years , SD = 1 . 1; seven female ) while they listened passively .", "Each phoneme was presented 90 times , and the presentation order was randomized .", "The nine phonemes were: /BA/ , /BI/ , /DA/ , /DI/ , /GA/ , /KI/ , /PA/ , /PI/ , and /TI/ .", "Each phoneme was ~200 ms in duration , presented at ~70 dB SPL , and was produced by the same male speaker .", "Waveforms showing the auditory-evoked potentials elicited by the nine phonemes are presented in Figure 6 .", "The waveforms are shown collapsed across electrodes FCz , Cz , and Fz .", "Of the nine different phonemes , /BA/ and /BI/were judged to be most similar in terms of their amplitude and overall shape , and hence these phonemes were chosen for use as the audible phonemes in both the inner and overt speech experiments ." ], [ "The present study used a novel experimental protocol to demonstrate that the production of an inner phoneme resulted in sensory attenuation of the auditory-evoked potential elicited by a simultaneously-presented audible phoneme , in the absence of any overt motor action .", "Crucially , the production of inner speech did not result in equal sensory attenuation to all sounds; sensory attenuation was dependent on the content of the inner phoneme matching the content of the audible phoneme .", "These results suggest that inner speech production is associated with a time-locked and content-specific internal forward model , similar to the one believed to operate in the production of overt speech ( Hickok , 2012; Tourville and Guenther , 2011; Hickok and Poeppel , 2004; Houde et al . , 2013 ) .", "In short , the results of this study suggest that inner speech alone is able to elicit an efference copy and cause sensory attenuation of audible sounds .", "The key finding of the present study was that the production of inner speech , by itself , led to N1-suppression to an audible sound .", "N1-suppression has been reported many times previously in response to overt speech ( Ford et al . , 2007a; Curio et al . , 2000; Heinks-Maldonado et al . , 2005; Oestreich et al . , 2015; Houde et al . , 2002 ) .", "There is strong evidence that the mechanistic basis of N1-suppression to overt speech involves an efference-copy mediated IFM ( Eliades and Wang , 2003; Crapse and Sommer , 2008; Rauschecker and Scott , 2009 ) .", "It thus seems reasonable to assume that a similar mechanism underlies sensory attenuation in the case of inner speech .", "The idea that inner speech shares mechanistic features with overt speech is consistent with the conceptualization of inner speech ‘as a kind of action’ ( Jones and Fernyhough , 2007 , p . 396 ) and , more generally , with Hughlings Jackson’s belief that thinking is merely our most complex motor act: ‘sensori-motor processes… form the anatomical strata of mental states’ ( Hughlings Jackson , 1958 , p . 49 ) .", "In the words of Oppenheim and Dell ( 2010 , p . 1158 ) , ‘inner speech cannot be independent of the movements that a person would use to express it’ .", "It was notable that inner speech production resulted in N1-suppression if – and only if – the content of the inner phoneme matched the content of the audible phoneme; that is , only in the Match condition .", "That said , we note that comparisons between the Match/Mismatch conditions on the one hand , and the Passive condition on the other , should be treated with a degree of caution .", "This is because data for these conditions came from different trial blocks , and ( most importantly ) differed in terms of the task that participants were required to perform ( i . e . , produce a covert/overt phoneme at the precise sound-time , versus passively listen to the audible phoneme ) .", "Notwithstanding the fact that the N1-suppression effect has previously been found to be robust to variations in attention ( Timm et al . , 2013; Saupe et al . , 2013 ) and trial structure ( Baess et al . , 2011 ) , this nevertheless raises the possibility that differences in participants’ attention or task-preparation may have contributed to the observed differences between the ‘active’ conditions and the passive condition .", "We note that this limitation is not restricted to the current study – it potentially applies to any procedure that attempts to measure sensory suppression by comparing active and passive conditions , which constitutes the vast majority of studies that have examined sensory suppression to overt speech .", "Critically , however , this limitation does not apply to the key contrast between the Match and Mismatch conditions .", "This is because data for these conditions came from the same trial blocks , in which participants were required to perform exactly the same task on each trial .", "For example , in blocks in which participants were required to produce the inner phoneme /ba/ , they experienced both Match trials ( in which the audible phoneme was /BA/ ) and Mismatch trials ( in which the audible phoneme was /BI/ ) , and there was no way for participants to predict whether the current trial would be a Match or Mismatch trial prior to the onset of the audible phoneme .", "Hence attention , task-preparation , etc . , during the pre-stimulus period could not differ systematically between Match and Mismatch trials .", "Our factorial design also ensured that the differences between the Match and Mismatch conditions were not driven by differences in the inner phoneme ( i . e . , /ba/ vs /bi/ ) or differences in the audible phoneme ( i . e . , /BA/ vs /BI/ ) .", "Consequently , we can conclude with confidence that the observed difference in N1 amplitude between Match and Mismatch trials reflects the impact of participants’ inner speech on their sensory response to the audible phoneme .", "It is this contrast that demonstrates that inner speech , like overt speech , is associated with a precise , content-specific efference copy .", "The results of the inner speech experiment mirror those of previous studies which have found sensory attenuation to overt speech to be reduced or eliminated if auditory feedback deviates from what would normally be expected; e . g . , by pitch-shifting the feedback or providing foreign-language feedback ( Heinks-Maldonado et al . , 2006; Behroozmand and Larson , 2011; Behroozmand et al . , 2011; Behroozmand et al . , 2016; Larson and Robin , 2016 ) .", "To confirm this pattern using the current procedure , we performed a supplementary experiment in which participants were required to overtly vocalize the phoneme at the sound-time .", "The key result from the overt speech experiment was that N1-amplitude elicited by an audible phoneme was significantly smaller when participants simultaneously produced the same phoneme in their overt speech ( Match condition ) , compared to when they produced a different phoneme in their overt speech ( Mismatch condition ) .", "This result , which is consistent with numerous previous studies in the sensory attenuation literature ( Heinks-Maldonado et al . , 2006; Behroozmand and Larson , 2011; Behroozmand et al . , 2011; Behroozmand et al . , 2016; Larson and Robin , 2016 ) , was identical to the corresponding contrast in the inner speech experiment , in which participants had to produce phonemes in inner , as opposed to overt , speech .", "A second notable finding of the overt speech experiment was that N1-amplitude was significantly smaller in the Passive condition compared to both active conditions ( i . e . , Match and Mismatch ) .", "A potential ‘low-level’ explanation for this result lies in the fact that participants were required to make an overt motor action in the overt speech experiment but not the inner speech experiment .", "While the marked difference in pre-stimulus activity between the active and passive conditions ( Figure 5a ) is consistent with this explanation , the fact that between-condition differences remained when correcting for the motor-associated activity ( Figure 5b ) stands against this possibility ( though see Horváth , 2015; Sams et al . , 2005 for a discussion of the challenges associated with motor correction when comparing active and passive conditions in studies of sensory attenuation ) .", "An alternative explanation for why N1 was smallest in the Passive condition is based on the idea that the auditory N1-component is ( in addition to sound intensity , discussed previously ) also sensitive to stimulus predictability , with predictable sounds evoking a smaller N1 than unpredictable sounds ( Behroozmand and Larson , 2011; Bäss et al . , 2008; Lange , 2011 ) .", "Our task differed from other willed vocalization tasks in the literature in that the audible phoneme delivered to the headphones was:", "( a ) of a different person’s voice , and", "( b ) much louder than the actual vocalization , as participants were instructed to vocalize quietly in order to minimize bone conduction .", "In other words , a substantial discrepancy between the predicted and actual sound existed , even in the Match condition .", "This sensory discrepancy was even larger in the Mismatch condition , as the content of the sound was also different .", "Consistent with the idea that N1-amplitude is sensitive to stimulus predictability , it is possible that the larger N1-amplitude in the active compared to the passive conditions was due to prediction-errors as to the expected quality of the audible phoneme .", "It is further possible that such detailed predictions as to phoneme quality do not occur in the context of inner speech ( a suggestion for which there is some empirical support – Oppenheim and Dell , 2008 ) , which may account for why N1-amplitude was not reduced in the passive condition in the inner speech experiment .", "In summary , both the inner speech and overt speech experiments showed the same basic pattern of results with respect to the key contrast: N1-magnitude was smaller if the phoneme generated by the participant ( either covertly or overtly ) matched the audible phoneme than if it mismatched .", "These findings suggest that inner speech – like overt speech – is associated with a precise , content-specific efference copy , as opposed to a generic and non-specific prediction .", "Taken together , our results provide support for the contention that inner speech is a special case of overt speech , which does not have an associated motor act .", "The notion that inner speech generates an IFM in the absence of an overt motor act has been hypothesized previously across several different literatures ( Jones and Fernyhough , 2007; Feinberg , 1978; Scott , 2013; Guenther and Hickok , 2015; Ford et al . , 2001b; Sams et al . , 2005; Kauramäki et al . , 2010 ) .", "However , this hypothesis has been notoriously difficult to test empirically , due to the covert nature of inner speech .", "Ford et al . , ( Ford and Mathalon , 2004 ) played participants repeated sentences over 30 s and asked them to reproduce the same sentences in inner speech .", "Ford et al . , found that the sentences elicited a smaller N1-component when participants engaged in inner speech compared to when they did not , consistent with the results of the present study .", "More recently , Tian and Poeppel ( Tian and Poeppel , 2010 ) used MEG to show that the auditory cortex was activated immediately following production of an inner phoneme in the absence of auditory feedback , which they took as evidence of an inner-speech-initiated efference copy .", "In a subsequent study , which was a strong influence on our own , Tian and Poeppel ( Tian and Poeppel , 2013 ) asked participants to produce an inner phoneme within a 2 . 4 s window .", "This window was followed by an audible phoneme that could either match or mismatch the content of the inner phoneme .", "The authors found no difference in the amplitude of the M100 between the match and mismatch conditions , inconsistent with the results of the present study .", "However , given the width of the temporal window in which participants were asked to produce their inner phoneme ( 2 . 4 s ) , the efference copy and auditory feedback would not necessarily be expected to coincide under these conditions , in which case M100-suppression would not be expected to occur .", "Tian and Poeppel ( Tian and Poeppel , 2015 ) asked participants to signal their production of an inner phoneme via a button-press , and measured the amplitude of the M100 component evoked by a pre-recorded audible phoneme of their own voice which matched the content of the inner phoneme , but which could be pitch-shifted or delayed .", "They found evidence of M100 suppression to unshifted , undelayed audible phonemes relative to a passive baseline condition , consistent with the results of the present study .", "Pitch-shifting or delaying the auditory phonemes was found to increase M100 amplitude above baseline levels .", "While this study’s design enabled the timing of the inner phoneme to be precisely specified , the fact that it was specified by means of an overt motor action ( i . e . , a button-press ) , which is known to be associated with N1-suppression per se ( Hughes et al . , 2013 ) , raises the possibility of the motor action and inner speech being confounded .", "Finally , Ylinen et al . , ( Ylinen et al . , 2015 ) asked participants to mentally rehearse tri-syllabic pseudowords in inner speech .", "After several mental repetitions , an audible pseudoword was played which had either matching or mismatching beginnings and endings to the rehearsed pseudoword .", "The results revealed that audible syllables that were concordant with participants’ inner speech elicited less MEG activity than discordant syllables , a result which is broadly consistent with the results of the present study .", "The current experiment holds some methodological advantages over previous designs .", "Firstly , the experimental stimuli ( animation , audible phonemes , rating-scale , etc . ) were physically identical across all conditions , as was the nature of participants’ task ( i . e . , to fixate on the screen and produce an inner phoneme at a designated time ) .", "The only thing that differed between the different trial-types was the inner phoneme that participants were asked to produce .", "This meant that the observed differences in sensory attenuation could not have been due to any physical differences between the conditions ( Luck , 2005 ) .", "Secondly , the fact that it was impossible to predict which of the two audible phonemes would be presented on any given trial meant that it was impossible to distinguish Match from Mismatch trials a priori .", "This meant that the observed results could not have been due to between-condition differences in , for example , demand characteristics .", "Thirdly , the ‘ticker tape’ feature of the current protocol enabled participants to very accurately time-lock the onset of their inner phoneme to match the onset of the external sound .", "In the current protocol , the position of the trigger line refreshed every 8 . 3 ms , which presumably enabled participants to time the onset of their inner phoneme far more accurately than would be possible with a countdown sequence , mental rehearsal or open temporal window , such as have been used in previous studies ( Tian and Poeppel , 2013; Ford et al . , 2001b; Ylinen et al . , 2015 ) .", "Finally , the current protocol did not require participants to make an active movement to signal the onset of their inner phoneme , such as by pressing a button .", "This is a significant advantage over previous studies which have employed a motor condition to signal the onset of covert actions , as it avoids the potential confound associated with having temporally-overlapping auditory-evoked and motor-evoked potentials – see Horvath ( Horváth , 2015 ) and Neszmélyi and Horvath ( Neszmélyi and Horváth , 2017 ) for a discussion of the challenges associated with ‘correcting’ for motor activity in studies of sensory attenuation .", "In light of its methodological features , the present study provides arguably the strongest evidence to date that inner speech results in sensory attenuation of the N1-component of the auditory-evoked potential , even in the absence of an overt motor response .", "Perhaps the most important strength of this paradigm is that all the above issues were controlled within a single task , thereby removing any reliance on cross-experimental inferences .", "This study’s focus on the N1 component is consistent with the majority of the existing literature on electrophysiological sensory attenuation .", "The rationale for focusing on N1 lies in the fact that the amplitude of this component is volume dependent; that is , other things being equal , loud sounds evoke N1-components of larger amplitude than do soft sounds ( Näätänen and Picton , 1987; Hegerl and Juckel , 1993 ) .", "In prior studies of N1-suppression , participants have typically generated sounds through overt actions such as overt speech , button-presses etc .", "The observation of N1-suppression in such studies thus implies that the brain processes self-generated sounds as though they were physically softer than identical external sounds .", "The N1-suppression demonstrated in the present study extends this idea by suggesting that the brain also processes imagined sounds as though they were physically softer than identical , unimagined sounds .", "In addition to providing evidence that inner speech is associated with an IFM of similar nature to overt speech , this finding provides evidence that mental state influences perception at a fundamental level ( Gregory , 1997 ) .", "With regards to the question of what mechanism could underlie sensory attenuation to inner speech: a recent study by Niziolek , Nararajan and Houde ( Niziolek et al . , 2013 ) on sensory attenuation in the context of overt speech production found that the degree of sensory attenuation was stronger when participants produced vowel sounds that were closer ( in terms of their acoustic properties ) to their median production of these sounds , compared to when they produced vowel sounds that were , for them , less typical .", "These results suggests that the efference copy associated with overt speech production represents a sensory goal ( i . e . , ‘a prototypical production at the center of a vowel’s formant distribution’ , p . 16115 ) , and that the distance ( in formant space ) of any given utterance from this ‘sensory prototype’ determines its degree of sensory attenuation .", "If inner speech is , in fact , a special case of overt speech ( as we have suggested above ) , then this raises the question of the nature of the sensory goal in the context of inner speech production .", "One possibility , based on the results of Niziolek et al . , is that in the present study ( in ‘imagine /ba/’ trials , for example ) the sensory goal was of a prototypical /ba/ , which was presumably covertly ‘spoken’ in the participant’s own voice ( though see below for a discussion of the validity of this assumption ) .", "In this case , the participant’s prototypical /ba/ would never match perfectly with the audible phoneme , as the audible phoneme would never be the participant’s own voice .", "The fact that the present study observed N1-suppression in the Match condition but not the Mismatch condition is nevertheless consistent with the Niziolek et al . , 2013 account , in that the distance , in formant space , between an inner /ba/ and an audible /BA/ would be smaller than the distance between an inner /bi/ and an audible /BA/ , even though the inner phoneme did not match the audible phoneme perfectly in either case ( as the audible phoneme was always produced by the same unknown speaker ) .", "The fact that while Niziolek et al . , 2013 observed maximal levels of sensory attenuation to prototypical vowel sounds , they still observed significant ( i . e . , non-zero ) levels of sensory attenuation to atypical vowel sounds is also consistent with this idea .", "A prediction of this account is that participants should show even greater levels of sensory attenuation in the Match condition if the audible phoneme is presented in their own voice rather than the voice of an unknown stranger; testing this prediction may be a worthwhile endeavor in future studies .", "In regards to the assumption that the sensory goals of inner speech are the same as overt speech , and that a person’s inner voice is the same as their actual voice , there is some evidence in support of this conjecture: Filik and Barber ( 2011 ) provided evidence that people produce inner speech in the same regional accent as their overt speech .", "However , other studies have reported evidence suggesting that inner speech has impoverished acoustic properties relative to overt speech ( Oppenheim and Dell , 2008 ) .", "It is also possible that inner speech can consist of several distinct ‘voices’ , with each having specific auditory properties; the fact that people with auditory-verbal hallucinations often report hearing multiple voices with different auditory properties ( McCarthy-Jones et al . , 2014 ) is consistent with this idea , if – as discussed further below – auditory-verbal hallucinations ultimately reflect inner speech being misperceived as overt speech .", "Finally , it is also possible that the acoustic properties of inner speech are not fixed .", "Specifically , in the context of the present study , it is possible that the acoustic properties of the audible phonemes began to influence the inner phonemes , such that after numerous repetitions , participants began to imagine themselves producing an inner phoneme with the acoustic properties of the audible phoneme .", "Testing these possibilities may also be worthwhile in future studies .", "While the primary focus of the paper was on the N1-component of the auditory-evoked potential , between-condition differences were also observed in the amplitude of the P2 and P3 components ( see Figures 3 and 4 ) .", "A likely explanation for the observed results in these later components involves another ERP component , the N2 , whose spatial and temporal distribution typically overlaps with that of the P2 ( Griffiths et al . , 2016 ) .", "The N2 and P3 components are among the most heavily investigated components in the ERP literature ( Näätänen and Picton , 1986; Polich , 2007 ) , and are typically elicited by tasks – such as the auditory oddball and Go/NoGo tasks – in which the participant is asked to identify ( by means of a button-press , for example ) ‘target’ stimuli which are nested among ‘non-target’ stimuli ( Smith et al . , 2010; Spencer et al . , 1999 ) .", "Critically , the N2 and P3 can also be elicited by tasks in which a mental response is required , such as when target stimuli have to be mentally counted as opposed to signaled with a button-press ( Mertens and Polich , 1997 ) .", "We suggest that , in the two inner speech conditions of our study , participants made a mental response – possibly a ‘template-matching response’ along the lines of whether the audible phoneme matched their inner phoneme ( Griffiths et al . , 2016 ) – which they did not make in the Passive condition .", "In this case , the audible phoneme in the inner speech conditions might be expected to elicit an N2 and a P3 component , which would not be present in the Passive condition .", "The occurrence of a ( negative-going ) N2 in the inner speech condition would then interact and compete with the expression of a ( positive-going ) P2 component elicited by the audible phoneme .", "The result would be the absence of a distinct P2 , but presence of a P3 , in the inner speech conditions but not the Passive condition – as observed empirically .", "It is also worth noting that Tian and Poeppel ( Tian and Poeppel , 2013 ) observed a larger M200 component in their match vs . mismatch comparison , consistent with the enhanced P2 observed in the Match vs . Mismatch comparison in the present study .", "Taken together , these results suggest that the M200/P2 component may index something other than a sensory prediction , possibly involving a cognitive ‘template matching’ process .", "The implied existence of an efference copy to inner speech holds important implications for how to best understand some of the psychotic symptoms associated with schizophrenia .", "Some of the most characteristic of these symptoms seem to reflect the patient misattributing , to external agents , self-generated motor actions ( e . g . , delusions of control ) and self-generated thoughts ( e . g . , delusions of thought insertion , auditory-verbal hallucinations – Feinberg , 1978; Frith , 1987 ) .", "An influential account of these experiences argues that they arise because of an abnormality in the IFM associated with both physical and mental actions ( Feinberg , 1978; Frith , 1992 ) .", "This IFM abnormality leads to an inability to predict and suppress the consequences of self-generated actions , which leads to confusion as to their origins .", "This hypothesis has a strong theoretical foundation: for example , the distinctive symptom of thought echo , in which the patient hears their own thoughts spoken out loud by an external voice , can be well explained as the patient’s own inner speech being misattributed and misperceived as an external voice ( Frith , 1992 ) .", "However , while numerous studies have provided empirical evidence showing that schizophrenia patients exhibit subnormal levels of sensory attenuation to their own physical actions ( Whitford et al . , 2011; Blakemore et al . , 2000b; Shergill et al . , 2005 ) , including subnormal levels of N1-suppression to overt speech ( Ford et al . , 2001a; Ford et al . , 2007b; Ford et al . , 2001c ) , there is little empirical evidence that schizophrenia patients show sensory attenuation deficits to self-generated mental actions such as inner speech .", "Furthermore , the few studies that did report sensory attenuation deficits to inner speech in patients with schizophrenia did not include a ‘mismatch’ condition , raising the possibility that these sensory attenuation deficits ultimately reflect attentional deficits in the patient group ( Ford and Mathalon , 2004; Ford et al . , 2001d ) .", "We suggest that the failure to identify electrophysiological sensory attenuation deficits to inner speech in schizophrenia patients is not because the deficits do not exist , but rather because previous experimental protocols have been insufficiently sensitive to detect them .", "In order to maximize the chances of detecting sensory attenuation deficits to inner speech in schizophrenia patients , we suggest that future experiments should:", "( a ) ensure that the onset of inner speech is precisely time-locked to the audible sound , without reverting to using a willed action ( e . g . , a button-press ) to signal inner speech-onset , as this could potentially lead to the auditory and motor responses being confounded;", "( b ) limit the content of inner speech to phonemes rather than entire sentences as this enables the onset and content of the inner speech to be more tightly controlled;", "( c ) investigate patients exhibiting those symptoms that seem to most clearly reflect misperceived inner speech ( e . g . , thought echo , other auditory-verbal hallucinations ) , rather than grouping patients with different symptom profiles into a clinically-heterogeneous ‘schizophrenia’ group .", "By providing an optimized protocol for quantifying N1-suppression to inner speech , our hope is that the present study can provide a methodological framework for identifying and assessing sensory attenuation deficits in inner speech in patients with schizophrenia .", "In conclusion , the present study demonstrated that engaging in inner speech resulted in sensory attenuation ( specifically , N1-suppression ) of the electroencephalographic activity evoked by an audible phoneme , but only if the content of inner speech matched the content of the audible phoneme .", "These results suggest that inner speech evokes an efference-copy-mediated IFM , which is both content-specific and time-locked to the onset of inner speech , which is consistent with the existing literature on sensory attenuation to overt speech .", "Cumulatively , this implies that inner speech may ultimately be ‘a kind of action’ , and a special case of overt speech , as long suggested by prominent models of language .", "Accordingly , these findings not only provide insight into the nature of inner speech , but also provide an experimental framework for investigating sensory attenuation deficits in inner speech , such as have been proposed to underlie some psychotic symptoms in patients with schizophrenia ." ], [ "Forty-two healthy individuals participated in the inner speech experiment .", "Participants’ mean age was 23 . 4 years ( SD = 7 . 3 ) and 24 were female .", "Fifty participants were originally recruited for the study , however eight participants generated ≤ 60 usable epochs in one or more conditions and were excluded from further analysis – see EEG Processing and Analysis for further details .", "The study was conducted at the University of New South Wales ( UNSW Sydney; Sydney , Australia ) , and approved by the UNSW Human Research Ethics Advisory Panel ( Psychology ) .", "Participants were seated in a quiet , dimly-lit room , approximately 60 cm from a computer monitor ( BenQ XL2420T , 1920 × 1080 pixels , 144 Hz ) , and were fitted with headphones ( AKG K77 Perception ) and an EEG recording cap .", "EEG was recorded with a BioSemi ActiveTwo system from 64 Ag/AgCl active electrodes placed according to the extended 10–20 system .", "A vertical electro-oculogram was calculated by recording from an electrode placed below the left eye , and subtracting its activity from that of electrode FP1; a horizontal EOG was recorded by placing an electrode on the outer canthus of each eye .", "We also placed an electrode on the tip of the nose , on the left and right mastoid , and on the masseter muscle to detect jaw movements .", "During data acquisition , the reference was composed of CMS and DRL sites , and the sampling rate was 2048 Hz .", "In regards to the animation that participants viewed on each experimental trial: the ticker tape moved at a constant velocity of 6 . 5°/s , which meant that it took 3 . 75 s until the trigger line intersected the fixation line .", "The ticker tape was marked with labels ‘3’ , ‘2’ and ‘1’ that passed the fixation line 3 s , 2 s , and 1 s prior to the trigger line ( see Figure 1b ) .", "The two audible phonemes , /BA/ and /BI/ , were selected on the basis of a pilot study which indicated that amongst nine candidate audible phonemes ( /BA/ , /BI/ , /DA/ , /DI/ , /GA/ , /KI/ , /PA/ , /PI/ , /TI/ ) , the two audible phonemes /BA/ and /BI/ produced auditory-evoked potentials that were most similar in terms of their amplitude and overall shape ( see Figure 6 ) .", "The two audible phonemes were produced by the same male speaker , and were similar in terms of their loudness ( ~70 dB SPL ) and duration ( ~200 ms ) .", "There were 60 trials in each trial block .", "Participants were instructed to fix their gaze on the fixation line on every trial .", "At the start of each block , participants were told that on every trial of that block they should produce a particular inner phoneme ( either /ba/ or /bi/ ) at the exact moment the trigger line interested the fixation line , or ( in Passive blocks ) that they should simply listen to the audible sound and not try to imagine anything .", "Each audible phoneme ( /BA/ and /BI/ ) was presented on 50% of trials within each trial block , and the order was randomized for each participant .", "This meant that , in those blocks in which participants were instructed to generate a particular inner phoneme ( i . e . , active blocks ) , on half of trials their inner phoneme matched the audible phoneme , while on half of trials it mismatched .", "Following each trial , participants were asked to rate their success in imagining the instructed inner phoneme at the sound-time ( or in not imagining anything in the Passive condition ) .", "These ratings were made on a scale from 1 ( ‘Not at all successful’ ) to 5 ( ‘Completely successful’ ) , and were reported using the computer keypad .", "Participants’ average ratings were 4 . 09 out of 5 ( SD = 0 . 65 ) for Match trials , 4 . 01 ( SD = 0 . 67 ) for Mismatch trials , and 4 . 87 ( SD = 0 . 40 ) for Passive trials .", "The order of the trial blocks ( imagine /ba/ , imagine /bi/ , or passive ) was randomized for each participant .", "Each block took approximately 7 min to complete , and was repeated twice over the course of the experiment .", "Stimulus presentation was controlled by MATLAB ( MathWorks , Natick , MA ) , using Psychophysics Toolbox extensions ( Brainard , 1997; Kleiner et al . , 2007 ) .", "The data pre-processing and analysis was performed in BrainVision Analyzer ( Brain Products GmbH , Munich , Germany ) .", "The EEG data were re-referenced offline to the nose electrode .", "Data were first notch filtered ( 50 Hz ) to remove mains artefact , and then band-pass filtered from 0 . 1 to 30 Hz using a phase-shift free Butterworth filter ( 48 dB/Oct slope ) .", "The filtered data were separated into 800 ms epochs ( 200 ms prior to sound onset , 600 ms post-onset ) , and baseline corrected to the mean voltage from –100 to 0 ms . The epochs were corrected for eye-movement artefacts , using the technique described in ( Gratton et al . , 1983 ) , and any epoch with a signal exceeding a peak-to-peak amplitude of 200 µV for any channel was excluded .", "To ensure data quality , epochs were classified as unusable and excluded prior to analysis if they failed to meet the above criterion , or if the participant rated their success on the trial as ≤2 out of 5 .", "The remaining usable epochs were included in the analysis and used to make the average waveforms for the three conditions .", "There were an average of 103 . 1 ( SD = 14 . 8 ) usable epochs in the Match condition ( of a max . possible 120 ) , 97 . 0 ( SD = 18 . 3 ) in the Mismatch condition , and 107 . 2 ( SD = 17 . 6 ) in the Passive condition .", "The amplitude of the N1-component of the auditory-evoked potential was the primary dependent variable .", "The N1 peak was identified on each participant’s average waveform ( Whitford et al . , 2011; Ford et al . , 2007b ) as the most negative local minimum in the window 25–175 ms post-stimulus onset .", "The auditory N1-component typically has a fronto-central topography ( Näätänen and Picton , 1987 ) , which was verified in the current data: N1 was maximal at electrode FCz ( see Figure 2a ) .", "Supplementary analyses were also performed on the P2 and P3 components in the inner speech experiment .", "As it was not possible to use a peak-detection approach for these components ( as not all conditions exhibited a clear P2 and P3 peak ) , time-windows were identified for the P2 ( 150–190 ms ) and P3 ( 250–310 ms ) components .", "Average voltage within these time-windows was the dependent variable in these supplementary analyses .", "Data were analyzed using repeated-measures ANOVA , with one factor Condition ( three levels: Match , Mismatch and Passive ) .", "In the case of a main effect of Condition , contrasts were used to unpack the simple effects .", "The Greenhouse-Geisser correction was used in the case of a violation in the assumption of sphericity .", "N1 was maximal at electrode FCz for all three conditions , however in order to improve the reliability of the analysis , the data was averaged across FCz and neighboring electrodes Fz and Cz ( Näätänen and Picton , 1987; Woods , 1995 ) .", "All of the relevant statistics remained significant when analysis was restricted to electrode FCz .", "With regards to the supplementary analyses on the P2 and P3 components: the P2 component ( 150–190 ms ) was maximal at electrode Cz; the data were collapsed across Cz and neighboring electrodes FCz and CPz for the statistical analysis .", "The P3 component ( 250–310 ms ) was maximal at electrode CPz; the data were collapsed across CPz and neighboring electrodes Cz and Pz for the statistical analysis .", "Due to the novelty of the paradigm , it was not possible to obtain precise estimates of the expected effect size .", "Thus we powered our study to detect a small effect size , based on the heuristic provided by ( Cohen , 1969 ) ; our sample size of 42 provided adequate power ( β = 0 . 8 ) to detect a small effect size ( ηp2 = 0 . 04 ) at α = 0 . 05 .", "The power analysis was conducted with G*Power software ( version 3 . 1 . 9 . 2; Faul et al . , 2007 ) .", "Each experiment was performed only once ." ] ]

[ "Efference copies refer to internal duplicates of movement-producing neural signals .", "Their primary function is to predict , and often suppress , the sensory consequences of willed movements .", "Efference copies have been almost exclusively investigated in the context of overt movements .", "The current electrophysiological study employed a novel design to show that inner speech – the silent production of words in one’s mind – is also associated with an efference copy .", "Participants produced an inner phoneme at a precisely specified time , at which an audible phoneme was concurrently presented .", "The production of the inner phoneme resulted in electrophysiological suppression , but only if the content of the inner phoneme matched the content of the audible phoneme .", "These results demonstrate that inner speech – a purely mental action – is associated with an efference copy with detailed auditory properties .", "These findings suggest that inner speech may ultimately reflect a special type of overt speech ." ]

[ "As you read this text , the chances are you can hear your own inner voice narrating the words .", "You may hear your inner voice again when silently considering what to have for lunch , or imagining how a phone conversation this afternoon will play out .", "Estimates suggest that we spend at least a quarter of our lives listening to our own inner speech .", "But to what extent does the brain distinguish between inner speech and the sounds we produce when we speak out loud ?", "Listening to a recording of your own voice activates the brain more than hearing yourself speak out loud .", "This is because when the brain sends instructions to the lips , tongue , and vocal cords telling them to move , it also makes a copy of these instructions .", "This is known as an efference copy , and it enables regions of the brain that process sounds to predict what they are about to hear .", "When the actual sounds match those predicted – as when you hear yourself speak out loud – the brain’s sound-processing regions dampen down their responses .", "But does the inner speech in our heads also generate an efference copy ?", "To find out , Whitford et al . tracked the brain activity of healthy volunteers as they listened to speech sounds through headphones .", "While listening to the sounds , the volunteers had to produce either the same speech sound or a different speech sound inside their heads .", "A specific type of brain activity decreased whenever the inner speech sound matched the external speech sound .", "This decrease did not occur when the two sounds were different .", "This suggests that the brain produces an efference copy for inner speech similar to that for external speech .", "These findings could ultimately benefit people who suffer from psychotic symptoms , for example as part of schizophrenia .", "Symptoms such as hearing voices are thought to reflect problems with producing and interpreting inner speech .", "The technique that Whitford et al . have developed will enable us to test this long-held but hitherto untestable idea .", "The results should increase our understanding of these symptoms and may eventually lead to new treatments ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "neuroscience" ]

[ [ "A major goal of neuroscience is explaining how nervous systems generate and organize behavior .", "This requires describing behavior in terms that can be correlated with neural activity .", "The dynamics of brain activity can be observed in whole brains at single-cell resolution ( Ahrens et al . , 2013; Ardiel et al . , 2017; Cong et al . , 2017; Lemon et al . , 2015; Nguyen et al . , 2016; Pulver et al . , 2015 ) , but behavioral dynamics has not been captured at a similar level of detail ( Datta et al . , 2019 ) .", "While progress in fine-mapping natural behavior , or ‘computational ethology’ ( Anderson and Perona , 2014 ) , has benefited from recent advances in visual tracking ( Johnson et al . , 2020 ) , 3D imaging ( Hong et al . , 2015 ) , machine vision ( Dankert et al . , 2009 ) , machine learning ( Kabra et al . , 2013; Machado et al . , 2015 ) , and image feature extraction ( Berman et al . , 2014; Mathis et al . , 2018; Wiltschko et al . , 2015 ) , the primary focus of these efforts has been kinematic , seeking to define anatomical movements at higher resolution .", "While movements are the essential components of behavior , they are complex products of motor neuron activity , which must balance the contractions of agonist and antagonist muscles and must also promote anatomical rigidity that supports movement .", "Here , we bridge the gap between motor neuron activity and movement by describing muscle activity at the single-cell level .", "Comprehensively monitoring muscle activity in behaving animals is achievable with genetically encoded Ca++ indicators and has been demonstrated at single-cell resolution in hydra ( Szymanski and Yuste , 2019 ) , roundworms ( Ardiel et al . , 2017 ) , and larval fruit flies ( Heckscher et al . , 2012; Zarin et al . , 2019 ) .", "However , the application of muscle Ca++ imaging to characterize more complex sequences has been constrained by the challenge of tracking behavior in freely moving animals .", "This problem is resolved in pupal fruit flies where behavior is restricted to the puparium ( Kim et al . , 2006 ) , which can be clarified for optical access to the confined animal .", "The pupa maintains a fixed position and orientation during movement , and all behavior is executed within a delimited field of view .", "Pupal Ca++ activity imaging of muscles has been previously demonstrated using GCaMP6s by Diao et al . , 2017 , who showed that the bulk Ca++ signal collected over ventral muscles exhibits temporal patterns that conform well to known body wall movements .", "The behavioral hallmark of pupal development is the Drosophila pupal ecdysis sequence , which is one of a general class of behavioral sequences used by insects to molt ( Truman , 2005; Zitnan and Adams , 2012 ) .", "These sequences typically divide into three principal phases during which the animal first loosens and then sheds its old exoskeleton before expanding its newly secreted one .", "Ecdysis sequences are strongly dependent on the action of hormones for their initiation and progression , and together with vertebrate reproductive behaviors , they have long served as a useful model of hormone-behavior interactions .", "Neural control of ecdysis behaviors is typically exercised by a conserved complement of hormones including eclosion hormone , ecdysis triggering hormone ( ETH ) , crustacean cardioactive peptide ( CCAP ) , and Bursicon ( Ewer and Reynolds , 2002; White and Ewer , 2014 ) .", "The sites of action of these hormones within the nervous system have been principally studied in Drosophila , but detailed neuroethological studies have been undertaken in larger insects , such as the locust , Schistocerca gregaria ( Hughes , 1980a; Hughes , 1980b; Hughes , 1980c ) , and cricket , Teleogryllus oceanicus ( Carlson , 1977; Carlson and Bentley , 1977 ) .", "Motor program organization of the adult ecdysis sequences in these insects is quite stereotyped , suggesting central control of motor execution , but sensory feedback can also modulate behavioral performance .", "While characterization of central nervous system ( CNS ) activity underlying ecdysis sequences has remained limited in larger insects , progress has been made in Drosophila where a fictive ecdysis sequence can be elicited in an excised pupal brain by exposure to ETH ( Diao et al . , 2017; Kim et al . , 2015; Kim et al . , 2006; Mena et al . , 2016 ) .", "Fictive activity imaged using Ca++ indicators grossly correlates with the motor patterns executed during pupal ecdysis , but comprehensively interpreting CNS activity remains a challenge .", "The Drosophila pupal ecdysis sequence occurs at the onset of metamorphosis ( Figure 1A ) and has been adapted to initiate transformation of the body plan ( Kim et al . , 2006 ) .", "Although it occurs in the context of the pupal molt and has three principal phases characterized by distinct motor programs , its function in molting is limited to casting off the cuticular linings of the gut and trachea .", "Its primary function is , instead , to create internal pressure at points along the body to evert adult parts such as the head , legs , and wings ( Denlinger and Zdarek , 1994 ) .", "ETH initiates the pupal ecdysis sequence after a long period of behavioral quiescence and targets neurons that express its two receptor isoforms , ETHRA and ETHRB ( Kim et al . , 2006 ) .", "Neurons expressing ETHRB are largely dispensable during larval life , but are essential for pupal ecdysis ( Diao et al . , 2016 ) .", "Neurons expressing ETHRA include the neuroendocrine cells that secrete CCAP and Bursicon .", "These cells are likewise essential at pupal , but not larval , ecdysis ( Clark et al . , 2008; Park et al . , 2003 ) .", "They become active approximately 10 min after the onset of pupal ecdysis , and their activation mediates the transition to the next behavioral phase .", "Understanding how the nervous system transforms such hormonal signals into temporally ordered behavioral sequences will require a more complete description of the motor neuron activity that dictates the behavioral sequences themselves .", "Here , we use body-wide fluorescence imaging from the dorsal , lateral , and ventral views to characterize the pupal ecdysis sequence at single-cell resolution .", "Using improved imaging methods—including a new pan-muscle LexA driver that permits dual imaging of muscle and neuron activity—we identify novel elements of the pupal ecdysis sequence , including previously undescribed movements and a phase of stochastic muscle activity preceding ecdysis .", "We find that muscle activity exhibits a high degree of variability , with individual muscles recruited stochastically into repeating small ensembles , which we call syllables .", "Syllable activity is synchronized over anatomical compartments to form movements , which are sufficiently stereotyped to be learned by a convolutional neural network ( CNN; for code see https://github . com/BenjaminHWhite/muscle_activity; copy archived at swh:1:rev:66456f6ff61e8faa9fe4b442b91ef3fce3b178f9 , White , 2021 ) .", "We can prevent synchronization at specific motor program transitions by suppressing neuromodulatory neurons , which is lethal .", "The suppression of proprioceptive neurons , which blocks initiation of pupal ecdysis , is also unexpectedly lethal .", "Overall , our analysis at single-cell resolution reveals a dynamical system in which movements are not rigidly specified but form from variable components subject to neuromodulatory reorganization ." ], [ "Previous characterization of pupal ecdysis has distinguished three principal phases ( Diao et al . , 2017; Kim et al . , 2006; Figure 1A–C , Video 1 ) .", "The first phase ( P1 ) consists of sustained longitudinal compression ( ‘lifting’ ) of posterior abdominal segments accompanied by unilateral , anteriorly propagating , ‘rolling contractions’ of the dorsal body wall that alternate left-to-right .", "The second ( P2 ) features left-to-right alternating lateral ‘swinging’ movements formed by unilateral , anteriorly directed contractions , while the third ( P3 ) consists of alternating left-right posteriorly directed contractions that change into bilaterally symmetric , backward peristaltic contractions .", "These phases always proceed in the same order .", "The movements increase internal pressure to evert the head ( i . e . , force it out of the body cavity ) and push the developing legs and wings to the body surface and elongate them ( Zdarek and Friedman , 1986 ) .", "Phalloidin labeling demonstrates that approximately half of larval muscles persist until pupal ecdysis , and all retain innervation by Ib synapses ( Prokop , 2006; Figure 1D , E ) .", "The most prominent loss of muscles occurs in the ventral and posterior compartments .", "Only 5 of 13 larval ventral muscles survive ( Figure 1F vs . G ) , and one of these ( M12 ) is absent in posterior segments ( Supplementary file 1 ) .", "Also missing from posterior segments are muscles M4 and M5 .", "All five dorsal longitudinal muscles , except M4 , are present in all segments , as are five of the six lateral transverse muscles .", "This was consistent across 17 animals , indicating that pupal ecdysis is executed by a standard set of persistent larval muscles .", "Although Drosophila behavior has been recorded at single-cell resolution , the drivers used to make such recordings express weakly at the pupal stage and cannot be used with Gal4 drivers targeting other cells such as neurons .", "We identified a striated muscle-specific gene , l ( 2 ) 01289 ( aka CG9432 ) , that expresses robustly from early larval stages through adulthood , which we call hulk ( hlk ) .", "We generated a Trojan exon insertion in the hlk gene that co-expresses LexA:QF and combined it with the Ca++ biosensor LexAop-GCaMP6s to report muscle activity .", "With this hlk-LexA>LexAop-GCaMP6s line ( i . e . , hlk>GCaMP6s ) , we imaged muscle Ca++ activity through the clarified puparium for approximately 90 min to capture pupal ecdysis behavior , in vivo ( see Materials and methods; Figure 2 , Figure 2—figure supplements 1 and 2 ) .", "The muscle activity patterns of animals imaged from the dorsal side match known behaviors , such as the bilateral posterior ‘Lifts’ and left-right alternating ‘rolling contraction’ ( RollCon ) movements of P1 ( Figure 2A , Video 2 ) .", "Temporal patterns of bulk Ca++ activity differentiated phases P1 , P2 , and P3 ( Figure 2B ) .", "Traces show phase-specific oscillations of varying amplitude and frequency , with individual oscillations conforming to bouts of movement ( see Materials and methods ) .", "Consistent with Diao et al . , 2017 , the alternating left-right oscillations of P1 persisted through P2 and P3 , with coordinated bilateral activity becoming dominant only in P3 .", "Bulk Ca++ activity imaged from the lateral side showed the same three phases ( Figure 2C ) , and oscillations reflected bouts that included identifiable movements ( Figure 2D ) .", "The Lift is performed by most muscles across the dorsal-ventral ( D-V ) axis in the posterior segments , while RollCons typically lack activity in the ventral longitudinal muscles 12 , 13 , and 15 ( Video 3 ) .", "Posterior-to-anterior ( P-to-A ) waves of activity in P1 reverse direction after head eversion in P2 ( Figure 2E ) , as previously shown ( Kim et al . , 2006 ) .", "These data confirm and refine previous observations and demonstrate that the hlk>GCaMP6s line accurately reports the ecdysis sequence behaviors .", "Ca++ imaging revealed a phase of random muscle activity prior to the onset of pupal ecdysis ( Figure 2B , C ) , which we call Phase 0 ( P0 ) .", "It begins approximately 3 hr before P1 and divides into distinct bouts of muscle activation ( Figure 3A , Video 4 ) .", "Individual muscle length changes during P0 are small ( ≤25% ) compared to P1–P3 ( 25–40%; Figure 3—figure supplement 1A ) and coincide with small body wall twitches rather than coherent movements .", "Such twitches are also observed during embryonic motor development and are initially myogenic , but later become neurogenic ( Crisp et al . , 2008; Crisp et al . , 2011 ) .", "To determine if random P0 muscle activation is myogenic or neurogenic , we created a dual-reporter fly line with hlk-LexA driving expression of the red fluorescent Ca++ biosensor jRGECO in the muscle and VGlut-Gal4 driving a Synaptotagmin-GCaMP6s fusion protein ( Syt-GCaMP6s ) in motor neurons , where it localizes to the neuromuscular junction ( NMJ , Figure 3B ) .", "In vivo imaging revealed synaptic Ca++ activity at the NMJ 30–40 min prior to the first muscle Ca++ response ( Figure 3C ) .", "As P0 progresses , coincident synaptic and muscle activity increases until the onset of P1 , when nearly all muscles and their synaptic inputs are synchronously active ( Figure 3D ) except M12 , which remains unresponsive to input until P2 ( Figure 3—figure supplement 1B ) .", "Near-complete muscle responsiveness may serve as a checkpoint for starting the ecdysis sequence and is possibly implemented by proprioceptive feedback .", "Class I dendritic arbor ( da ) neurons , dmd1 , vbd , and dbd act as proprioceptors during larval locomotion ( Vaadia et al . , 2019 ) and remain present at the pupal stage at least through ecdysis ( Figure 3E ) .", "Moreover , bulk Ca++ activity in sensory neurons is correlated with muscle activity during P0 ( Figure 3F ) , and sensory neurons commonly show correlated Ca++ activity with adjacent muscles ( Figure 3G ) .", "When class I da neurons were suppressed with UAS-Kir2 . 1 , Ca++ became sustained and widely distributed during P0 before twitching stopped ( Figure 3—figure supplement 2A , B ) .", "Unexpectedly , all animals died before P1 ( n = 10 ) .", "The transition from P0 to P1 is accompanied by the first overt movements , with bouts typically consisting of a Lift followed by a RollCon .", "The transition from P1 to P2 is demarcated by a sudden behavioral switch in which a P1 bout is followed by 4–5 bouts containing only a Swing .", "We used changing muscle activity patterns with associated body wall displacements to define five further canonical movements , all executed in characteristic anatomical compartments ( Figure 4A , Video 5 , Table 1 ) .", "We also define a precise onset for P3 , which had previously been difficult ( e . g . , see Diao et al . , 2017; Kim et al . , 2006 ) .", "Muscle activity in a Swing is coordinated across the dorsoventral axis as it travels anteriorly ( Figure 4B , C ) .", "While initial Swings are rapid , they slow after head eversion ( Kim et al . , 2006 ) and are then accompanied contralaterally by a movement we call the ‘Brace’ ( Figure 4A , D , orange box ) .", "The Brace is performed by concurrent contraction of lateral transverse muscles M21–23 and M8 in anterior hemisegments , followed by contraction of these same muscles in posterior hemisegments ( Figure 4D , lateral view ) .", "The Brace begins the shift of activity from P-to-A waves in P1 to A-to-P waves in P3 .", "Onset of P3 is indicated by a movement we call the ‘Crunch’ ( Figure 4A ) .", "The first Crunch follows the last P2 bout after a relatively long interbout interval and combines ventral contractions in the posterior compartment with dorsal contractions in the anterior compartment .", "The compartmentalized and complex movements that follow the Crunch include what we call the ‘Anterior Compression’ ( AntComp ) , ‘Posterior Contraction’ ( PostCon ) , and ‘Posterior Swing’ ( PostSwing ) .", "The first two comprise what have been termed ‘stretch compressions’ ( Kim et al . , 2006 ) .", "All of these movements are unique to P3 .", "Each of the elementary movements defined in Figure 4A and Video 5 is associated with the activity of specific muscles , and we trained a CNN to recognize and annotate the movements ( Figure 4—figure supplement 1A ) .", "Using the CNN to measure movement durations ( Figure 4—figure supplement 1B , C ) , together with measurements of bout and phase durations ( see Materials and methods ) , we characterized the variability of pupal ecdysis behavior at the level of phases , bouts , and movements .", "The relative stereotypy of the pupal ecdysis sequence can be seen from bulk Ca++ traces ( Figure 5A ) , but variation exists in the bout and interbout interval durations of all phases ( Supplementary file 2 , Figure 5B ) , and in the bout number of P1 and P2 ( Figure 5C ) .", "Coefficients of variation ( CV ) were lowest for P2 for all phase and bout parameters examined ( Supplementary file 2 ) .", "This finding is consistent with the developmental importance of P2 and suggests that its execution is the most tightly regulated of all the phases .", "Movement durations also showed variability , with CVs exceeding 50% ( Supplementary file 2 ) .", "However , the order in which movements were executed as determined by the SequenceMatcher algorithm ( see Materials and methods ) indicated considerable stereotypy .", "Sequence similarity scores ( SS ) for movements were computed pairwise for all bouts within each animal by phase and compared across animals .", "The mean SSs of the sequences for P1 , P2 , and P3 were all above 0 . 6 , a threshold for similarity ( Figure 5D ) , with CVs of 20–44% .", "P3 had the lowest SS and P2 the highest .", "To evaluate the stereotypy of the muscle activation patterns used to generate individual movements , we also used the SequenceMatcher algorithm .", "The order in which muscles were activated in bouts of P1 yielded an SS of 0 . 44 ± 0 . 17 , indicating low similarity .", "However , this SS differed significantly from that of shuffled sequences ( 0 . 32 ± 0 . 12 , p<0 . 001 ) .", "The sequences are thus not entirely random .", "Variability was evident between animals ( Figure 6A ) and for individual P1 movements across animals ( Figure 6B , blue plots ) .", "The low similarity of muscle activity patterns suggested that movements may be generated by muscles that are consistently active together even when their individual activation times vary between bouts .", "Co-active muscle groups have been shown to be important for larval locomotion ( Zarin et al . , 2019 ) ; we thus identified groups of muscles for which ( 1 ) all muscles were co-active in three or more consecutive frames , ( 2 ) the group was identified in at least 80% of the movements in a given animal , and ( 3 ) the group was identified in at least 80% of animals .", "We found eight co-active muscle groups , which we call ‘pupal muscle ensembles’ ( PMEs; Figure 6D ) .", "Muscles forming a PME are not recruited in a consistent order ( Figure 6C ) .", "In addition , we found several muscles that individually satisfied criteria", "( b ) and", "( c ) , but not as part of a group .", "Collectively with the PMEs , we call these movement-associated muscles ‘syllables , ’ and those active in the eight pupal ecdysis movements are listed in Table", "1 . We used the SequenceMatcher algorithm to evaluate the stereotypy of syllable activation during movements in each of the three phases ( Figure 6E ) .", "Surprisingly , the order in which syllables were recruited was quite variable , with SSs below 0 . 5 .", "However , those of P2 and P3 movements were significantly more consistent than the sequence of recruited individual muscles ( Figure 6E ) .", "Syllables associated with P1 movements showed SSs similar to those obtained using the muscle sequences ( see also Figure 6B , orange plots ) .", "This suggests that random muscle activations outside of syllables may contribute to the P1 movements , and such idiosyncratic activations were common in movements of all phases ( Figure 6F ) .", "Because syllables are often confined to particular anatomical compartments , we also compared the activity sequences in the D-V and A-P compartments across P1–P3 .", "P1 bouts exhibit only modest similarity but the bouts of P2 and P3 are intermediate in similarity to those of movements and syllables ( compare Figures 6E and 5D ) .", "Thus , the observed stereotypy for ordered motor execution in pupal ecdysis is highest for phases , and incrementally decreases with spatiotemporal scale .", "The least stereotypy ( SSs <40% , CVs >40% ) is seen in the recruitment order of individual muscles , which is less consistent than the activation of syllables .", "The SequenceMatcher results indicate variability in the recruitment of syllables into movements .", "To more comprehensively examine their contribution to movement , we sought to examine the phasic relationships between syllables that participate in the movements of P1 and early P2 ( i . e . , prior to the brace ) .", "These syllables are shown in Figure 7A–C for the lateral , dorsal , and ventral views .", "The phasic activation of the syllables that can conveniently be monitored from the lateral view during P1 bouts is represented in Figure 7D .", "As expected , these bouts initiate in the posterior compartment ( see key , Figure 7D , E , right ) with the activation of syllables in A6 driving the Lift ( Figure 7D , bottom ) .", "This activity precedes the activity of the syllables driving the RollCon in A4 ( Figure 7D , top ) .", "The syllables initially activated in A6 are predominantly located in the ventral compartment and their activity is followed by prolonged activity in the dorsal compartment by PME4 , which comprises dorsal longitudinal muscles M1–3 .", "Together , the ventral and dorsal contractions span the P1 bout and serve to compress posterior segments .", "In anterior segments , compression is transient , with roughly coincident activity of syllables M2 and PME6 in the dorsal and ventral compartments , respectively .", "This activity overlaps with and is outlasted by contractions of the strictly lateral transverse muscles of PME2 and M8 .", "Contraction of the latter muscles constricts the body wall , effectively pulling the dorsal surface away from the puparium .", "Separation of the dorsal body wall may be facilitated by reduced surface tension as the RollCons push air anteriorly between the puparium and dorsal body wall .", "In contrast to P1 , syllable activation in P2 bouts is considerably more uniform across hemisegments , body axes , and time ( Figure 7E ) .", "Syllables representing all dorsoventral compartments activate together in each hemisegment , compressing the animal longitudinally and along the D-V axis , with a wave of such compressions traversing the body wall in the P-to-A direction as can be seen by the delayed activation of syllables in A4 relative to A6 ( compare dotted lines in Figure 7E top vs . bottom ) .", "Full details of the compression wave constituting the Swing can be achieved by integration of information about muscle Ca++ activity from the dorsal and ventral views , which permits the reconstruction of the movement from the posterior to anterior end of the animal ( Figure 7—figure supplement 1 ) .", "In general , the phasic patterns of syllable activation provide a framework for understanding the evolution of pupal ecdysis movements and behavior .", "Although the hemisegmental patterns of muscle Ca++ activity represented by the PMEs provide a general description of the pupal ecdysis movements , not all body wall movement is a consequence of local muscle contractions .", "This is because the pliable cuticle of the pupa provides little rigidity .", "Like other animals reliant on a hydroskeleton ( Kier , 2012; Kristan et al . , 2000 ) , the pupa uses muscle contractions not only to produce local movement in the body wall , but also to increase hydrostatic pressure of the internal fluid to produce movement in distant parts of the body wall .", "Isometric contractions of antagonistic muscles also create body wall rigidity to resist body wall distortion so that pressure is appropriately directed .", "Directing pressure to particular parts of the body is , in fact , a central function of pupal movements , which thus rely on two types of muscle Ca++ activity: activity that results in muscle shortening to deflect the body wall and generate local movement and pressure changes , and isometric activity that does not result in length changes and promotes body wall rigidity to direct pressure .", "To better characterize how Ca++ activity in muscles of the pupal syllabary generates movement and facilitates and responds to pressure changes , we measured the normalized maximum shortening ( ΔL/L ) and peak fluorescence intensity ( ΔF/F ) for each muscle contraction in segments A3–A5 for each ecdysis phase .", "Increases in fluorescence only moderately correlated with muscle shortening ( r = –0 . 54; Figure 8—figure supplement 1A ) .", "To determine which muscles shorten the body wall , we calculated the average ΔL/L for each muscle over each phase , focusing first on P2 because of its role in promoting morphological change .", "For P2 , M12 and the muscles comprising PMEs 1 ( M26 , M13 , M8 ) , 2 ( M21–23 ) , 3 ( M2 , M3 ) , and 4 ( M1–M3 ) shorten the most ( Figure 8A ) .", "As noted above , these contractions create a wave of hemisegmental compressions in the P-to-A direction during the Swing ( Figure 8C , Video 5 ) .", "The greatest constriction across the D-V axis occurs in posterior segments , consistent with pronounced shortening in PME2 muscles ( M21–23 ) in A5 .", "Progressively decreased shortening of PME2 muscles is observed in A4 and A3 .", "Shortening of the ventral and dorsal longitudinal muscles ( M12 , M13 , M1–3 ) is more uniform across hemisegments , but the absence of M12 in posterior hemisegments and somewhat greater shortening of the dorsal longitudinal muscles anteriorly is consistent with the greater longitudinal compression of anterior hemisegments ( Figure 8C ) .", "Comparing ΔL/L with the corresponding average ( ΔF/F ) reveals hemisegmental differences ( Figure 8B vs . Figure 8A ) including an increase in peak Ca++ activity of PME2 muscles ( M21–23 ) , moving from A5 to A3 ( Figure 8B ) .", "This trend runs opposite to muscle shortening , which means that in successive anterior hemisegments , the PME2 muscles work harder to generate a smaller length change .", "This suggests counterforces on the anterior body wall , consistent with increased pressure to evert the head ( Figure 8D ) as has been measured in blowflies ( Zdarek and Friedman , 1986 ) .", "Each Swing is initiated posteriorly when the hemolymph is uniformly distributed throughout the body cavity and internal pressure is low .", "Ascending compression of the body wall on one side pushes the opposite side of the body against the static puparium .", "This prevents further body wall distension on that side and the compression wave drives hemolymph forward , like squeezing a tube of toothpaste from the bottom up .", "This creates pressure anteriorly , which is maintained by isometric contractions so that the head is pushed out ( Figure 8E ) .", "While a bilaterally coordinated compression might evert the head more efficiently , the unilateral Swing has a second function: it extrudes the larval tracheal linings on each side of the body .", "These are deposited in ascending segments on the puparium with each Swing ( Figure 8F ) .", "The two movements of P1 share features of the Swing and likely prepare the animal for P2 .", "The Lifts draw out the dorsal tracheal trunks , which remain attached to the posterior spiracles ( Robertson , 1936 ) ; the RollCons may help fragment the linings of the stretched trunks in anterior segments so that they are efficiently extruded at P2 .", "Essential to the Lift is compaction of the posterior hemisegments , which is accomplished by bilateral contraction of almost the same syllables as the Swing ( Table 1 ) .", "The RollCon , like the Swing , is performed unilaterally in a P-to-A direction .", "However , it fails to compact hemisegments as it traverses them and only deflects the dorsal body wall .", "Single-muscle changes in ΔL/L and ΔF/F underlying RollCons are much smaller than those for the Swing ( compare Figure 8—figure supplement 1B , C with Figure 8A , B ) .", "In addition , RollCons engage only a subset of the syllables comprising the Swing ( Table 1 ) .", "Rare Ca++ activity of M12 is idiosyncratic in P1 and does not contribute to ΔL/L .", "The coordinated contractions of M12 with other muscles during the Swing , together with the recruitment of additional syllables and higher Ca++ activities , explain why RollCons result only in deflections of the dorsal body wall while Swings cause hemisegmental compaction to bend the entire animal ( compare Figure 8—figure supplement 1D with Figure 8C ) .", "Overall , the active elements of P1 appear to merge in P2 , combining into one robust concerted P-to-A movement .", "In P3 , coordinated movement across anatomical compartments separates into the multiple compartmentalized movements introduced in Figure 4A and elaborated in Figure 8—figure supplement", "2 . These movements form activity patterns without strictly repeating units .", "Bulk Ca++ activity imaged from the lateral side shows an initial oscillatory pattern of variable frequency and amplitude that evolves into a more fixed pattern of alternating wide peaks and slim double peaks ( see Figure 2C , arrows ) .", "The initial variable period of activity is heralded by the Crunch , which is generated by the contralateral activation of PME1 in ventral hemisegments posterior to segment 4 and M2 in anterior dorsal segments .", "These contractions slightly lift the posterior segments and compact the anterior segments .", "The Crunch is typically followed by a Brace or an AntComp .", "The latter movement compacts the dorsal and anterior compartments via contractions of PME7 , PME3 , and M1 and with ventral activity in PME6 .", "Realignment of the body is achieved by the execution of a PostCon followed by a PostSwing , each composed of syllables in Table", "1 . The block of movements containing sequentially a Crunch , Brace , AntComp , PostCon , and PostSwing yield an A-to-P flow of activity .", "They constitute a fairly regular repeating unit with some variation in the order .", "This block forms the wide peak leading the double peaks seen in the bulk Ca++ trace ( Figure 2C , arrows ) , and as P3 evolves it increasingly alternates with a modified block that lacks the PostSwing and is followed by long interbout intervals .", "The double peaks typically consist of a Brace-AntComp-PostCon block followed quickly by a Crunch-Brace combination .", "In addition , later P3 blocks also include M12 contractions in PostSwings and sometimes Crunches , which visibly increase the compaction of the anterior segments .", "The increased compaction may increase pressure posteriorly , driving hemolymph into the appendages to lengthen them ( Figure 8—figure supplement 2B , C ) .", "We used the inwardly rectifying K+ channel , Kir2 . 1 , to selectively silence neurons that critically regulate entry into P1 and P2 ( Diao et al . , 2016; Kim et al . , 2015 ) .", "Specifically , we suppressed neurons expressing the B isoform of the ETH receptor ( NETHRB ) , and two overlapping populations of neurons expressing CCAP and Bursicon .", "The former manipulation disrupts P1 initiation by blocking abdominal lifting , while the latter blocks initiation of P2 ( Diao et al . , 2017 ) .", "We monitored the effects on Ca++ activity using hlk>GCaMP6s .", "In animals in which NETHRB neurons are suppressed , the baseline increase in bulk muscle Ca++ during P1 is severely attenuated relative to WT ( Figure 9A , B ) .", "Although PMEs 2 , 3 , and 6 characteristic of P1 ( Table 1 ) appear 10–15 min prior to P2 ( Figure 9C ) , activity in M15 and M1 ( and thus PME4 ) is missing .", "Muscles of PME1 are also not simultaneously active and thus do not exhibit ensemble activity ( Figure 9D ) .", "Finally , activity in the D-V compartments is not usually synchronized in the posterior segments .", "These data indicate that a principal population of ETH-targeted neurons coordinates muscles into syllables to produce the lift movement .", "Components of the Lift also remain absent from later movements .", "For example , M1 activity remains disrupted during Swings ( Figure 9—figure supplement 1A ) .", "Suppressing CCAP-secreting neurons ( NCCAP ) results in normal Ca++ activity during P0 and P1 , but P2 and P3 activity is absent ( Figure 9E ) .", "Although repetitive P2 swinging is absent , a single , partial swing-like movement is observed after numerous P1 bouts , suggesting that the transition to P2 may be attempted but is not maintained ( Video 6 ) .", "M1–3 activate asynchronously in anterior segments so that PME4 fails to form correctly .", "Consequently , the P-to-A wave on the dorsal side is disrupted by anterior contractions occurring too early ( Figure 9—figure supplement 1B ) .", "The partial swing propagates only through segment A5 and accompanying segmental compression is limited to A6 and A7 ( Figure 9—figure supplement 1C ) .", "The lateral muscles comprising PME2 are unsynchronized with the dorsal and ventral longitudinal muscles ( Figure 9F ) .", "Finally , the dorsal and ventral longitudinal muscles in segments A4–A5 change less in fluorescence ( ΔF/F = 157 . 2 ± 33 . 2 ) and length ( ΔL = 29 . 4 μm ± 6 . 23 ) than in WT animals ( ΔF/F = 272 . 3 ± 92 . 6; ΔL = 42 . 2 μm ± 2 . 52 ) .", "While NCCAP-suppression is lethal ( Diao et al . , 2016 ) , there is persistent activity resembling P1 Lifts and RollCons with no significant reversal in P-to-A direction before death .", "There are no obvious transitions and our CNN detected few P3-specific movements ( Figure 9—figure supplement 1D ) .", "We conclude that NCCAP modulates several aspects of the transition to P2 , including ( 1 ) generalized increase in muscle activity during P2; ( 2 ) coordination of syllables along the A-P axis that facilitates full body swings; and ( 3 ) coordination of activity across the D-V axis , as indicated by the desynchronization of PME2 activity .", "The loss of synchronous activity across D-V compartments led us to investigate the innervation pattern of motor neurons that express the CCAP receptor ( CCAP-R , Diao et al . , 2017 ) .", "Intersectional labeling of CCAP-R-expressing motor neurons using the Split Gal4 system ( Luan et al . , 2006 ) showed that these neurons innervate approximately half of the pupal muscles via Ib synapses , including all dorsal and three of the six ventral muscles ( Figure 10A , C ) .", "Although none of the motor neurons innervating the lateral transverse muscles express CCAP-R , the transverse muscles M21–23 of PME2 are labeled by the CCAP-R-Gal4 driver ( Figure 10B , C , Supplementary file 1 ) .", "This suggests that CCAP centrally modulates motor neuron output to dorsal and ventral muscles , while directly modulating lateral transverse muscles .", "At the larval stage , CCAP is co-released with Bursicon from type III terminals on muscles M12 and M13 , which straddle the muscles of PME2 ( Veverytsa and Allan , 2011 ) .", "Anti-Bursicon staining established the persistence of type III terminals on M12 ( Figure 10B , magenta , arrow ) .", "In addition , we confirmed the responsiveness of M21–23 to CCAP in fillet preparations treated with bath-applied peptide ( Figure 10D , E , Video 7 ) .", "Our results demonstrate a role for NCCAP in coordinating syllable activity across both the A-P and D-V axes and a peripheral role for CCAP in directly modulating lateral muscles .", "Genetic data suggest that CCAP and Bursicon act synergistically at pupal ecdysis ( Lahr et al . , 2012 ) , and neurons expressing the Bursicon receptor have been identified as essential for ecdysis motor programs ( Diao et al . , 2017 ) .", "Consistent with Bursicon’s colocalization with CCAP in central neurons , we find that suppressing the subset of Bursicon-expressing neurons ( NBurs ) has effects similar to NCCAP suppression: P1 activity is normal , but P2 and P3 are not correctly executed ( Figure 11A ) and the animals die without everting their heads .", "Animals also execute a single swing-like movement after numerous bouts of P1 , but in this case it consists of an entire anteriorly directed wave and some subsequent patterned activity is observed that resembles AntComp , Crunch , Brace , and PostSwing movements , which can be identified by our CNN ( Figure 11B ) .", "This activity lacks organization and remains desynchronized during the swing-like movement activity in the D-V compartments ( Figure 11C ) .", "Additional swing-like movements also occur , but always separated by other types of movement .", "We conclude that D-V synchronization for abdominal swinging requires NBurs , as does sustained execution of swinging behavior , and that full coordination of activity across the A-P axis additionally requires non-Bursicon-expressing neurons in NCCAP .", "The results of suppressing neuromodulatory signaling support both central and peripheral roles for the ecdysis hormones in promoting syllable coordination across the A-P and D-V axes .", "This coordination promotes the observed coherence of behavioral execution despite the variable timing of activation of individual muscles ." ], [ "Our results significantly extend previous descriptions of pupal ecdysis and illustrate the power of pan-muscle Ca++ imaging .", "Behavioral fine-mapping at single-cell resolution permits the definition and automated detection of elemental movements , the identification of a syllabary of movement-associated muscles and muscle ensembles , and the analysis of their sensitivity to neuronal manipulations .", "Importantly , single-cell analysis permits the identification of muscle activity that is not consistently associated with movements .", "The most salient example of such idiosyncratic activity occurs in P0 , a previously undescribed phase of muscle activity lacking coordinated movement .", "Variability persists in phases P1–P3 , which exhibit idiosyncratic muscle activation comingled with stereotyped movement syllables .", "Furthermore , muscle recruitment into syllables , and recruitment of syllables into movements , exhibits considerable variability both within and across animals .", "All observations suggest that variability in the order of recruitment of behavioral elements is a pervasive feature of the pupal ecdysis sequence with stereotypy emerging only at higher levels of behavioral description .", "Variability in pupal ecdysis behavior may arise from the need to adjust movement to changing forces on the body wall , both from inside and outside .", "Inside the animal , hydrostatic pressure varies globally in response to local contractions of the body wall .", "This pressure may need to be countered to maintain control of movement .", "Outside the animal , the wall of the puparium may form an inhomogeneous substrate as the distribution of molting fluid and air at different places varies .", "A notable feature of pupal ecdysis is that it is heralded by the appearance of a large air bubble in the abdomen , which is expelled into the puparium by the movements executed during P1 ( Bainbridge and Bownes , 1981; Chadfield and Sparrow , 1984 ) .", "After expulsion from the body , air is displaced by the animal’s movements , first posteriorly and then anteriorly .", "In the presence of residual molting fluid , pockets of air likely cause fluctuations in surface tension between the body wall and puparium .", "Forces exerted both by internal pressure and by substrate interactions within the puparium may thus require that motor output be dynamically adjusted , presumably by sensory cues .", "The importance of sensory cues to pupal ecdysis is evident from our finding that animals lacking proprioceptive input die at P0 without initiating the behavioral sequence .", "The cause of death remains to be determined , but its timing suggests that proprioceptive feedback may signal muscle responsiveness to neural input during the period of muscle reactivation and thus provide a readiness signal for ecdysis initiation .", "Alternatively , pressure on the body wall due to air bubble growth may trigger pupal ecdysis .", "Sensory cues have been shown to gate behavioral transitions in the adult ecdysis sequences of crickets and also to adjust motor program execution when extrication from parts of the old cuticle fails ( Carlson , 1977 ) .", "For the pupal ecdysis sequence , more work will be required to determine the sources of the observed variability .", "Notably , sources that arise frequently in the context of other behaviors , such as external environmental stimuli ( i . e . , stimuli outside the puparium ) and competing physiological needs , are absent for pupal ecdysis .", "In addition , proprioceptive cues , while they may tune ecdysis behavior , are not essential for generating it in that a fictive sequence is generated by an excised pupal brain treated with ETH ( Diao et al . , 2017; Kim et al . , 2006; Mena et al . , 2016 ) .", "Finally , our evidence suggests that at least some behavioral variability derives from the operation of the ecdysis neural network itself since stochasticity is clearly evident at P0 and then appears to extend to other phases .", "The variability of P0 muscle bouts is reminiscent in some ways of the neurogenic bursts of muscle activity observed in Drosophila embryos prior to hatching ( Crisp et al . , 2008 ) .", "Initial embryonic bursts consist of uncoordinated muscle activity that becomes increasingly organized over several hours as the locomotor networks mature ( Crisp et al . , 2011 ) .", "By the time of hatching , complete peristaltic motor sequences are regularly performed .", "Similar precocious network activity is also found in a variety of other systems ( Blankenship and Feller , 2010; O'Donovan , 1999 ) , including pupal moths where the developing circuitry for flight drives low-threshold neuromuscular responses in muscles that fail to elicit contractions ( Kammer and Kinnamon , 1979; Kammer and Rheuben , 1976 ) .", "Such activity has also been proposed to support network maturation .", "P0 motor activity may share this function as patterns of muscle activity become increasingly complex during P0 and recognizable syllables emerge ( Table 1 ) .", "However , in contrast to other systems , fully coordinated activity does not appear until the phase ends with the first P1 Lift and even after this muscle and syllable recruitment remain irregular , suggesting continued network variability .", "This apparently intrinsic variability may relate to the tradeoffs required of any multifunctional system .", "Pupal ecdysis , like most behaviors , depends on the integration of signals from CPGs , proprioceptors , neuromodulators , and possibly higher-order command systems—all of which are likely used in the context of larval behavior .", "For example , circuits that generate waves of activity along the anteroposterior axis are required for both larval locomotion and pupal ecdysis , but while they generate coordinated bilateral activity in locomotion ( Heckscher et al . , 2012; Lemon et al . , 2015; Pulver et al . , 2015; Zarin et al . , 2019 ) , they generate mostly alternating activity during ecdysis .", "This degree of flexibility may be bought at the cost of reproducibility of execution .", "Indeed , precise reproducibility may not be prioritized by nervous systems , which may instead favor solutions that are ‘good enough’ as has been generally proposed for biological control systems ( Partridge , 1982 ) .", "According to this view , further optimization of pupal ecdysis—in the form of behavioral stereotypy—may incur costs on performance in the execution of other behaviors that rely on the same circuit elements .", "Variability in pupal motor output is substantially altered by the neuromodulatory action of the ecdysis hormones ETH , CCAP , and Bursicon .", "Previous evidence indicates that these hormones induce the motor activity characteristic of P1 and P2 , even in an isolated pupal nervous system ( Diao et al . , 2017; Kim et al . , 2006 ) .", "Consistent with the ability of neuromodulators to reconfigure motor networks ( Marder , 2012 ) , this induction likely represents reorganization and possibly stabilization of network activity , and also increased network coherence .", "P1 is distinguished from P0 by the presence of coherent movements and P2 is more coherent than P1 in recruitment of muscles , syllables , and compartmental activity .", "The reduced stereotypy in P3 may result from waning neuromodulatory action .", "The ecdysis hormones thus reorganize motor output and increase the stereotypy of its execution .", "This conclusion is supported by suppression of neuromodulatory signaling , which disrupts muscle activity at multiple levels , as expected from the broad distribution of ecdysis hormone receptors ( Diao et al . , 2017; Diao et al . , 2016; Kim et al . , 2006 ) .", "What causes the release of Bursicon , CCAP , and ETH at pupal ecdysis is unknown , but our results suggest that ecdysis activity itself may be a factor .", "Release of ETH occurs only after muscle responsiveness to neural stimulation is ensured , suggesting that the latter may represent a checkpoint for release .", "Similarly , the aborted swing-like activity occurring in the absence of NCCAP and NBurs activity indicates that entry into P2 has a hormone-independent component that may act as a checkpoint for hormone release , which then sustains P2 network activity .", "One possible mechanism might be that as the animal pulls back during P1 and creates an anterior space , sensory feedback from the head signals the readiness for P2 .", "Similar checkpoint control mechanisms have been proposed to operate in the adult ecdysis sequence of locusts and crickets ( Carlson , 1977; Hughes , 1980b ) .", "The granular analysis of muscle activity presented here also reveals how neuromodulators may serve to coordinate action across anatomically and functionally distinct compartmental boundaries .", "ETHRB neuron suppression blocks the Lift , a movement of the posterior compartment , while suppressing CCAP neurons prematurely terminates the first ( and only ) swing-like movement by blocking its progression into the anterior compartment .", "Additionally , the distribution of CCAP-R appears to reflect mechanisms for selectively regulating distinct compartments across the D-V axis .", "CCAP-R is expressed selectively in motor neurons that innervate dorsal and ventral muscles .", "These motor neurons have dendrites that occupy similar positions on the myotopic map and share similar synaptic inputs ( Landgraf et al . , 2003; Zarin et al . , 2019 ) .", "This distinguishes them from motor neurons that innervate the lateral transverse muscles , which do not express CCAP-R .", "However , a subset of lateral transverse muscles ( i . e . , M21–23 ) themselves express CCAP-R , and the pattern of CCAP-R expression may thus represent a mechanism for synchronizing activity across the D-V axis during the swing movements of P2 when CCAP is released .", "Notably , muscle synchrony across compartments in posterior segments is lost in the one , partially executed Swing when CCAP-expressing neurons are suppressed ( Figure", "9 ) and no further Swings are executed .", "The putative source of CCAP for the muscles of PME2 are the type III terminals on muscle M12 ( Veverytsa and Allan , 2011 ) , which is selectively retained in the anterior compartment ( i . e . , segments 1–4 ) .", "CCAP-R-expressing muscles in the anterior compartment may thus receive CCAP modulation prior to those in the posterior compartment .", "This may facilitate the delayed appearance of the contralateral brace , which is formed principally by segmental PME2s and which changes the character of the Swing after head eversion .", "Interestingly , the anatomical asymmetry in the distribution of M12 is one of several we observed in pupal muscles across the D-V and A-P body axes .", "These have only been partially described previously ( Liu et al . , 2010 ) and result from the selective degradation of larval muscles in the ventral and posterior compartments .", "The asymmetries in muscle distribution correlate with several pupal movements that are physically partitioned across the A-P axis , including the Lift , which occurs only in the posterior compartment during P1 , and the AntComp , which is restricted to the anterior compartment during P3 .", "Synaptic mechanisms for controlling movements across the A-P boundary in the larva have been described by Tastekin et al . , 2018 , and similar mechanisms may synergize with anatomical asymmetries and neuromodulatory mechanisms in the pupa to spatially and temporally constrain muscle activity to specific compartments .", "It is worth noting that the expression of CCAP-R by the lateral transverse muscles indicates the potential importance of muscle modulation in shaping behavior .", "Whether modulation of muscle properties also underlies the generalized failure of muscles to respond to synaptic input at the onset of P0 is unclear .", "Animals stop moving shortly after pupariation and do not resume until pupal ecdysis , but neuromuscular activity may be maintained throughout this period .", "We observed neuromuscular activity without muscle responses at the earliest pre-pupal stages we examined ( approximately stage P2 of Bainbridge and Bownes , 1981; data not shown ) .", "However , more detailed imaging would be required to determine the extent , continuity , and pattern of the input .", "Drosophila muscles are targets of a variety of neuropeptides including myoinhibitory peptides , which have also been implicated in pupal ecdysis behavior ( Kim et al . , 2015 ) , and it is possible that these play a role in suppressing muscle responses to neural input .", "Alternatively , neuromuscular signaling may be progressively potentiated during P0 .", "Such a mechanism has been proposed to operate in pupal moth flight muscles , which respond electrically—but fail to contract—to synaptic input corresponding to flight motor patterns ( Fitch and Kammer , 1986; Klaassen and Kammer , 1985 ) .", "As animals approach eclosion , rising octopamine levels upregulate neuromuscular efficacy so that flight muscles activate in response to input .", "A goal of computational neuroethology ( Datta et al . , 2019 ) is to describe behavior at a level of resolution that permits the identification of its neural determinants .", "The muscle-level description provided here lends itself naturally to this purpose .", "For example , the activity patterns of the muscles comprising PME2 have likely neural correlates within the synaptic and neuromodulatory networks that govern pupal ecdysis behavior .", "At the neuromodulatory level , the NCCAP cells that terminate on M12 are likely sources of CCAP modulation of PME2 muscles , perhaps to help reverse their P-to-A activation at P2 .", "At the synaptic level , the regular , intersegmental pattern of activation of PME2 muscles in nearly all pupal movements and behavioral bouts ( Table 1 ) suggests that PME2 motor neurons are driven by CPG neurons that generate anteroposterior rhythms .", "Our results thus provide testable predictions about the patterns of neuromodulatory and synaptic connectivity between muscles , motor neurons , and premotor interneurons of various types .", "Testing these predictions will be facilitated by data emerging from reconstruction of the larval CNS at synaptic resolution ( Clark et al . , 2018; Kohsaka et al . , 2019; Zarin et al . , 2019 ) .", "Although pupal behaviors differ from those of the larva , broad similarities suggest that at least some neural substrates are shared .", "The initial P-to-A activity flow of the pupal ecdysis sequence is reminiscent of larval forward peristalsis , and its reversal after alternating bilateral Swing movements resembles the switch to backward peristalsis following sensory stimuli ( Carreira-Rosario et al . , 2018; Tastekin et al . , 2018 ) .", "An important difference , however , is that the basic features of larval locomotion are identifiable in the default activity of the excised larval brain ( Lemon et al . , 2015; Pulver et al . , 2015 ) , whereas the pupal nervous system produces patterned activity resembling pupal ecdysis only in response to ETH .", "Pupal ecdysis will thus permit investigation of the mechanisms by which intrinsic neuronal activity is organized by neuromodulatory control .", "Analyzing behavior at single-muscle resolution , as demonstrated here , will facilitate this investigation and should be applicable to other translucent preparations of neuroscientific interest , such as larval fruit flies , roundworms , and larval zebrafish ." ], [ "Further information and requests for resources and reagents should be directed to and will be fulfilled by Benjamin White ( benjaminwhite@mail . nih . gov ) .", "All neuronal suppression experiments were conducted using two copies of UAS-Kir2 . 1 in a parental line generated by combining insertions on chromosomes II and III with hlk-LexA::QFAD and LexAOP-GCaMP6s , respectively .", "Control animals bearing the UAS-Kir2 . 1 transgene , but no Gal4 driver , perform the ecdysis sequence normally , and animals in which the 410-Gal4 , CCAP-Gal4 , ETHRB-Gal4 , and Burs-Gal4 drivers express mCD8-GFP are healthy and viable to adulthood .", "P2 stage pupae ( Bainbridge and Bownes , 1981 ) were cold-anesthetized , washed in PBS , and dissected as for immunohistochemistry .", "The brain was removed by severing ventral nerve cord ( VNC ) projections and removing the brain and VNC .", "Once filleted , the PBS was replaced with 1 mL of Schneider’s insect medium ( SIM , Sigma-Aldrich ) .", "Imaging was performed for 30 min on a Nikon SMZ25 stereomicroscope with a 1×/0 . 3 NA objective and sampled at 2 Hz .", "After the first 5 min , 1 μM CCAP solution in SIM was added to the bath , for a final effective concentration of 0 . 5 μM , and image collection continued for the remaining 25 min .", "The vehicle control was SIM without CCAP .", "Ca++-free controls were conducted with SIM without Ca++ ( Sigma-Aldrich ) .", "CCAP ( BACHEM , Torrence , CA ) stock solutions were prepared by dilution to 1 mM in water .", "For experiments requiring GCaMP6s and jRGECO1a fluorescence , animals were prepared as intact animals above ( pupal stage P2 for NMJ/muscle experiments and pupal stage P4 for sensory/muscle experiments ) and imaging was conducted on a Nikon Ti epifluorescence microscope with a 10×/0 . 5 NA air objective , Cairn twin-cam , two PCO edge 4 . 2 sCMOS cameras , and filters for GFP and RFP .", "Images were acquired at 2 Hz for 60–90 min .", "Unless otherwise noted , all live experimental image series were background subtracted in Fiji ImageJ2 ( Schindelin et al . , 2012 ) .", "Where values of length and fluorescence are indicated , the line tool ( width , 3 px ) was used to manually measure intensity and length for muscles 1 , 2 , 3 , 5 , 12 , 13 , 15 , 22 , 26 , and 28 in hemisegments 3 , 4 , and 5 for each of the ecdysis sequence phases at the onset of muscle activity ( visible fluorescence ) , the peak of the muscle activity ( brightest fluorescence ) , and the offset of muscle activity ( fluorescence returns to baseline ) .", "Single-muscle identification was facilitated by the histolysis of half of the larval musculature and the occlusion of opposing side muscles by thick and highly scattering internal tissue .", "Data were collected sub-saturation but most videos and figures presented are contrast-enhanced to aid the eye .", "Bulk Ca++ traces were extracted from ROIs defined as the entire animal excluding the puparium , or as single muscles/neurons where indicated , and normalized to F0 , defined here as the average fluorescence intensity over the first 50 frames in the image series .", "Raster plots were generated from activity peaks identified from Ca++ traces using a peak finding algorithm in Python with manually determined thresholds for GCaMP6s and jRGECOa1 .", "A subset of four animals from the total hlk>GCaMP6s experimental dataset ( N = 16 ) were annotated frame-by-frame by expert raters to identify the muscles newly activated in each frame .", "These data were used to determine the frequency and order of activation for each muscle .", "Movements were annotated manually for these four animals plus one more by visual inspection of muscle activity pattern and deflections of the body wall with respect to the puparium from raw image series ( before background subtraction ) that were contrast-enhanced to allow visualization of autofluorescence from the body wall .", "Bouts were identified manually from a subset of 10 hlk>GCaMP6s animals as the start of a sequence of muscle activations flanked on either side by ≥ 2 frames of no new activations , which were defined as inter-bout intervals .", "The criteria for defining PMEs were determined empirically based on frame-by-frame analysis of 10 complete image series of the pupal ecdysis sequence .", "Groups of muscles that were repeatedly observed ( ≥80% of bouts in ≥8 animals ) to be co-active in a regular pattern were subsequently analyzed for the duration of co-activity .", "A group in which the activity of all muscles overlapped for three or more frames was defined as a PME .", "Post-hoc analysis revealed that three frames on average represent approximately 15% of the total period of co-active duration for PMEs .", "To find direction of activation ( A->P or P->A ) ( Figure 1K ) , we first computed a MIP along the x ( horizontal ) axis of the hlk>GCaMP6s image .", "For every frame , the MIP was a 1D vector of length H for an HxW image .", "Then we computed the location of the mode of the MIP vector on the y axis .", "An intensity mode indicates the location of the most dominant muscle activation .", "Therefore , the locations of the modes give an estimate of the direction of motion during the muscle activity period .", "Note that we are interested in finding the location of maximum motion , which is efficiently captured by the mode of the intensities , not by the mean .", "Once the intensity modes were identified for every frame using MIPs , we computed the number of modes above and below a threshold , indicating P->A and A->P motion , respectively .", "The threshold is automatically computed as the median of all modes .", "We then calculated the ratio of the number of modes below the median line to the number of modes above the median line and used this ratio to determine if the direction of activation is posteriorly or anteriorly directed .", "We used a CNN to automatically estimate the type of motion in each hlk>GCaMP6s image frame .", "The network model is a modified version of Inception-v3 ( Szegedy et al . , 2015 ) .", "To fit the model in available GPU memory based on the image size , we removed the final Dense layer and replaced it with a GlobalAveragePooling2D layer .", "In addition , a dropout layer was also added to introduce stochasticity into the model to avoid overfitting .", "There were totally 21 . 8M parameters in the model .", "The detailed network is described in the train . py provided as supplementary material .", "The model was trained to predict nine motions ( Anterior Compression , Posterior Swing , Brace , Crunch , Rolling Contraction , Lift , Posterior Contraction , Rest , Swing ) from the hlk>GCaMP6s images of five animals .", "For each animal , the images were resized to 403 × 129 × N , where N is the variable number of frames based on the duration of the phases .", "Each frame was obtained at 2 Hz .", "Every frame , starting from phase 1 , was assigned to one of the above-mentioned motions and was derived by consensus of three expert raters .", "Since the motion cannot possibly be derived by looking at a single frame , we used windows of 25 frames ( 12 previous and 12 following ) to estimate the motion at one frame .", "Therefore , the training data for each frame consisted of a 403 × 129 × 25 matrix .", "The CNN model was then trained by predicting the motion of the center ( 13th ) frame .", "Since there were only five animals available for training , we used all possible overlapping frames to augment the training data .", "Categorical cross-entropy with the Adam ( Kingma and Ba , 2015 ) optimizer was used with learning rate of 0 . 0001 to obtain a probabilistic estimation of motion for each frame .", "An early termination criterion was used to prevent the model from overfitting , where the training was stopped if the categorical cross-entropy did not increase for 10 consecutive training epochs .", "Figure 4—figure supplement 1A shows the accuracy of motion prediction averaged over all available frames for each of the five training animals in a leave-one-out cross-validation .", "The final ( magenta ) curve shows the training accuracy where frames from all five animals were aggregated and then the model was trained on the aggregated frames .", "Similar accuracy between cross-validation and aggregated training shows that the CNN model did not overfit the data .", "However , to use information from all training animals , we applied the aggregated trained model ( i . e . , magenta ) on the remaining 11 animals .", "For every frame of a test animal image , the motion with maximum probability was used as the final motion .", "For better accuracy , the predicted motions of the 11 animals were corrected by an expert rater .", "The annotated muscle datasets were sorted by onset frame number and then by muscle .", "Then , cells were concatenated into strings by bout , movement , or syllable , and assessed pairwise via the Python SequenceMatcher algorithm to score for similarity in string order .", "Other behavioral features were similarly analyzed using the onset times of syllables , movements , or compartments to order the sequences .", "Documentation for this algorithm is provided by the difflib library ( https://docs . python . org/3/library/difflib . html ) ." ] ]

[ "Identifying neural substrates of behavior requires defining actions in terms that map onto brain activity .", "Brain and muscle activity naturally correlate via the output of motor neurons , but apart from simple movements it has been difficult to define behavior in terms of muscle contractions .", "By mapping the musculature of the pupal fruit fly and comprehensively imaging muscle activation at single-cell resolution , we here describe a multiphasic behavioral sequence in Drosophila .", "Our characterization identifies a previously undescribed behavioral phase and permits extraction of major movements by a convolutional neural network .", "We deconstruct movements into a syllabary of co-active muscles and identify specific syllables that are sensitive to neuromodulatory manipulations .", "We find that muscle activity shows considerable variability , with sequential increases in stereotypy dependent upon neuromodulation .", "Our work provides a platform for studying whole-animal behavior , quantifying its variability across multiple spatiotemporal scales , and analyzing its neuromodulatory regulation at cellular resolution ." ]

[ "How do we find out how the brain works ?", "One way is to use imaging techniques to visualise an animal’s brain in action as it performs simple behaviours: as the animal moves , parts of its brain light up under the microscope .", "For laboratory animals like fruit flies , which have relatively small brains , this lets us observe their brain activity right down to the level of individual brain cells .", "The brain directs movements via collective activity of the body’s muscles .", "Our ability to track the activity of individual muscles is , however , more limited than our ability to observe single brain cells: even modern imaging technology still cannot monitor the activity of all the muscle cells in an animal’s body as it moves about .", "Yet this is precisely the information that scientists need to fully understand how the brain generates behaviour .", "Fruit flies perform specific behaviours at certain stages of their life cycle .", "When the fly pupa begins to metamorphose into an adult insect , it performs a fixed sequence of movements involving a set number of muscles , which is called the pupal ecdysis sequence .", "This initial movement sequence and the rest of metamorphosis both occur within the confines of the pupal case , which is a small , hardened shell surrounding the whole animal .", "Elliott et al . set out to determine if the fruit fly pupa’s ecdysis sequence could be used as a kind of model , to describe a simple behaviour at the level of individual muscles .", "Imaging experiments used fly pupae that were genetically engineered to produce an activity-dependent fluorescent protein in their muscle cells .", "Pupal cases were treated with a chemical to make them transparent , allowing easy observation of their visually ‘labelled’ muscles .", "This yielded a near-complete record of muscle activity during metamorphosis .", "Initially , individual muscles became active in small groups .", "The groups then synchronised with each other over the different regions of the pupa’s body to form distinct movements , much as syllables join to form words .", "This synchronisation was key to progression through metamorphosis and was co-ordinated at each step by specialised nerve cells that produce or respond to specific hormones .", "These results reveal how the brain might direct muscle activity to produce movement patterns .", "In the future , Elliott et al . hope to compare data on muscle activity with comprehensive records of brain cell activity , to shed new light on how the brain , muscles , and other factors work together to control behaviour ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "biochemistry and chemical biology" ]

[ [ "Nutrient availability naturally fluctuates , and animals have a variety of physiological responses that allow them to cope with nutrient stress .", "Starvation resistance is critical to evolutionary fitness , and fasting is an important clinical intervention with broad-ranging effects on aging and disease ( Longo and Mattson , 2014 ) .", "The roundworm Caenorhabditis elegans often faces starvation in the wild ( Félix and Braendle , 2010 ) , and it has developmental adaptations that facilitate starvation survival at multiple points in its lifecycle ( Angelo and Van Gilst , 2009; Baugh , 2013; Golden and Riddle , 1984; Johnson et al . , 1984; Schindler et al . , 2014 ) .", "In particular , dauer larvae develop as an alternative to the third larval stage in response to high population density and limited food ( Golden and Riddle , 1984 ) .", "Notably , dauer larvae form in anticipation of starvation ( food is actually required for dauer development ) , provisioning fat and developing morphological modifications that support survival .", "In contrast , worms that hatch in the absence of food ( Escherichia coli in the laboratory ) remain in a state of developmental arrest known as ‘L1 arrest’ ( or ‘L1 diapause’ ) until they feed ( Baugh , 2013 ) .", "Unlike dauer larvae , arrested L1 larvae have no morphological modification and are provisioned maternally .", "C . elegans L1 arrest provides a powerful organismal model to investigate gene regulatory and metabolic mechanisms that enable animals to cope with acute starvation .", "Insulin-like signaling is a critical regulator of starvation survival during L1 arrest ( Baugh and Sternberg , 2006; Muñoz and Riddle , 2003 ) .", "Feeding promotes insulin-like signaling through the receptor daf-2/InsR ( Chen and Baugh , 2014 ) , which antagonizes the transcription factor daf-16/FoxO ( Lin et al . , 1997; Ogg et al . , 1997 ) .", "In the absence of insulin-like signaling , daf-16 is active and promotes developmental arrest and starvation survival ( Baugh and Sternberg , 2006 ) .", "daf-16 promotes developmental arrest by inhibiting the dbl-1/TGF-β and daf-12 steroid hormone receptor pathways , but these pathways do not affect starvation survival ( Kaplan et al . , 2015; Lee and Ashrafi , 2008 ) .", "That is , the effects of daf-16 on developmental arrest and starvation resistance are distinct , and how daf-16 promotes starvation resistance is unknown .", "daf-16 also promotes longevity in fed adults ( Kenyon et al . , 1993 ) , and understanding how it does so has received considerable attention .", "A variety of studies have identified daf-16 target genes in the context of aging ( Dong et al . , 2007; Kaletsky et al . , 2016; Lee et al . , 2003; Murphy et al . , 2003 ) , resulting in identification of over 3000 genes affected directly or indirectly by daf-16 ( Tepper et al . , 2013 ) .", "However , these experiments typically examined the effect of constitutive daf-16 activation in fed daf-2/InsR mutant adults rather than conditional effects of daf-16 in response to nutrient availability .", "We examined the effect of daf-16 on gene expression in L1 larvae starved for ~12 hr ( Kaplan et al . , 2015 ) , but this is well after the starvation response is mounted ( Baugh et al . , 2009 ) , and this experiment did not include nutrient availability as a factor .", "Consequently , the immediate-early targets of daf-16 involved in the conditional response to starvation have not been identified .", "Metabolic adaptations in dauer larvae represent a ‘microaerobic’ metabolism , with reduced reliance on respiration and oxidative phosphorylation , instead oxidizing fatty acids as fuel for cellular maintenance ( Braeckman , 2009; Burnell et al . , 2005; O'Riordan and Burnell , 1989; 1990; Wadsworth and Riddle , 1989 ) .", "Expression analysis suggests that dauer larvae are metabolically similar to long-lived daf-2/InsR mutant adults , with expression of enzymes involved in the glyoxylate shunt ( a ‘shortcut’ through the TCA cycle ) , gluconeogenesis , and trehalose synthesis upregulated in both ( Depuydt et al . , 2014; Fuchs et al . , 2010; McElwee et al . , 2004; 2006; Penkov et al . , 2015 ) .", "However , the acute starvation response that occurs during L1 arrest or other non-dauer stages has not been compared to dauer larvae .", "Given the unique features of dauer larvae , it is unclear whether their metabolic adaptations represent a universal starvation response .", "Trehalose is a disaccharide of glucose formed by an α–α , 1–1 glycosidic bond .", "Trehalose buffers unicellular and multicellular organisms from osmotic stress , desiccation , heat stress , and freezing , and it can improve proteostasis ( Behm , 1997; Elbein et al . , 2003; Elliott et al . , 1996; Erkut et al . , 2011; François et al . , 2012; Honda et al . , 2010; Jain and Roy , 2009; Lamitina and Strange , 2005; Newman et al . , 1993; Page-Sharp et al . , 1999; Singer and Lindquist , 1998; Tapia and Koshland , 2014; Tapia et al . , 2015; Yoshida et al . , 2016 ) .", "Trehalose is not synthesized in vertebrates , but it confers desiccation tolerance on human cells ( Guo et al . , 2000 ) .", "In C . elegans , simultaneous disruption of both trehalose 6-phosphate synthase genes ( tps-1 and tps-2 ) reduces desiccation tolerance in dauer larvae ( Erkut et al . , 2011 ) .", "The glyoxylate shunt also supports desiccation tolerance in dauer larvae ( Erkut et al . , 2016 ) .", "However , regulation of these metabolic adaptations is not understood .", "Furthermore , it remains unclear what role trehalose or trehalose synthesis plays in mediating starvation resistance .", "The protective role of trehalose in conditions where water is limiting is attributed to its function as a compatible solute and its ability to replace water molecules at the surface of proteins and lipid bilayers , stabilizing polar interactions and preserving membrane organization and protein structure ( Erkut et al . , 2011 , 2012; Leekumjorn and Sum , 2008 ) .", "We refer to this biochemical mechanism of trehalose function as that of a ‘stress protectant’ .", "However , trehalose can also be used as an energy source to fuel glycolysis .", "For example , trehalose is the primary circulating sugar in insects , satisfying the high-energy demands of flight ( Clegg and Evans , 1961; Wyatt and Kale , 1957 ) .", "Given multiple reported physiological roles of trehalose and variation across taxa , there is debate over the relative importance of the different roles of trehalose in different contexts ( Crowe , 2007; Hohmann et al . , 1996 ) .", "We used a combination of genome-wide expression analysis and metabolomics to determine the nutrient-dependent effects of daf-16/FoxO in recently hatched L1-stage larvae of C . elegans .", "We report that during acute starvation , daf-16 promotes carbon flux through the glyoxylate shunt and gluconeogenesis towards trehalose synthesis , similar to the metabolic adaptations that occur in dauer larvae .", "Furthermore , we demonstrate that this metabolic shift is physiologically significant and supports starvation resistance .", "Multiple lines of evidence show that trehalose promotes starvation survival through two distinct mechanisms: as a stress protectant without being catabolized and as an energy source for glycolysis .", "We also show that trehalose and glucose interconvert and that interconversion is necessary for the dual function of trehalose .", "This work elucidates how daf-16/FoxO promotes starvation resistance , and it reveals a central role of trehalose metabolism in maintaining organismal energy homeostasis and stress resistance during acute starvation ." ], [ "We performed genome-wide expression analysis to determine the immediate effects of daf-16/FoxO activity in L1-stage larvae upon starvation .", "We used a ‘double-bleach’ procedure to ensure synchronized hatching ( Baugh , 2013; Baugh et al . , 2009 ) .", "We measured expression in wild-type ( WT ) and daf-16 mutant larvae shortly after hatching with and without food ( E . coli HB101 , Figure 1—figure supplement 1 ) .", "These data compare favorably with a published time series of WT gene expression using the same staging procedure ( Baugh et al . , 2009 ) , confirming a robust effect of nutrient availability and that the samples represent recently hatched larvae ( Figure 1—figure supplement 2A ) .", "Nutrient availability had a substantially larger effect on mRNA expression than genotype , with ~4000 genes displaying differential expression between fed and starved conditions in both WT and daf-16 mutants ( Figure 1A; false-discovery rate [FDR]<0 . 05 ) .", "Given the daf-16 mutant phenotypes during L1 starvation ( starvation-sensitive and arrest-defective ) , it was surprising that daf-16 mutants did not have a larger effect on the starvation response ( Figure 1—figure supplement 1 ) , with only 650 genes affected in starved larvae ( Figure 1A ) .", "That is , the starvation response was largely intact in daf-16 mutants ( Figure 1—figure supplement 1 , Figure 1—figure supplement 2A , B ) .", "This result demonstrates that other pathways are critical for the starvation response , and it is consistent with capturing relatively early , direct effects of daf-16 .", "Indeed , DAF-16 binds approximately half of the genes affected by it during starvation based on modENCODE ChIP-seq results ( hypergeometric enrichment p=3 . 3E-8 ) ( Niu et al . , 2011 ) ( Figure 1B ) , suggesting direct regulation of these genes .", "Notably , daf-16 had much less of an effect on fed larvae , affecting only 17 genes ( Figure 1A ) .", "This is consistent with daf-16 functioning in starved but not fed larvae , where it is localized to the cytoplasm due to antagonism from insulin-like signaling ( Weinkove et al . , 2006 ) .", "Moreover , the differential effect of daf-16 in fed and starved larvae confirms that we captured the effects of conditional regulation by daf-16 .", "Expression analysis suggests that daf-16/FoxO has a pervasive effect on central carbon metabolism in response to starvation .", "We used a two-factor analysis to formally identify genes with a significant interaction between condition ( fed vs . starved ) and genotype ( WT vs . daf-16 ) .", "This analysis addresses our experimental design explicitly , but it has less statistical power than a pairwise test , and it identified only 103 genes that are regulated by nutrient availability in daf-16-dependent fashion ( FDR < 0 . 05 ) .", "As a positive control , this stringent statistical analysis identified the superoxide dismutase gene sod-3 , a known direct target of DAF-16 ( Henderson et al . , 2006; Zhang et al . , 2013 ) ( Figure 1—figure supplement 2C , interaction FDR = 0 . 002 ) .", "Like sod-3 , the majority of these 103 genes were upregulated by starvation in WT , but not in daf-16 mutants ( Figure 1C ) .", "Gene ontology ( GO ) term enrichment analysis for these 103 genes revealed substantial bias towards metabolism terms , particularly carbohydrate metabolism ( Figure 1D ) .", "A schematic representation of carbohydrate metabolism shows 16 enzymes that are differentially expressed between WT and daf-16 during starvation ( Figure 1E in red ) .", "Notably , 15 of these differentially expressed genes were downregulated in daf-16 mutants ( all but tre-5 ) , and ChIP-seq suggests that DAF-16 binds each of them ( Niu et al . , 2011 ) , suggesting DAF-16 directly activates transcription of these metabolic genes during starvation .", "Time-series analysis revealed variation in dynamics of these daf-16-regulated metabolic enzymes but broadly confirmed sustained upregulation during L1 starvation ( Figure 1—figure supplement 2D ) ( Baugh et al . , 2009 ) .", "daf-16/FoxO appears to promote metabolic flux through the glyoxylate shunt , gluconeogenesis , and trehalose synthesis in response to starvation .", "icl-1 , the gene encoding the isocitrate lyase/malate synthase enzyme essential for the glyoxylate shunt , was upregulated during starvation in daf-16-dependent fashion ( Figure 1F ) .", "Many glycolysis enzymes are bidirectional and also catalyze the reverse gluconeogenic reactions , but several genes are exclusive to glycolysis or gluconeogenesis , allowing us to infer how gene expression changes affect carbon flux .", "Hexokinase and pyruvate kinase catalyze unidirectional glycolytic reactions , and none of the five genes encoding these two enzymes were affected by daf-16 .", "In contrast , the gluconeogenic enzymes pyruvate carboxylase ( pyc-1 ) and phosphoenolpyruvate carboxykinase ( PEPCK , pck-1 and pck-2 ) were upregulated during starvation in WT , but not in daf-16 mutants .", "Trehalose synthesis genes tps-1 , tps-2 , and gob-1 were also upregulated during starvation in daf-16-dependent fashion ( Figure 1F ) .", "Collectively , these results suggest that increased flux through the glyoxylate shunt and gluconeogenesis provides glucose for trehalose synthesis .", "daf-16/FoxO-dependent changes in metabolic gene expression during L1 starvation translate into changes in carbon metabolism .", "We conducted targeted metabolomics for panels of amino acids , organic acids , and acyl carnitines using the same experimental design as for expression analysis .", "Principal component analysis of these data showed that metabolic profiles are significantly affected by nutrient availability in WT , but the difference between conditions is reduced in daf-16 mutants ( Figure 2A ) , consistent with daf-16 activity contributing to the difference between conditions in WT .", "However , none of the individual metabolites targeted for analysis was significantly affected by genotype during starvation after correction for multiple testing ( Figure 2—figure supplement 1 ) .", "We suspect our inability to detect significant individual differences is due to lack of statistical power since an apparent effect on the overall metabolic profile was observed ( Figure 2A ) .", "In contrast , non-targeted metabolomic analysis revealed a 5 . 7-fold increase in disaccharide levels during starvation in WT but a mere 1 . 8-fold increase in daf-16 mutants ( Figure 2B ) .", "Because expression of trehalose synthesis genes was increased during starvation in daf-16-dependent fashion ( Figure 1 ) , we suspected this peak was due to increased trehalose levels .", "Indeed , specifically measuring trehalose in a biochemical assay revealed a 3 . 9-fold increase in WT during starvation and a mere 1 . 5-fold increase in daf-16 mutants ( Figure 2C ) .", "Time-series analysis revealed a difference in trehalose levels between fed and starved larvae within 4 hr of hatching ( Figure 2D ) .", "These results demonstrate that the metabolic gene expression changes caused by daf-16 during L1 starvation are physiologically significant , with the net effect of increasing steady-state levels of trehalose .", "The metabolic shift mediated by daf-16/FoxO promotes starvation resistance .", "We measured L1 starvation survival of mutants that specifically affect glycolysis , gluconeogenesis , and the glyoxylate shunt ( Figure 3A , Supplementary file 1 ) .", "daf-16 mutants were extremely sensitive to starvation , as expected ( Baugh and Sternberg , 2006 ) .", "Consistent with our hypothesis that gluconeogenesis contributes to starvation survival , the phosphoenolpyruvate carboxykinase mutant pck-1 was sensitive to starvation , though not to the same extent as daf-16 .", "The glyoxylate shunt is disrupted in icl-1/isocitrate lyase/malate synthase mutants , and icl-1 mutants were also starvation-sensitive , consistent with the glyoxylate shunt feeding into gluconeogenesis during starvation .", "In contrast , we hypothesized that glycolysis is less important to starvation survival than gluconeogenesis based on daf-16-dependent changes in gene expression ( Figure 1 ) .", "Indeed , survival of pyruvate kinase ( pyk-1 ) mutants , which specifically affect glycolysis , was indistinguishable from WT ( Figure 3A , Supplementary file 1 ) .", "These results are consistent with the glyoxylate shunt and gluconeogenesis being particularly important to starvation survival .", "Glycolysis is relatively more important to fed larvae than starved larvae .", "We measured size ( length ) after 48 hr of larval growth in well-fed metabolic mutants ( Figure 3B ) .", "In contrast to their effect on starvation survival , daf-16 and icl-1 mutants did not have a significant effect on growth rate .", "These results are consistent with daf-16 and the glyoxylate shunt being relatively starvation-specific .", "However , pck-1 and pyk-1 mutants grew relatively slow .", "These results suggest that fed larvae rely more on glycolysis than starved larvae , and that gluconeogenesis is important to fed and starved larvae .", "Furthermore , like daf-16 , the differential effects of pyk-1 and icl-1 on starvation survival and larval growth suggest their phenotypes are not simply due to general sickness .", "Pharmacological analysis corroborates the results of genetic analysis of metabolic mutants .", "We assayed the effect of the glycolytic inhibitor 2-deoxy-D-glucose ( 2-DG ) in starved and fed larvae , measuring survival and growth rate , respectively .", "Dose–response curves for starvation survival and growth rate are clearly distinct ( Figure 3C , Supplementary file 1 ) .", "Doses of 2-DG up to 20 mM had no effect on starvation survival ( p=0 . 84 , unpaired t-test , n = 4 ) but inhibited growth ( p=0 . 0002 , unpaired t-test , n = 3 ) .", "These results are consistent with larvae relying more on glycolysis for growth when fed than survival when starved .", "3-mercaptopicolinic acid ( 3 MPA ) is an inhibitor of PEPCK and has been shown to disrupt gluconeogenesis in plants and mammals ( DiTullio et al . , 1974; Ray and Black , 1976 ) , and we expect it to do the same in C . elegans .", "3 MPA reduced starvation survival and growth comparably ( Figure 3D ) .", "Similarity in 3-MPA dose–response curves corroborates results with pck-1 mutants ( Figure 3A , B ) , suggesting that gluconeogenesis is relatively important in fed and starved larvae .", "In summary , genetic and pharmacological analyses suggest that a shift in metabolism away from glycolysis toward the glyoxylate shunt and gluconeogenesis is a physiologically significant aspect of adaptation to starvation .", "Trehalose synthesis promotes starvation survival .", "Trehalose-6-phosphate synthase catalyzes the first step of trehalose synthesis by converting glucose-6-phosphate and UDP-glucose into trehalose 6-phosphate , and two genes ( tps-1 and tps-2 ) encode this enzyme in C . elegans ( Pellerone et al . , 2003 ) .", "tps-1 mutants were starvation-sensitive , and tps-2 mutants were marginally sensitive ( p=0 . 07 , Figure 4A ) .", "A tps-1; tps-2 double mutant was also starvation-sensitive , but no more sensitive than the tps-1 single mutant .", "These results suggest that elevated levels of trehalose , or trehalose synthesis itself , supports starvation survival .", "Reporter gene analysis confirmed transcriptional upregulation of tps-1 during L1 starvation and suggested that induction occurs in the intestine .", "Using promoter–GFP fusions ( McKay et al . , 2003 ) , we observed significant upregulation of Ptps-1::GFP in whole L1 larvae when starved compared to fed ( Figure 4B–D ) .", "In contrast , upregulation of Ptps-2::GFP during starvation was not significant in whole larvae ( Figure 4E–G ) .", "Lack of significance may be due to absence of regulatory elements in the reporter gene or whole-worm analysis , with tissue-specific differences being obscured .", "Indeed , each reporter was visible primarily in hypodermis and neurons in fed L1 larvae , and expression appeared to be induced specifically in the intestine in response to starvation ( Figure 4B–F; Figure 4—figure supplement 1 ) .", "Ptps-1::GFP had at least some visible intestinal expression in 75% of fed larvae but clear intestinal expression in 100% of starved larvae , with apparently increased intensity ( n = 44 and 31 worms , respectively ) .", "Likewise , Ptps-2::GFP was visible in the intestine of 19% of fed larvae and 90% of starved larvae ( n = 32 and 30 worms , respectively ) .", "These results suggest trehalose synthesis is upregulated in the intestine of starved L1 larvae .", "Both tps-1 and tps-2 contribute to trehalose synthesis in starved L1 larvae .", "Each single mutant had significantly reduced trehalose content with some residual trehalose ( Figure 4H ) .", "In contrast , trehalose levels were at background in the tps-1; tps-2 double mutant , which was significantly different from each single mutant .", "These results show that tps-1 and tps-2 both contribute to trehalose synthesis during L1 arrest ( Figure 4A ) .", "Supplementation of otherwise starved L1 larvae with trehalose in the buffer increases survival substantially .", "1 mM ( not shown ) and 10 mM supplementation did not affect survival , but 20 mM significantly increased survival with a clear dose response that reaches a maximum around 50 mM ( Figure 4I , Supplementary file 1 ) .", "50 mM and higher concentrations of trehalose doubled survival during L1 arrest .", "The dose response for survival was mirrored by measurement of internal levels of trehalose after supplementation , also reaching a maximum around 50 mM ( Figure 4J ) .", "This result confirms that larvae can internalize trehalose from the buffer , and it suggests that saturation of internalization limits the effect of supplementation on survival .", "L1 larvae decrease in length during starvation , but worms supplemented with 50 mM trehalose are longer than control worms after 8 d of starvation ( Figure 4K ) .", "Reduction in the rate of shrinkage during starvation further supports the conclusion that trehalose supports starvation resistance .", "Changes in insulin-like signaling act through trehalose synthesis to affect starvation survival .", "L1 starvation survival analysis of tps-1; tps-2; daf-2 triple mutants revealed quantitative epistasis between tps-1; tps-2 and daf-2 ( Figure 4L , Supplementary file 1 , two-way ANOVA pint = 0 . 02 ) .", "This genetic interaction is consistent with daf-2/InsR mutants increasing starvation resistance by upregulating trehalose synthesis via DAF-16 .", "Likewise , supplementing starvation-sensitive daf-16/FoxO mutants with 50 mM trehalose significantly increased survival , but not to the extent of WT ( Figure 4M , Supplementary file 1 , two-way ANOVA pint < 0 . 0001 ) .", "Supplementation of daf-16 mutants with 50 mM trehalose increased internal trehalose levels to WT levels without supplementation ( Figure 4N ) , despite supplementation incompletely restoring survival of daf-16 ( Figure 4M ) .", "This result together with incomplete suppression of daf-2 starvation resistance by tps-1; tps-2 ( Figure 4L ) indicates that insulin-like signaling affects more than just trehalose synthesis to regulate starvation resistance .", "We used a variety of approaches to distinguish between possible physiological roles of trehalose in supporting starvation survival .", "Trehalose is known to preserve membrane organization and protein structure during various forms of abiotic stress ( Erkut et al . , 2011; Guo et al . , 2000; Jain and Roy , 2009; Matsuda et al . , 2015; Singer and Lindquist , 1998; Tapia et al . , 2015 ) .", "It is possible that trehalose functions similarly as a stress protectant during starvation , preserving integrity of proteins , membranes or other cellular components to support survival .", "It is also possible that trehalose serves as a carbon source during starvation to provide energy via glycolysis in support of survival .", "Notably , these two hypothetical roles of trehalose are not mutually exclusive .", "Catabolism of trehalose and use as an energy source requires trehalase activity .", "We disrupted trehalase activity to test the relative contributions of trehalose to starvation survival as an energy source and a stress protectant .", "Reporters for tre-2 , tre-3 , tre-4 and tre-5 were expressed in relatively distinct patterns ( Figure 5—figure supplement 1A–D ) .", "No individual trehalase mutant significantly altered starvation survival ( Figure 5—figure supplement 1E , Supplementary file 1 ) .", "We generated a tre-1; tre-5; tre-2; tre-3; and tre-4 quintuple mutant to eliminate all trehalase activity .", "We confirmed that the quintuple mutant has elevated trehalose levels ( Figure 5A ) , consistent with a failure to catabolize trehalose .", "Surprisingly , starvation survival of the trehalase quintuple mutant was not significantly different than WT ( Figure 5B , Supplementary file 1 ) .", "However , starvation survival of the quintuple mutant was only partially extended by supplementation with 50 mM trehalose compared to WT ( Figure 5B , Supplementary file 1 ) .", "This intermediate effect is consistent with a role as stress protectant , but it also suggests that trehalose , at least when supplemented exogenously , must be metabolized in order to maximally support survival .", "Supplementation with a non-hydrolyzable form of trehalose corroborated genetic ablation of trehalase activity .", "In contrast to naturally occurring α–α trehalose , α–β trehalose cannot be hydrolyzed by trehalases but should retain function as a stress protectant .", "We confirmed that the biochemical assay we use to measure trehalose levels does not detect α–β trehalose ( Figure 5—figure supplement 1F ) .", "In contrast to α–α trehalose , supplementation with 50 mM α–β trehalose did not significantly increase internal α–α trehalose levels ( Figure 5C ) , indicating that α–β trehalose is not substantially converted to α–α trehalose in vivo .", "Nonetheless , supplementation with 50 mM α–β trehalose increased starvation survival ( Figure 5D ) .", "This clearly suggests a role for trehalose as a stress protectant .", "However , 50 mM α–β trehalose did not increase survival to the same extent as α–α trehalose ( Figure 5D ) .", "Consistent with the results of trehalose supplementation in the trehalase quintuple mutant , this intermediate effect suggests that trehalose functions as a stress protectant but also must be metabolized to maximally support survival .", "Trehalose is used for glycolysis to support starvation survival .", "Inhibiting glycolysis with 20 mM 2-DG did not affect starvation survival of WT ( Figures 3C and 5E ) .", "However , 20 mM 2-DG reduced survival in worms supplemented with 50 mM trehalose ( Figure 5E ) .", "This intermediate effect of trehalose supplementation in worms with inhibited glycolysis further supports the conclusion that trehalose is used as an energy source during starvation .", "Metabolism of trehalose would presumably cause a decrease in levels as it is catabolized and resources to synthesize more of it are depleted .", "Indeed , trehalose levels decreased over time during extended L1 starvation ( Figure 5F ) , consistent with it being used as an energy source .", "In summary , multiple lines of evidence suggest trehalose functions as both a stress protectant and an energy source during L1 starvation .", "Trehalose supplementation does not fully complement the tps-1; tps-2 mutant .", "tps-1; tps-2 had reduced starvation survival , as expected ( Figure 4A , K ) , and trehalose supplementation increased survival ( Figure 6A ) .", "However , survival of tps-1; tps-2 with supplementation was less than WT with supplementation .", "Trehalose supplementation also increased heat shock survival on the first day of L1 arrest , and again survival of tps-1; tps-2 with supplementation was less than WT with supplementation ( Figure 6B ) .", "This discrepancy in survival between genotypes with supplementation was reconciled by measuring corporeal trehalose levels with and without supplementation .", "The tps-1; tps-2 mutant had no detectable trehalose ( Figure 6C ) , as expected ( Figure 4H ) , consistent with it being null for trehalose synthesis activity .", "50 mM trehalose supplementation increased WT levels , as expected ( Figure 4N ) , but had no effect on tps-1; tps-2 ( Figure 6C ) .", "This result reveals that trehalose synthesis is required to maintain an internal pool of trehalose even with supplementation , as if ingested and possibly endogenous trehalose is rapidly catabolized during starvation .", "Given the inability of the tps-1; tps-2 mutant to maintain significant levels of internal trehalose , we believe that the trehalose supplemented in the medium is used primarily as an energy source but does not itself substantially contribute to survival as a stress protectant .", "Consequently , supplementation of tps-1; tps-2 with trehalose produces an intermediate survival effect , analogous to other conditions where its function as a stress protectant and an energy source are uncoupled ( Figure 5 ) .", "Synthesis of trehalose from other sugars supports starvation survival and heat resistance .", "Supplementation with 50 mM maltose or 100 mM glucose extends starvation survival to the same extent as 50 mM trehalose ( Figure 6A ) .", "100 mM glucose was used because trehalose and maltose are disaccharides of glucose .", "Supplementing tps-1; tps-2 mutants with maltose or glucose also increased starvation survival but not to the same extent as supplementation in WT .", "Furthermore , maltose and glucose supported heat shock survival , but not to the same extent in tps-1; tps-2 as WT ( Figure 6B ) .", "We hypothesized these sugars increase survival because they are used to fuel glycolysis and synthesize trehalose , both of which contribute to survival .", "Indeed , supplementation with glucose increased endogenous trehalose levels in WT , comparable to trehalose supplementation , but not in tps-1; tps-2 ( Figure 6C ) .", "Maltose supplementation also increased trehalose levels in WT , but to a lesser extent .", "Thus , we conclude that sugars supplemented in the medium of otherwise starved larvae interconvert between glucose and trehalose , that trehalose synthesis is necessary to maintain high steady-state levels of trehalose even with supplementation , and that in the absence of trehalose ( re- ) synthesis sugar supplementation contributes to survival primarily as an energy source .", "Gene expression analysis corroborates the conclusion that trehalose synthesis is necessary for the full physiological effect of trehalose supplementation .", "We sequenced mRNA from WT and tps-1; tps-2 mutants on the first day of L1 starvation with and without 50 mM trehalose supplementation .", "Notably , the gene expression profile associated with the trehalose synthesis defect of the tps-1; tps-2 mutant is not fully complemented by trehalose supplementation: 116 genes were differentially expressed in tps-1; tps-2 compared to WT when both were supplemented with trehalose ( Figure 7A ) .", "285 genes were differentially expressed in response to trehalose supplementation in WT , but only 99 genes were affected by supplementation in tps-1; tps-2 .", "That is , there was a common response to supplementation in the two genotypes , but it was dampened in the mutant so the two expression profiles remained distinct ( Figure 7B , Figure 7—figure supplement 1 ) .", "This incomplete effect of supplementation in tps-1; tps-2 on gene expression is analogous to the effect of supplementation on survival ( Figure 6A ) , and it too suggests that conversion of supplemental trehalose into glucose and back into trehalose is necessary to produce a complete physiological effect .", "Expression analysis provided an opportunity to identify genes whose expression is affected by the use of trehalose as an energy source during starvation .", "The effects of trehalose supplementation in tps-1; tps-2 mutants suggest that supplementary trehalose functions as an energy source rather than a stress protectant in this background ( Figure 6 ) .", "We examined the set of genes differentially expressed in tps-1; tps-2 mutants in response to trehalose supplementation , and they are enriched for GO terms related to DNA synthesis and replication ( Figure 7C ) , an energetically expensive process .", "All eight genes driving this association were upregulated by trehalose supplementation .", "We hypothesized that trehalose is used as an energy source to fuel DNA synthesis and possibly cell division .", "We examined two cell lineages that divide during the L1 stage in fed larvae .", "Hypodermal seam cells of the V lineage divide about 5 hr after hatching with food ( Sulston and Horvitz , 1977 ) .", "daf-16 mutants fail to arrest seam cell divisions during L1 arrest if they are supplemented with ethanol , but penetrance is incomplete and divisions are much slower than in fed larvae ( Baugh and Sternberg , 2006; Kaplan et al . , 2015 ) .", "We potentiated cell division using a daf-16 mutant without ethanol , and we found that trehalose supplementation promoted seam cell division in these permissive conditions ( Figure 7D ) .", "The M mesoblast cell lineage begins dividing about 9 hr after hatching in fed larvae ( Sulston and Horvitz , 1977 ) .", "Like the seam cells , M lineage cell divisions occur in daf-16 mutants during L1 arrest if supplemented with ethanol ( Baugh and Sternberg , 2006; Fukuyama et al . , 2015; Kaplan et al . , 2015 ) .", "Trehalose supplementation did not promote M lineage divisions in otherwise starved WT larvae , even with ethanol , but the number of M lineage divisions was increased by α–α trehalose supplementation in daf-16 mutants ( Figure 7E , Figure 7—figure supplement 2 ) .", "daf-16 mutants supplemented with α–α trehalose were also more likely to have multiple M lineage divisions: 16% had two or more divisions ( at least four cells ) , compared to 6% in daf-16 without supplementation ( n = 1200 and 1205 animals , respectively ) .", "Furthermore , supplementation with non-hydrolyzable α–β trehalose did not promote M lineage division ( Figure 7E ) , confirming that this effect on cell division reflects the use of trehalose as a glycolytic input .", "Endogenous trehalose promotes cell division in permissive conditions .", "Mutation of tps-1 and tps-2 in a daf-16/FoxO background reduced the number of M-cell divisions with ethanol but no trehalose supplementation ( Figure 7F , p=0 . 005 , unpaired t-test , n = 4 ) , suggesting endogenous trehalose promotes cell division in permissive conditions .", "Similar to trehalose supplementation , glucose supplementation promoted M lineage divisions in daf-16 with ethanol ( Figure 7F , p=0 . 04 , unpaired t-test , n = 4 ) .", "However , in tps-1; tps-2; daf-16 mutants , these sugars had no effect on cell division ( Figure 7F ) .", "This result shows that cell divisions promoted by sugar supplementation require endogenous trehalose synthesis , implying that supplementary sugar must be converted to trehalose to promote cell division ." ], [ "We define a set of early targets of daf-16/FoxO in the ecologically and developmentally relevant context of L1 starvation ( Baugh , 2013 ) .", "We show that enzymes of the glyoxylate shunt , gluconeogenesis , and trehalose synthesis are transcriptionally upregulated during L1 starvation .", "These findings corroborate expression analysis of dauer larvae , suggesting a conserved starvation response across developmental stages despite unique features of dauer larvae ( Braeckman , 2009; Erkut et al . , 2016; McElwee et al . , 2006; Wang and Kim , 2003 ) .", "Furthermore , we show that changes in expression have physiological consequence , affecting trehalose levels and starvation resistance .", "We provide evidence that starved larvae are sensitive to disruption of the glyoxylate shunt , which feeds into gluconeogenesis , in contrast to fed larvae .", "Conversely , starved larvae are relatively insensitive to disruption of glycolysis , in contrast to fed larvae .", "Thus , metabolic flux shifts from the TCA cycle and electron transport chain to the glyoxylate shunt and from glycolysis to gluconeogenesis during starvation .", "We show that this metabolic shift culminates in increased levels of trehalose , and that trehalose supports starvation survival .", "Expression of icl-1 , the isocitrate lyase/malate synthase enzyme central to the glyoxylate shunt , is upregulated by starvation at other developmental stages as well ( Liu et al . , 1997 ) , suggesting the metabolic shift we describe is part of a general starvation response rather than being specific to L1 or dauer larvae .", "Critically , we show that this metabolic shift is under direct regulation of insulin-like signaling via DAF-16/FoxO .", "That is , DAF-16 binds directly to the tps-1 promoter and the 14 other metabolic enzymes whose transcription is activated by daf-16 ( Niu et al . , 2011; Schuster et al . , 2010; Zhang et al . , 2013 ) .", "Mammals do not synthesize trehalose , but mammalian FOXO1 also upregulates gluconeogenesis during nutrient stress , suggesting broad conservation of the effects of insulin-like signaling on metabolic adaptation ( Matsumoto et al . , 2007; Puigserver et al . , 2003 ) .", "Intermediary metabolism is subject to extensive post-translational regulation ( Arif et al . , 2017 ) , and such regulation likely reinforces the patterns of transcriptional regulation we describe for L1 starvation .", "Although the changes in mRNA abundance we report are statistically significant with concerted effects on carbon metabolism , in many cases the fold-changes are relatively modest .", "It is likely that these patterns of transcriptional regulation are reinforced by post-translational control , possibly even under the control of insulin-like signaling .", "Indeed , enzymes involved in glycolysis and gluconeogenesis are known to be subject to post-translational modification ( Tripodi et al . , 2015 ) .", "Thus , the expression patterns we report are consistent with the changes in metabolic flux observed during starvation , but transcription is unlikely to be the sole cause for these physiological changes .", "The integration of transcriptional and post-translational regulation of metabolism during starvation is an exciting area for further research .", "We show that trehalose synthesis is an effector mechanism by which reduced insulin-like signaling and daf-16/FoxO activity promote starvation resistance .", "Disruption of trehalose synthesis reduces starvation survival , and supplementation of otherwise starved worms with trehalose increases survival .", "We also show that trehalose synthesis is required for heat resistance during L1 starvation , and that supplemental trehalose increases resistance .", "Extended starvation survival of a daf-2/InsR mutant depends on trehalose synthesis , indicating that daf-16/FoxO-dependent induction of the three enzymes that mediate trehalose synthesis ( tps-1 , tps-2 , and gob-1 ) contributes to starvation resistance and likely cross-tolerance to other stressors such as heat , high salt , and freezing ( Baugh , 2013 ) .", "However , disruption of trehalose synthesis does not completely eliminate increased survival of daf-2 mutants .", "Likewise , trehalose supplementation of a daf-16 mutant increases survival , but not to the level of WT worms supplemented with trehalose .", "We believe supplementation of daf-16 mutants with trehalose does not have more of an effect on survival because induction of tps-1 and tps-2 is abrogated by disruption of daf-16 , and trehalose synthesis is necessary for the full physiological effect of supplementation .", "Furthermore , these results imply that trehalose synthesis is not the only effector of reduced insulin-like signaling and DAF-16 activation , suggesting functional contribution of other DAF-16 targets to starvation resistance .", "Nonetheless , trehalose synthesis is a potent target of DAF-16 with substantial effects on starvation physiology .", "Genetic , pharmacological , and biochemical approaches reveal dual function of trehalose as a glycolytic input and stress protectant .", "The glyoxylate shunt and trehalose synthesis are required for desiccation tolerance in dauer larvae ( Erkut et al . , 2011; Erkut et al . , 2016 ) .", "Likewise , mutants with reduced insulin-like signaling are resistant to osmotic stress , and disruption of tps-1 and tps-2 suppresses this effect ( Lamitina and Strange , 2005 ) .", "In addition , trehalose supplementation improves viability during cryopreservation of C . elegans ( Kevin O'Connell , personal communication; see Worm Breeder's Gazette ) .", "These results suggest that trehalose supports viability in conditions where water is limiting , as in other organisms .", "Trehalose functions as such a stress protectant by preserving membrane organization and protein structure ( Erkut et al . , 2011; 2012; Leekumjorn and Sum , 2008 ) .", "Supplementation of starved L1 larvae with non-hydrolyzable α–β trehalose increases survival , suggesting similar function as a stress protectant during starvation , despite ample water .", "However , supplementation with α–β trehalose does not increase survival to the extent that hydrolyzable α–α trehalose does , suggesting catabolism of trehalose and use as a glycolytic input also contributes to starvation survival .", "Likewise , trehalose supplementation in worms with all five trehalase genes mutated extends survival , providing additional evidence of trehalose function as a stress protectant .", "However , extension of survival is limited in the quintuple mutant compared to WT , further showing that catabolism of trehalose is necessary for its full effect .", "In addition , pharmacological inhibition of glycolysis limits the increase in survival provided with trehalose supplementation in WT , consistent with the glucose produced by catabolism being used for glycolysis .", "By uncoupling the effects of trehalose , these results provide multiple lines of evidence that supplemental trehalose supports starvation survival by functioning as an input for glycolysis as well as a stress protectant .", "These results also indicate that starvation survival requires maintenance of molecular and cellular integrity as well as adequate energy .", "Furthermore , analysis of the trehalose synthase double mutant and trehalase quintuple mutant show that cycling of glucose and trehalose is necessary for the dual function and complete physiological effects of trehalose .", "The relative physiological significance of endogenous trehalose as an energy source and a stress protectant is difficult to discern .", "The trehalose synthase double mutant , which lacks trehalose entirely , is starvation sensitive , but it is unclear if its lack of function as a stress protectant , an energy source , or both is responsible .", "In contrast , the trehalase quintuple mutant , which maintains abnormally high levels of trehalose ( higher than those observed in WT with supplementation ) but cannot catabolize it for energy , survives starvation similar to WT .", "This result suggests that abnormally high levels of trehalose and the inability to catabolize it offset each other in their effects on survival .", "We did observe a reduction in endogenous trehalose levels relative to protein content during long-term starvation , consistent with depletion of it as an energy source .", "Furthermore , endogenous trehalose ( inferred from the tps-1; tps-2 mutant ) promotes cell division in a permissive background , presumably reflecting function as an energy source .", "However , disruption of glycolysis had a relatively minor effect on starvation survival .", "Notably , we assessed survival in laboratory conditions , without examination of other organismal properties , like movement or foraging behavior , that could be supported by glycolysis and contribute to survival in the wild .", "It is somewhat surprising that gluconeogenesis is upregulated during starvation .", "One might naively imagine that sugars are catabolized for energy during starvation and that fat and other nutrient stores are more directly utilized for energy than by fueling synthesis of sugar .", "That is , a cycle of sugar synthesis and catabolism during starvation is seemingly futile .", "Function of trehalose as a stress protectant during starvation provides some resolution to this paradox , but upregulation of gluconeogenesis during starvation also occurs in mammals ( Puigserver et al . , 2003 ) , though they do not produce trehalose .", "In humans , fasting and starvation induce hepatic gluconeogenesis ( Rothman et al . , 1991 ) , and dysregulation of hepatic glucose production is a hallmark of diabetes ( Consoli , 1992; Lin and Accili , 2011; Titchenell et al . , 2017 ) .", "Glucose produced in the liver is circulated to other organs with high-energy demand such as skeletal muscle and the brain .", "Thus , in mammals , glucose transport between organs resolves the paradox of sugar synthesis during starvation .", "We speculate that in nematodes trehalose is synthesized in the hypodermis and intestine during starvation and transported to other organs to satisfy energy demand .", "Indeed , insects transport trehalose instead of glucose: trehalose synthesized in the fat body is circulated through the hemolymph to fuel flight and other metabolic processes ( Candy and Kilby , 1961; Clegg and Evans , 1961; Saito , 1963; Thompson , 2003; Treherne , 1958; Wyatt and Kale , 1957 ) .", "Given the apparent absence of a glucose-6-phosphatase enzyme in C . elegans , there has been speculation that trehalose is the primary transport sugar ( McElwee et al . , 2006 ) .", "We observe expression of tps-1 and tps-2 in the intestine and hypodermis , which are also fat depots and primary sites of energy storage ( Mullaney and Ashrafi , 2009 ) .", "The phosphatase gob-1 , which converts trehalose 6-phosphate to trehalose , is also expressed in the intestine ( Kormish and McGhee , 2005 ) .", "In addition , catabolism of trehalose in neurons may support glycolysis and neuronal function during starvation ( Jang et al . , 2016 ) , which may be required for appropriate foraging behavior and adaptation to starvation in the wild ( Kniazeva et al . , 2015 ) .", "In summary , we propose conservation of an integrated organismal response to starvation among nematodes , insects , and mammals that is defined by FoxO transcription factors promoting carbon flux from fatty acids through gluconeogenesis to produce sugar that is transported throughout the animal to support glycolysis ." ], [ "The N2 Bristol strain was used and is referred to as WT .", "Worm stocks were maintained according to standard culture methods with nematode growth medium ( NGM ) and OP50 E . coli .", "Strains used include: GR1307 daf-16 ( mgDf50 ) , PS5150 daf-16 ( mgDf47 ) , BC14885 dpy-5 ( e907 ) ; sEx14885 , BC14876 dpy-5 ( e907 ) ; sEx14876 , PD4667 ayIs7[Phlh-8::GFP] , LRB101 daf-16 ( mgDf50 ) ; ayIs7[Phlh-8::GFP] , RB766 icl-1 ( ok531 ) , RB1688 pck-1 ( ok2098 ) , VC1265 pyk-1 ( ok1754 ) , tps-1 ( ok373 ) , tps-2 ( ok526 ) , tps-1 ( ok373 ) ; tps-2 ( ok526 ) , tps-1 ( ok373 ) ; tps-2 ( ok526 ) ; daf-2 ( e1370 ) , RB728 tre-1 ( ok327 ) , RB789 tre-2 ( ok575 ) , RB1400 tre-3 ( ok394 ) , LRB327 tre-4 ( gk298765 ) ( 2x backcrossed VC40057 ) , RB806 tre-5 ( ok612 ) , LRB334 tre-1 ( ok327 ) ; tre-5 ( ok612 ) ; tre-2 ( ok575 ) ; tre-3 ( ok394 ) ; tre-4 ( gk298765 ) , BC14863 dpy-5 ( e907 ) ; sEx14863 , BC12475 dpy-5 ( e907 ) ; sIs11667 , BC15383 dpy-5 ( e907 ) ; sEx15383 , BC14865 dpy-5 ( e907 ) ; sEx14865 , PS4657 syIs78[Pajm-1::AJM-1::GFP and unc-119+] , LRB240 daf-16 ( mgDf50 ) ; syIs78[Pajm-1::AJM-1::GFP] , tps-1 ( ok373 ) ; tps-2 ( ok526 ) ; daf-16 ( mgDf50 ) ; ayIs7[Phlh-8::GFP] .", "Embryos of WT ( N2 ) and GR1307 daf-16 ( mgDf50 ) worms were subjected to a double-bleach procedure to isolate staged embryos as described elsewhere ( Baugh , 2009; Baugh et al . , 2009 ) .", "Embryos were cultured in S-basal for 16 hr at 20°C , washed with S-basal by centrifugation and frozen in liquid nitrogen .", "At this collection time , all embryos had hatched and L1 larvae had been arrested for 2–4 hr .", "RNA was isolated using TRIzol ( Invitrogen , Carlsbad , CA ) according to the manufacturer’s specifications .", "RNA was further purified using RNeasy micro kit ( Qiagen , Valencia , CA ) .", "Spectrophotometry was used to assess RNA purity and concentration , and gel electrophoresis was used to confirm its integrity .", "100 ng of total RNA was amplified and labeled as cRNA using the MessageAmp II-Biotin Enhanced kit ( Ambion , Foster City , CA ) according to the manufacturer’s protocol .", "Molecular weight and concentration of the biotin-labeled cRNA product was assessed with an Agilent BioAnalyzer and spectrophotometer .", "12 . 5 μg of cRNA was fragmented , hybridized to the C . elegans expression array ( Affymetrix , Santa Clara , CA ) , and scanned according to the manufacturer’s instructions .", "Microarray data were analyzed using the LIMMA package in R . Each probe set was mapped to WS180 as in ( Baugh et al . , 2009 ) .", "Normalized log2 GCRMA values were used to assess significance of expression changes with the LIMMA/GCRMA empirical Bayes test .", "Genes significantly different between conditions were hierarchically clustered with Euclidian distances using Gene Cluster 3 . 0 ( Figure 1—figure supplement 1 ) .", "This same group of genes was used for principal component analysis ( Figure 1A ) .", "To measure metabolites in WT and daf-16/FoxO mutant worms , both strains were grown on 10 cm NGM plates with OP50 E . coli .", "These populations were bleached to isolate embryos .", "Embryos were arrested in S-complete and were either maintained in S-complete ( starvation ) or had 25 mg/mL HB101 E . coli added to culture upon hatching ( fed ) .", "Worms were washed in S-basal and flash frozen in 0 . 6% formic acid .", "Worms were lysed by sonication with a Bioruptor at 4°C for 15 min with 30 s on , 30 s off at high power .", "Sonicated samples were stored at −80°C .", "To extract metabolites , all samples were thawed on ice and then sonicated on ice , using ten 30 s on–off pulses at 30% power using a Series 60 Sonic Dismembrator ( Model F60 , Fisher Scientific ) .", "An aliquot was saved for total protein analysis via bicinchoninic acid assay ( BCA Sigma , St . Louis , MO ) .", "Remaining sample was mixed with acetonitrile ( 1:1 ratio ) , vortexed , and divided into five parts .", "Three parts of the sample was used for the quantification of amino acid and acyl carnitine , one part for organic acids , and from the remaining one part sample equivalent to 500 µg of protein was used for non-targeted metabolomics analysis .", "Amino acids and acylcarnitines were analyzed using MS/MS as previously described ( An et al . , 2004; Wu et al . , 2004 ) and organic acids were quantified using GC/MS as described in the study by Jensen et al . ( 2006 ) .", "Non-targeted metabolomics analysis was performed using GC/MS metabolomics as described in the study by Banerjee et al . ( 2015 ) : Raw data were normalized to protein levels , and converted to log2 fold change values relative to fed WT worms .", "Embryos were isolated by standard hypochlorite treatment and arrested at a density of 1/μL in virgin S-basal ( no ethanol added ) in glass test tubes .", "Tubes were kept on a roller drum at 21–22°C .", "For survival experiments with 2-DG , 3-mercaptopicolinic acid , trehalose , glucose , and maltose , these compounds were added to virgin S-basal before addition of embryos .", "To score survival , 100 μL samples of each culture were plated next to a lawn of OP50 E . coli on 6 cm NGM plates .", "The total number of worms plated ( Tp ) was counted after plating and 2 d later the number of worms alive and able to move to the bacterial lawn ( Ta ) was scored .", "Survival was calculated as Ta/Tp , logistic survival curves were fit to the data , and median survival time was calculated ( Artyukhin et al . , 2013; Hibshman et al . , 2016; Kaplan et al . , 2015 ) .", "We used the Megazyme Trehalose Assay Kit ( K-Treh ) to measure the levels of trehalose in worms .", "Briefly , washed worms were flash frozen in 100 μL of 0 . 6% formic acid .", "Worms were sonicated for lysis , as with metabolomics analysis .", "Samples were split for protein analysis and trehalose analysis .", "Trehalose was measured according to the protocol provided with the Megazyme kit .", "Absorbances were measured on a Nanodrop .", "Protein concentration was quantified with a Qubit protein assay kit ( Thermo Fisher Scientific , Waltham , MA ) .", "Levels of trehalose were normalized to protein .", "Normalizing trehalose content by worm number instead of protein concentration provided qualitatively similar results .", "The Wormsizer plugin for FIJI was used to measure the length of worms ( Moore et al . , 2013 ) .", "Length after 48 hr of development was measured in several genotypes and in the presence of a range of 2-DG and 3-mercaptopicolinic acid concentrations ( Figure 3B–D ) .", "Length of starved L1 larvae was similarly measured ( Figure 4K ) .", "Worms were washed in S-basal , plated on unseeded 10 cm NGM plates and imaged on a Zeiss Discovery . V20 stereomicroscope .", "Images were processed with Wormsizer .", "To quantify fluorescence of Ptps-1::GFP and Ptps-2::GFP strains ( Figure 4B–G ) images were acquired at 40x with consistent exposure times ( 400 ms ) on a Zeiss Imager . A1 outfitted with an Axiocam 506 Mono .", "Analysis was conducted in Fiji by outlining worms and calculating the average pixel intensity per worm .", "Background was subtracted from these measurements .", "Figure 4D , G show the distribution of relative intensities of individual worms from three independent biological replicates .", "P-values were calculated from unpaired t-tests on the mean fluorescent intensities from three biological replicates .", "Reporter strains were observed at 100x to determine sites of expression .", "Embryos were obtained through standard hypochlorite treatment and arrested in 16 mm glass tubes with virgin S-basal buffer and virgin S-basal with addition of various sugars .", "L1 worms in liquid culture were kept at 35°C for 9 hr with shaking at 180 rpm .", "As with starvation survival , 100 μL of aliquots were plated onto 6 cm plates with NGM and OP50 .", "The total number of worms plated ( Tp ) was counted .", "After 2 d the total number of worms alive ( Ta ) was scored .", "Survival was calculated as Ta/Tp .", "N2 and tps-1; tps-2 worms were cultured on standard NGM plates seeded with OP50 .", "Cultures were bleached to isolate embryos , which were suspended 1/μL in virgin S-basal or virgin S-basal with 50 mM trehalose .", "Samples were flash frozen on liquid nitrogen 24 hr after bleach .", "RNA was extracted with trizol and chloroform .", "Libraries were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina ( E7530 ) with 500 ng of RNA per library and 12 cycles of polymerase chain reaction .", "Libraries were sequenced using Illumina HiSeq4000 , acquiring single end 50 bp reads .", "Bowtie was used to map reads ( Langmead et al . , 2009 ) .", "EdgeR was used to assess differential expression from count tables ( Robinson et al . , 2010 ) .", "An ANOVA-like test in EdgeR identified 750 genes with variability across conditions ( FDR < 0 . 05 ) .", "These were hierarchically clustered with Euclidian distances using Gene Cluster 3 . 0 ( Figure 7—figure supplement 1 ) .", "Pairwise comparisons between conditions were calculated with the edgeR exact test .", "Genes different in any pairwise comparison were included in CA ( Figure 6C ) .", "The GOrilla gene ontology enrichment analysis and visualization tool was utilized to determine significant enrichment of processes , functions , and components ( Eden et al . , 2009 ) .", "In each case , a list of differentially expressed genes was tested against the background set of detected genes .", "The PS4657 and LRB240 strains were used to visualize seam cells .", "The number of cell divisions after 4 d of starvation in virgin S-basal was scored on a Zeiss Imager . A1 compound microscope .", "The ayIs7[Phlh-8::GFP] transgene was used to visualize M cells in different genetic backgrounds , and divisions were scored after 8 d of starvation in S-basal buffer ( with ethanol and cholesterol added ) .", "Statistics were calculated based on independent biological replicates performed on different days with all experimental methods performed independently of other replicates .", "Statistical analysis was performed in Microsoft Excel and R Studio .", "Statistical tests and the number of replicates are described when p-values are reported .", "Throughout the figures a single asterisk indicates p<0 . 05 , double asterisks indicates p<0 . 01 , and triple asterisks indicate p<0 . 001 ." ] ]

[ "daf-16/FoxO is required to survive starvation in Caenorhabditis elegans , but how daf-16IFoxO promotes starvation resistance is unclear .", "We show that daf-16/FoxO restructures carbohydrate metabolism by driving carbon flux through the glyoxylate shunt and gluconeogenesis and into synthesis of trehalose , a disaccharide of glucose .", "Trehalose is a well-known stress protectant , capable of preserving membrane organization and protein structure during abiotic stress .", "Metabolomic , genetic , and pharmacological analyses confirm increased trehalose synthesis and further show that trehalose not only supports survival as a stress protectant but also serves as a glycolytic input .", "Furthermore , we provide evidence that metabolic cycling between trehalose and glucose is necessary for this dual function of trehalose .", "This work demonstrates that daf-16/FoxO promotes starvation resistance by shifting carbon metabolism to drive trehalose synthesis , which in turn supports survival by providing an energy source and acting as a stress protectant ." ]

[ "Most animals rarely have access to a constant supply of food , and so have evolved ways to cope with times of plenty and times of shortage .", "Insulin is a hormone that travels throughout the body to signal when an animal is well fed .", "Insulin signaling inhibits the activity of a protein called FoxO , which otherwise switches on and off hundreds of genes to control the starvation response .", "The roundworm , Caenorhabditis elegans , has been well studied in the laboratory , and often has to cope with starvation in the wild .", "These worms can pause their development if no food is available , or divert to a different developmental path if they anticipate that food will be short in future .", "As with more complex animals , the worm responds to starvation by reducing insulin-like signaling , which in turn activates a FoxO protein called daf-16 .", "When the worms stop feeding , daf-16 is switched on , which is crucial for survival .", "It was known how daf-16 stops the roundworm’s development , but it was not known how it helps the worms to survive starvation .", "Now , Hibshman et al . have compared normal roundworm larvae to larvae that are missing the gene for daf-16 to determine how this protein influences the roundworm’s ability to survive starvation .", "The worms were examined with and without food , to look for which genes were switched on and off by daf-16 during starvation .", "This revealed that daf-16 controls metabolism , activating a metabolic shortcut that makes the worms produce glucose and begin turning it into another type of sugar , called trehalose .", "This sugar usually promotes survival in conditions where water is limiting , like dehydration and high salt , but it can also be broken down to release energy .", "The levels of trehalose in the worms rose within hours of the onset of starvation .", "To confirm the importance of trehalose in surviving starvation , roundworms with mutations in genes involved in glucose or trehalose production were examined , as was the effect of giving starving worms glucose or trehalose .", "Disrupting the production of sugars caused the worms to die sooner of starvation , while supplementing with sugar had the opposite effect meaning the worms survived for longer .", "Taken together , these findings reveal that daf-16 protects against starvation by shifting metabolism towards the production of trehalose .", "This helps worms to survive by both protecting them from stress and providing them with a source of energy .", "These findings not only extend the current understanding of how animals respond to starvation , but could also lead to improved understanding of diseases where this response goes wrong , including diabetes and obesity ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "evolutionary biology", "computational and systems biology" ]

[ [ "When important cellular functions are inactivated , for example by genetic mutations , long ranging disruptions of cellular networks can occur , which poses a major adaptive challenge to cells .", "Recent studies investigated evolution of E . coli upon inactivation of non-essential enzymes of carbon metabolism that lead to re-wiring through less efficient pathways ( Krüsemann et al . , 2018; Long et al . , 2018; McCloskey et al . , 2018a; McCloskey et al . , 2018b; McCloskey et al . , 2018c; McCloskey et al . , 2018 ) .", "These studies highlight a crucial interplay between regulatory responses and imbalances in metabolite concentrations resulting from gene knockouts , which can be subsequently corrected by mutations elsewhere .", "Importantly all these studies involved gene-knockout so that adaptation by reversal to wild type genotype was not possible .", "Less is known about the adaptation mechanisms that follow inactivation of unique cellular processes that are deemed indispensable in microbes .", "The genes that perform such functions , often classified as essential , are believed to face higher selective pressure and thus evolve slower ( Luo et al . , 2015 ) .", "It is thus expected that functional disruption of essential genes either by mutation or by antibiotic stress can create a major barrier to the adaptability of bacteria .", "Nevertheless , a comprehensive study involving knock outs of >1000 genes classified as essential in yeast has shown that a small percentage of mutants could recover viability after laboratory evolution ( Liu et al . , 2015 ) , revealing that essentiality can , in some cases , be overcome through adaptation .", "Loss of essential biosynthetic genes have also been observed to occur naturally , under evolutionary conditions where pathway products are provided externally ( D'Souza and Kost , 2016 ) , highlighting a context-dependent nature of essentiality .", "Nevertheless , while adaptation upon inactivation of essential genes in microbes has been demonstrated , its mechanisms remain unknown .", "Here we study evolutionary adaptation upon functional inactivation of dihydrofolate reductase ( DHFR ) , an essential E . coli enzyme .", "As the cellular target of the antibiotic trimethoprim , DHFR has been repeatedly observed to accumulate mutations leading to extremely high drug resistance levels in various experimental evolution studies ( Rodrigues et al . , 2016; Tamer et al . , 2019; Toprak et al . , 2012 ) .", "Past efforts to link fitness effects of chromosomal variation in the folA locus encoding DHFR and their biophysical effects on DHFR protein allowed us to develop an accurate quantitative biophysical model of DHFR fitness landscape ( Bershtein et al . , 2015a; Bershtein et al . , 2013; Bershtein et al . , 2012; Bershtein et al . , 2015b; Rodrigues et al . , 2016 ) .", "The availability of a clear genotype-phenotype relationship makes DHFR an excellent model to study the dynamics and outcomes of evolution to recover from its inactivation by point mutations .", "In contrast to previous studies that used gene knockouts , here we inactivate the chromosomal E . coli DHFR by introducing mutations in the folA locus at a key catalytic residue in position 27 , generating strains that express inactive DHFR ( with at least 4 orders of magnitude lower catalytic efficiency than the wild type ) .", "These strains are viable only with external metabolic compensation , allowing the mutants to adapt to lack of DHFR function by decreasing supplement concentration .", "The advantage of this approach is that it presents cells with an obvious evolutionary ‘solution’ of reverting the mutant back to wild type variant without massive rewiring that could lead to potentially lesser fit variants .", "However , an actual outcome may depend on evolutionary dynamics which could revert to wild type or other form of active DHFR ( higher fitness peak ) or converge to a potentially more accessible solution of rewiring to a less efficient metabolic pathways that do not require DHFR function .", "Therefore , this setup allows us to assess relative roles of height and accessibility of fitness peaks in determining the outcome of evolutionary dynamics .", "We show that , depending on starting DHFR variant , partial reversion of DHFR phenotype may indeed occur .", "However , adaptation to low concentration of external metabolites through metabolic rewiring is the prevalent evolutionary solution due to the availability of a greater number of trajectories leading to consecutive gene inactivation events in two key loci , thyA and deoB .", "Using omics analysis , we observe global perturbations in metabolites and proteins levels in both naïve D27 mutant strains and in evolved strains .", "Finally , we show how adaptation to loss-of-function mutations in DHFR , the cellular target of the antibiotic trimethoprim , can lead to high levels of drug resistance ." ], [ "We selected single or double nucleotide base mutations to replace a key catalytic residue Asp 27 either for Asn , Gly or Phe .", "The presence of a carboxylate side chain in position 27 is strictly conserved among 290 bacterial DHFR orthologues , where Asp and Glu are present at a frequency of 82% and 18% , respectively ( Figure 2—figure supplement 1A ) .", "These replacements were chosen to explore how different degrees of structural perturbations could potentially lead to alternative mutational pathways; while asparagine is structurally similar to aspartate , the introduction of residues with side chains that are either bulky and hydrophobic ( phenylalanine ) or absent ( glycine ) is expected to create significantly more structural perturbations , at least locally at the active site .", "Purified D27 mutant proteins were characterized ( Table 1 ) .", "The catalytic activity measurements confirm the lack of significant DHFR function in these variants; catalytic efficiencies ( kcat/KM ) are several orders of magnitude lower than wild type .", "However , thermal denaturation data obtained for the D27 mutant proteins indicates that their stability is mostly unaffected ( or even significantly increased in the case of D27F mutant; Tian et al . , 2015 ) showing that , despite the lack of catalytic activity , these proteins retain the ability to fully fold .", "Importantly , this provides a pathway for restoration of DHFR function over the course of evolution , either by revertant mutations at the D27 locus or , potentially , compensatory mutations elsewhere in the protein .", "D27 mutant strains were generated by lambda red recombination ( Bershtein et al . , 2012 ) and plated in folAmix-containing agar media , however , growth was only visible after 48 hr and the colonies formed were miniscule .", "When DHFR function was assayed in cell lysates of D27 mutant strains , no DHFR activity could be detected ( Figure 2—figure supplement 1B–C ) , confirming that these mutations inactivate DHFR function in the cell .", "As expected , growth experiments showed that D27 mutants grow only at high concentrations of folAmix ( Figure 2A ) .", "Interestingly , D27N and D27G mutants show slightly better growth at lower concentrations of folAmix than D27F .", "Given that the catalytic efficiencies of D27G and N , albeit extremely low , are 2 orders of magnitude higher than that of D27F , it is possible that such difference could have a beneficial impact at low folAmix concentrations .", "In addition , potential acquisition of a slightly advantageous mutation elsewhere upon genetic manipulation to introduce D27G/N mutation in the folA locus can also lead to growth differences ( see Discussion ) .", "We tested D27 mutants for growth in the presence of individual components of folAmix and their different combinations and found that only thymine was essential , although growth with thymine alone is slower compared to growth with all folAmix components ( Figure 2—figure supplement 2 ) .", "The growth rates measured for all D27 mutants at high folAmix fall in the range 70–80% of wild type and lag times were typically 1 . 5–2 fold longer ( Figure 2B ) .", "The previous observation that D27 mutant strains show severe growth defects suggests that DHFR inactivation imposes considerable homeostatic imbalance .", "We focused on the strain D27F to carry out a detailed characterization of the systems-level effects of DHFR inactivation .", "To that end we carried out high throughput proteomics analysis of D27F mutant strains using LC/MS TMT approach as described earlier ( Bershtein et al . , 2015a ) .", "The method based on differential labeling provides abundances of proteins in the proteome relative to a reference strain which in our case was wild type ( Bershtein et al . , 2015a ) .", "We computed Z-scores of the log of relative ( to wild type as reference ) abundance according to the following equation: ( 1 ) zistrain/ref=Yistrain/ref−⟨Ystrain/ref⟩σYstrain/ref , where index i refers to protein , Yistrain/ref=Log10 ( AistrainAiref ) where Aistrain and Airef are the protein i abundances obtained for the mutant and reference ( wild type ) strains , respectively , ⟨ Ystrain/ref ⟩ denotes a quantity Yistrain/ref averaged over all proteins for a given strain , and σYstrain/ref is the standard deviation of Ystrain/ref ( see Supplementary file 1 ) .", "Then , we classified the proteins by their cellular function according to Sangurdekar et al . ( 2011 ) and performed a Student t-test to determine which groups of proteins had statistically significant variation of protein levels in D27F strain , with respect to wild type ( see Supplementary file 2 ) .", "Numerous cellular processes were significantly altered , reflecting broad genome-wide effects of DHFR inactivation ( Figure 2—figure supplement 3 ) .", "Proteins involved in central processes were significantly downregulated in D27F strain , including energy metabolism ( aerobic respiration , TCA cycle ) , metabolism of nitrogen , several amino acids , pyrimidines and lipopolysaccharides .", "On the other end , we found significant increase in the expression of proteins involved in stress responses , peptidoglycan recycling and salvage of guanine and xanthine .", "We then performed both targeted and untargeted metabolomic analysis of D27F mutant strain and wild type ( see experimental details ) to characterize significant changes at the level of metabolites .", "Likewise , Z-scores for metabolite levels were computed for D27F mutant , with respect to wild type ( see Supplementary file 3 ) .", "The metabolites with the highest absolute Z-scores ( >1 . 96 ) were selected for an enrichment test using MBRole online software ( López-Ibáñez et al . , 2016 ) to identify pathways in which altered metabolites are overrepresented .", "The analysis revealed that the metabolism of purines , pyrimidines , beta-alanine , histidine and sulfur were the most significantly changed in D27F mutant comparatively to wild type ( Figure 2—figure supplement 4A–B ) .", "A detailed scheme representing the changes in metabolites and proteins levels of nucleotide synthesis pathways is shown in Figure 2C .", "Not surprisingly , we observe build-up of metabolites upstream the reactions that require reduced folate cofactors ( PurT , PurH , and ThyA ) , whereas metabolites downstream of those reactions are found to be strongly depleted .", "Overall , weaker growth and small colony phenotype show that D27 mutants are severely compromised even in presence of folAmix .", "Using the evolutionary scheme described previously ( Figure 1C ) , we allowed six replicates derived from the same colony of each mutant strain D27F/N/G to evolve in parallel for about 50 days .", "Cultures of D27G mutant strains were first grown in the presence of folAmix diluted to 0 . 1 fold with respect to the initially defined folAmix composition .", "Throughout the course of the experiment the concentration of supplement mixture further decreased ( Figure 3A ) in response to increasing fitness of the mutant strains , as imposed by the feed-back control loop discussed previously .", "Thus , the change in folAmix concentration over time reflects the effect of adaptation .", "Three trajectories ( 1 , 3 and 6 ) stand out by reaching the ability to grow in the complete absence of folAmix .", "Trajectories 2 and 5 reached a point where growth dropped below detection limit , and ultimately could not be recovered , whereas trajectory four had adapted to grow at low concentrations of folAmix .", "We hypothesized that mutations in folA locus could have restored DHFR catalytic activity in the cultures where reversion of phenotype was observed .", "Accordingly , Sanger sequencing analysis of this region revealed that in all these three trajectories ( 1 , 3 and 6 ) , but not trajectory 4 , residue Gly27 had mutated to cysteine .", "This finding was surprising because alignment of multiple known DHFR sequences shows that cysteine does not occur naturally in position 27; only aspartate or glutamate are observed in this locus ( Figure 2—figure supplement 1A ) .", "To assess whether Cys27 mutant is functional , this variant was purified and characterized in vitro , and its catalytic properties are compared with wild type and other mutants in Table 1 .", "Although stability-wise D27C mutant is similar to wild type protein , it shows much weaker catalytic efficiency , mostly in terms of high KM , retaining only about 0 . 1% kcat/KM of wild type .", "To assess if this residual catalytic function is sufficient to explain the reversion of phenotype in the evolved strain we first predicted fitness based on a previously established model ( Rodrigues et al . , 2016 ) .", "Taking as input the in vitro biophysical properties of purified D27C mutant , namely kcat/KM and bis-ANS fluorescence values , the model predicts a growth rate of about 30% of the wild type strain .", "To check the validity of this prediction , the mutation D27C was reconstituted in the wild type background by lambda red recombination and cells were plated in folAmix-containing agar plates to lift any selective pressure for DHFR activity .", "This strain was able to grow in the absence of folAmix supplementation , and the measured growth rate was 92% of the wild type , which is in fair agreement with the in vitro-based prediction which does not take into account a possibility of upregulation in response to DHFR deficiency ( Bershtein et al . , 2015a ) ( see Material and methods section Predicting fitness of D27 mutants for discussion ) ( Figure 3B–D ) .", "This analysis shows that D27C mutation alone explains a significant fraction of fitness recovery of the evolved strains .", "Of note is also the fact that the significant loss in catalytic efficiency in D27C mutant is associated with a dramatic 4 orders of magnitude increase in the inhibition constant for trimethoprim , and consequent nearly 100-fold increased resistance of the D27C strain to trimethoprim inhibition in comparison with the wild type ( Figure 3E ) .", "This is in line with previous observations that active site mutations compromise catalytic function but also disrupt pocket interactions with the drug , resulting in high levels of resistance ( Bader et al . , 2006; Rodrigues et al . , 2016 ) .", "Evolutionary trajectories of D27F and D27N mutants represented in Figure 4A and B , respectively , show a marked decrease in folAmix necessary to sustain growth , in most cases reaching concentrations nearly three orders of magnitude lower than initially required for naïve strains .", "To verify that these strains adapted to grow at low folAmix concentrations we first plated evolved cultures ( from −80 °C glycerol stocks ) in agar medium supplemented with 1x concentration of folAmix and then randomly selected individual colonies from different trajectories to measure growth at various concentrations of folAmix .", "Plating the cultures at a high folAmix concentration ensures that there is no biased selection for high-fitness phenotypes when colonies are randomly picked from agar media .", "We found that individual colonies taken among all trajectories that adapted to low folAmix concentrations all showed a strikingly similar phenotype - folAmix-dependent growth profile , irrespective of the initial variant at D27 locus ( F/N and also trajectory four in D27G ) ( Figure 4—figure supplement 1A–C ) .", "Figure 4C shows representative growth profiles obtained with colonies taken from trajectories 1 and 6 of D27F and D27N , respectively , showing a marked decrease in growth rate at concentrations of folAmix comparable to the lowest concentrations achieved during the evolution experiment .", "However , these strains cannot grow in the complete absence of folAmix .", "This result indicates that , unlike some trajectories in D27G evolution , adaptation of D27N and D27F strains did not involve reversion of the folA- phenotype .", "Accordingly , no mutations were found in folA locus in evolved D27N and D27F strains ( see Supplementary file 4 ) .", "Other mechanisms must therefore be responsible for the ability to grow at low folAmix concentrations .", "The growth rate of the evolved strains measured at high concentrations of folAmix reached values comparable to wild type , showing a clear fitness increase with respect to naïve strain .", "However , at lower concentrations of folAmix the growth rate of evolved strain becomes markedly reduced to nearly half of the wild type value and appears to plateau in an intermediate range of folAmix concentrations .", "The growth rate value at this plateau coincides with that measured for cells grown in thymine alone ( Figure 4—figure supplement 1D ) , which is indicative that thymine is the growth-limiting component at these concentrations of folAmix and that cells utilize thymine less efficiently in the absence of the other folAmix components .", "We also evaluated sensitivity to trimethoprim for both naïve and evolved D27F strains .", "Not surprisingly , in the absence of a functional DHFR and in the presence of folAmix D27F mutant strains are extremely resistant to trimethoprim ( Figure 4—figure supplement 2A ) .", "We noted , however , that while no decrease in growth was evident in naïve D27F strain up to 1000 µg/mL , the growth rate of the evolved strain dropped abruptly above 500 µg/mL trimethoprim , suggesting that the drug is acting on an unknown target in the cell which is essential in the evolved but not the naïve strain .", "We then tested how the resistance of evolved D27F strain would compare with wild type at very low concentrations folAmix ( Figure 4—figure supplement 2B ) .", "We found that in those conditions IC50 for the wild type is similar to that measured in the absence of supplement , yet the evolved D27F strain still shows an extremely high resistance to trimethoprim ( IC50 = 656 ± 78 µg/mL ) .", "Whole genome sequence ( WGS ) analysis was performed to identify the genetic basis for the adaptation mechanisms of the evolved strains .", "We analyzed naïve D27F/N/G strains and individual clones representative of adaptation to low folAmix ( evolved D27F - trajectories 1 and 2 and evolved D27N - trajectory 6 ) , and one clone representative of adaptation through DHFR reversion ( D27G- trajectory 6 ) .", "In addition , to characterize the dynamics of mutation fixation , individual colonies from intermediate evolutionary time points of D27F trajectory two were also sequenced .", "Figure 5A–B summarizes the WGS results obtained for each clone of evolved D27 and naïve strains .", "We found that D27N and D27G strains , but not D27F , had additional background mutations that must have been acquired prior to starting the evolution experiments; although all D27 strains were constructed from the same wild type E . coli parent strain , we could not prevent the appearance of mutations at any given stage of genetic manipulation prior to the evolution experiments .", "Strain D27N had a point mutation ( P74S ) in the gene nanM that encodes N-acetylneuraminate mutarotase , which supports efficient growth form sialic acid as carbon source ( Severi et al . , 2008 ) , and a point mutation ( L44Q ) in the gene thyA ( discussed below ) .", "Strain D27G had a point mutation ( A101V ) in the gene ymfD that encodes a putative SAM-dependent methyltransferase and an early stop codon in the gene upp , that encodes the enzyme uracil phosphoribosyltransferase that participates in pyrimidine salvage pathway .", "It is possible that these mutations may provide some fitness advantage with respect to ‘pure’ D27F strain ( see below and discussion ) .", "Comparison between evolved and naïve D27G strains reveals that no other mutation was fixed upon evolution besides the one nucleotide change at the D27 locus of the folA gene , corresponding to the Gly->Cys substitution described earlier .", "This is fully consistent with the earlier conclusion that D27C mutation alone explains the fitness recovery observed in D27G evolution .", "We asked if the presence of mutations in upp and ymfD genes originally found in D27G strain could have an impact on fitness of the cells , and thus could have conditioned the evolutionary fate of this strain .", "We note the potential role of the mutation in upp gene which protein product levels appeared depleted in proteomics analysis of naïve D27F strain .", "To address this , we engineered additional D27G strains and , after confirming the absence of mutations in thyA , upp and ymfD , compared their growth properties at various folAmix concentrations with D27G upp/ymfD strain that was used in the evolution experiments .", "No difference was found in the growth rate profiles of these strains ( Figure 5—figure supplement 1 ) , indicating no obvious impact of mutations in upp and ymfD in favoring the reversion of DHFR function over thyA inactivation .", "From the analysis of evolved D27F and D27N we can identify two common hotspots for mutations , the genes thyA and deoB , encoding thymidylate synthase and phosphopentomutase , which are involved in the synthesis of dTMP via de novo and salvage pathways , respectively .", "For that reason , the sequence of these two loci were determined by Sanger analysis for all other trajectories that evolved to grow at low folAmix concentrations , including trajectory four in D27G evolution .", "Strikingly , in a total of 12 trajectories all were found to have mutations in thyA and 11 had deoB mutated ( Figure 5C ) .", "We noted as well that evolved D27F strains also carried mutations in other genes , crr ( 7 bp deletion ) , rpe and yhdH ( 4 bp deletion ) , however , these were not observed among other trajectories indicating that these are most likely either random passenger mutations or provide only marginal advantage on a specific genetic background .", "On the other hand , the occurrence of deletions and deleterious point mutations ( see Supplementary file 5 ) in both thyA and deoB strongly implies that functional inactivation of these gene products arises as a main mechanism of adaptation to the lack of DHFR function .", "We reasoned that complementation with either thymidylate synthase or phosphopentomutase should create a significant fitness disadvantage to the evolved cells , which can be reverted if these enzymes become inactivated .", "To verify this prediction , we transformed evolved D27F strains with plasmids expressing either wild type ThyA or DeoB proteins and compared the ensuing growth rates of these strains with transformants carrying the same plasmids expressing functionally inactive ThyA and DeoB , respectively .", "Expression of functional ThyA and especially DeoB were found to be highly deleterious in evolved D27F strains at low folAmix concentrations , as shown Figure 5 D-E , whereas no significant change in growth was observed upon expressing inactive mutants .", "Overall , these results show two competing adaptation mechanisms to the loss of DHFR activity , either involving a reverting mutation at the D27 locus or , more prevalently , consecutive inactivation of two genes .", "The gene deoB is part of an operon responsible for deoxyribose degradation , that includes deoA , deoC , and deoD , which is under the control of 2-deoxyribose-5-phosphate-inducible deoR repressor ( Hammer-Jespersen and Munch-Ptersen , 1975 ) .", "DeoB catalyzes the interconversion of 2-Deoxy-D-ribose-1-phosphate and 2-Deoxy-D-ribose-5-phosphate , and , while the former is necessary for synthesis of dTMP from thymine by DeoA , the latter can be further degraded in glycolysis , through conversion into acetaldehyde and D-glyceraldehyde 3-phosphate by DeoC ( Figure 6A ) .", "Therefore , inactivating deoB or deoC is an ‘economy’ solution preventing key metabolite in thymine uptake to be wasted in energy production , allowing cells to efficiently use small amounts of thymine available from media supplement for nucleotide synthesis ( Dale and Greenberg , 1972 ) .", "In agreement with the key role of deoxyribose in limiting thymine uptake , while deoB+ cells cannot grow at low concentrations of thymine in the media , replacing this precursor by thymidine or dTMP , which provide a deoxyribose moiety , improves the growth of evolved D27F strains transformed with plasmid expressing active DeoB ( Figures 6B-C ) .", "Moreover , to confirm that the route for deoxyribose degradation into energy production is blocked in evolved mutants , we measured the growth of naïve and evolved cells in the presence of thymidine as the sole carbon source and verified that deoB- cells cannot grow unless complemented with a plasmid expressing wild type DeoB ( Figure 6D ) .", "Next , we carried out LC/MS TMT proteomics analysis of evolved strains ( D27F , D27N and D27G ) to help identify systems-level changes emerging from adaptation to lack of DHFR function .", "Since we observed that all clones randomly selected from trajectories that adapted to low folAmix concentrations showed very similar fitness profiles , we focused on individual clones arbitrarily chosen among D27F and D27N trajectories as representatives of adaptation to low folAmix .", "Specifically , clone F51T1#1 from D27F trajectory one and clone N51T6#1 from D27N trajectory six were selected .", "Likewise , clone G33T6#1 from D27G trajectory six was chosen as representative of adaptation through reversion of DHFR function .", "To get a glimpse of global proteomics changes we applied PCA analysis which revealed that both wild type and evolved D27G occupy the same quadrant in the space of two principal components , whereas D27 mutants that are folAmix dependent cluster more closely in another quadrant ( Figure 7—figure supplement 1A ) .", "We then computed the Z-scores for the proteomic changes in evolved strains with respect to naïve D27F ( ZD27_evo/D27F_naïve ) , to assess the effect of evolution on the proteomic levels and plotted these values against ZD27F_naïve/WT to compare with the initial changes caused by D27F mutation ( see also Supplementary file 1 and Supplementary file 2 ) .", "A clear anticorrelation was observed for all mutants ( Figure 7A ) , being the strongest for evolved D27G strain .", "These results indicate that adaptation leads to proteomic changes that generally oppose the immediate effects caused by DHFR inactivation .", "In the case of D27G , the strong global proteomic shift towards wild type levels is somewhat expected since in the evolved strain , the DHFR catalytic activity is partly restored by G27- > C mutation .", "To better characterize the proteomic changes that were specific to evolution of folAmix dependence we searched for classes of proteins grouped by function with significantly altered protein abundance levels in both evolved D27F and D27N with respect to naïve D27F strains ( Figure 7—figure supplement 1B ) , as described previously .", "We found a significant increase in the expression levels of proteins involved in glycolysis , fermentation , sugar alcohol degradation and response to low pH , suggesting a metabolic shift towards mixed acid fermentation as a result of adaptation that blocked using derivative of thymidine for glycolysis to save them for DNA synthesis .", "On the other hand , the levels of proteins involved in DNA restriction/methylation and D-ribose uptake were significantly decreased with respect to naïve D27F in both evolved D27F and D27N strains .", "Next , we focused on evolved D27F strain to perform a detailed metabolomic analysis and characterize the changes occurring at the level of metabolites ( see also Supplementary file 3 ) .", "We computed Z-scores for metabolite changes with respect to naïve D27F ( ZD27_evo/D27F_naïve ) and we found significant anticorrelation with Z-scores obtained for naïve D27F ( ZD27F_naïve/WT ) ( Figure 7B ) , indicating that metabolite concentrations in evolved strain generally change to partially recover wild type levels , as observed with proteomics .", "We found that the pathways significantly enriched in metabolites with the highest ZD27_evo/D27F_naïve scores ( >1 . 96 ) in evolved D27F were the same as those that had the most significant changes in naïve D27F , with respect to wild type ( Figure 2—figure supplement 4A–B ) .", "Overall these results show that adaptation to low folAmix concentrations is accompanied by an overall shift in the abundance of metabolites and proteins to partially revert the system-level changes that are caused by DHFR inactivation .", "Metabolites of purine and pyrimidine biosynthetic pathways were among the most strongly depleted in naïve D27F strain and showed highest increase upon evolution ( Figure 7C , D and Figure 2—figure supplement 4A–B ) .", "We reasoned that these depleted metabolites could be limiting growth of the D27 mutants , and that supplementing the culture medium with these metabolites would improve growth .", "Surprisingly , supplementation with purines strongly inhibited growth of the naïve D27F strain , whereas pyrimidines addition had , at most , a slight stimulatory effect ( Figure 7E ) .", "The detrimental effect of individual purine supplementation appears to be associated with regulatory responses and/or with toxic effects caused by metabolite imbalance ( e . g . purines vs pyrimidines ) upon supplementation .", "In the case of evolved D27F strain , the effect of supplementation of individual nucleotides on growth was comparably weaker or non-existent , indicating that the inhibitory effects were relaxed upon adaptation .", "Since we found no mutations directly associated with regulatory genes , it seems that the observed changes result solely from the re-wiring of metabolic pathways .", "In this regard our examples are particularly noteworthy .", "Evolution of D27F and D27N strains blocked supplementary glycolysis pathway by rerouting Deoxyribose towards DNA synthesis while the ensuing metabolic changes lead to significant increase in abundance of glycolysis proteins , presumably to compensate for diminished flux of sugar metabolites along the glycolysis pathway ( see Supplementary file 2 ) .", "Another example of metabolite-induced rewiring is purR operon whereby purR is inhibited by purine hypoxanthine .", "Evolved strains show significant increase in abundance of hypoxanthine giving rise to significant drop in abundance of proteins controlled by purR ( see Supplementary file 2 ) .", "Overall , these results show that two key loss of function mutations that are responsible for the partial adaptation of D27F to loss of DHFR activity result in significant metabolic , proteomic and regulatory shifts observed in the evolved strains ." ], [ "In this study we set to explore the evolutionary mechanisms that follow inactivation of the essential gene that encodes DHFR .", "Such scenario is akin to antibiotic-mediated target inhibition , but distinct in that there is no selection for mutations that affect interactions with the drug , such as target modification and drug efflux mechanisms .", "For example , when treated with DHFR inhibitor trimethoprim , bacterial cells recurrently acquire resistance by high-fitness mutations near the active site of the target protein that decrease drug binding affinity , even at the expense of catalytic efficiency ( Rodrigues et al . , 2016; Tamer et al . , 2019; Toprak et al . , 2012 ) .", "In contrast , a genetic perturbation in the same target gene provides the opportunity to study other potential unexplored mechanisms of adaptation in the absence of drug and evaluate their impact in the context of antibiotic resistance .", "We found that , in the conditions studied here , evolution is constrained towards two solutions that appear to be mutually exclusive , either ( partial ) restoration of DHFR catalytic activity or adaptation to lack of DHFR function .", "While D27F , D27N and one trajectory in D27G convergently adapted to lack of DHFR function , other trajectories in D27G mutant reached reversion from thymine auxothroph phenotype .", "It is important to address the possible reasons for the observed outcome .", "A potentially influential factor could be the presence of background mutations observed in both D27N and D27G .", "The point mutation L44Q in thyA , reported to affect the activity of a nearly identical orthologue enzyme ( Nur-E-Kamal et al . , 1994 ) , could have skewed the solution towards further inactivation of thyA by subsequent point mutations in other loci of that gene that were later observed in different trajectories .", "On the other hand , mutations in the genes upp and ymfD found in D27G strains did not impact the growth rate , thus the role of these mutations in altering the accessibility of the DHFR functional reversion G27- > C seems to be neutral .", "The codon structure of each mutant studied here and bias in the frequency of each mutation type can also affect the likelihood that phenotype-reverting mutations may arise .", "We note that the nearest accessible high-fitness Asp/Glu codon for D27F mutant is two nucleotide substitutions away ( although one nucleotide away from one D27C codon ) , whereas both D27G and D27N could be rescued by a single G->A or A->G transition , respectively .", "Only D27G was found to revert , however , not to wild type codon , but to a less catalytically efficient cysteine residue caused by a G->T transversion .", "In this respect , it is rather surprising that D27C mutation was fixed in three trajectories when transversions are generally regarded to be less likely than transitions .", "That this mutant was able to fix , despite its poor enzymatic activity , brings important insights on how deleterious mutations can be maintained when the selection pressure on protein function is alleviated by particular environmental conditions , in this case the presence of folAmix , and may provide a rapid route to the development of resistance .", "In addition , these results may hint at a possible mechanism for the divergence of the key residues in the active sites of enzymes across long evolutionary timescales , where fixation of non-consensus mutations , such as D27C , may be allowed by transient adaptation to permissive environments , and subsequent compensatory mutations that restore protein function at later stages .", "While the selection pressure regime used in our study was solely based on the feedback loop strategy to control the concentration of folAmix , it remains to be explored how the implementation of alternative selection protocols could influence the course of evolution .", "Nonetheless , it appears that the system studied in this work provides an excellent framework for future theoretical and experimental studies to evaluate the role of environmental pressure dynamics in shaping evolution .", "The most crucial factor in determining the fate of D27 mutant evolution appears to be the higher accessibility of mutational routes for inactivating thyA that lead to their fixation very early in the evolution experiment .", "Thymidylate synthase is the only known enzyme in E . coli able to synthetize dTMP de novo from dUMP , and inactivation of thyA inevitably commits the cell to thymine auxothrophy; reversion of DHFR function on the thyA- background is not expected to change this phenotype , as it is known that folA+ thyA- strains are thymine-dependent ( Bertino and Stacey , 1966; Schober et al . , 2019 ) .", "Competition between DHFR reversion and thyA inactivation thus appears to be decisive in determining the evolutionary solution .", "However , since inactivation of a gene can be achieved by multiple ways , there is greater number of accessible evolutionary trajectories leading to thyA knockout than to reversion of DHFR function .", "This view has major implications for our understanding of how the relative impact of height and accessibility of fitness peaks shapes the evolutionary dynamics short-term adaptation .", "Upon thyA inactivation , the only available route for dTMP synthesis involves DeoA-catalyzed formation of thymidine from thymine and 2-deoxy-D-ribose-1-phosphate ( Figure 6F ) .", "However , DeoA is part of the deo operon that is induced by high levels of 2-deoxy-D-ribose-5-phosphate .", "This regulatory scheme allows cells to recycle excess metabolites into energy production .", "However , on the background of thyA inactivation , the co-expression of deo operon proteins creates simultaneously a solution and a problem for the uptake of thymine .", "Now DeoA becomes an essential protein , but its function is opposed by the roles of DeoC and DeoB which divert 2-Deoxy-D-ribose-1-phosphate into degradation via the glycolysis pathway .", "The evolutionary solution found in D27 strains converged to the inactivation of a single gene of the operon , deoB , an event that is sufficient to block the degradation pathway without disrupting the regulatory structure .", "This critical example illustrates how evolution can be constrained by regulatory circuits that provide conflicting responses when the optimal directionality of metabolic routes is switched due to genetic perturbations .", "Understanding these processes is critical for guidance towards new strategies for antibiotic resistance prevention .", "Perhaps not surprisingly , we found that pathways of adaptation to genetic inactivation of an antibiotic target provide a route to high levels of resistance .", "Likewise , a recent study also reported that E . coli cells challenged with increasing concentrations of trimethoprim in the presence of thymidine repeatedly evolved high levels of resistance by thyA loss-of-function mutations besides acquisition of commonly observed resistant mutations in DHFR ( Schober et al . , 2019 ) .", "Strikingly , mutations in thyA are also known to confer trimethoprim resistance in bacterial clinical isolates of S . aureus ( Chatterjee et al . , 2008; Kriegeskorte et al . , 2014 ) , and H . influenza ( Rodríguez-Arce et al . , 2017 ) , which emphasizes the need of laboratory studies of microbial adaptation as useful models to tackle serious global threat .", "More generally this study highlights the evolutionary conundrum between ‘survival of the fittest’ and ‘survival of the fastest’ .", "Unlike previous studies that followed evolution of adaptation to gene knockouts , the current setup provides an ‘easy’ highest fitness solution by genetic reversion of single amino acid substitution .", "However , for most evolutionary trajectories the dynamics quickly converged to a ‘quick and dirty’ solution – a dead-end local fitness peak which provided an immediate relief from stress but blocked the path to highest fitness peak .", "Future studies will reveal how general this scenario is for evolutionary dynamics of adaptation to mutational inactivation and/or antibiotic induced inhibition of essential bacterial enzymes and beyond ." ], [ "A portion of chromosomal apaH:apaG genes was amplified by PCR using primers P4_chrom_flanking_for and PCRseq_apaH_rev .", "Then the pKD13 plasmid was linearized by PCR using the primers P4_chrom_flanking_for and PCRseq_apaH_rev .", "Both PCR products were purified and combined in a new PCR reaction using phosphorylated primers pKD13_post_downstream_for and PCRseq_apaH_rev .", "The linear product was ligated using T4 ligase to make plasmid Plasmid pKD13-cmR-folA ( wt ) -kanR-apaH-apaG .", "Then a portion of kefC gene was amplified by PCR using primers PCRseq_KefC_for2 and CapR-Chrom-Flanking rev . Then the plasmid pKD13-cmR-folA ( wt ) -kanR-apaH-apaG was linearized by PCR using primers Upstream_capR_for and pKD13_post_upstream_rev .", "Both products were combined in a new PCR reaction using phosphorylated primers PCRseq_KefC_for2 and pKD13_post_upstream_rev .", "Finally , the linear product was ligated using T4 ligase to make pKD13-kefC ( 897–1863 ) -cmR-folA ( wt ) -kanR-apaH-apaG .", "Plasmid pKD13-kefC ( 897–1863 ) -cmR-folA ( wt ) -kanR-apaH-apaG was linearized using phosphorylated mutation primers D27Xmut_For and primer D27_rev , and ligated using T4 Ligase .", "Inactive D27 mutants were created by lambda-red recombination ( Datsenko and Wanner , 2000 ) according to a previously described procedure ( Bershtein et al . , 2012 ) with some modifications .", "Briefly , a pKD13 plasmid was modified to contain the entire regulatory and coding sequence of folA gene , flanked by two different antibiotic markers ( genes encoding kanamycin ( kanR ) and chloramphenicol ( cmR ) resistances ) and approximately 1 kb homologous region of both upstream and downstream chromosomal genes flanking folA gene ( kefC and apaH , respectively ) .", "A list of relevant primers ais shown in Supplementary file 6 .", "The entire cassette was amplified using primers PCRseq_KefC_for2 and PCRseq_apaH_rev and transformed into BW25113 cells with induced Red helper plasmid pKD46 , and cells were recovered in SOC medium containing 1x folAmix ( adenine 20 ug/mL , inosine 80 ug/mL , thymine 200 ug/mL , methionine 20 ug/mL and glycine 20 ug/mL ) .", "Transformants were plated in agar media containing both antibiotics and folAmix .", "To confirm correct integration of the desired mutations , folA locus was amplified by PCR ( primers Ampl_RRfolA_for , Ampl_RRfolA_rev ) and Sanger-sequenced using primer PCRseq_RRfolA_rev .", "Plasmid pKD46 was removed by plating cells at 37°C twice in the absence of antibiotic selection .", "Prior to starting the evolution experiments , the cultures had gone through three bottlenecks that involved randomly picking single colonies from agar plates for the subsequent step required for genetic manipulation .", "A liquid handling instrument Tecan Freedom Evo 150 equipped with Tecan Infinite M200 Pro plate reader and Liconic shaker was used in this work .", "The experiments were done at 30 °C and using M9 minimal media supplemented with 2 g/L glucose , 34 µg/mL chloramphenicol and 50 µg/mL kanamycin .", "The liquid handling worktable has two 100 mL troughs ( Tecan ) with either fresh M9 medium + antibiotics or 40% glycerol .", "In addition , solutions of folAmix at various concentrations ( prepared in M9 + antibiotics ) are provided in 50 mL falcon tubes .", "The general procedure involves up to four 96-well plates that are used for serial dilutions of bacterial cultures .", "Each working plate can carry eight trajectories that are positioned in a single column .", "In this work we used the first and eight wells in the column with media alone to control for contamination .", "The experiment starts by placing the 200 µL cultures/control in the first column of the 96-well plate .", "All the four plates are incubated in a shaker at 30 °C and at every 30 min the OD of each plate is determined alternately .", "The growth rate of each culture is calculated from OD measurements over time .", "To ensure proper comparison , the growth rate was computed only from OD values within a specified range ( 0 . 1–0 . 25 ) .", "When the average OD of the six experimental replicates in a plate exceeds a threshold of 0 . 30 each culture is diluted into the next adjacent column by mixing a calculated volume of both culture and fresh medium and folAmix so that the initial OD is 0 . 01 in a total of 200 µL .", "At this point , the remaining portion of the previous culture is mixed with glycerol in an auxiliary plate and subsequently frozen at −80 °C .", "The very first cultures to be frozen at −80 °C ( at passage zero ) , are considered to be the naïve strains , and referred as such in the text to contrast with evolved strains which are picked at later passages .", "The cycle is repeated throughout the entire experiment .", "At every passage , the growth rate is compared with a threshold value ( 0 . 16 h−1 ) and whenever a culture exceeds this value the concentration of folAmix is halved .", "The volume of folAmix added in each passage is 10 , 5 or 2 . 5 µL .", "Cell cultures were recovered by inoculating fresh M9 media medium supplemented with 0 . 2% glucose and 1x folAmix with a portion of −80 °C glycerol stocks taken at different passages .", "After recovery for several hours , the cultures were plated in agar media containing 1x folAmix , 34 µg/mL chloramphenicol and 50 µg/mL kanamycin and incubated at 30 °C .", "Plating evolved cultures at high folAmix concentrations ensures that the clones growing in agar media are not biased towards high fitness phenotypes .", "Individual colonies were randomly picked , grown in M9-media+folA mix and stored in glycerol at −80 °C for later analysis .", "Clones selected in this manner were labeled using the following mask: XdTt#i , where X refers to the amino acid letter in D27 locus ( F/N/G ) , d is day at which the culture was passaged in the evolution experiment , t is the trajectory number ( 1-6 ) and i is the unique number of different colonies taken from the same culture .", "Cell cultures grown overnight were diluted in fresh M9 minimal media containing 1x folAmix and antibiotics and were grown for additional 4–6 hr .", "Cultures were then pelleted by centrifugation and washed three times in fresh M9 without folAmix .", "Microplates containing 150 µL of M9 minimal with 0 . 8 g/L glucose , antibiotics , and varying concentrations of folAmix were then inoculated with each culture at a starting OD of 0 . 0005 .", "Growth measurements were performed in a Infinite 200 PRO plate reader for 48 hr at 30 °C with constant shaking .", "Growth rate values are represented as mean ± SD from at least three biological replicates .", "D27 mutant fused to C-terminal ( 6x ) His-tag were overexpressed using pFLAG expression vector under isopropyl β-D-1-thiogalactopyranoside ( IPTG ) inducible T7 promoter .", "The recombinant proteins were purified from lysates on Ni-NTA columns ( Qiagen ) as described previously ( Rodrigues et al . , 2016 ) .", "DHFR kinetic parameters were derived from the analysis of progress-curve kinetics of NADPH oxidation in the presence of different concentrations dihydrofolate using the software Kintek Explorer ( Johnson , 2009 ) as described before ( Rodrigues et al . , 2016 ) .", "The data correspond to the mean ± S . E . of at least three measurements .", "DHFR protein solutions ( 2 μM ) in the presence of 12 μM of bis-ANS were prepared in 50 mM phosphate and 1 mM DTT at pH 7 . 0 and placed in a 1 cm path-length quartz cuvette .", "The samples were equilibrated for 5 min at 37°C and the fluorescence emission spectra between 460 and 600 nm were recorded upon excitation at 395 nm .", "The emission band was integrated and the background of bis-ANS fluorescence in the absence of protein was subtracted .", "The area of the band was normalized to wild type E . coli .", "DHFR .", "The data corresponds to the mean ± S . E . of three measurements .", "DHFR solutions ( 5 μM ) were prepared in 50 mM phosphate buffer and 1 mM DTT at pH 7 . 0 in the presence of 100 μM NADPH .", "A temperature ramp of 1 °C/min was set between 25°C and 90°C , and the tryptophan fluorescence was recorded by measuring the intensity at 370 and 320 nm upon excitation at 280 nm .", "Thermal denaturation curves were also performed in the presence of 5x concentration of Sypro Orange and 20 μM .", "Melting temperature and confidence intervals at 95% were determined from the simultaneous analysis of the thermal denaturation curves obtained with both tryptophan and Sypro Orange fluorescence using the online software Calfitter ( Mazurenko et al . , 2018 ) .", "Cells from 14 mL cultures grown at 30°C at an OD of 0 . 5 were pelleted by centrifugation and lysed with 200 µL of lysis buffer containing 1 × Pop Culture reagent ( Merck Millipore ) , 100 mM MES ( 2- ( N-morpholino ) ethanesulfonic acid ) at pH 7 . 0 in the presence of 1 mM DTT in the presence of 1 × complete protease inhibitor mixture ( Roche ) and 1 mg/mL lysozyme and incubated for 20 min at room temperature .", "The lysate was sonicated with 10 pulses of 1 s , cleared by centrifugation and then 85 µL of the soluble fraction was transferred to 96-well white plates for total activity measurements using a total assay volume of 100 μL .", "The lysate was preincubated with 100 μM NADPH and the reaction was started with the addition of 100 μM dihydrofolate and mixing .", "The decrease in fluorescence ( excitation at 300 nm and emission at 440 nm ) was measured over 60 min at 25°C , and the initial velocities were computed .", "Measurements were also done in the presence of 1 mM TMP to control for the non-DHFR-specific reaction .", "Data corresponds to the mean ± S . E . of three measurements .", "The fitness of E . coli DHFR mutants can be predicted from in vitro biophysical parameters using a simple metabolic model described in an earlier work ( Rodrigues et al . , 2016 ) .", "The metabolic flux through DHFR reaction in DHFR mutant strains , normalized to wild type , can be approximated to: ( 2 ) Vdhfrnorm=kcatmutKMmut⋅[ DHFR ]mutkcatEcoliWTKMEcoliWT⋅[ DHFR ]WT⋅1 ( 1+ α⋅[ TMP ]mediumKimut ) Where , kcatKM , Ki and [ DHFR ] are , respectively , the catalytic efficiency , trimethoprim inhibition constant and intracellular protein abundance of the mutant or of the wild type , and [ TMP ]medium and α are , respectively , the concentration of trimethoprim in the culture medium and the ratio of intracellular vs extracellular concentrations of trimethoprim , defined previously to be 0 . 1 ( Rodrigues et al . , 2016 ) .", "To connect flux to fitness , we first measure the growth rate of wild type E . coli cells at various concentrations of DHFR inhibitor trimethoprim .", "Then fitness is plotted against the flux through DHFR reaction calculated at each concentration of inhibitor using Equation 2 , and catalytic parameters determined in vitro ( Table 1 ) .", "Protein abundance is determined by measuring the total catalytic activity in cell lysates , as described earlier ( Rodrigues et al . , 2016 ) .", "Finally , we use Equation 3: ( 3 ) Grnorm~Vdhfrnorm ( B+Vdhfrnorm ) to fit the fitness vs Vnorm data points to determine the parameter B , which represents the normalized flux at which growth rate is reduced by half .", "From this equation , the in vitro parameters determined for D27 mutants are used in Equation 2 and 3 to predict fitness .", "The protein abundance of D27 mutants , however , cannot be determined from enzymatic assays in cell lysates , as these are inactive variants .", "Instead , protein abundance was predicted using an inverse relationship with relative bis-ANS fluorescence of those variants ( Bershtein et al . , 2013; Rodrigues et al . , 2016 ) .", "In qualitative terms , our model accurately predicted that the D27C variant is able to grow in the absence of folAmix supplement .", "However , the measured fitness was somewhat higher than the value predicted by the calculations .", "It is possible that some assumptions are not entirely met .", "One important assumption is that the concentration of DHFR and substrate dihydrofolate are either always constant or change only as a function of the flux calculated by Equation 2 .", "The latter implies that , for every given value of flux , the changes in DHFR and dihydrofolate concentration will always be the same , and thus always comparable regardless of what parameters are used as input in Equation 2 .", "However , this might not be always true .", "For example , a tight feedback control regulates the DHFR promoter activity , in response to the metabolic needs of the cell ( Bershtein et al . , 2015a; Bershtein et al . , 2015b ) .", "Likewise , a decrease in DHFR catalytic function is also expected to result in a considerable level of substrate build up .", "In these conditions , the magnitude of substrate KM for each variant might become more relevant .", "In our calculations , however , substrate saturation is not considered .", "This may lead to deviations , especially when KM values differ significantly , as we find in this work .", "How much these dynamic processes may differently affect the flux through DHFR reaction in each mutant is still to be determined .", "The genes deoB and thyA and corresponding mutants deoB p . 309Stop and thyA 755-762del were cloned into a modified pTRC plasmid in which the laqI and promoter region were replaced by the tetR repressor gene and promotor region derived from plasmid Plasmid pEM-Cas9HF1-recA56 ( addgene Addgene plasmid # 89962; Moreb et al . , 2017 ) .", "Transformed strain cells were grown in M9 minimal media + folAmix , then washed with fresh M9 media without folAmix and plated at different concentration of folAmix .", "Growth rate values are the mean ± SD of at least three biological replicates .", "Cultures of mutant strains ( naïve and evolved D27F ) and wild type ( 30 mL ) were grown in parallel in M9 minimal media supplemented with 2 g/L glucose in a 250 mL flask at 30C .", "At an OD of 0 . 2–0 . 25 the cultures were centrifuged .", "The volume of culture to be pelleted was calculated so that the product OD ×volume of cell culture ( mL ) = 5 .", "The pellet was mixed with 300 µl of 80:20 ratio of methanol:water that had been pre-chilled on dry ice .", "Samples were vortexed and incubated in dry ice for 10 min followed by centrifugation at 4 °C for 10 min at maximum speed .", "The supernatant was collected , and the pellet was processed by repeating the procedure .", "Samples were stored at −80 °C until analyzed by mass spectrometry .", "At least three independent biological replicates were analyzed for each strain .", "LC-MS analysis in the positive and negative mode was performed as previously described ( Bhattacharyya et al . , 2016 ) .", "Retention times of several metabolites of the nucleotide biosynthesis pathway were determined from the analysis of pure compounds .", "Cell cultures from isolated colonies were grown at 30 °C in M9 minimal media supplemented with 0 . 2% glucose and were collected by centrifugation during mid-exponential phase ( OD ~0 . 2–0 . 25 ) , well below saturation due to oxygen limitation ( typically at OD >0 . 8–0 . 9 ) .", "Global proteomic analysis was performed as described previously ( Bershtein et al . , 2015a ) .", "Sequencing was performed on isolated colonies on Illumina MiSeq in 2 × 150 bp paired-end configuration ( Genewiz , Inc , South Plainfield , NJ ) .", "The raw data were processed with the with the breseq pipeline ( Deatherage et al . , 2014 ) on default settings , using the E . coli K-12 substr .", "BW25113 reference genome ( GenBank accession no . CP009273 . 1 ) .", "The experiment accession numbers for the raw reads deposit are as follows: Naïve D27F: SRX6873308 , evolved F15T2#1: SRX6873309 , evolved F22T2#1: SRX6873310 , evolved F27T2#1: SRX6873311 , evolved F32T2#1: SRX6873312 , evolved F51T1#1: SRX6873313 , naïve D27N: SRX6873314 , evolved N51T6#1: SRX6873315 , naïve D27G: SRX6873316 , Evolved G33T6#1: SRX6873317 .", "The BioProject accession number for the whole project is PRJNA566398 .", "Data analysis was performed using the software packages MzMatch ( Scheltema et al . , 2011 ) and IDEOM ( Creek et al . , 2012 ) for untargeted analysis .", "For peak assignment in untargeted analysis , IDEOM includes both peak m/z values and predicted retention times calculated based on chemical descriptors ( Creek et al . , 2011 ) .", "A list of 32 experimentally measured retention times was initially used to calibrate retention time predictions .", "The retention time of putatively identified metabolites were found to correlate fairly well with the values included in IDEOM ( R2 = 0 . 73 ) and published in other studies ( Pluskal et al . , 2010 ) ( R2 = 0 . 88 and R2 = 0 . 61 , respectively ) ee .", "For this reason , additional metabolites from those sources that closely matched IDEOM assignments were treated as standards in the identification routine .", "Following identification , Z-scores with respect to wild type ( ZD27F_naïve/WT ) or to naïve D27F strain ( ZD27F_evo/D27F_ naïve ) were calculated using Equation 1 .", "Z-scores determined for each set i of n biological replicates were combined for each metabolite met using the expression zcombinedmet=∑inzimetn .", "The set of metabolites with highest absolute combined Z-scores ( >1 . 96 ) was selected to perform an overrepresentation ( enrichment ) analysis of categorical annotations using MBrole2 . 0 ( López-Ibáñez et al . , 2016 ) , using as reference the metabolic pathways of E . coli from KEGG database .", "Z-scores were computed using Equation 1 .", "For grouping analysis , we used the functional and regulatory classification group sets as ( Sangurdekar et al . , 2011 ) .", "For each set of genes belonging to a group we employed a one-sample t-test which provides the p-value against the null hypothesis that the group of genes was drawn from a normal distribution and considered that a given group of genes is upregulated or downregulated with respect to a reference if the average of Z-scores of that group is positive or negative , respectively ." ] ]

[ "The mechanisms of adaptation to inactivation of essential genes remain unknown .", "Here we inactivate E . coli dihydrofolate reductase ( DHFR ) by introducing D27G , N , F chromosomal mutations in a key catalytic residue with subsequent adaptation by an automated serial transfer protocol .", "The partial reversal G27- > C occurred in three evolutionary trajectories .", "Conversely , in one trajectory for D27G and in all trajectories for D27F , N strains adapted to grow at very low metabolic supplement ( folAmix ) concentrations but did not escape entirely from supplement auxotrophy .", "Major global shifts in metabolome and proteome occurred upon DHFR inactivation , which were partially reversed in adapted strains .", "Loss-of-function mutations in two genes , thyA and deoB , ensured adaptation to low folAmix by rerouting the 2-Deoxy-D-ribose-phosphate metabolism from glycolysis towards synthesis of dTMP .", "Multiple evolutionary pathways of adaptation converged to a suboptimal solution due to the high accessibility to loss-of-function mutations that block the path to the highest , yet least accessible , fitness peak ." ]

[ "Predicting how viruses and bacteria evolve remains a challenge .", "The ability to anticipate when and how bacteria might develop drug resistance would make treating life-threatening diseases easier and could potentially help prevent drug resistance altogether .", "Studying bacterial evolution under different conditions and cataloguing all possible DNA mutations that allow these bacteria to survive are crucial steps in predicting the appearance of drug resistance .", "Studies have revealed that bacteria can adapt to sources of stress , such as antibiotics , in different ways – each involving mutations in distinct genes .", "However , not all the mutations provide the same benefits to the organism , and the factors that influence how bacteria will adapt are unclear .", "Now , Rodrigues and Shakhnovich have used a new approach to push the adaptation ability of the bacterium Escherichia coli to the limit .", "First , they genetically engineered different E . coli strains by introducing distinct mutations to an enzyme the bacterium needs to make DNA .", "These mutations make the resulting strains dependent on external molecules to synthesize new DNA .", "Next , the cells were grown in conditions where the supply of these DNA precursors was progressively decreased , forcing them to adapt .", "The obvious way for bacteria to adapt to these conditions would be to ‘revert’ the mutations that Rodrigues and Shakhnovich introduced in the first place .", "By using this approach , Rodrigues and Shakhnovich were able to test how often the obvious evolutionary solution happens compared with presumably less-preferred alternative routes .", "In rare cases , a specific mutation did restore the activity of the enzyme that enabled DNA synthesis .", "However , in most cases the bacteria found a different evolutionary solution whereby they all adapt to the decrease in external DNA precursors in the same way , but not by reverting the original mutation .", "Instead , further mutations disrupt the activity of two metabolic genes , allowing the cells to use the external DNA precursors more efficiently , and keep building DNA .", "These cells are therefore able to survive even when the levels of the external DNA components are very low , but they will die in the complete absence of these precursor molecules .", "This evolutionary solution leads to a non-optimal effect: mutations that restore the activity of the original enzyme , which are the best solution when the two metabolic genes are intact , are no longer as effective .", "This finding represents a clear example of interactions between genes determining evolutionary outcomes .", "Rodrigues and Shakhnovich showed that , since it is more likely for a random mutation to disrupt a gene than to revert a previous mutation , adaptations that are less-than-optimal but still work might predominate simply because they happen faster .", "Understanding why certain evolutionary adaptations prevail is an important step in predicting evolution and may lead to breakthroughs in many areas .", "For example , if scientists can identify mutations likely to make bacteria resistant to drugs , it may be possible to act proactively against the bacterial strains that carry those mutations .", "Eventually , if the factors that lead to specific adaptations are known , it may be possible to exploit this knowledge to create weaknesses in the bacteria’s own defences ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "microbiology and infectious disease" ]

[ [ "N6-methyladenosine ( m6A ) is the most prevalent internal modification of eukaryotic messenger RNAs ( mRNAs ) .", "Despite the identification of this modification over four decades ago , only recently have major breakthroughs in our understanding of this modification been made due to the development of m6A-seq , which allows immunoprecipitation of fragmented m6A-modified RNAs followed by next-generation sequencing ( NGS ) analysis .", "m6A-seq and subsequent enhanced versions of the technique led to transcriptome-wide maps of cellular m6A modification ( Dominissini et al . , 2012; Meyer et al . , 2012; Patil et al . , 2016 ) .", "m6A writers have also been identified , a catalytically active methyl-transferase , METTL3 ( Wang et al . , 2016 ) , together with adapter components , catalyses m6A addition onto specific RNA sequences containing the DRm6ACH motif , where D = A , G or U; R = A or G; H = A , C or U . The RNA demethylases FTO ( Jia et al . , 2011 ) and ALKBH5 ( Zheng et al . , 2013 ) can erase m6A marks offering a dynamic regulation of m6A status in RNA .", "Importantly , a family of effector m6A readers have also been identified comprising five YT521-B homology ( YTH ) domain-containing proteins .", "YTH readers directly bind m6A in target RNAs through an aromatic cage ( Liao et al . , 2018 ) , directing their targets towards different biological fates .", "YTHDF2 recruits the CCR4-NOT deadenylase complex and promotes degradation of m6A-containing RNAs ( Du et al . , 2016; Wang et al . , 2014 ) while YTHDF1 , YTHDF3 and YTHDC2 stimulate mRNA translation of their targets ( Wang et al . , 2015; Shi et al . , 2017; Hsu et al . , 2017 ) and YTHDC1 regulates mRNA splicing ( Xiao et al . , 2016 ) .", "Whether other proteins can directly recognise m6A has remained a question to date .", "Other RNA-binding proteins can read m6A indirectly , in a so-called ‘m6A switch’ mechanism , in which m6A destabilises the local RNA structure in hairpins allowing recruitment of either hnRNPC to U-tracts ( Liu et al . , 2015 ) or hnRNPG to a purine-rich motif ( Liu et al . , 2017 ) , both readers then regulate alternative splicing .", "Recently , IGF2BP proteins were reported to enhance mRNA stability and translation of m6A-containing RNAs and proposed to be a novel class of m6A readers ( Huang et al . , 2018 ) .", "Due to recent transcriptome-wide m6A mapping of multiple viruses ( Kennedy et al . , 2016; Lichinchi et al . , 2016a; Tirumuru et al . , 2016; Gokhale et al . , 2016; Lichinchi et al . , 2016b; Courtney et al . , 2017; Tsai et al . , 2018; Hesser et al . , 2018; Tan et al . , 2018 ) , it is becoming evident that there is an interplay between m6A-decorated viral RNA and cellular m6A machinery , resulting in modulation of viral replication output .", "Kaposi’s sarcoma-associated herpesvirus ( KSHV ) is a DNA virus responsible for several Acquired Immuno Deficiency Syndrome-associated aggressive malignancies ( Baquero-Pérez and Whitehouse , 2015; Schumann et al . , 2017 ) .", "KSHV has a biphasic life cycle , with a latent phase and a productive lytic phase .", "During latency , the KSHV episome is dormant in the nucleus of endothelial progenitor cells and B-lymphocytes ( Giffin and Damania , 2014 ) , with few viral latent genes expressed .", "Under various stimuli , the KSHV episome can be reactivated into lytic replication , leading to the ordered expression of more than 80 viral proteins .", "The replication and transcription activator ( RTA ) viral protein , which is encoded from open reading frame 50 ( ORF50 ) , is the first lytic protein produced and is essential for the latent-lytic switch ( Guito and Lukac , 2012 ) .", "Here we accurately decipher the KSHV m6A epitranscriptome using a novel m6A peak-calling algorithm and characterise m6A readers that specifically interact with KSHV m6A-modified RNA sequences found in ORF50 and ORF37 .", "Through the use of RNA affinity , in addition to YTH readers , eight members from the Tudor domain ‘Royal family’ , including SND1 ( Staphylococcal nuclease domain-containing protein 1 ) , were specifically enriched in m6A-modified KSHV sequences .", "The ‘Royal family’ is a well-characterised family that reads methylated residues in histones through the use of an aromatic cage ( Chen et al . , 2011 ) which is structurally similar to the one found in YTH readers ( Luo and Tong , 2014 ) .", "Electromobility shift assays ( EMSAs ) demonstrate the ability of specific Royal domains to selectively bind m6A-modified RNA .", "As these domains do not bind all m6A-modified RNA sequences it suggests this binding may occur in a RNA secondary structure/sequence-dependent manner .", "We further developed a modified RIP-seq technique , which significantly improves the resolution of standard RIP , accompanied by a unique bioinformatic analysis to characterise for the first time the transcriptome-wide binding profile of SND1 to cellular and KSHV mRNAs , revealing SND1 as a bona fide RNA-binding protein that targets m6A-modified RNAs in KSHV-infected cells , including the extensively m6A-modified ORF50 RNA .", "SND1 eCLIP ( enhanced crosslinking immunoprecipitation ) analysis using publically available datasets deposited in the ENCyclopedia Of DNA Elements ( ENCODE ) further confirmed that SND1 has a binding profile similar to other m6A reader proteins .", "Importantly , depletion of SND1 in KSHV-infected cells significantly reduced the stability of unspliced ORF50 RNA and led to markedly reduced levels of RTA protein together with a global impairment of KSHV lytic replication .", "Furthermore , we show that m6A-modification in ORF50 RNA regulates SND1 binding to this RNA , particularly to the unspliced form .", "These data identify SND1 as an essential m6A reader for KSHV lytic replication and implicate the ‘Royal family’ as a family which comprises m6A readers .", "This , considerably expands the landscape of m6A readers and the epigenetic functions of Royal members beyond protein methylation ." ], [ "We have previously developed dedicated software ( m6aViewer ) which implements a novel m6A peak-calling algorithm that identifies high-confidence methylated residues with more precision than previously described approaches ( Antanaviciute et al . , 2017 ) .", "Utilising this software we mapped m6A modifications in the KSHV transcriptome by performing m6A-seq in TREx BCBL1-Rta cells , a BCBL1-based , primary effusion lymphoma B-cell line containing latent KSHV episomes capable of doxycycline-inducible reactivation of lytic replication .", "We carried out m6A-seq in latent cells and cells undergoing lytic replication for 8 hr and 20 hr post-induction in two biological replicates .", "In latent cells , we consistently observed m6A peaks in six viral RNAs , including ORF72 and ORF73 .", "At 8 hr post-reactivation , 33 m6A peaks were identified in 21 KSHV ORFs in both biological replicates ( Figure 1—figure supplement 1a ) .", "At 20 hr post-reactivation , 75 m6A peaks were mapped in 42 KSHV ORFs ( Figure 1a ) .", "The positions of these m6A peaks were highly reproducible across the different time points and replicates ( Figure 1—figure supplement 1b ) .", "12 viral m6A peaks were further validated by two-step quantitative reverse transcription PCR ( qRT-PCR ) ( Figure 1—figure supplement 2 ) .", "m6A peaks in cellular RNAs were also consistently called , with 18 , 946 m6A peaks common to both replicates in latent cells and 18 , 935 and 13 , 926 m6A peaks in cells reactivated for 8 hr and 20 hr , respectively ( Figure 1b , Supplementary file 4 ) .", "Cellular m6A peaks remained mostly unchanged between latent and 8 hr of lytic replication .", "Lower detection of m6A peaks at 20 hr post-reactivation was observed in part due to KSHV-mediated host cell shut-off ( Conrad , 2009 ) ( Figure 1c ) .", "However , the majority of uniquely identified m6A peaks at this time point were found in RNAs whose expression was increased during lytic replication ( Figure 1c ) .", "Cellular m6A peaks were enriched in the coding region ( CDS ) and 3’ untranslated region ( UTR ) throughout KSHV infection ( Figure 1d ) .", "In viral mRNAs , the majority of m6A peaks were located in the CDS ( Figure 1e ) .", "We also confirmed the DRm6ACH motif both in cellular and viral mRNAs ( Figure 1f and g , respectively ) .", "Approximately 60% of viral m6A peaks contained the GGm6AC[G/U] motif , a significant over-representation of the motif when compared to the expected DRm6ACH frequency .", "A further comparison using our dedicated software was performed between our m6A-seq datasets and previously published m6A-seq studies carried out in TREx BCBL1-Rta cells ( Tan et al . , 2018 ) and in a renal carcinoma cell line infected by recombinant KSHV BAC16 ( iSLK cells ) ( Hesser et al . , 2018; Tan et al . , 2018 ) .", "All raw sequencing data were subjected to quality control and processing as described in methods and m6A peaks were identified with m6aViewer .", "Peaks were filtered to keep only those with a minimum of 20 mean reads , 1% FDR ( benjamini-hochberg ) and an enrichment of 4-fold m6A-IP/input .", "After applying these cut-offs , our TREx BCBL1-Rta cells datasets contained twice as many m6A peaks as compared to the Tan dataset in the same cell line .", "~17 , 000 and~10 , 000 cellular m6A peaks were identified in our dataset during latency and lytic replication , respectively , while the Tan dataset yielded ~9 , 000 m6A peaks during latency and ~4 , 500 m6A peaks during lytic replication ( Figure 2a ) .", "The most cellular m6A peaks were identified in the Hesser datasets in iSLK cells with ~25 , 000 m6A peaks .", "A direct one-to-one reciprocal overlap comparison between each of our m6A peaks and those found in the other datasets was not possible , mostly due to vastly different peak widths ( with the other datasets having narrower peaks and containing short read length coupled to single end data ) .", "In such situations , many peaks cannot be mapped one-to-one , with multiple peaks often overlapping a single one when comparing datasets , which leads to overlap quantifications that are not easily interpretable .", "Therefore , we compared the overlap of m6A-modified transcripts identified between all three studies .", "In TREx BCBL1-Rta cells , 72% of our 5 , 830 m6A-modified transcripts in latent cells were also present in the same cell line during latency in the Tan dataset , while 52% of our 4 , 059 m6A-modified transcripts in lytic cells were common to the lytic Tan dataset ( Figure 2b ) .", "~80% of m6A-modified transcripts in our TREx BCBL1-Rta dataset were also identified in the Hesser dataset which mapped m6A in iSLK cells ( Figure 2b ) .", "This analysis shows a high reproducibility identifying m6A-modified transcripts in KSHV-infected cell lines by three different groups .", "Regarding viral m6A peaks , again , after applying the same data processing and cut-offs , we identified almost twice as many m6A peaks during lytic replication than the Tan dataset ( Figure 2a ) .", "We then compared the m6A-IP coverage maps of the KSHV transcriptome in lytic TREx BCBL1-Rta cell lines , overall , viral m6A peaks were strikingly similar in both studies ( Figure 2c ) .", "Of note , m6A peaks in ORF50 RNA were consistently mapped in similar regions in both iSLK and TREx BCBL1-Rta cell lines ( Figure 2—figure supplement 1 ) , with the highest similarity found between TREx BCBL1-Rta cell lines .", "Although , in particular instances , m6A peaks differed between studies , for example ORF56 and ORF64 had a distinct m6A peak in this study but not in Tan et al . ( Figure 2c and d , purple boxes ) , while K12 was m6A-modified in Tan et al . but not in our study ( Figure 2c and d , green box ) .", "Intriguingly , when we compared the m6A-IP coverage maps between our lytic TREx BCBL1-Rta cells and lytic iSLK cells from Tan et al . , although clear common peaks were present , these maps differed more than the TREx BCBL1-Rta cells ( Figure 2c and d , blue lines ) suggesting that the KSHV transcriptome may be differentially modified depending on the cell type , suggesting potential epitranscriptomic remodelling of silencing and activating m6A motifs may take place differently between cell types ( Hesser et al . , 2018; Tan et al . , 2018 ) .", "We were intrigued to determine whether any m6A readers uniquely interact with methylated viral mRNAs .", "Of particular interest were the m6A peaks found in the second exon of ORF50 RNA , as this RNA encodes the latent to lytic switch RTA protein .", "To this end , RNA affinity coupled to mass spectrometry analysis was performed .", "Viral RNA baits were centred on the closest GGACU motif to the m6A peak positions of the largest peak of ORF37 and the first and fourth m6A peaks of the second exon of ORF50 ( Figure 1h ) ( hereafter referred to as ORF37 , ORF50-1 and ORF50-4 baits respectively ) .", "See Figure 3—figure supplement 1 for m6A peak positions from all m6A-seq time points .", "Mass spectrometry analysis revealed that all three m6A baits enriched for YTH readers ( Figure 3a ) .", "An intriguing observation was that of the three viral baits , exclusively the methylated ORF50-1 ( m6A-ORF50-1 ) distinctly enriched SND1 , a Tudor domain-containing protein .", "SND1 was in the top thirteen enriched protein hits , together with YTHDF1 , YTHDF2 and YTHDF3 .", "Further binding validation of YTHDF1 and SND1 in RNA affinity experiments demonstrated that YTHDF1 was greatly enriched in all three methylated baits while SND1 showed a modest but clear enrichment exclusively in m6A-ORF50-1 bait ( Figure 3—figure supplement 2 ) .", "SND1 binds symmetrically dimethylated arginines ( sDMA ) via its Tudor domain ( Liu et al . , 2010 ) , thus it harbours the ability to selectively recognise methyl groups .", "In addition to SND1 , m6A-ORF50-1 bait also prominently pulled down three spliceosomal proteins known to interact with the Tudor domain of SND1 , snRNP200 , snRNP116 and PRPF8 ( Yang et al . , 2007 ) .", "These proteins were within the top fifteen enriched protein hits .", "Multiple proteins related to RNA processing were also enriched in this bait ( Supplementary file 1 ) .", "SND1 belongs to the Tudor domain ‘Royal family’ , comprising five subfamilies: Tudor , plant agenet , chromo , PWWP and MBT ( Maurer-Stroh et al . , 2003 ) .", "Members from each subfamily share a structurally related β-barrel that harbours an aromatic cage implicated in binding methylated arginines and lysines ( Chen et al . , 2011 ) .", "Intriguingly , the aromatic cage used for m6A recognition by YTH readers is structurally similar to the one present in the ‘Royal family’ ( Luo and Tong , 2014; Li et al . , 2014; Xu et al . , 2015; Xu et al . , 2014 ) .", "Thus , further scrutiny for Royal members was carried out in the mass spectrometry data .", "Strikingly , several Royal members were also enriched in methylated viral baits .", "All three plant agenet members , which were recently identified as RNA sequence-dependent m6A readers ( Edupuganti et al . , 2017; Arguello et al . , 2017 ) , were exclusively bound to m6A-ORF50-1 bait ( Figure 3a ) .", "Three PWWP domain-containing proteins were also enriched in m6A-ORF50-1 bait while m6A-ORF37 bait retrieved the chromo protein CBX3 ( Figure 3a ) .", "These results suggest that Royal domains may be capable of reading methyl-decorations not only in proteins but also in RNA .", "None of the indirect m6A readers , hnRNPA2B1 , hnRNPC or hnRNPG , or IGF2BP proteins were enriched in any of the baits ( Supplementary file 2 ) .", "Eight proteins with methyl-transferase activity were also recruited to methylated baits ( Supplementary file 2 ) , of these , METTL16 is the second m6A methyltransferase identified to date ( Pendleton et al . , 2017 ) , which methylates structured RNAs where the adenosine is present in a bulge in the nonamer sequence UACAGAGAA , where A is modified ( Mendel et al . , 2018 ) .", "The remaining identified proteins may play a previously uncharacterised role in the m6A RNA metabolism .", "Taken together , these results identify several members from the ‘Royal family’ as putative m6A readers .", "We next determined whether the Royal domains from the Royal members enriched in methylated viral RNAs display affinity for m6A-modified RNA in the absence of any other protein interaction .", "The complete Royal Tudor domain of SND1 ( residues 650–910 ) is required for sDMA-binding ( Liu et al . , 2010 ) , consequently , a GST-recombinant protein containing these residues ( 548-910 ) , referred here as SND1-C-terminus , was used in native electromobility shift assays ( EMSAs ) .", "Notably , SND1-C-terminus shifted m6A-ORF50-1 bait in contrast to the control bait ( Figure 3b ) .", "Neither ORF50-4 nor ORF37 methylated baits were selectively bound by SND1-C-terminus ( Figure 3c and d , respectively ) .", "Note that the membranes had to be overexposed to obtain a good shift signal due to the small amount of shifted RNA in comparison with the free bait , consistent with the modest enrichment of SND1 in m6A-ORF50-1 bait ( Figure 3—figure supplement 2 ) .", "Similarly , a weak shift has also been previously observed in EMSAs for FMRP protein ( Edens et al . , 2019 ) and IGF2BP proteins ( Huang et al . , 2018 ) .", "RNA secondary structure prediction of all baits indicates that they form strongly base-paired hairpins with an apical loop in which m6A is exposed and an additional large unpaired bulge; however , ORF50-4 and ORF37 feature this unpaired bulge in the middle of their stem ( Figure 3—figure supplement 3 ) .", "This may imply that SND1 cannot bind when the unpaired bulge is present at this position irrespective of m6A .", "In contrast , the beginning of the stem of ORF50-1 shows weak base-pairing with four unpaired bases ( Figure 3—figure supplement 3 , black box ) that may allow opening of the hairpin and selective SND1-binding when m6A is present .", "Curiously , when running m6A-ORF50-1 bait on its own ( without any protein ) , two distinct bands were visualised , one lower band which migrated at the same speed of A-ORF50-1 bait and another higher band migrating slower ( Figure 3—figure supplement 4 ) .", "Electrospray ionisation ( ESI ) of the ORF50-1 baits showed that there were no truncated forms and that the correct molecular weight of a methyl group had been added ( 15 daltons ) , demonstrating the purity of the baits ( Figure 3—figure supplement 4 ) .", "As m6A can destabilise local RNA structure in hairpins ( Liu et al . , 2015 ) , it seems plausible that m6A addition to ORF50-1 destabilises the hairpin which then migrates slower compared with the non-methylated bait .", "To test this hypothesis , a cropped version of ORF50-1 bait ( cORF50-1 ) with strong base-pairing throughout the stem ( Figure 3—figure supplement 3 ) was tested in EMSAs .", "Remarkably , SND1-C-terminus did not shift this bait ( Figure 3e ) , highlighting that structural features of the RNA ligand are critical for SND1-binding and that the beginning of the m6A-ORF50-1 hairpin ( Figure 3—figure supplement 3 , boxed region ) may be required for an interaction with SND1 .", "We further mutated seven nucleotides to make this region very structured and stable , as demonstrated by the increase in free energy of this stable hairpin ( sORF50-1 ) .", "No specific selectivity for m6A-sORF50-1 by SND1-C-terminus was seen ( Figure 3f ) , indicating that destabilisation of this region is required for SND1 binding .", "Shorter exposure of the free m6A-sORF50-1 bait did not reveal two distinct bands as the ones present in m6A-ORF50-1 hairpin ( Figure 3—figure supplement 4 ) .", "To further support the hypothesis that Royal domains harbour selectivity for m6A-modified RNA , GST-recombinant proteins containing the Royal domains of FXR1 , PSIP1 and CBX3 were tested in EMSAs .", "Coomassie staining for all recombinant proteins can be seen in Figure 3—figure supplement", "5 . The Royal domains of FXR1 and PSIP1 selectively shifted m6A-ORF50-1 bait , in contrast , none of the other baits were bound ( Figure 3—figure supplement 6a and b , respectively ) .", "The CBX3 chromodomain displayed a very faint shift for m6A-ORF50-1 bait , however this shift was not consistent between protein preparations ( Figure 3—figure supplement 7a ) indicating that this domain either may not be able to read m6A , or other protein partners could be required for its interaction with m6A in vivo .", "Control glutathione S-transferase ( GST ) , a protein with no RNA-binding properties , was also tested in EMSA showing no selectivity for m6A-ORF50-1 bait ( Figure 3—figure supplement 7b ) .", "EMSAs were also performed in the presence of excess herring sperm DNA as a source of non-specific DNA .", "In the presence of sperm DNA no shift was detected and the free bait remained uncomplexed ( Figure 3—figure supplement 7c ) , suggesting that non-specific DNA prevents the interaction between SND1 and m6A-ORF50-1 bait .", "This is not surprising as excess of DNA may sequester SND1 which is a known DNA-binding transcription factor .", "Together , these results confirm that Royal domains in the absence of any other protein interaction display selectivity for m6A-modified RNA , our data also suggests that this selectivity may occur in a RNA secondary structure-dependent manner .", "Next , we aimed to characterise the RNA-binding sites of endogenous SND1 transcriptome-wide during KSHV infection by performing RIP-seq in two biological replicates .", "Latent and lytic TREx BCBL1-Rta cells that had been reactivated for either 8 or 20 hr were used .", "We aimed to obtain the best protein binding site resolution by sonicating the majority of the total RNA to <200 bp ( Figure 4—figure supplement 1a and b ) .", "Unexpectedly , after construction of the cDNA library from the SND1-RNA immunoprecipitated ( RIP ) samples , the majority of the fragments ranged from 150 to 1000 bp ( Figure 4—figure supplement 1c ) .", "Thus , this technique has a binding site resolution of ~1 kB .", "A transcript-wide analysis enabled us to identify SND1 RNA targets , using a false discovery rate ( FDR ) < 1% and a fold change of RIP reads over input reads >2 , this analysis uncovered SND1 as a bona fide RNA-binding protein , yielding 5061 target transcripts ( Supplementary file 5 ) .", "Of these , 3319 transcripts were mRNAs and 748 were long non-coding RNAs ( lncRNAs ) .", "These target RNAs were consistently bound by SND1 during latency and lytic KSHV replication ( both at 8 and 20 hr ) .", "Gene ontology ( GO ) analysis revealed that these transcripts code for proteins that are involved in regulating GTPase activity , nervous system development and cell morphogenesis ( Figure 4a ) .", "Next , all highly expressed transcripts identified by RIP-seq ( FDR < 1% ) were divided into high-confidence targets ( those which had at least twice the normalised coverage in RIPs than input ) and high-confidence non-targets ( those which had at least twice as much coverage in inputs than RIPs ) and the proportion of m6A-bearing RNAs in each group was determined .", "Strikingly , we observed that ~50% of high-confidence targets and ~24% of high-confidence non-targets were m6A-modified RNAs ( Figure 4b ) , representing a marked enrichment of m6A-bearing RNAs in the target group .", "It is worth noting that this transcript-wide analysis will not identify all SND1 targets , as when looking at the SND1-binding profile at the transcript level , it was evident that some RNAs are bound by SND1 at a specific region only ( Figure 4c ) .", "A positive correlation between the number of m6A peaks in a given transcript and the SND1-fold enrichment was not found ( Figure 4d ) .", "These results are not unexpected as they are in agreement with the finding that SND1 does not bind m6A indiscriminately .", "We then mined previously processed PAR-CLIP and m6A-seq data sets ( Wang et al . , 2014; Wang et al . , 2015; Shi et al . , 2017 ) from HeLa cells and calculated the percentage of total RNA targets of YTH readers that contain m6A-modified transcripts .", "~ 65% of all RNA targets contained m6A-modified transcripts ( Figure 4—figure supplement 2 ) .", "In addition , we compared the overlap of target genes containing or lacking m6A modification between SND1 and each heterologously expressed YTH reader .", "To our surprise , despite comparing TREx BCBL1-Rta and HeLa cells , we found that ~50% , ~32% and~37% of SND1 m6A-modified targets are common to YTHDF1 , YTHDF2 and YTHDF3 , respectively ( Figure 4e ) .", "When we compared the SND1 targets that lack m6A , only ~4% , ~1% and ~3% were shared with YTHDF1 , YTHDF2 and YTHDF3 , respectively ( Figure 4f ) .", "These results show that SND1 does not merely co-precipitate with YTH readers and that it has a distinct RNA-binding profile .", "To identify SND1 RNA targets that could be missed by transcript-wide analysis , a novel localised enrichment analysis was developed similar to that used in ChIP-seq analysis ( see Materials and methods ) .", "In brief , both spliced transcripts and introns were segmented into a total of ~750 , 000 transcriptomic regions based on changes in their fold change RIP/input ( Figure 4g ) .", "Applying a fold change >2 and >50 mean reads per region ( FDR < 1% ) , 32 , 314 transcriptomic regions were significantly bound by SND1 .", "These regions encompassed 12 , 915 Ensembl transcripts and after applying a cut-off of >2 fold m6A-IP/input enrichment and a minimum RIP read depth of 50 , ~40% of these transcripts were m6A-modified .", "4872 of these total targets showed SND1-binding throughout 5’UTR , CDS and 3’UTR ( Figure 4h ) .", "We then focused on analysing SND1-enriched intronic regions , as introns tend to be longer than spliced RNAs and RIP-seq has a low resolution , we hypothesised that if SND1 is an m6A reader we might observe m6A peaks overlapping with SND1-enriched regions in introns .", "Out of the total 32 , 314 SND1-enriched regions , 2563 regions were found in introns .", "Of the latter , 516 intronic regions ( 20% ) , directly overlapped with m6A peaks ( Figure 5a and e ) .", "As a control for random overlap , 8 , 328 SND1-unbound intronic regions were used , these were defined using a fold change RIP/input < 0 . 5 and >50 mean reads per region ( FDR < 1% ) .", "Of these , only ~1 . 3% ( 109 ) directly overlapped with m6A peaks ( Figure 4e ) .", "SND1-enriched regions overlapping m6A peaks in UTRs ( Figure 5b ) and lncRNAs ( Figure 4—figure supplement 3a ) were also observed .", "In 5’UTRs and 3’UTRs we found ~40% overlap with m6A peaks in SND1-enriched regions compared to only ~20% overlap of SND1-unbound regions ( Figure 5e ) .", "Next , we set out to investigate the SND1-enriched intronic regions for enriched motifs .", "Interestingly , a U-tract was the most significant motif identified ( Figure 4—figure supplement 3b ) .", "In addition , several GAC-containing motifs appeared as significantly enriched ( Figure 4—figure supplement 3c ) .", "When searching for motifs that were 30 nucleotides long in SND1-bound intronic regions containing m6A peaks , a U-tract immediately followed by an m6A motif was identified ( Figure 4—figure supplement 3d ) .", "In SND1-bound intronic regions that do not have m6A peaks , a U-tract was also the most enriched motif .", "Notably , U-rich motifs found adjacent to m6A residues are also targeted by RBM15/15B and hnRNPC ( Patil et al . , 2016; Liu et al . , 2015 ) .", "RIP-seq also enabled us to identify differential SND1-binding to target RNAs that correlated to both differentially m6A-modified RNAs and the status of KSHV infection .", "During latency , one of the introns in TPO transcript was not m6A-modified and failed to bind SND1 .", "In contrast , at 8 hr and 20 hr post-reactivation , several adenosines in this intron are m6A-modified and SND1 binding ensues ( Figure 5c ) .", "The reverse scenario was also observed for example in the overlapping DUSP5P1-RHOU transcripts , in which specific m6A peaks and SND1-enriched regions present at 0 hr and 8 hr post-reactivation disappeared at 20 hr of lytic reactivation ( Figure 5d ) .", "A list of these differential SND1-binding events to target RNAs can be accessed in Supplementary file", "6 . Taken together , these results demonstrate that SND1 targets m6A-modified regions in KSHV-infected cells at the transcriptome level .", "To further address the binding profile for SND1 we re-analysed multiple eCLIP datasets from the ENCODE consortium for established m6A reader proteins and SND1 ( Van Nostrand , 2017 ) .", "Since the eCLIP datasets were derived from HepG2 cells , we firstly assessed the overlap between the m6A profile in latent TREx BCBL1-Rta cells and HepG2 cells using an existing m6A-seq dataset from HepG2 cells ( Huang et al . , 2018 ) ( Figure 6a ) .", "This revealed an extensive overlap of transcripts which contain m6A sites in these two cells lines , with 77% of TREx BCBL1-Rta m6A-modified transcripts being present in HepG2 cells .", "We then investigated the overlap between transcripts which are m6A-modified and bound by SND1 from RIP-seq and eCLIP datasets ( Figure 6b ) .", "This showed an overlap of 1166 transcripts bound by SND1 of which 88% contained m6A sites .", "We next compared the extent of overlap between m6A and eCLIP peaks on transcripts ( Figure 6c ) .", "We found comparable binding site overlap for SND1 and established readers with m6A peaks , whereas a control protein ( TIAL1 ) showed reduced overlap between m6A peaks and its binding sites .", "We investigated the repertoire of transcripts bound by SND1 and other reader proteins and found extensive binding to protein coding transcripts ( Figure 6d ) .", "SND1 showed a similar distribution to m6A across transcripts with a bias towards coding region binding in common with most other reader proteins , whereas the control TIAL1 protein displayed a strong 3’ UTR bias ( Figure 6e ) .", "We used HOMER to identify motifs bound by SND1 and other established reader proteins using the ENCODE eCLIP datasets .", "This analysis , using peaks called from all transcripts , revealed a common sequence GGAC , the core of the consensus motif surrounding m6A sites .", "For SND1 , this motif is further enriched from the eight highest scoring motif , when performed on all eCLIP peaks ( 30 , 121 peaks ) , to the second highest scoring motif , upon restricting the analysis to SND1 binding sites on exons which contain the m6A modification ( 7965 peaks ) , of which 58% ( 4 , 637 ) directly overlap an m6A site ( Figure 6f ) .", "Together , this analysis reveals that SND1 recognises the consensus m6A motif in vivo and has a similar binding profile to other established reader proteins .", "Regarding KSHV viral mRNAs , we were able to confirm ORF50 RNA as a high-confidence SND1 target .", "As a comparison , a highly expressed viral lytic RNA , ORF57 , did not reach the cut-off to be considered a high-confidence target ( Figure 4—figure supplement 3e ) .", "We additionally identified 33 , 23 and 14 KSHV transcripts as high-confidence SND1 targets at 0 hr , 8 hr and 20 hr post-reactivation , respectively ( Figure 7a ) .", "Deep-sequencing coverage for high-confidence SND1 targets can be seen in Figure 7—figure supplements 1 and 2 .", "Validation of SND1-binding to the different regions containing m6A peaks in the second exon and the intron of ORF50 RNA was performed by RIP followed by RT-qPCR .", "A ~ 40 fold SND1 enrichment was detected in the second exon of ORF50 and a ~ 10 fold enrichment in the intron compared with the SND1 enrichment detected on the non-target 18S rRNA ( Figure 7b ) .", "We further tested by RIP the binding of endogenous FXR1 , FXR2 , PSIP1 , YTHDF1 and YTHDF3 to ORF50 RNA , while non-specific rabbit immunoglobulin ( IgG ) was used as negative control antibody .", "The YTHDF1 RNA target SON ( Wang et al . , 2014 ) was used as a binding control RNA for YTH readers .", "Both YTH readers and FXR2 bound ORF50 RNA , showing a ~ 8 fold enrichment ( Figure 7b ) and ~15 fold enrichment was observed for FXR1 .", "PSIP1 showed a limited but consistent enrichment ( ~3 fold ) above the negative control IgG .", "These results highlight that all these Royal members can target ORF50 RNA , SND1 displaying the highest affinity .", "Having established the SND1 RNA-binding topology , we further investigated the role of SND1 in KSHV infection and its relationship with ORF50 RNA .", "Here , two TREx BCBL1-Rta cell lines with stable shRNA knockdown of SND1 ( SND1 KD1 and SND1 KD2 ) and a shRNA scramble cell line were generated .", "Surprisingly , following 24 hr of lytic replication there were no significant differences between the scramble and the knockdown cell lines in the amount of KSHV lytic transcripts produced ( Figure 7c ) .", "In accordance with mRNA levels , RTA and ORF57 proteins were not reduced ( Figure 7d ) .", "Similarly , the early lytic protein ORF54 was not decreased ( Figure 7d ) .", "These data were also confirmed by RNA-seq performed in scramble and SND1 KD2 cells from two biological replicates .", "Following 24 hr of lytic reactivation , expression of the KSHV transcriptome in SND1-depleted cells was not significantly altered from scramble cells , with the exception of upregulation of ORF71 and ORF72 mRNAs ( Figure 7e ) .", "qRT-PCR analysis confirmed a ~ 4 fold-induction of these transcripts in SND1-depleted cells during the lytic cycle ( data not shown ) .", "These results possibly suggest that SND1 plays a role in maintaining low expression of these latent RNAs during lytic replication .", "Importantly , in the presence of overexpressed RTA protein , KSHV lytic replication is essentially unchanged in SND1-depleted TREx BCBL1-Rta cells , thus these cells still fulfil all viral functions necessary for triggering the lytic cascade .", "TREx BCBL1-Rta cells are reactivated by expression of Myc-tagged RTA which is integrated in the host genome in a spliced form and its expression is under the control of a doxycycline-inducible promoter .", "In contrast , the parental BCBL1 cell line lacks Myc-tagged RTA and reactivation is achieved by addition of the histone deacetylase inhibitor sodium butyrate ( NaB ) , which leads to the expression of unspliced ORF50 RNA .", "This native RNA matures to the spliced form giving rise to RTA protein , which then transactivates the ORF50 promoter in a positive transcriptional feedback mechanism ( Guito and Lukac , 2012 ) .", "However , as SND1 does not take part in the splicing of ORF50 RNA ( Figure 7c ) , it therefore suggests a potential role in the stabilisation of ORF50 RNA , which could be masked by constitutive overexpression of Myc-tagged RTA in TREx BCBL1-Rta cells .", "Thus , the same lentiviral shRNAs targeting SND1 were used to knockdown SND1 in BCBL1 cells .", "Remarkably , both SND1 knockdown cell lines showed a dramatic decrease in viral RNAs , including ORF50 RNA ( Figure 7f ) , and RTA , ORF57 and ORF54 protein levels ( Figure 7g ) .", "These data were also confirmed by RNA-seq performed in scramble and SND1 KD2 BCBL1 cells from two biological replicates .", "After 24 hr of lytic reactivation , 48 KSHV RNAs were significantly downregulated in SND1-depleted cells compared with scramble cells ( FDR < 5% ) , including ORF50 RNA ( Figure 7h ) .", "These results indicate that in the absence of SND1 there is a global impairment of lytic KSHV replication downstream of RTA and uncover SND1 as an essential protein for lytic KSHV replication .", "Although SND1 did not affect splicing of the ORF50 RNA , we assessed whether SND1 regulated splicing events in cellular RNA processing , due to the previously reported role of SND1 in the regulation of splicing ( Jariwala et al . , 2015 ) .", "Thus we hypothesised that SND1 may play a role in splicing through binding methylated intronic regions .", "Splicing analysis revealed multiple significant differential splicing events between scramble latent cells and scramble lytic TREx BCBL1-Rta cells , however , there were no significant splicing differences between scramble and SND1-depleted cells , when analysing cellular or viral transcripts from either latent or lytic cells ( Figure 8a ) .", "The same results were also observed in BCBL1 cells ( data not shown ) .", "As SND1 has previously been described as a transcriptional activator ( Jariwala et al . , 2015 ) we determined whether it could associate with the ORF50 promoter by carrying out chromatin immunoprecipitation ( ChIP ) with the ChIP-grade antibody we previously used for RIP-seq experiments .", "RNA polymerase II ( RNAPII ) antibody ( clone CTD4H8 ) and non-specific immunoglobulin ( IgG ) were used respectively as positive and negative control antibodies .", "Whilst RNAPII was enriched at the GAPDH promoter and multiple viral promoters including ORF50 , there was no significant enrichment for either SND1 or the non-specific IgG ( Figure 7—figure supplement 3 ) .", "Moreover , RNA-seq analysis of TREX BCBL1-Rta-SND1 depleted cells showed no significantly downregulated viral gene expression ( Figure 7e ) .", "Consequently , we conclude that SND1 does not participate in the activation of the ORF50 promoter .", "The strikingly different knockdown phenotype between TREX BCBL1-Rta and BCBL1 cells directly points to a potential regulatory role of SND1 on the essential lytic ORF50 RNA , and suggests that SND1 may stabilise the ORF50 RNA .", "To test this hypothesis without the possible interference of NaB on ORF50 RNA decay , the decay of native ( unspliced ) ORF50 RNA was monitored in scramble and SND1-depleted TREx BCBL1-Rta cells that had been reactivated into the lytic cycle for 24 hr before addition of actinomycin D . Strikingly , native ORF50 RNA was significantly more unstable in SND1-depleted cells ( Figure 8b ) with ~80% of ORF50 RNA remaining at 6 hr post-transcription inhibition in scramble cells and ~50% in depleted cells .", "To further determine whether SND1 may also play a role in regulating the stability of ORF71 and ORF72 RNAs during the KSHV lytic cycle , the turnover of these transcripts together with other high confidence SND1 KSHV RNA targets was also investigated .", "In contrast to ORF50 RNA , these transcripts decayed in a similar manner in scramble and depleted cells ( Figure 8b ) .", "Due to the broad and overlapping m6A-seq peaks across the ORF50 RNA and the high frequency of DRm6ACH motifs throughout ORF50 RNA , we attempted to deplete global m6A levels in ORF50 RNA by stably depleting METTL3 in BCBL1 cells and assess its effect on lytic replication , however limited depletion was achieved and no significant effect on KSHV lytic replication was observed ( data not shown ) .", "The same METTL3 knockdowns were repeated in BCBL1 cells but transiently .", "After five days post-lentiviral transduction , cells were reactivated for 24 hr and viral and protein levels analysed .", "Here , one METTL3 knockdown cell line ( METTL3 KD2 ) achieved a higher level of depletion of METTL3 protein and viral transcript and protein levels were both significantly reduced compared with the scramble cell line ( Figure 7—figure supplement 4a and b ) .", "In contrast , a second cell line ( METTL3 KD1 ) showed no depletion of METTL3 protein and no significant differences in viral replication between these cells and the scramble were observed ( Figure 7—figure supplement 4a and b ) .", "In addition , we generated stable BCBL1 cell lines harbouring shRNA knockdown of either FTO , YTHDF1 or YTHFD3 .", "In contrast , despite consistently achieving ~75% depletion of YTHDF2 at the mRNA level , following 12 days post-transduction these depleted cells would dramatically lose their knockdown at the mRNA level , suggesting that YTHDF2 may be essential for BCBL1 cells , therefore we performed transient transductions for YTHDF2 .", "Following reactivation , FTO-depleted cells displayed increased viral mRNA and protein levels ( Figure 7—figure supplement 4c and d ) , including increased levels of ORF50 RNA and RTA protein .", "Depletion of YTHDF1 or YTHDF3 readers resulted in decreased ORF50 RNA together with other lytic RNAs ( Figure 7—figure supplement 5a-b and e-f ) , particularly , in YTHDF1-depleted cells , which also showed a marked reduction of viral protein levels .", "Depletion of YTHDF2 did not affect viral RNA levels , with the exception of PAN RNA , however a clear decrease in RTA protein and a slight reduction in ORF57 protein levels were evident ( Figure 7—figure supplement 5c and d ) .", "Taken together , these results indicate that m6A modification has a pro-viral role in the KSHV lytic replication cycle , in agreement with previous studies performed in the same cell line ( Ye et al . , 2017 ) .", "Next , we set out to determine whether the m6A status of ORF50 RNA regulates SND1 binding .", "Firstly , single-point mutations were performed in the GGACU motifs present in ORF50 baits to elucidate whether the chosen motifs were m6A-modified in the context of the full length ORF50 RNA .", "Wild type ( WT ) FLAG-ORF50 -containing both ORF50 exons and the intron- and mutant plasmids were transfected into HEK-293 cells and m6A enrichment quantified by m6A-IP-qPCR ( Figure 8c ) .", "In transfected cells , ORF50 RNA was particularly m6A-modified in ORF50-1 and ORF50-4 regions , suggesting cell type differences between TREx BCBL1-Rta and HEK-293 cells .", "Point mutation in the motif contained in ORF50-1 bait , but not in ORF50-4 bait , significantly reduced m6A enrichment , confirming that this site is m6A-modified .", "Next , the binding of SND1 to WT FLAG-ORF50 and to a FLAG-ORF50 plasmid with a point mutation in the motif contained in ORF50-1 bait was evaluated by RIP .", "No significant decrease in SND1 binding was observed ( Figure 8d ) , indicating that other SND1 binding sites are necessary for ORF50 RNA-SND1 interaction .", "We finally assessed the binding of SND1 to ORF50 RNA in the absence or presence of m6A modification .", "For this purpose , we made use of the drug 3-deazaadenosine ( DAA ) , which inhibits m6A deposition by reducing levels of the methyl donor S-adenosylmethionine ( SAM ) .", "HEK-293 cells were transfected with WT FLAG-ORF50 plasmid and 4 hr after transfection , control 0 . 25% ( v/v ) DMSO or 200 µM DAA was incubated for an additional 24 hr .", "Note that DAA did not result in cytotoxicity at this concentration after 26 hr post-treatment ( Figure 8—figure supplement 1 ) .", "DAA effectively reduced m6A enrichment in all m6A-modified regions of ORF50 RNA ( Figure 8c ) and led to a significant decrease of SND1 binding across ORF50 RNA ( Figure 8d ) .", "Interestingly , DAA completely abolished SND1 binding to the native ORF50 transcript , indicating that m6A inhibition on ORF50 RNA regulates SND1 binding to it , however the exact points of interaction between SND1 and ORF50 RNA remain to be elucidated at single nucleotide resolution .", "In summary , these experiments propose a model where in the absence of SND1 , unspliced ORF50 RNA is more unstable resulting in reduced RTA protein levels which will further reduce activation of the RTA promoter in a negative feedback loop that culminates in lytic replication impairment ." ], [ "SND1 is a multi-functional protein .", "It functions as a transcriptional co-activator of Epstein-Barr virus nuclear protein 2 , STAT5 , STAT6 and c-Myb ( Jariwala et al . , 2015 ) .", "Additionally , SND1 has multiple roles modulating gene expression at a post-transcriptional level .", "SND1 is a component of the RISC complex ( Jariwala et al . , 2015 ) and acts in the processing of specific miRNAs ( Heinrich et al . , 2013 ) .", "m6A promotes processing of primary miRNAs ( pri-miRNAs ) ( Alarcón et al . , 2015 ) , thus SND1 may also process m6A-modified pri-miRNAs , which could explain the SND1-binding in methylated introns we observed .", "In agreement with our finding that SND1 stabilises ORF50 RNA , SND1 binds to the 3′UTR of angiotensin II type one receptor ( AT1R ) and stabilises this mRNA leading to elevated protein AT1R levels ( Jariwala et al . , 2015 ) .", "Intriguingly , AT1R mRNA contains a single m6A site which is located in the 3’UTR region ( Sun et al . , 2016 ) .", "Finally , SND1 also participates in RNA editing ( Jariwala et al . , 2015 ) .", "Under stress conditions , in mammalian cells SND1 co-localises with G3BP protein in stress granules ( Gao et al . , 2010 ) and stabilises AT1R and IGFBP2 mRNAs ( Gao et al . , 2015 ) .", "Similarly , in plants SND1 is essential for the stabilisation of a subset of stress-responsive mRNAs ( Frei dit Frey et al . , 2010 ) .", "It will be of interest to address to what extent SND1 regulates the stability of its target RNAs .", "Curiously , the readers YTHDF1-3 , FXR1 , FXR2 and FMR1 have also been identified in mammalian stress granule cores ( Jain et al . , 2016 ) .", "We performed RNA-lifetime profiling in scramble and SND1-depleted cells using latent and lytic TREx BCBL1-Rta and BCBL1 cells; however the NGS data were extremely noisy and this precluded us from identifying a consistent phenotype between replicates .", "To date , very little research has been performed on deciphering the role of SND1 during viral infections .", "SND1 binds the 3’UTRs of transmissible gastroenteritis coronavirus ( TGEV ) ( Galán et al . , 2009 ) and dengue virus ( DENV ) .", "Moreover , SND1 silencing reduced the levels of viral RNA and protein in DENV-infected cells ( Lei et al . , 2011 ) .", "Intriguingly , the 3’UTR of DENV contains m6A sites ( Gokhale et al . , 2016 ) , thus it would be of considerable interest to examine whether this modification enables SND1 recruitment to the 3’UTR to stabilise the RNA DENV genome .", "Differences in the binding affinities for the different RNA baits used in this study between YTH readers and the Royal members can be inferred from previous structural studies .", "Interestingly , in SND1 ( Chen et al . , 2009a ) , plant agenet members ( Adams-Cioaba et al . , 2010 ) , PSIP1 , HDGFRP2 and MSH6 ( Qin and Min , 2014 ) , the aromatic pocket is hydrophobic and surrounded by both positive and negative residues thus , it seems plausible that binding only ensues when matching electrostatic interactions are achieved between the RNA sequence and the Royal domain , consequently , methylated proteins would adopt a different orientation to interact with negatively charged residues while m6A-decorated RNAs would interact with positively charged residues .", "This model would explain the RNA structure dependency observed for these proteins in binding m6A-modified RNA .", "In contrast , the aromatic cage of YTH readers resides in a hydrophobic pocket surrounded by a positively charged surface , rendering the YTH domain favourable to bind any methylated RNA sequence .", "Of note , plant agenet members contain two plant agenet domains ( Agenet1 and Agenet2 ) in tandem , each harbouring an aromatic cage .", "Whilst the aromatic cage of Agenet2 is proposed to be the site that binds methylated lysines ( Adams-Cioaba et al . , 2010 ) , Agenet1 exhibits a basic surface patch with potential for RNA binding ( Myrick et al . , 2015 ) .", "The plant Agenet domain of FMRP ( 1-134 ) has already been shown to harbour RNA-binding ability to RNA homopolymers , with progressively increased affinity when testing longer FMRP constructs ( 1–180 and 1–214 ) , suggesting a cooperative effect between the positively charged residues distributed along the N-terminus region ( Milosevich and Hof , 2016 ) .", "In a similar manner , the other domains present in SND1 cannot be disregarded in considering how SND1 may bind its target RNAs .", "Our RIP-seq data and eCLIP analysis reveals for the first time that SND1 is indeed a bona fide RNA-binding protein acting at the transcriptome level .", "Structural and biochemical analysis had already proposed that the N-terminal region of SND1 , specifically SN3 and SN4 , possess RNA binding ( Li et al . , 2008 ) , thus , in addition to the Tudor domain interacting with methylated RNA , the N-terminal region of SND1 most likely also contributes to RNA binding and may play a significant role in determining which RNAs are targeted by SND1 , including those that are methylated .", "Elucidating Royal domains structures in complex with m6A-ORF50-1 hairpin will help reveal the reason for the distinct selectivity between YTH readers and Royal members and to elucidate to which extent the aromatic cage of Royal domains plays a role in recognising m6A-modified RNA .", "Moreover , these studies could lead to the development of small molecule inhibitors that specifically block the aromatic cage of SND1 to hinder recognition of methylated ORF50 RNA for the treatment of KSHV-related malignancies .", "Pioneering inhibitors for some methyl-lysine readers of the Tudor subfamily and other Royal members are currently being investigated with success ( Milosevich and Hof , 2016 ) .", "Our findings underscore the potential of other members from the ‘Royal family’ as putative new regulators of m6A .", "Of the Tudor subfamily , the Tudor domain-containing ( TDRD ) proteins , AKAP1 , SMN and SPF30 display methyl-arginine-binding and are involved in RNA metabolism ( Chen et al . , 2011 ) , therefore these are ideal candidates to reveal more m6A readers .", "In contrast to the ubiquitously expressed SND1 , most of the mammalian TDRD proteins display male germline-enriched expression and are essential for spermatogenesis ( Chen et al . , 2011 ) .", "It will be of interest to examine whether m6A-decorated RNAs are regulated post-transcriptionally via TDRD proteins during germ cell differentiation .", "Notably , spermatid perinuclear RNA-binding protein ( STRBP ) was within the top ten enriched proteins in m6A-ORF50-1 bait .", "The remainder members of the Tudor protein subfamily bind methyl-lysine residues and contain other domains related to chromatin biology , consequently , these proteins are unlikely to participate in RNA metabolism .", "The discovery of PSIP1 , HDGFRP2 and MSH6 as putative m6A readers is surprising as the PWWP domain is a well-established nucleosome-binding domain and these proteins participate in DNA repair ( Qin and Min , 2014 ) .", "However , FMRP takes part in the DNA damage response ( Alpatov et al . , 2014 ) but FMRP is also a RNA-binding protein that regulates the translation of its m6A-containing target RNAs ( Edupuganti et al . , 2017 ) .", "An m6A RNA-mediated response to UV-induced DNA damage was recently reported ( Xiang et al . , 2017 ) , as such , these PWWP proteins could represent a link between methylation of RNA and DNA damage .", "In support of the putative m6A reading ability of these proteins is the fact that mass spectrometry identification of PSIP1 short ( p52 ) isoform , which includes the PWWP domain , revealed that ~95% of interactors function in pre-mRNA processing .", "Moreover this isoform co-localised with SRSF2 in nuclear speckles and modulated alternative splicing ( Pradeepa et al . , 2012 ) , thus the implication of these proteins in m6A RNA metabolism requires further investigation .", "Finally , it is worth pointing out the possibility that SND1 may be able to read other RNA methyl-modifications in addition to m6A .", "Further studies characterising these methyl-modifications and their corresponding readers will help resolve this matter .", "In conclusion , our data supports the hypothesis that highly specialised domains such as the Royal domains , which harbour a structurally-related aromatic cage to the one found in the YTH domain , may be required for the selective and direct recognition of m6A , while proteins without aromatic cages may not be able to directly read m6A ." ], [ "HEK-293T and HEK-293 cells were purchased from ATCC ( American Type Culture Collection ) and cultured in Dulbecco’s modified Eagle’s medium with glutamine ( DMEM , Lonza ) supplemented with 10% ( v/v ) fetal calf serum ( FCS ) ( Gibco ) and 1% ( v/v ) penicillin-streptomycin ( P/S ) ( Gibco ) .", "TREx BCBL1-Rta cells , a BCBL1-based , primary effusion lymphoma ( PEL ) B cell line that has been engineered to inducibly express exogenous Myc-tagged RTA by the addition of doxycycline , were a gift of JU Jung ( University of Southern California , USA ) .", "BCBL1 cells were a gift from Dr Andrew Hislop ( University of Birmingham , UK ) .", "BCBL1 cells were grown in RPMI1640 growth medium with glutamine ( Gibco ) supplemented with 10% ( v/v ) FCS ( Gibco ) and 1% ( v/v ) P/S ( Gibco ) .", "TREx BCBL1-Rta cells were grown in RPMI1640 growth medium with glutamine ( Gibco ) supplemented with 10% ( v/v ) FCS , ( Gibco ) , 1% P/S ( v/v ) ( Gibco ) and 100 μg/mL hygromycin B ( Thermo Scientific ) .", "All cell lines were tested negative for mycoplasma .", "For virus reactivation , TREx BCBL1-Rta cells were induced using 2 μg/mL doxycycline hyclate ( Sigma-Aldrich ) and BCBL1 cells were induced using 2 mM sodium butyrate ( Sigma-Aldrich ) .", "Antibodies used in Western blotting are listed below: anti-SND1 ( Proteintech , 10760–1-AP , 1:1 , 000 ) ; anti-SND1 ( Proteintech , 60265–1-Ig , 1:1 , 000 ) ; anti-METTL3 ( Bethyl , A301-567A , 1:500 ) ; anti-FTO ( Abcam , ab126605 , 1:5 , 000 ) ; anti-YTHDF1 ( Proteintech , 17479–1-AP , 1:1 , 000 ) ; anti-YTHDF2 ( Abclonal A9639 , 1:500 ) ; anti-YTHDF3 ( Abclonal , A8395 , 1:500 ) ; anti-ORF57 ( Santa Cruz , sc-135746 1:1 , 000 ) ; anti-GAPDH ( Abcam , ab8245 1:5 , 000 ) ; anti-PARP ( CST , 9542 1:2 , 500 ) , the rabbit polyclonal anti-RTA was a gift from Professor David Blackbourn ( University of Surrey , UK ) and used at 1:1000 .", "The mouse monoclonal anti-ORF54 was a gift from Friedrich Grässer ( University of Homburg , Germany ) and used at 1:1000 .", "Rabbit anti-m6A antibody ( ABE572 ) ( Merck Millipore ) was used in m6A-immunoprecipitations .", "Antibodies used in RIP are as follows: anti-SND1 ( Proteintech , 10760–1-AP ) ; anti-FXR1 ( Proteintech , 13194–1-AP ) ; anti-FXR2 ( Proteintech , 12552–1-AP ) ; anti-PSIP1 ( Proteintech , 25504–1-AP ) ; anti-YTHDF1 ( Proteintech , 17479–1-AP ) ; anti-YTHDF3 ( Abclonal , A8395 ) and normal rabbit IgG ( Merck Millipore , 12–370 ) .", "3-deazaadenosine ( DAA ) was purchased from Cambridge Bioscience .", "For RIPs , either 2 µg of anti-SND1 , FXR1 , FXR2 , PSIP1 or normal rabbit IgG were used per RNA immunoprecipitation .", "For YTHDF1 and YTHDF3 , 1 and 5 . 8 µg were used per immunoprecipitation respectively .", "For ChIP experiments , 2 µg of α-RNAPII ( clone CTD4H8 ) antibody ( Merck Millipore ) , anti-SND1 ( Proteintech , 10760–1-AP ) or normal rabbit IgG ( Merck Millipore , 12–370 ) were used per chromatin immunoprecipitation .", "The same lot number ( 00020506 ) of SND1 antibody ( Proteintech , 10760–1-AP ) was used for western blot , RIP-seq , RIP-qPCR and ChIP experiments .", "Total RNA from TREx BCBL1-Rta cells was extracted using TRIzol ( Thermo Scientific ) according to the supplier’s protocol .", "DNA-free DNA Removal Kit ( Ambion ) was used to remove any contaminating DNA from RNA samples .", "Isolated RNA was purified by standard ethanol precipitation and 100 μg of total RNA was fragmented with RNA fragmentation reagent ( Ambion ) according to the manufacturer’s protocol .", "Fragmented RNA was ethanol-precipitated and re-suspended in 10 μl of RNase-free water and stored at −80°C .", "2 μg of total fragmented RNA was saved as input RNA for later use in cDNA library construction .", "For each m6A-immunoprecipitation ( m6A-IP ) , 25 μl of slurry of Magna ChIP Protein A+G magnetic beads ( Merck Millipore ) were washed twice with IP/wash buffer [20 mM Tris HCl pH 7 . 4 , 150 mM NaCl , and 0 . 1% NP-40 ( v/v ) ] .", "Beads were re-suspended in 100 μl of IP/wash buffer and coated with 5 μg of rabbit anti-m6A antibody ( ABE572 ) ( Merck Millipore ) for 45 min at room temperature with rotation .", "Beads were then washed three times with IP/wash buffer and IPs were prepared by mixing the antibody-coated beads with 900 μl of IP/wash buffer , 35 μl of 0 . 5 M EDTA pH 8 . 0 , 4 μl of RNasin Plus ( Promega ) and 100 μg of fragmented RNA .", "IPs were incubated overnight at 4°C with rotation .", "Beads were then washed six times with IP/wash buffer .", "IP samples were further incubated with 126 μl of IP/wash buffer , 15 μl of 10% SDS ( v/v ) and 9 μl of PCR-grade proteinase K ( 20 mg/mL ) ( Thermo Scientific ) for 30 min at 55°C .", "After incubation , the supernatant containing the RNA was transferred to a new microcentrifuge tube and 250 μl of IP/wash buffer was added to each sample .", "RNA was purified with the use of phenol:chloroform:isoamyl alcohol ( Sigma-Aldrich ) and finally sodium acetate/ethanol-precipitated together with 1 µl of RNA-grade glycogen ( Thermo Scientific ) to allow visualisation of the RNA pellet .", "Several m6A-IPs ( four to six ) were pooled to provide enough sample for cDNA library construction and next-generation sequencing ( NGS ) as described below .", "1 . 5 to 3 ng of RNA from input and m6A-IPs were used for NGS library production using NEBNext Ultra kit ( NEB ) according to the manufacturer’s protocol .", "Libraries for the first biological replicate were sequenced on a HiSeq 2500 platform ( Illumina ) with 101 bp paired-end lane .", "Libraries from the second biological replicate were sequenced on a HiSeq 3000 platform ( Illumina ) with 151 bp paired-end lane .", "For m6A-seq two independent biological replicates were prepared for each time point analysed ( 0 hr , 8 hr and 20 hr post-reactivation ) .", "TREx BCBL1-Rta cells remained unreactivated or were reactivated for 8 or 20 hr .", "At the desired time point a fraction of the cells was removed to serve as input RNA to control for RNA expression and stored in TRIzol ( Thermo Scientific ) at −80°C .", "For each RNA immunoprecipitation ( RIP ) , 7 × 106 cells were used .", "Cells were fixed with 1% ( v/v ) formaldehyde ( Calbiochem ) for 10 min at room temperature .", "Crosslinking was stopped by addition of glycine at a final concentration of 125 mM for 5 min .", "Cells were washed with PBS ( Lonza ) and re-suspended in 200 μl of ice-cold shearing buffer [50 mM Tris HCl pH 7 . 4 , 100 mM NaCl and 0 . 1% ( v/v ) NP-40] supplemented with Complete , EDTA-free protease inhibitors ( Roche ) and 1 µl of murine RNase inhibitor ( NEB ) .", "Samples were then sonicated with an EpiShear multi-sample sonicator ( Active Motif ) with pulses of 30 s of sonication followed by a 30 s rest for a total of 12 min at 30% amplitude .", "Polystyrene sonication tubes ( Active Motif ) were used to achieve efficient sonication .", "After sonication , samples were centrifuged at 12 , 000 x g for 10 min at 4°C and the supernatant was used immediately in RIPs .", "For each RIP , 25 μl of slurry of Magna ChIP Protein A+G magnetic beads ( Merck Millipore ) were coated with the antibody of interest as previously described for m6A-seq .", "RIPs were prepared by mixing the antibody-coated beads with 800 μl of IP/wash buffer [20 mM Tris HCl pH 7 . 4 , 150 mM NaCl , and 0 . 1% NP-40 ( v/v ) ] , 35 μl of 0 . 5 M EDTA pH 8 . 0 , 4 μl of murine RNase inhibitor ( NEB ) and 200 μl of lysate containing fragmented RNA .", "RIPs were incubated for 3 hr at 4°C with rotation .", "Beads were then washed six times as previously described ( Gilbert and Sj , 2006 ) .", "RIP samples were then further incubated with 200 μl of IP/wash buffer , 2 μl of PCR-grade proteinase K ( 20 mg/mL ) ( Thermo Scientific ) , 4 μl of 5M NaCl and 0 . 5 μl of murine RNase inhibitor ( NEB ) for 1 hr at 60°C .", "After incubation , the supernatant containing the RNA was transferred to a new microcentrifuge tube and 50 μl of IP/wash buffer was added to each sample .", "RNA was purified as described for m6A-seq but instead of using phenol:chloroform:isoamyl alcohol for purification , TRIzol LS ( Thermo Scientific ) was used according to the manufacturer’s instructions .", "DNA-free DNA Removal Kit ( Ambion ) was used to remove any contaminating DNA from RIP RNA samples .", "Total RNA from input samples was isolated with the use of TRIzol ( Thermo Scientific ) according to the supplier’s protocol and treated with DNase I using the DNA-free DNA Removal Kit ( Ambion ) .", "Input RNA was saved without applying sonication because we observed lower quality sequencing libraries when using sonicated input RNA than when using input RNA that was heat-fragmented .", "Saved input RNA was therefore heat-fragmented for 8 min at 94°C before proceeding with library construction .", "Due to the large size of RNA fragments observed after SND1 immunoprecipitation , isolated RNA from RIP samples was further fragmented for 7 min at 94°C before first strand synthesis .", "100 to 200 ng of each input and RIP sample was used for NGS libraries which were made using the TruSeq Stranded Total RNA library production kit ( Illumina ) according to the manufacturer’s protocol .", "All samples were multiplexed and sequenced on two 151 bp paired-end lanes on a HiSeq 3000 instrument ( Illumina ) .", "For RIP-seq , two independent biological replicates were prepared for each time point analysed ( 0 hr , 8 hr and 20 hr post-reactivation ) .", "Additionally , for one biological replicate , two technical replicates were deep-sequenced for all RIP samples .", "A third biological replicate RIP sample at 0 hr was also deep-sequenced .", "NGS libraries were generated from two independent biological replicates using scramble and SND1 KD2 TREx BCBL1-Rta cells both during latency and after 24 hr of lytic reactivation .", "Two independent biological replicates from scramble and SND1 KD2 BCBL1 cells both during latency and after 24 hr of lytic reactivation were also deep-sequenced .", "Libraries were made using TruSeq Stranded Total RNA library preparation kit ( Illumina ) according to the manufacturer’s protocol .", "Libraries were sequenced on 151 bp paired-end lanes on a HiSeq 3000 instrument ( Illumina ) .", "m6A-IPs and RIPs were carried out as described above respectively , with the following modification .", "1% input samples ( 10 μl from the 1 mL IP reaction ) were removed before immunoprecipitation and stored at −80°C .", "Input samples were processed together with immunoprecipitated samples from the proteinase K treatment onwards .", "Purified input and immunoprecipitated RNA was resuspended in 10 μl of RNase-free water and reverse transcribed as described in the RT-qPCR analysis section .", "qPCR normalisation was performed similarly to chromatin immunoprecipitation ( ChIP ) coupled to detection by qPCR analysis using ΔΔCt method ( relative quantification ) .", "An example for m6A-IPs follows .", "Each m6A-IP sample Ct value for each primer used was normalised to the corresponding input Ct value: ΔCt normalised m6A-IP = Ct m6A-IP – [Ct Input –log2 ( Input dilution factor ) ] .", "Input dilution factor = ( fraction of the input reaction saved ) −1 .", "As 1% input was saved , the dilution factor is 100 or 6 . 644 cycles ( i . e . log2 of 100 ) .", "Percentage of recovery from the initial reaction ( % input ) was then calculated for each primer as 100 x Amplification efficiency ( AE ) ( -ΔCt normalised m6A-IP ) .", "m6A enrichment was finally calculated as the fold change between the % input for a region containing m6A peaks over the % input for a negative control region .", "SND1 enrichment was calculated as the fold change between the % input for a region of interest over the % input for a region of a non-target control RNA such as 18S rRNA .", "For HEK-293 cells , RIP-qPCR was performed the same way as described for TREx BCBL1-Rta cells with the exception that the sonication time was reduced to eight min and 50 μg of fragmented RNA were used per IP .", "All m6A-seq , RIP-seq and RNA-seq data were generated at the next-generation sequencing facility of the University of Leeds , United Kingdom .", "Data were extracted and de-multiplexed using bcl2fastq Conversion software ( Ilumina ) , which exports a matched pair ( read 1 and read 2 ) of compressed fastq files per sample .", "Quality control of all sequence data , including publicly available datasets , was carried out using FastQC software ( Andrews , 2010 ) , which allowed the identification of sequence adapter contamination , overrepresented sequences , estimation of the PCR and optical duplicate rate and overall sequencing quality .", "All raw sequence data were then processed using Cutadapt software ( Martin , 2011 ) in order to remove poor quality bases ( quality score less than 20 ) as well as Illumina universal sequencing adapter sequence ( AGATCGGAAGAG ) from the 3’ end of reads .", "Orphan read pairs were discarded .", "Reads shorter than 25 bp after trimming were discarded to limit ambiguous alignments in downstream processing .", "The KSHV reference genome sequence was downloaded in FASTA format from the NCBI website , while a gene transfer format ( GTF ) file containing genomic feature coordinates ( ORFs , genes , exons , UTRs ) was assembled manually using data from the KSHV 2 . 0 annotation dataset ( Arias et al . , 2014 ) .", "The human hg38 reference genome sequence was downloaded from the FTP-UCSC genome browser ( Kent et al . , 2002 ) in FASTA format .", "The human hg38 genome annotation was downloaded using the UCSC Table Browser Tool ( Karolchik , 2004 ) .", "KSHV data were manually added to the human reference FASTA and GTF files as an additional contig .", "The genome sequences in the merged FASTA file were indexed for alignment using STAR software ( Dobin et al . , 2013 ) .", "Paired-end sequence data was subsequently aligned to this index using the splice-aware read aligner STAR , in paired-end , two-pass mode .", "Aligned reads in binary alignment map ( BAM ) format were sorted by coordinate and indexed using Samtools ( Li et al . , 2009 ) and PCR and optical duplicates flagged for additional QC checks ( but not removed ) using Picard Tools software .", "Additional QC metrics were assembled and assessed using multiQC software ( Ewels et al . , 2016 ) .", "m6A peaks were called using m6aViewer software version 1 . 6 ( Antanaviciute et al . , 2017 ) with default settings and exported to tab-delimited format for additional analyses in R . To define significantly enriched m6A peaks in both viral and cellular RNAs , a minimum fold change of m6A-IP reads over input reads of ≥1 . 5 in addition to a false discovery rate of 5% ( FDR < 5% ) was required in both biological replicates .", "Peaks positions were considered overlapping between replicates if the calls were within 100 nucleotides between corresponding positions .", "To determine the number of SND1 RNA targets identified by transcript-wide analysis that are m6A-modified , a more stringent m6A peak calling cut-off was used to compliment a more stringent SND1 RIP cut-off , with a minimum of 100 read paired at the tallest point in the m6A peak and a 2-fold enrichment of m6A-IP reads over input reads using a FDR < 1% .", "For target overlap between heterologously expressed YTH readers and SND1 , high-confidence SND1-bound genes ( summarised at HGNC gene symbol annotation level , where multiple Ensembl genes mapped to a symbol , the longest was used ) were defined at a cut-off of FDR < 1% and a minimum of 2-fold RIP enrichment over input , while m6A peaks were used as before ( FDR < 5% , 1 . 5 fold minimum enrichment in both replicates ) .", "Peak motif discovery was performed by exporting the flanking 100 base of RNA sequence surrounding peaks in KSHV methylome to a FASTA file using m6aViewer software .", "Sequences containing repetitive viral sequence were removed .", "The remaining data was then used for enriched sequence motif detection using the MEME software ( Bailey et al . , 2009 ) , with scrambled sequences used as a control .", "KSHV methylome maps were produced using custom Java code .", "SND1 binding sites were initially identified at transcript-level resolution by counting reads in the SND1 immunoprecipitated ( RIP ) and input ( control ) sample data that mapped to each RefSeq and KSHV transcripts .", "R package Rsubread ( Liao et al . , 2014 ) was used to obtain raw read counts as follows .", "Each uniquely mapping read pair was counted towards the total transcript count for each sample , while multi-mapping reads were counted as partial reads , based on the number of mapped positions .", "Since the library preparation protocol preserved the strand of the original RNA molecule , only ‘correctly’ stranded read pairs were counted for each transcript .", "DESeq2 R package ( Love et al . , 2014 ) was used to normalise the data and identify transcripts that showed a significant increase in the coverage of the normalised RIP samples when compared to the input controls .", "In order to increase the resolution of the SND1-bound regions , custom Java code was used that identified transcriptome regions that were enriched in the RIP data when compared to the control data .", "Initially , the application segmented regions into intronic or exonic sequences: a region was classified as exonic if the sequence was present in at least one mature transcript .", "The per-base raw read coverage was determined for both intronic and exonic sequences and normalised using the size factors determined from the earlier normalisation step ( DESeq2 ) to account for library compositions and sizes .", "Using a sliding window approach , first each mature transcript or intron was divided into contiguous segments that loosely showed putative enrichment in the RIP data ( >1 fold enrichment ) compared to the input data and those that putatively were depleted in ( or equal to ) RIP ( ≤1 fold enrichment ) .", "Low coverage segments ( <20 reads in RIP ) were filtered out as quality control .", "All data from the different samples in the analysis were then merged to generate a single dataset that contained read depth data for all consensus segments identified this way , both intronic and exonic .", "Each segmented region was subsequently treated as an individual ‘gene’ for re-analysis using DESeq2 , specifically testing for RIP signal enrichment over input ( alternative hypothesis: normalised segment read coverage in RIP greater than in input ) in order to identify regions with a significant increase in RIP over input signal .", "Differential expression analyses were performed in R using DESeq2 package .", "Functional enrichment analyses were performed using the clusterProfiler R package ( Yu et al . , 2012 ) , using the significance cut-off of <0 . 05 ( Benjamini-Hochberg corrected p-values ) , with all expressed/detected genes ( at least one mapped read pair ) used as a background control .", "Alternative splicing events were detected using Comprehensive AS Hunting ( CASH ) ( Wu , 2017 ) .", "In addition , the lack of statistically significant splicing events between scramble and SND1-depleted TREx BCBL1-Rta cells highlighted by CASH was also confirmed using Spladder software ( Kahles et al . , 2016 ) and DEXSeq R package ( Anders et al . , 2012 ) for detecting differential exon usage ( data not shown ) .", "Read coverage and splicing graphs were visualised using Integrative Genomics Viewer ( IGV ) ( Robinson et al . , 2011 ) .", "Data downloaded from public repositories were aligned as above , except without the addition of KSHV sequence to the reference genome .", "The following data were obtained from NCBI’s GEO database as raw FASTQ files: HeLa cell line YTHDF1 PAR-CLIP data , two replicates ( accessions: GSM1553242 , GSM1553243 ) ; HeLa cell line YTHDF2 PAR-CLIP data , three replicates ( accessions: GSM1197605 , GSM1197606 , GSM1197607 ) ; HeLa cell line YTHDF3 PAR-CLIP data , three replicates ( accessions: GSM2424844 , GSM2424845 , GSM2424846 ) .", "HepG2 cell line m6A-seq data , four replicates ( accessions: GSM2409802 , GSM2409803 , GSM2715523 , GSM2715524 ) .", "Two replicates of each eCLIP experiment were obtained from the ENCODE database as narrowPeak bed files: HepG2 FXR2 ( accessions: ENCFF702QGF , ENCFF638WRZ ) ; HepG2 SND1 ( ENCFF471JAQ , ENCFF761CYV ) ; IGF2BP1 ( accessions: ENCFF705SDK , ENCFF145YYK ) ; IGF2BP3 ( accessions: ENCFF076GHL , ENCFF998WZW ) ; TIAL1 ( accessions: ENCFF302ROS , ENCFF467UOO ) .", "Binding sites from eCLIP experiments were downloaded from ENCODE as narrowPeak bed files .", "Clusters from replicate one which directly overlapped clusters in replicate two were extracted and kept for downstream analyses .", "To look at overlaps between eCLIP sites and m6A peaks , HepG2 RIP-seq data was processed with m6aViewer 1 . 6 software , using default settings , and overlapping peaks , containing a ≥ 1 . 5 fold increase of m6A-IP reads over input with a FDR < 5% across both replicates were kept .", "De novo motif analysis of HepG2 m6A-seq and eCLIP sites was performed by feeding peaks into the findMotifsGenome . pl function within the HOMER suite ( version 4 . 9 . 1 ) using parameters ‘-rna -len 5’ .", "For SND1 motif discovery over m6A exonic regions , exons containing m6A-seq peaks were identified and these whole exon sequences were screened for SND1 peaks .", "The SND1 peaks within this set of m6A-modified exons were used for motif discovery .", "Excel data sheet for all m6A peaks called in both biological replicates is supplied as Supplementary file", "4 . Excel data sheet for all SND1 targets identified by transcript-wide analysis is supplied as Supplementary file", "5 . All deep-sequencing data discussed in this publication have been deposited in NCBI’s GEO Database , GEO accession number GSE119026 .", "All identified peptides/PSMs for each RNA bait can be found in Supplementary file 7–15 .", "The following biotin-labelled RNA oligonucleotides were centred on the closest GGACU motif to the m6A peaks detected in ORF37 and ORF50 transcripts .", "For each oligo , one oligo was m6A-modified at the GGACU motif while the control bait remained unmodified .", "For the ORF37 bait the sequences used were: 5´-biotin-CGGAAAGCUGGCACUGAAGG-m6A-CUUCUUCUAUAGCAUUUCCA-3´ and 5´-biotin-CGGAAAGCUGGCACUGAAGGACUUCUUCUAUAGCAUUUCCA-3´ .", "Baits spanning an m6A consensus in the first m6A peak identified in ORF50 ( ORF50-1 ) were: 5´-biotin-UUUGCCAAUCCUGGAGCCAGG-m6A-CUGUUGCCGGCUUCCAUGGUA-3´ and 5´-biotin-UUUGCCAAUCCUGGAGCCAGGACUGUUGCCGGCUUCCAUGGUA-3´ .", "The sequences for baits spanning an m6A consensus in the fourth m6A peak identified in ORF50 ( ORF50-4 ) were: 5´-biotin-GUUGUCCAGUAUUCUGCAAGG-m6A-CUGUACCAGCUGGACACGCCA-3´ and 5´-biotin-GUUGUCCAGUAUUCUGCAAGGACUGUACCAGCUGGACACGCCA-3´ .", "Cropped versions of ORF50-1 ( cORF50-1 ) were: 5´-biotin-GGAGCCAGG-m6A-CUGUUGCCGGCUUC-3´ and 5´-biotin-GGAGCCAGGACUGUUGCCGGCUUC-3´ .", "Stable versions of ORF50-1 ( sORF50-1 ) were: 5´-biotin-UUGGCCCAUCCCGGAGCCAGG-m6A-CUGUUGCCGGCUUCCGGGGCC-3´ and 5´-biotin-UUGGCCCAUCCCGGAGCCAGGACUGUUGCCGGCUUCCGGGGCC-3´ .", "All baits were purchased from Integrated DNA Technologies ( IDT ) .", "TREx BCBL1-Rta cells were reactivated with doxycycline for 24 hr followed by lysis of the cells for 25 min in lysis buffer [10 mM NaCl , 2 mM EDTA , 0 . 5% ( v/v ) triton X-100 , 0 . 5 mM DTT and 10 mM Tris HCl , pH 7 . 4] containing complete protease inhibitor cocktail ( Roche ) and phosphatase inhibitor cocktail 2 ( Sigma-Aldrich ) .", "Lysates were centrifuged at 4°C for 10 min at 12 , 000 x g to pellet cell debris and the supernatant was kept .", "1000 µg of protein per pull-down in an approximate volume of 200 µl were supplemented with 40 units of RNasin Plus ( Promega ) and 50 µg of yeast tRNA ( Sigma-Aldrich ) and pre-cleared with 30 µl ( resin volume ) of streptavidin-conjugated agarose beads ( Merck Millipore ) for 3 hr at 4°C with rotation .", "The final volume of the binding reaction was topped to 1 mL with binding buffer ( 150 mM KCl , 1 . 5 mM MgCl2 , 0 . 05% ( v/v ) NP-40 , 0 . 5 mM DTT , 10 mM Tris HCl , pH 7 . 4 ) .", "While pre-clearing , 30 µl ( resin volume ) of streptavidin-conjugated agarose beads per pull-down were blocked with 1% ( w/v ) BSA ( Sigma-Aldrich ) in PBS ( Lonza ) and 50 µg of yeast tRNA ( Sigma-Aldrich ) .", "Pre-cleared lysates were then mixed with the blocked beads and 4 µg of each biotinylated RNA oligo were added per pull-down .", "Input samples were immediately collected and stored at −80°C until further use .", "RNA baits were incubated for 2 hr at 4°C with rotation followed by five washes with binding buffer .", "Proteins were released from the beads by heating at 95°C for 5 min in 30 µl of 2 X Laemmli sample buffer .", "Input and pull-down samples were used for either Western blotting or LC-MS/MS analysis .", "LC-MS/MS was performed at the proteomics facility of the University of Bristol , United Kingdom .", "Samples were separated using SDS-PAGE until the dye front had migrated approximately one centimetre into the separating gel .", "Each gel lane was then excised and subjected to in-gel tryptic digestion using a DigestPro automated digestion unit ( Intavis Ltd . ) .", "The resulting peptides were fractionated using an Ultimate 3000 nano-LC system in line with an LTQ-Orbitrap Velos mass spectrometer ( Thermo Scientific ) .", "In brief , peptides in 1% ( v/v ) formic acid were injected onto an Acclaim PepMap C18 nano-trap column ( Thermo Scientific ) .", "After washing with 0 . 5% ( v/v ) acetonitrile 0 . 1% ( v/v ) formic acid , peptides were resolved on a 250 mm ×75 μm Acclaim PepMap C18 reverse phase analytical column ( Thermo Scientific ) over a 150 min organic gradient , using seven gradient segments ( 1–6% solvent B over 1 min . , 6–15% B over 58 min . , 15–32%B over 58 min . , 32–40%B over 5 min . , 40–90%B over 1 min . , held at 90%B for 6 min and then reduced to 1%B over 1 min . ) with a flow rate of 300 nl min−1 .", "Solvent A was 0 . 1% formic acid and Solvent B was aqueous 80% acetonitrile in 0 . 1% formic acid .", "Peptides were ionised by nano-electrospray ionisation at 2 . 1 kV using a stainless-steel emitter with an internal diameter of 30 μm ( Thermo Scientific ) and a capillary temperature of 250°C .", "Tandem mass spectra were acquired using an LTQ- Orbitrap Velos mass spectrometer controlled by Xcalibur 2 . 1 software ( Thermo Scientific ) and operated in data-dependent acquisition mode .", "The Orbitrap was set to analyse the survey scans at 60 , 000 resolution ( at m/z 400 ) in the mass range m/z 300 to 2000 and the top twenty multiply charged ions in each duty cycle selected for MS/MS in the LTQ linear ion trap .", "Charge state filtering , where unassigned precursor ions were not selected for fragmentation , and dynamic exclusion ( repeat count , 1; repeat duration , 30 s; exclusion list size , 500 ) were used .", "Fragmentation conditions in the LTQ were as follows: normalised collision energy , 40%; activation q , 0 . 25; activation time 10 ms; and minimum ion selection intensity , 500 counts .", "The raw data files were processed and quantified using Proteome Discoverer software v1 . 4 ( Thermo Scientific ) and searched against the UniProt Human database ( downloaded October 2015; 131351 sequences ) plus KSHV protein sequences using the SEQUEST algorithm .", "Peptide precursor mass tolerance was set at 10ppm , and MS/MS tolerance was set at 0 . 8 Da .", "Search criteria included carbamidomethylation of cysteine ( +57 . 0214 ) as a fixed modification and oxidation of methionine ( +15 . 9949 ) as a variable modification .", "Searches were performed with full tryptic digestion and a maximum of 1 missed cleavage was allowed .", "The reverse database search option was enabled and all peptide data was filtered to satisfy false discovery rate ( FDR ) of 5% .", "Comparative reports were produced between methylated and control RNA baits , the data were filtered at 5% FDR and had also removed any proteins that were only matched by a single peptide .", "In addition , the data was sorted based on the number total number of peptide spectrum matches ( PSM’s ) identified , such that those proteins at the top of the list were only identified in the methylated samples ( e . g . no peptides matching that protein were detected in the non-methylated sample ) .", "Comparative reports for ORF50-1 , ORF50-4 and ORF37 baits are supplied as Supplementary file 7 , 8 and 9 respectively .", "To uncover putative m6A readers , all proteins identified for a given methylated and control bait were sorted by total number of PSM’s for the m6A bait .", "Proteins were then classified as enriched in methylated baits if the number of PSM’s assigned to the protein was at least double in the methylated bait compared with the control bait .", "All identified unique peptides and PSMs for all RNA baits can be accessed on Supplementary file 10–15 .", "Gene-annotation enrichment analysis were performed with The Database for Annotation , Visualisation and Integrated Discovery ( DAVID ) v6 . 8 .", "RNA secondary structure prediction of baits was carried out using UNAfold web server ( Zuker , 2003 ) .", "All recombinant proteins were gene synthesised ( NovoPro Bioscience ) , cloned into pGEX-4T-1 vector ( NovoPro Bioscience ) and expressed in E . coli strain BL21-DE3 ( Thermo Scientific ) .", "GST-recombinant proteins contained the FXR1 plant agenet domain ( residues 2–132 ) which includes Agenet1 and Agenet2 in tandem , the PSIP1 PWWP domain ( residues 3–100 ) or the CBX3 chromodomain ( residues 29–86 ) .", "SND1-C-terminus comprises of residues 548–910 .", "Recombinant GST plasmid was commercially available ( GE healthcare ) .", "Recombinant proteins were produced by lowering the temperature to 18°C and inducing the culture with 0 . 2 mM IPTG for 20 hr .", "Bacteria pellet from 1 L culture was re-suspended with 30 mL of lysis buffer [50 mM Tris HCl , pH 7 . 5 , 150 mM NaCl , 0 . 05% ( v/v ) NP-40 and freshly added 0 . 25 mg/mL lysozyme ( Sigma-Aldrich ) ] and incubated on ice for 45 min .", "The lysate was then sonicated using a MSE Soniprep 150 sonicator ( 12 cycles of 20 s pulse-on and 20 s pulse-off ) .", "The lysate was centrifuged at 12 , 000 x g for 15 min at 4°C .", "The supernatant was incubated with glutathione agarose beads ( GE healthcare ) for 2 hr at 4°C .", "The resin was washed twice with lysis buffer and once with 50 mM Tris HCl , pH 7 . 4 .", "Protein was eluted from the beads using 50 mM Tris HCl with 20 mM reduced glutathione ( Sigma-Aldrich ) that had been adjusted to a final pH of 7 . 4 .", "EMSAs were carried out using LightShift chemiluminescent RNA EMSA Kit ( Thermo Scientific ) according to the manufacturer’s instructions .", "Binding reactions consisted of kit supplied 1 X binding buffer ( 10 mM HEPES , pH 7 . 3 , 20 mM KCl , 1 mM MgCl2 and 1 mM DTT ) supplemented with 5% ( v/v ) glycerol .", "For EMSAs in the presence of herring sperm DNA ( Promega ) , DNA was mixed and incubated in the binding reaction .", "Note that this sperm DNA is provided after phenol-chloroform extraction , ethanol precipitation and sonication , which produces single-stranded fragments .", "Binding reactions were incubated with increasing amounts of recombinant protein which was freshly isolated ( e . g . no more than two days after purification from bacteria and stored at 4°C ) .", "The same biotinylated baits used in the RNA affinity experiments were used for EMSAs .", "All baits were used at 3 . 75 nM oligo final concentration , except for cORF50-1 which was used at 16 nM .", "Binding reactions were incubated for 30 min at room temperature .", "10 µl reactions were mixed with 2 µl of 5 X loading dye and 10 µl were loaded onto a 6% ( w/v ) non-denaturing polyacrylamide gel in 1 X TAE buffer ( 40 mM Tris , 20 mM acetate and 1 mM EDTA/NaOH pH 8 . 0 ) .", "Gels were ran for 45 min at 100V and transferred to a nylon membrane ( GE healthcare ) via wet transfer with 1 X TAE as transfer buffer for 45 min at 400 mA .", "RNA was crosslinked to the membrane at 120mJ/cm2 using a CL-1000 ultraviolet crosslinker ( UVP ) .", "The rest of the protocol was performed as described in the kit manual .", "Biotinylated baits were visualised after exposure to an ultra-sensitive ECL ( SuperSignal west femto maximum sensitivity substrate ) ( Thermo Scientific ) and exposed to Amersham hyperfilm ECL ( GE Healthcare ) .", "A FLAG tag ORF50 gene which included both ORF50 exons and the ORF50 intron cloned into a pCDH-CMV-MCS-EF1-Puro vector was purchased from NovoPro Bioscience .", "Single point mutations in this plasmid were generated using QuickChange II site-directed mutagenesis kit ( Stratagene ) according to the manufacturer’s instructions .", "All plasmids containing the desired mutation were confirmed by DNA sequencing ( Eurofins Genomics ) .", "To mutate the GGACT motif present in ORF50-1 bait the following primers were used: TCCTGGAGCCAGGATTGTTGCCGG ( forward ) , CCGGCAACAATCCTGGCTCCAGGA ( reverse ) .", "To mutate the GGACT motif present in ORF50-4 bait these primers were used: TCCAGTATTCTGCAAGGATTGTACCAGCTGGACAC ( forward ) , GTGTCCAGCTGGTACAATCCTTGCAGAATACTGGA ( reverse ) .", "Lentiviruses were generated by transfection of HEK-293T cells seeded in 12-well plates using a three-plasmid system .", "Per 12-well , 4 µl of lipofectamine 2000 ( Thermo Scientific ) were used together with 1 µg of pLKO . 1 plasmid expressing shRNA against the protein of interest , 0 . 65 µg of pVSV . G , and 0 . 65 µg psPAX2 .", "pVSV . G and psPAX2 were a gift from Dr . Edwin Chen at the University of Leeds .", "8 hr post-transfection , medium was changed into 1 . 5 mL of DMEM supplemented with 10% ( v/v ) FCS .", "Two days post-transfection viral supernatants were harvested , filtered through a 0 . 45 µm filter ( Merck Millipore ) and immediately used for transductions of TREx BCBL1-Rta or BCBL1 cells .", "One mL of each 12-well plate was used to infect 500 , 000 cells by spin inoculation for 60 min at 800 x g at room temperature , in the presence of 8 μg/mL of polybrene ( Merck Millipore ) .", "Virus supernatant was removed after 7 hr post-spin inoculation and cells were maintained in fresh growth medium for 48 hr before undergoing 3 µg/mL puromycin ( Sigma-Aldrich ) selection .", "Stable cell lines were generated after 8 days post-selection when cell stocks were frozen .", "The following shRNAs were used in the experiments: SND1 KD1 ( TRCN0000245143 ) , SND1 KD2 ( TRCN0000049656 ) , METTL3 KD1 ( TRCN0000289742 ) , METTL3 KD2 ( TRCN0000289812 ) , FTO KD1 ( TRCN0000246247 ) , FTO KD2 ( TRCN0000246250 ) , YTHDF1 KD1 ( TRCN0000062771 ) , YTHDF1 KD2 ( TRCN0000286871 ) , YTHDF2 KD1 ( TRCN0000254411 ) , YTHDF2 KD2 ( TRCN0000265510 ) , YTHDF3 KD1 ( TRCN0000365164 ) and YTHDF3 KD2 ( TRCN0000167772 ) .", "All shRNA plasmids were purchased from either Sigma-Aldrich or Dharmacon .", "Scramble shRNA was a gift from Professor David Sabatini ( Addgene plasmid # 1864 ) .", "Western blots were performed as previously described ( Schumann et al . , 2017 ) .", "In brief , protein samples were run on SDS-PAGE gels and transferred onto nitrocellulose membrane ( GE healthcare ) via wet transfer .", "Membranes were blocked with TBS + 0 . 1% ( v/v ) Tween 20 ( TBST ) and 5% ( w/v ) dried skimmed milk powder for 30 min , and then incubated for 1 hr with relevant primary and secondary antibodies diluted in 5% ( w/v ) milk TBST .", "Membranes were treated with either ECL Western blotting substrate ( Promega ) or SuperSignal West Femto Maximum Sensitivity Luminol/Enhancer solution ( Thermo scientific ) and exposed to Amersham hyperfilm ECL ( GE Healthcare ) .", "Secondary antibodies were horseradish peroxidase ( HRP ) -conjugated polyclonal goat anti-mouse ( Dako ) and polyclonal goat anti-rabbit ( Dako ) , both used at 1:5000 dilution .", "qRT-PCR was performed as previously described ( Baquero-Pérez and Whitehouse , 2015 ) .", "In brief , total RNA from cells was extracted using TRIzol ( Thermo Scientific ) according to the supplier’s protocol .", "DNA-free DNA Removal Kit ( Ambion ) was used to remove any contaminating DNA from RNA samples .", "Reverse transcription was performed with ProtoScript II ( NEB ) , murine RNase inhibitor ( NEB ) , random hexamers ( Bioline ) and 1 μg of total RNA .", "Quantitative PCR ( qPCR ) reactions ( 20 μl ) included 1 X SensiMix SYBR green master mix ( Bioline ) , 0 . 5 μM of each primer and 5 μl template cDNA .", "Cycling was performed in a RotorGene Q 2plex machine ( Qiagen ) .", "The cycling programme was a 10 min initial preincubation at 95°C , followed by 40 cycles of 95°C for 15 s , 60°C for 30 s and 72°C for 20 s .", "After qPCR , a melting curve analysis was performed between 65°C and 95°C ( with 0 . 2°C increments ) to confirm amplification of a single product .", "To assess primer amplification efficiency ( AE ) , for each gene of interest a standard curve was constructed using a pool of cDNA derived from unreactivated and reactivated cells .", "At least four different dilutions of pool cDNA were quantified to generate a standard curve .", "The slope of the standard curve was used to calculate the AE of the primers using the formula: AE = ( 10−1/slope ) .", "For gene expression analysis all genes of interest were normalised against the housekeeping gene GAPDH ( ΔCT ) .", "A summary of all the primers used in this study is provided in Supplementary file 3 .", "Primers specific to METTL3 have previously been described ( Liu et al . , 2014 ) .", "TREx BCBL1-Rta cells were treated with 2 . 5 μg/ml of actinomycin D ( Thermo Scientific ) and samples were collected at the desired time points .", "Total RNA was extracted using TRIzol ( Thermo Scientific ) according to the supplier’s protocol .", "DNA-free DNA Removal Kit ( Ambion ) was used to remove any contaminating DNA from RNA samples and qRT-PCR was carried out as described above .", "The gene of interest was normalised against 18S rRNA , as this RNA , but not GAPDH , was stable after 6 hr of actinomycin D treatment .", "ChIP experiments were carried out as previously described ( Baquero-Pérez and Whitehouse , 2015 ) .", "Formaldehyde-crosslinked chromatin was prepared using the Pierce Chromatin Prep Module ( Thermo Scientific ) following the manufacturer’s protocol with the following modified steps .", "2 × 106 BCBL1 cells were used per immunoprecipitation and digested with 15 units of micrococcal nuclease ( MNase ) per 100 μl of MNase Digestion buffer in a 37°C water bath for 15 min .", "Nuclei were lysed for 30 min with lysis buffer two in addition to vortexing for 30 s every 5 min .", "Immunoprecipitations were carried out using EZ-ChIP kit ( Millipore ) according to the supplier’s instructions .", "Immunoprecipitations were done overnight at 4°C and contained 50 μl of digested chromatin ( 2 × 106 cells ) , 450 μl of ChIP dilution buffer and 2 µg of the antibody of interest .", "Primers for the KSHV promoter regions of ORF50 , Ori-Lyt , PAN RNA , ORF59 and K12 have been previously reported ( Chen et al . , 2009b; Chen et al . , 2012; Hughes et al . , 2015 ) .", "Primers for the cellular GAPDH promoter were supplied with the EZ-ChIP kit .", "qPCR normalisation was performed similarly to RIP-qPCR experiments as described above .", "Determination of the cellular metabolic activity was performed using a non-radioactive CellTiter 96 AQueous One Solution Cell Proliferation Assay ( MTS ) ( Promega ) , according to the manufacturer's manual .", "10 , 000 HEK-293 cells were seeded in quadruplicate in a flat 96-well culture plate ( Corning ) .", "After 26 hr inhibitor exposure , 20 µl of CellTiter 96 AQueous One Solution Reagent was added and cells were incubated for 1 hr in a humidified incubator in 5% CO2 at 37°C .", "Absorbance was measured at 490 nm using a PowerWave XS2 ( BioTek ) plate reader ." ] ]

[ "N6-methyladenosine ( m6A ) is the most abundant internal RNA modification of cellular mRNAs .", "m6A is recognised by YTH domain-containing proteins , which selectively bind to m6A-decorated RNAs regulating their turnover and translation .", "Using an m6A-modified hairpin present in the Kaposi’s sarcoma associated herpesvirus ( KSHV ) ORF50 RNA , we identified seven members from the ‘Royal family’ as putative m6A readers , including SND1 .", "RIP-seq and eCLIP analysis characterised the SND1 binding profile transcriptome-wide , revealing SND1 as an m6A reader .", "We further demonstrate that the m6A modification of the ORF50 RNA is critical for SND1 binding , which in turn stabilises the ORF50 transcript .", "Importantly , SND1 depletion leads to inhibition of KSHV early gene expression showing that SND1 is essential for KSHV lytic replication .", "This work demonstrates that members of the ‘Royal family’ have m6A-reading ability , greatly increasing their epigenetic functions beyond protein methylation ." ]

[ "When a cell needs to make a protein , it reads from the master copy of the gene in the DNA and prints out temporary duplicates called mRNA .", "These duplicates then act as templates for protein production .", "Both DNA and mRNA can be further modified by adding on chemical tags that recruit specific proteins .", "While chemical modifications in DNA are known to control the activity of genes , their role in mRNA is only just being uncovered .", "One of the most common chemical modifications in mRNA is the addition of a methyl group called m6A .", "This methyl group has also been found in the mRNA of certain viruses , including the Kaposi’s sarcoma-associated herpesvirus ( KSHV ) which causes cancer .", "Recent work has shown that a family of proteins , known as the YTH family , can recognise and bind to this specific methyl group and regulate the rate at which mRNA degrades .", "To investigate whether other proteins can recognise m6A , Baquero-Pérez et al . mapped the m6A residues of mRNAs encoded by KSHV genes and looked at which proteins the methyl mark interacts with .", "The experiments revealed that a family of proteins – nicknamed the ‘Royal family’ – that recognise methyl groups in proteins , can also bind to mRNA that contains m6A .", "Baquero-Pérez et al . showed that a member of this family , SND1 , can read the m6A methyl mark on mRNAs from both the virus and the host cell .", "Further experiments showed that SND1 binds to and stabilises a viral mRNA which provides the template for a protein that the virus needs to replicate .", "When SND1 was removed from human immune cells infected with KSHV , this caused the virus to replicate less efficiently .", "The discovery that the Royal family of proteins can recognise methylated mRNA as well as methylated proteins suggests that there may be a common feature that allows proteins to read methylation .", "Understanding the shape of this feature could lead to new treatments that block viruses from making the proteins they need to replicate ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "developmental biology", "cell biology" ]

[ [ "The mesenchymal condensation , first recognized in limb bud condensations and named ‘precartilage condensates’ by Dame Honor Fell ( Fell , 1925 ) , is a tissue-level structure preceding organ development .", "Since then , mesenchymal condensations have been described in the precursors of several organs , occurring in most ectodermal appendages ( tooth , hair , mammary gland , feather , scales ) as well as in bone and muscle ( Widelitz and Chuong , 1999; Hall and Miyake , 2000; Newman and Bhat , 2007; da Rocha-Azevedo and Grinnell , 2013; Biggs and Mikkola , 2014 ) .", "The condensation has been suggested to be the basic cellular unit of a tissue , and to function as the driver of morphogenesis ( Atchley and Hall , 1991 ) , yet the underlying molecular and cellular mechanisms remain largely unknown .", "Condensations are morphologically distinguishable and are defined as a local increase in cell density .", "Characteristics of condensing mesenchymal cells include a change in cell shape , close surface contact between adjacent cells , and increased nucleus-to-cytoplasm ratio ( Thorogood and Hinchliffe , 1975; Searls et al . , 1972 ) ( for review see [Hall and Miyake , 2000] ) .", "Signals from the epithelium are critical for mesenchymal cell condensation .", "The question of how a local increase in cell density is achieved remains to be addressed .", "Several modes of cell condensation have been proposed , including", "i ) increase in mitotic activity ,", "ii ) active migration of cells , and", "iii ) failure of cells to disperse due to changes in cell-cell and/or cell-extracellular matrix ( ECM ) interactions ( Hall and Miyake , 1992 ) .", "The hair follicle ( HF ) is an excellent model to study the early attributes of mesenchymal condensation .", "HFs of the mouse dorsum develop in three waves as a result of reciprocal epithelial-mesenchymal signaling events with the first subset initiating morphogenesis at embryonic day ( E ) E13 . 5 and eventually producing guard hairs ( Hardy , 1992 ) .", "Within 24 hr , the previously homogenous epidermis exhibits discrete , focal thickenings , known as placodes , which are accompanied by condensation of the adjacent mesenchyme ( Biggs and Mikkola , 2014 ) .", "Coincident with dermal fibroblast condensation , their transcriptome profoundly alters .", "In particular , genes involved in cell-cell signaling such as Bmp4 and Wnt pathway components , p75 neurotrophin receptor , and many transcription factors including Sox2 , one of the earliest markers of incipient dermal condensates ( DC ) , are upregulated ( Driskell et al . , 2009; Sennett et al . , 2015; Jones et al . , 1991; Botchkareva et al . , 1999 ) .", "Another hallmark of DC formation is the differential expression of ECM molecules including tenascin , NCAM , and chondroitin sulfate proteoglycan , as well as syndecan-1 ( Richardson et al . , 2009 ) .", "As HF morphogenesis continues , the DCs become enveloped by the down-growing follicular epithelium and subsequently mature into the permanent , mesenchymal component of the HF termed the dermal papilla ( DP ) ( Morgan , 2014 ) .", "The DP directs HF cycling throughout life and its miniaturization or absence results in a thinner or absent hair shaft ( Chi et al . , 2013; Rompolas et al . , 2012 ) .", "Of note , the DP has inductive capacity demonstrated by transplantation of freshly isolated or cultured ( low passage ) rodent DP cells under glabrous epithelium , which results in induction of hair follicle development ( Oliver , 1970; Jahoda et al . , 1984 ) .", "By comparison , human DP cells lose their inductive capacity even faster in vitro but some activity can be sustained in 3D culture in aggregates ( Higgins et al . , 2013 ) , suggesting that cellular condensation is tightly linked with DP cell fate specification and maintenance .", "The molecular regulators of DC/DP morphogenesis have slowly begun to emerge .", "Absence of platelet-derived growth factor A ( Pdgf-A ) results in smaller DPs ( Karlsson et al . , 1999 ) ; however , the entire dermis is thinner , likely accounting for the DP phenotype as indicated by more recent studies in which the PDGF receptors were conditionally deleted in the dermis ( Rezza et al . , 2015 ) .", "Another placode-derived factor implicated in DC formation is Sonic hedgehog ( Shh ) ( Karlsson et al . , 1999; St-Jacques et al . , 1998; Chiang et al . , 1999 ) .", "However , conditional dermal deletion of Shh receptor Smoothened indicates a role in DC maintenance rather than induction ( Woo et al . , 2012 ) .", "Several other known DC markers such as Sox2 ( Clavel et al . , 2012 ) , Tbx18 ( Grisanti et al . , 2013a ) , Cxcr4 ( Sennett et al . , 2014 ) , and Enpp2/autotaxin ( Grisanti et al . , 2013b ) have also been conditionally ablated , but none of them results in absence of the DC .", "Perhaps surprisingly , the most informative genetic studies uncovering the molecular basis of DC formation have been those targeting the epithelium .", "Wnt signaling is believed to be the at the top of the hierarchy of signaling factors guiding HF morphogenesis , and expression of stabilized β-catenin in the epidermis results in broad adoption of placode fate as well as condensed mesenchyme concomitant with DC marker expression throughout the upper dermis ( Närhi et al . , 2008; Zhang et al . , 2008; Suzuki et al . , 2009 ) .", "The ectodysplasin ( Eda ) /Edar pathway is another essential pathway for placode morphogenesis: in its absence only rudimentary primary hair placodes form transiently and DCs are missing ( Headon and Overbeek , 1999; Laurikkala et al . , 2002; Schmidt-Ullrich et al . , 2006 ) .", "Downstream of Wnt and Eda signaling , placodal factor Fgf20 is expressed early during HF morphogenesis ( Huh et al . , 2013; Lefebvre et al . , 2012 ) .", "Deletion of Fgf20 results in absent DC in guard hairs and many secondary ( awl and auchene ) hairs as shown by morphological and molecular analyses , as well as failure in placode invagination , and ultimately , absent hairs ( Huh et al . , 2013 ) .", "Moreover , the condensed mesenchyme observed in embryos expressing stabilized epithelial β-catenin was ablated in the absence of Fgf20 further confirming the indispensable function of Fgf20 in DC induction .", "Although these molecular players have been investigated , the cellular mechanism of DC development and the role that Fgf20 plays in DC morphogenesis remain unknown .", "We therefore aimed to define the hallmarks of DC morphogenesis and the function that Fgf20 plays in this process .", "We applied a multifaceted approach to determine the cellular and molecular changes leading to DC morphogenesis using murine back skin primary hair follicle as the model due to its feasibility for manipulation and ex vivo imaging ( Ahtiainen et al . , 2014 ) .", "We identify cell shape changes and cycle exit as early hallmarks of DC morphogenesis .", "Live confocal imaging and lineage tracing showed that fibroblasts were recruited from the near vicinity and not from a pre-specified pool of Sox2+ cells , and migrate toward placodal epithelium .", "Further , Fgf20 induced fibroblast migration and cell shape change , as well as transcriptional responses that suggest its involvement in cell cycle exit .", "Collectively , our data show that Fgf20 governs multiple cellular and molecular events critical for DC formation ." ], [ "To determine to what extent the fibroblasts within the dermal condensate are more densely packed than the interfollicular fibroblasts , we examined primary HFs in E14 . 5 whole skin in 3D using confocal microscopy .", "The volume of the DC was determined by using a transgenic Sox2-GFP reporter ( Figure 1A , left panel ) , whose expression correlates well with endogenous Sox2 ( [Driskell et al . , 2009]; Figure 1A ) , and the same volume was used on an interfollicular area of the upper dermis .", "Quantification of nuclei confirmed that cells were nearly twice as dense within the DC than in the interfollicular area ( p=0 . 000106 ) ( Figure 1B ) .", "We have previously shown that Fgf20β-Gal/β-Gal ( hereafter Fgf20-/- ) mice lack all molecular signs of primary DC formation including Sox2 ( Huh et al . , 2013 ) ( Figure 1A , right panel ) , and quantification of dermal fibroblast density directly underneath the Fgf20-/- placode revealed that these fibroblasts exhibit density similar to that of the wild-type interfollicular dermis ( p=0 . 962425 ) ( Figure 1B ) .", "Next , we wanted to quantify DC formation in more detail .", "Primary hair placode induction is a continuous process first occurring near the mammary line and proceeding dorsally and caudally ( Dhouailly et al . , 2004 ) , and hence the same embryo contains hair placodes in different developmental stages .", "To better capture the dynamic nature of DC formation , we categorized hair placodes in four developmental stages .", "Follicular epithelium was identified by the Fgf20β-gal knock-in allele , one of the earliest placode markers ( [Huh et al . , 2013]; Figure 1C ) .", "Stage I was defined as a single layered placode , and stage II as a multilayered placode .", "Stage III is a placode that has invaginated into the dermis , and stage IV placodes have an anterior pocket where DC cells reside ( Figure 1C ) .", "The number and distance from placodes of Sox2+ cells was analyzed in 3D .", "Throughout these stages , the number of Sox2+ cells associated with each placode increased significantly ( p<0 . 0001 ) ( Figure 1D ) .", "Quantification of their distance from the placode revealed a progressive decrease in median distance ( p<0 . 0001 ) ( Figure 1E ) .", "At stage I , some dispersed Sox2+ cells were observed , which by stage II were preferentially oriented on the anterior side of the placode .", "By stage III , the cells appeared to be closer to the placode , and at stage IV , the Sox2+ cells maintained their proximity but increased in number ( Figure 1D , E ) .", "Mesenchymal cell condensation is often accompanied by cell shape changes ( Ray and Chapman , 2015; Mammoto et al . , 2011 ) .", "Our 3D analysis ( Figure 1A , C ) also suggested that DC formation correlates with cell shape changes .", "Transmission electron microscopy ( TEM ) revealed that E14 . 5 DC cells have an elongated , convex shape and display a characteristic alignment next to each other ( Figure 1F ) .", "3D rendering of nuclear shapes in whole mount and subsequent quantifications of nuclear sphericity during DC formation showed that the change in nuclear shape is an early indicator of DC formation ( all stages p<0 . 0001 ) ( Figure 1G , H ) .", "Consistent with the TEM and nuclear data , 3D rendering of cell shapes based on a ubiquitous cell membrane marker confirmed that Sox2+ DC cells exhibited a convex shape , whereas the non-DC fibroblasts were relatively spherical ( Figure 1—video 1 ) .", "Given that the number of Sox2+ cells increased while their distance to placode decreased over time , and that the dermis contains Sox2+ Schwann cell precursors ( Jessen and Mirsky , 2005 ) , we next asked whether the DC cells were recruited from a pre-existing pool of Sox2+ cells or whether they acquired Sox2 expression de novo .", "To this end , we utilized Sox2creERT2;R26RtdTomato/+ embryos and analyzed tdTomato expression after 24 or 48 hr of tamoxifen ( TAM ) exposure beginning at E12 . 5 ( before morphological and molecular appearance of HF ) , E13 . 5 ( earliest molecular sign of HF ) , and at E14 . 5 ( placode stage of HF ) ( Figure 2A−F ) .", "To minimize the effect of variation in HF development , we examined hair follicles from the same region in all embryos ( ventro-lateral skin ) and quantified the total number of Sox2+ cells using Sox2 antibody and analyzed the proportion of tdTomato-labeled cells amongst them ( Figure 2G , H ) .", "When labeling was induced at E14 . 5 and cells analyzed 24 hr later , 65% of Sox2+ cells were tdTomato+ , indicating that the TAM dosage used results in a relatively high labeling efficiency ( Figure 2D , G ) .", "Strikingly , when TAM was administered at E13 . 5 , only 20% of Sox2+ cells were tdTomato 24 hr later ( Figure 2C , G ) .", "This proportion , however , increased substantially when mice were analyzed at E15 . 5 ( Figure 2F , G ) .", "Finally , when TAM was administered at E12 . 5 and DC cells analyzed at E13 . 5 and 14 . 5 , only 5% and 8% of Sox2+ cells were tdTomato+ , respectively ( Figure 2B , E , G ) indicating that there is no pre-specified pool of Sox2+ cells .", "Analysis of secondary hair placodes ( that form at E15 . 5 ) in mice injected with TAM at E13 . 5 or E14 . 5 revealed that 5% and 12% of Sox2+ cells were labeled , respectively , further confirming that DC cells acquire Sox2 expression de novo ( Figure 2—figure supplement 1 ) .", "While quantifying the Sox2+;tdTomato+ cells in the DC , we noticed that they exhibited a preferential location adjacent to the placode , while cells that recently acquired DC fate ( Sox2+ , tdTomato- ) appeared further away from the epithelium ( Figure 2 ) .", "We quantified this phenomenon in E15 . 5 HFs labeled at E14 . 5 ( where 65% of the Sox2+ were tdTomato+ ) ( Figure 2G , H ) and measured the median distance of the tdTomato+ cells to the center of the placode surface .", "Our analysis revealed that the Sox2+;tdTomato+ cells were significantly closer to the placode than Sox2+;tdTomato- cells ( p=0 . 0105 ) ( Figure 2I ) .", "Nearest neighbor analysis showed that 87% of tdTomato positive cells had a tdTomato+ neighbor and hence were not randomly distributed ( p<0 . 0001 ) ( Figure 2J ) .", "Together , these data indicate that DC cells gain Sox2 expression just prior to becoming a DC cell .", "Further , we find that cells do not randomly assort after entering the DC , thus the ‘oldest’ DC cells are most likely to be located closest to the placode .", "An increase in cell number can be achieved in a number of ways .", "Our quantifications revealed that when TAM was administered at E13 . 5 , on average 20% and 53% of Sox2+ cells were tdTomato+ at E14 . 5 and 15 . 5 , respectively ( Figure 2G ) .", "Given that the average number of Sox2+ DC cells increased only by seven cells ( from 67 to 74 ) from E14 . 5 to E15 . 5 in the same dataset ( Figure 2H ) , our findings suggest that either TAM remains active for longer than 24 hr as previously suggested ( Hayashi and McMahon , 2002 ) , or that Sox2+ cells labeled between E13 . 5 and E14 . 5 increase in number by proliferation .", "To test whether locally enhanced proliferation could drive DC formation , we assessed cell cycle dynamics during DC morphogenesis with the aid of a bitransgenic cell cycle indicator Fucci mouse model ( Sakaue-Sawano et al . , 2008 ) in which mKusabira Orange ( mKO ) is expressed during the G1/G0 phase ( hereafter G1 ) and mAzami Green ( mAG ) during the S/G2/M phase ( Figure 3A ) .", "The interfollicular dermal cells ( Sox2- ) were equally distributed between G1 and S/G2/M phases at all stages of placode morphogenesis analyzed ( Figure 3B ) .", "In contrast , the Sox2+ cells showed progressive exit from the cell cycle .", "At stage I , the Sox2+ cells were nearly evenly distributed between proliferative and non-proliferative phases .", "By stage II , the majority of Sox2+ cells were in G1 , which persisted through stages III and IV ( pI = 0 . 0166 , pII = 0 . 002 , pIII , IV < 0 . 001 , Figure 3B ) .", "Further , the percent non-proliferating cells in the stage I DC was significantly lower than the following stages ( pIvsII = 0 . 0211 ) , suggesting that DC fate acquisition occurs before cell cycle exit .", "This cell cycle exit was not transient as the vast majority of DC cells remained in G1 through E15 . 5 ( Figure 3B , C ) .", "To determine whether this cell cycle exit is dependent on Fgf20 , we analyzed the cell cycle status of E14 . 5 Fgf20-/- fibroblasts .", "Immediately below the placode , in a volume equivalent to a wild-type stage IV DC , the proportion of G1 and S/G2/M cells did not differ from that of the interfollicular dermal cells ( p=0 . 473 , Figure 3B and D ) .", "To further substantiate our findings , we examined the cell cycle distribution in mice overexpressing Eda under the Keratin14 promoter ( K14-Eda ) , a model of enlarged DC .", "Not only are the placodes bigger at E14 . 5 than in the wild type ( Mustonen et al . , 2004; Ahtiainen et al . , 2014 ) , but there are also more Sox2+ cells ( Huh et al . , 2013 ) .", "Similar to control DC , about 95% of the Sox2+ DC cells were in the G1 phase ( Figure 3B , E ) indicating that increased DC size can be achieved without enhancing cellular proliferation .", "Similar findings were observed with the R26Fucci2aR cell cycle indicator mouse model ( Figure 3—figure supplement 1 ) .", "To confirm our observations , we further assessed cell proliferation using a uridine-analogue EdU incorporation assay at different stages of DC development ( Figure 3F ) .", "Concordantly with the Fucci-reporter analysis , at stage I we observed that 18% of the Sox2+ DC cells were EdU positive , but at stage II the proportion of EdU-positive cells dropped significantly to 5 . 2% ( pIvsII = 0 . 003 ) and remained unchanged through stage IV ( pIIvsIII = 0 . 277 , pIIIvsIV = 0 . 591 ) .", "Collectively , these data suggest that cell cycle exit is a hallmark of dermal condensate morphogenesis .", "Having ruled out proliferation as a mechanism for the increased cell density in DC cells , we next assessed the contribution of cellular migration by using live , confocal 3D imaging .", "We utilized Sox2-GFP;FuccimKO skins to monitor the behavior of DC-forming cells and as a control , tracked interfollicular , non-DC fibroblasts ( Sox2-GFP− ) at the corresponding cell cycle phase ( FuccimKO+ cells ) .", "Cultures were initiated at E13 . 75 , a time point when incipient DCs were visible ( Figure 4A and Video 1 ) .", "To characterize cell movement in detail , we quantified velocity , net velocity , straightness , and directionality .", "We initially tracked all Sox2-GFP+ DC cells ( Figure 4A−C ) , but observed that many of the cells that were present in the condensates at the beginning of tracing displayed low velocity suggesting that their movement is restricted ( Figure 4D ) .", "However , cells that were initially further than 30 µm ( the average DC diameter ) from the center of the condensate at the beginning of tracing behaved differently .", "Notably , these cells showed a preferential movement direction towards the condensate center , while interfollicular cells exhibited no directionality ( Figure 4B , C ) .", "We observed that the condensate-forming fibroblasts also moved at a significantly higher velocity than interfollicular cells ( p=0 . 0051 ) ( Figure 4D ) yet there was no significant difference between the straightness of cell movement or net velocity ( p=0 . 8945 and p=0 . 1390 , respectively ) ( Figure 4E , F ) .", "Furthermore , when we analyzed migration of condensate-forming fibroblasts only until they enter the dermal condensate , not only were they preferentially moving toward the DC center but also migrated on a significantly straighter track ( p=0 . 0232 ) and had higher net velocity than the interfollicular cells ( p=0 . 0006 ) ( Figure 4—figure supplement 1 ) .", "As cell migration is associated with remodeling of the actin cytoskeleton ( for review see [Svitkina , 2018] ) , we investigated whether Sox2-GFP cells presumably en route to the DC could be distinguished from other dermal fibroblasts or Sox2-GFP cells already present in the DC based on the organization of their actin cytoskeleton .", "The non-DC Sox2-GFP cells displayed a slightly higher intensity of F-actin than dermal fibroblasts , which may be indicative of their migratory status ( Figure 4—figure supplement 2A , B ) .", "However , the Sox2-GFP cells within the DC displayed even higher levels of F-actin intensity compared to non-DC Sox2-GFP cells ( Figure 4—figure supplement 2A , B ) .", "Further , the intensity of F-actin increased as the DC progressed through morphogenesis ( Figure 4—figure supplement 2D , E ) .", "As these cells exhibited reduced motility , this suggests that the F-actin is involved in maintaining the 3D configuration of the DC .", "Together , these results suggest that directed migration of the dermal fibroblasts is driving DC formation , but once the cells have entered the DC , their motile behavior changes , likely due to limitations posed by increased cell density , a finding in line with our lineage tracing/nearest neighbor analysis ( Figure 2I , J ) .", "In order to address the function of Fgf20 in DC formation , we first aimed to identify its immediate transcriptional targets .", "Isolated E13 . 5 Fgf20-/- dermises were cut into two pieces: one half was incubated in recombinant FGF20 for 3 hr , and the other half in BSA .", "RNA sequencing was carried out on five pairs of biological replicates ( Figure 5A ) .", "Differential gene expression analysis revealed 40 protein-coding genes including many known Fgf/MAPK pathway target genes and feedback regulators ( Ornitz and Itoh , 2015; Murphy et al . , 2010 ) , such as those within the Dual-specificity phosphatase ( Dusp ) , Sprouty and Sprouty-related Spred gene families ( Table 1 ) .", "We validated the RNAseq by qRT-PCR analysis of a subset of these genes in independently generated samples ( Figure 5B ) .", "Of the 31 upregulated genes , 7 belong to the recently identified DC signature , and an additional 5 genes were >2 x more highly expressed in DC compared to non-DC fibroblasts in the same study ( Sennett et al . , 2015 ) .", "Notably , several genes implicated in cell cycle regulation such as Bcl6 and Cdkn1a ( p21 ) , a cyclin-dependent kinase inhibitor , which we previously identified as an early DC marker ( Huh et al . , 2013 ) , were among the upregulated genes ( Table 1 ) .", "To assess whether Fgf20 could also drive the expression of the potential transcriptional target genes in vivo we attempted to create a gain-of-function mouse line expressing Fgf20 under the K14 promoter .", "Unfortunately , we were unsuccessful in generating K14-Fgf20 lines that would display detectable Fgf20 overexpression , and we therefore took advantage of a mouse model overexpressing Edar , the upstream regulator of Fgf20 , under the K14 promoter ( Pispa et al . , 2004 ) .", "We confirmed upregulation of Edar expression in the entire basal epithelium by in situ hybridization ( Figure 5—figure supplement 1 ) .", "Accordingly , Fgf20 , visualized by the Fgf20β-gal knock-in reporter , was ectopically expressed in the basal layer from E15 . 5 onwards ( Huh et al . , 2013 ) ( Figure 5C ) .", "Consistent with our RNAseq analysis , we observed ectopic Cdkn1a expression in the mesenchyme immediately adjacent to the Fgf20-expressing epithelium on the K14-Edar background , whereas in the control dermis its expression was confined to the DC ( Figure 5C ) , as reported previously ( Huh et al . , 2013 ) .", "Importantly , Cdkn1a upregulation was Fgf20-dependent , as shown by its absence on the K14-Edar;Fgf20-/- background ( Figure 5C ) .", "Further , Sox2 which was not upregulated by Fgf20 in our RNAseq data , was not detected in the K14-Edar or K14-Edar;Fgf20-/- embryos , but was readily observed in control embryos ( Figure 5C and Figure 5—figure supplement 1 ) .", "Collectively , these data suggest that Fgf20 regulates a subset of DC genes including Cdkn1a , a well-characterized inducer of G1 arrest of the cell cycle .", "Our analyses of 3D live imaging data showed that directional migration drives dermal condensate morphogenesis .", "This observation led us to ask whether Fgf20 signaling plays a role in this process .", "First , we analyzed the effect of Fgf20 signaling on migration of a monolayer of growth-arrested E13 . 5 primary dermal fibroblasts in a scratch wound healing assay over 24 hr .", "FGF20 induced significantly faster wound closure compared to control fibroblasts ( p=0 . 0177 ) , an effect that was abolished in the presence of an Fgfr inhibitor , SU5402 ( p=0 . 6100 ) , confirming the specificity of the response to FGF20 ( Figure 6A and B ) .", "SU5402 alone had no significant effect on wound closure ( p=0 . 2056 ) ( Figure 6A and B ) .", "Similar observations were made when cells were treated with FGF9 , a member of the same Fgf subfamily as Fgf20 ( Figure 6—figure supplement 1 ) .", "The scratch wound healing assay , however , does not distinguish between directional ( chemotaxis ) and non-directional cell migration ( chemokinesis ) .", "To assess whether Fgf20 could function as a chemotactic factor , we analyzed migration of growth-arrested E13 . 5 primary dermal fibroblasts in a transwell assay .", "When FGF20 was present in the lower chamber only , the number of migrating cells was significantly higher than in control wells ( p=0 . 0003 ) ( Figure 6C ) .", "When the FGF20 gradient was abolished by adding FGF20 also to the upper chamber , again a significantly higher number of cells migrated to the lower chamber ( p<0 . 0001 ) , however , there was no difference between the two types of FGF20 treatments ( p=0 . 2256 ) ( Figure 6C ) .", "Similar data was obtained when FGF9 was used instead of FGF20 ( Figure 6—figure supplement 1 ) .", "Taken together , these data indicate that Fgf20 induces migration of embryonic dermal fibroblasts in vitro and that this effect is likely chemokinetic .", "Next , we wanted to test the impact of Fgf20 in a more physiological setting .", "To this end , we introduced FGF20 locally via a bead on E13 . 5 dermis explants to mimic the in vivo situation in which Fgf20 is produced locally in the placode .", "First , we investigated the activity of FGF20 in this context by assessing its ability to upregulate the expression of Spry4 and Dusp6 , two known Fgf pathway feedback inhibitors also differentially expressed in our RNAseq data ( Figure 5 ) , using whole-mount RNA in situ hybridization .", "While we observed a robust induction of both genes after a 3 hr treatment , no consistent responses were detected after 8- and 16 hr treatments ( Figure 6H ) , suggesting that the FGF20 protein loses its activity in intact dermal explants over this period of time .", "Vehicle-loaded control beads showed no induction of gene expression after any treatment period ( Figure 6H ) .", "We further analyzed whether FGF20 bead incubation results in upregulation of Sox2 , but observed no induction at any time point analyzed ( Figure 6H ) .", "FGF9 beads led to a prominent induction of Spry4 and Dusp6 after all analyzed treatment periods ( Figure 6—figure supplement 1 ) .", "However , an overnight treatment also led to a robust increase in cell proliferation around the FGF9 bead ( Figure 6—figure supplement 1 ) .", "This response is in contrast to what we observed during dermal condensate formation in vivo ( Figure 3 ) indicating that this experimental set-up may not represent a physiologically relevant model to study DC cell behavior and thus we concentrated on FGF20 .", "We next used the bead assay to test the impact of FGF20 on fibroblast cell behavior .", "To do this , we quantified the density of nuclei in E13 . 5 dermal explants cultured 3 hr with FGF20 or BSA vehicle control beads ( Figure 6D , E ) .", "We observed a significant , 35% increase in cell density 15 µm around the FGF20 bead compared to the control bead ( p=0 . 002 ) , indicating that a localized Fgf20 source can induce aggregation of mesenchymal cells .", "We observed a similar increase in cell density upon treatment with FGF9 bead ( p=0 . 033 ) ( Figure 6—figure supplement 1 ) .", "Nuclear shape analysis showed that cells in the immediate vicinity of the FGF20 bead displayed a significant decrease in nuclear sphericity compared to control bead ( p<0 . 0001 ) ( Figure 6F , G ) , similarly to DC cells in vivo ( Figure 1G , H ) .", "We also tested whether cell cycle exit was induced upon local addition of FGF20 .", "We could not detect Cdkn1a induction and no significant increase in Fucci-mKO+ cells were observed around the bead ( Figure 6—figure supplement 2 ) .", "Together this data suggests that Fgf20 can induce some of the cellular changes observed during DC morphogenesis .", "Finally , we analyzed whether ectopic expression of Fgf20 could also lead to condensation of the mesenchyme in the K14-Edar model .", "We did not detect any significant difference in the cell density in the upper dermis between control , K14-Edar , and K14-Edar;Fgf20-/- genotypes ( p>0 . 115 ) ( Figure 5—figure supplement 1 ) .", "However , given that Cdkn1a was expressed throughout the upper dermis in K14-Edar embryos , a lack of condensation in this model could be due to a lack of supply of cells available to condense .", "The Fgf20-/- mouse model lacks all molecular and cellular signs of DC formation ( Huh et al . , 2013 ) ( Figure 1A , B ) and therefore offers limited tools to assess the effect of Fgf20 on cellular mechanisms governing DC formation .", "Therefore , we decided to inhibit Fgf signaling using SU5402 ex vivo which allows precise temporal manipulation of pathway activity followed by DC analysis in 3D .", "The same concentration of SU5402 that blocked the ability of Fgf20 to induce migration of E13 . 5 fibroblasts ( Figure 6 ) , also fully suppressed DC formation when applied to E13 . 5 skin cultured for 24 hr ( Figure 7—figure supplement 1 ) .", "Yet , it led to the expansion of Fgf20β-Gal knock-in allele expression into a stripe-like pattern ( Figure 7—figure supplement 1 ) , as also observed in E14 . 5 Fgf20-/- embryos in vivo ( Huh et al . , 2013 ) confirming the applicability of this approach .", "SU5402 can also inhibit VEGFR and thus we used an inhibitor more specific to VEGFR , XL184 , to test the effects of VEGFR-inhibition on DC formation .", "We added XL184 at an equivalent dose to inhibit VEGFR as 20 µM SU5402 ( Figure 7—figure supplement 2 and Figure 7—figure supplement 2—source data 1 ) as well as at five-times higher concentration and observed normal DC formation ( Figure 7—figure supplement 2D , E ) .", "Finally , we confirmed that the absence of DCs in the SU5402-treated samples was due to inhibition of FGFR-signaling by using BGJ398 , an inhibitor more specific to FGFR ( Figure 7—figure supplement 2 ) .", "An equivalent dose to inhibit FGFR as 20 µM SU5402 and a 2 . 5-times higher dose blocked formation of the DCs in the skins and altered the Fgf20β-Gal knock-in allele expression similar to the Fgf20-/- animals ( Figure 7—figure supplement 2 ) , confirming the effects of the SU5402 to be due to FGFR-inhibition .", "Next , we applied SU5402 slightly later , when DC formation had initiated .", "Skin explants were divided into two halves: one was used as the control and the other one treated with SU5402 ( Figure 7A and Figure 7—figure supplement 1 ) .", "At this stage , the inhibitor did not result in absence of dermal condensates , but the number of Sox2+ cells was significantly lower in the SU5402-treated samples compared to the controls ( p=0 . 0061 ) ( Figure 7B ) , indicating that Fgf signaling is necessary for further addition of Sox2+ cells .", "Analysis of the average distances of DC cells to their nearest neighbor , however , showed no difference between control and SU5402 treated samples ( p=0 . 7319 ) ( Figure 7C ) suggesting that inhibition of Fgf signaling does not affect the density of the existing DC .", "To assess whether Fgf20 could also play a role in the maintenance of DC cells , we compared dermal condensates at the start ( T0 ) of experiment with condensates after 12 hr SU5402 treatment ( Figure 7D and Figure 7—figure supplement 1 ) .", "A significant reduction in the number of Sox2+ DC cells was observed ( p=0 . 0036 ) ( Figure 7E ) .", "Again , Sox2+ DC cell density was not affected by SU5402 treatment ( p=0 . 3010 ) ( Figure 7F ) .", "Taken together , these results highlight the role of Fgf signaling both in recruitment and maintenance of DC cells ." ], [ "The origin of the DC/DP population is poorly understood .", "Tissue recombination and mouse mutant studies ( reviewed in [Biggs and Mikkola , 2014; Morgan , 2014] ) together with the ex vivo Fgf inhibitor experiments ( this study ) show that early DC fate is plastic and reversible .", "Yet , these findings do not preclude the existence of a predetermined pool of DC cells .", "Previous studies have shown that DPs of the dorsum largely derive from the early fibroblast precursor population marked by expression of Delta-like homologue 1 ( Dlk1 ) ( Driskell et al . , 2013 ) , and are polyclonal in origin ( Collins et al . , 2012 ) .", "Dlk1 lineage tracing from E12 . 5 ( when all dermal fibroblasts are Dlk1+ ) , but not after E16 . 5 , reveal a contribution to post-natal DP ( Driskell et al . , 2013 ) .", "Lineage tracing of neural crest cells with Wnt1-Cre or Ht-PA-Cre reveals that the whisker DP ( along with non-DC head/facial fibroblasts ) is neural crest-derived , but that neural crest cells or their progeny are rare in back skin DP ( Fernandes et al . , 2004; Wong et al . , 2006 ) .", "Our short-term lineage-tracing experiments show that Sox2 expression is de novo acquired in DC cells , confirming that the dorsal DCs do not arise from a pre-existing pool of Sox2+ cells , for example the neural crest-derived Schwann cell lineage of the skin ( Adameyko et al . , 2012; Sennett et al . , 2015 ) .", "Studies in an adult model of ectopic hair follicles via forced epithelial β-catenin also argue against a pre-specified DC/DP subpopulation of dermal fibroblasts ( Collins et al . , 2012 ) .", "Hence , all available data suggest that the unique attributes of DC/DP cells do not reflect a distinct fibroblast lineage but are induced by placode-derived signals .", "We show in 3D that HF DCs exhibit a less spherical shape in vivo , and that this shape change is an early event during DC morphogenesis .", "Previous studies in other organs ( bone , tooth , feather ) have also revealed an altered cell shape in condensed mesenchyme when compared to the adjacent non-condensed tissue ( Thorogood and Hinchliffe , 1975; Ray and Chapman , 2015; Mammoto et al . , 2011; Wessells , 1965 ) .", "However , these analyses were conducted in 2D and thus their similarity or difference with the HF DC is not apparent .", "What is of note is that the cells within condensed mesenchyme display a distinct morphology .", "Indeed , a critical role for actomyosin contractility has been proposed to drive cytoskeletal rearrangements , and that the resulting cell shape changes are required for mesenchymal condensation ( Ray and Chapman , 2015 ) .", "Here , we show that in the hair follicle DC the intensity of F-actin is increased in the DC compared to the surrounding mesenchyme , likely playing a critical role in maintaining cell shape .", "We show that in hair follicle DC , the cell shape changes depend on and can be induced by Fgf20 .", "The utility of this cell shape change along with cell compaction could be manifold , including increased cell-cell contacts to foster cell-cell communication and further to maintain the structure of the DC .", "Transcriptomic analysis has indicated the expression of cell-cell adhesion factor R-cadherin and cadherin11 ( encoded by Cdh4 and Cdh11 , respectively ) in the DC ( Sennett et al . , 2015 ) and cadherin-based junctions were detected between DP cells at E17 ( Nanba et al . , 2003 ) , but their functional importance for condensation remains to be tested .", "During DC morphogenesis , Sox2+ cells were found to exhibit a rapid exit from the cell cycle and maintain the G0/G1 status through E15 . 5 , a finding in line with a previous study showing that morphologically distinct DC cells fail to incorporate H3-thymidine ( Wessells and Roessner , 1965 ) .", "Even as the size of the DC is increased by genetic means ( K14-Eda ) , the DC cells remain quiescent supporting the idea that the increase in DC cell number is not a result of proliferation .", "Further , mitotic inactivity is a prominent feature of the mature HF DP ( Pierard and de la Brassinne , 1975 ) .", "During the growth ( anagen ) and rest ( telogen ) phases of the hair cycle , however , the DP dynamically increases and decreases in cell number .", "Yet , the increase in DP cell number upon reentering anagen phase does not arise via proliferation but instead DP cells are recruited from the proximal dermal sheath cells ( Tobin et al . , 2003; McElwee et al . , 2003; Chi et al . , 2010 ) .", "Furthermore , hair reconstitution experiments show that mitotically inhibited cells are fully competent to generate the DP ( Collins et al . , 2012 ) indicating that proliferation is not necessary for DC/DP formation .", "We have previously shown that expression of Cdkn1a is an early marker of DCs ( Huh et al . , 2013 ) , and recent transcriptome profiling study showed that Cdkn1c ( a . k . a . p57 ) , another cyclin dependent kinase inhibitor , is also expressed at high levels in the DC ( Sennett et al . , 2015 ) .", "Interestingly , Fgf20 target transcriptome analysis displayed upregulation of cell-cycle-related genes , such as Cdkn1a and Bcl6 , which suggests that Fgf signaling could provide the cue for the G0-arrest observed in the DC fibroblasts .", "Despite this , FGF20 was unable to induce cell cycle arrest in the dermis in a short-term experiment when supplied in a localized manner .", "However , Cdkn1a was ectopically expressed in our model of Fgf20 overexpression ( K14-Edar ) in an Fgf20-dependent manner .", "Similarly , Fgf signaling induces Cdkn1a-mediated cell cycle arrest in chondrocytes ( Aikawa et al . , 2001 ) and further , ectopic Fgf20 induces growth arrest of rat chondrosarcoma cells in vitro ( Buchtova et al . , 2015 ) .", "Bcl6 is also a DC signature gene ( Sennett et al . , 2015 ) and although its function is context-dependent , it has been shown to suppress proliferation of many primary cells including fibroblasts ( Ranuncolo et al . , 2008 ) .", "Our confocal time-lapse imaging of developing HF revealed that dermal fibroblasts initially outside of the future condensate migrate toward the placode , indicating directed migration as a mechanism of DC formation .", "A recent study tracking movements of Wnt-responsive cells using TCF/Lef::H2B-GFP reporter ( Glover et al . , 2017 ) is consistent with our findings .", "In vitro scratch wound assay revealed that Fgf20 enhances cell motility .", "Although transwell assays did not support a chemotactic role for Fgf20 , we showed that local delivery of FGF20 via beads on dermal explants induces an increase in cell density , suggesting that Fgf20 induces directed migration and in a tissue context , dermal condensation .", "Further , we find that inhibition of Fgfr signaling ex vivo blocks accumulation of Sox2+ fibroblasts in the DC while Fgf20 does not appear to directly regulate Sox2 expression .", "These data support the conclusion that Fgf20 signaling regulates directed movement of dermal fibroblasts .", "An additional factor that we did not test but may play a role in DC morphogenesis is the contribution of differential ECM composition surrounding the hair follicle ( Kaplan and Holbrook , 1994; Pflieger et al . , 2006 ) .", "It is possible that Fgf20 induces migration of fibroblasts and simultaneously , differential ECM composition around the hair follicle holds the DC cells in place .", "Previous studies have shown that Fgf signaling induces migration , both chemotactic and chemokinetic , in several different developmental contexts ( Mammoto et al . , 2011; Delfini et al . , 2005; Attia et al . , 2012 ) .", "Indeed , a general role for Fgf signaling in regulating dermal condensation via cell migration is an appealing idea .", "Our ex vivo manipulation experiments showed that inhibition of Fgfr signaling suppresses accumulation of new Sox2+ cells , but also compromises maintenance of nascent DC .", "The latter finding is suggestive for a role also in DC maintenance .", "In odontogenic mesenchyme , attractive Fgf8 and repellent Sema3f are thought to act in concert to induce migration-driven cell compaction ( Mammoto et al . , 2011 ) .", "Involvement of Fgf signaling in mammary mesenchyme condensation is an intriguing hypothesis , but has not been examined .", "Fgf20 is expressed in the epithelium of the mammary buds during the period of mesenchymal condensation ( Elo et al . , 2017 ) yet it seems to be dispensable for this process , but other epithelially expressed Fgfs may compensate for the loss of Fgf20 .", "In contrast , Fgf20 is necessary for feather development ( Houghton et al . , 2007; Wells et al . , 2012 ) .", "Although the exact function of Fgf20 in feather morphogenesis has not been studied , the ability of exogenous Fgfs to induce dermal cell aggregation ex vivo ( Lin et al . , 2009; Song et al . , 2004 ) and to elicit feather formation in Fgf20-deficient skin explants ( Song and Sawyer , 1996 ) argue for a conserved role for Fgf20 in DC induction .", "Although the transcriptional profile of the DC has been described ( Sennett et al . , 2015 ) , the molecular regulation of DC fate acquisition is not well understood .", "Fgf20 is necessary for DC marker expression ( Huh et al . , 2013 ) and here we show that Fgf20 is also necessary for dermal cell condensation .", "However , our RNAseq profiling of genes induced in the dermis upon short Fgf20 treatment revealed only a few DC signature genes ( Sennett et al . , 2015 ) , and for example Sox2 , was not upregulated by Fgf20 ex vivo , nor in our in vivo over-expression model of ectopic Fgf20 signaling .", "These data suggest that Fgf20 alone is sufficient to induce only a subset of DC markers .", "It is possible that other cues , molecular or mechanical , together with Fgf20 determine DC fate , and studies in dental and cartilage mesenchyme show that cellular condensation contributes to fate acquisition ( Mammoto et al . , 2015; Mammoto et al . , 2011; Ray and Chapman , 2015 ) .", "Alternatively , it is possible that Fgf20 functions mainly to regulate cell behaviors rather than fate .", "In conclusion , we show here that cell shape change , cell cycle exit and directed migration define HF DC formation .", "While altered cell shape and directed mesenchymal cell movement may be a shared characteristic , cell cycle exit appears to be a hair follicle specific feature , as cells within the condensing mammary ( Lee et al . , 2011 ) and tooth mesenchyme exhibit proliferation ( Mammoto et al . , 2011 ) at the same rate as the surrounding non-condensed mesenchyme .", "However , the function of DC cell cycle exit in hair follicle morphogenesis remains unknown .", "It appears not to be sufficient to induce DC fate , but it remains open whether it is necessary .", "To date , culturing methods for maintaining the hair-follicle inductive capacity of DC/DP remain elusive , and after a few passages , DP populations completely lose their inductive abilities .", "Although speculative , the obligate proliferation under in vitro culture may compromise DC fate maintenance .", "The challenge in future therapeutic efforts to generate hair inductive fibroblasts is to uncover culture conditions that induce DC cell fate de novo ." ], [ "All mouse studies were approved and carried out in accordance with the guidelines of the Finnish national animal experimentation board .", "The following mice were maintained on C57Bl/6 background .", "Fgf20β-Gal mice harbor an Fgf20-β-Galactosidase knock-in allele ( Huh et al . , 2013 ) ; Sox2CreER was obtained from Jackson Laboratory ( Stock 017593 ) ; R26RtdTomato was obtained from Jackson Laboratory ( Stock 007914 ) ; R26RmT/mG was obtained from Jackson Laboratory ( Stock 007576 ) .", "R26Fucci2aR mice ( Mort et al . , 2014 ) were obtained from the European Mouse Mutant Archive ( EM:08395 ) and upon derivation mated with ubiquitous cre line , Pgk1-cre ( Jackson Laboratory; Stock 020811 ) in order to obtain mice which heritably and constitutively express the Fucci2a construct in every cell .", "Sox2-EGFP , K14-Eda , and K14-Edar have been described ( Mustonen et al . , 2003; Pispa et al . , 2004 , D'Amour and Gage , 2003 ) .", "Fucci cell cycle indicator mice ( Sakaue-Sawano et al . , 2008 ) were maintained on a mixed background .", "Mice were kept in 12 hr light-dark cycles and food and water were available ad libitum .", "To label Sox2-expressing cells , pregnant wild-type dams mated with Sox2CreER/wt; R26RtdTomato/tdTomato males were given one intraperitoneal injection of 3 mg tamoxifen ( Sigma-Aldrich , Saint Louis , MO ) dissolved in corn oil ( Sigma-Aldrich ) at 12pm on the indicated day of pregnancy ( appearance of a vaginal plug was taken as embryonic ( E ) 0 ) .", "All embryos used in the study were staged according to limb and other external morphological criteria .", "Samples were fixed in 2 . 5% glutaraldehyde at room temperature for 2 hr , washed in 0 . 1 M NaPO4 , and subsequently fixed in 2% PFA in 0 . 1 M NaPO4 .", "The samples were then dehydrated through a graded series of ethanol and acetone before embedding in Epon .", "Ultra-thin sections were generated .", "Images were acquired with Jeol JEM-1400 electron microscope ( Jeol Ltd . , Tokyo , Japan ) .", "For whole-mount RNA in situ hybridization , cultured dermal explants were fixed to their filter with cold methanol for 2 min and then fixed in 4% PFA in PBS overnight at 4°C , washed with PBS and then dehydrated in a series of methanol .", "The hybridization was performed using InSitu Pro robot ( Intavis AG , Cologne , Germany ) as described before ( Fliniaux et al . , 2008; Huh et al . , 2013 ) .", "Briefly , the samples were rehydrated in a methanol series , treated with 10 µg/ml Proteinase K ( Roche , Mannheim , Germany ) for 5 min and post fixed with 4% PFA .", "The hybridization was performed with digoxigenin-labeled antisense RNA probes: Dusp6 ( Dickinson et al . , 2002 ) , Cdkn1a ( Jernvall et al . , 1998 ) , Spry4 ( Zhang et al . , 2001 ) , and Sox2 ( Ferri et al . , 2004 ) , 1 µg/ml in hybridization buffer at 65°C for 14 hr .", "After hybridization , the excess probe was removed in stringent washes , samples were blocked , the probe was detected with an alkaline phosphatase conjugated anti-digoxigenin antibody ( Roche , Mannheim , Germany ) , and a subsequent reaction with precipitating alkaline phosphatase substrate BM purple ( Roche , Mannheim , Germany ) .", "Samples were fixed with 4% PFA and imaged using Lumar . V12 stereomicroscope with 1 . 2x objective and AxioCam ICc camera ( Zeiss , Oberkochen , Germany ) .", "For radioactive section in situ hybridization , E16 . 5 embryos were collected , fixed in 4% PFA in PBS and processed into paraffin blocks using standard protocols and cut into 5 µm sagittal sections .", "Radioactive in situ hybridization with 35S-UTP-labeled probes: Edar ( Laurikkala et al . , 2001 ) , Sox2 ( Ferri et al . , 2004 ) , and Cdkn1a ( Jernvall et al . , 1998 ) , was performed as previously described ( Huh et al . , 2013 ) .", "Sections were imaged using Axio Imager M . 2 widefield microscope equipped with Plan-Neofluar 20x/0 . 5 objective and AxioCam HRc camera ( Zeiss ) using bright and dark field microscopy .", "The dark field images were inverted , thresholded linearly and superimposed on the bright field images using Adobe Photoshop software ( Adobe , San Jose , CA ) .", "Fgf20β-Gal/+ embryos were pre-fixed for 2 hr in 2% PFA , 0 . 2% glutaraldehyde ( Sigma-Aldrich , St . Louis , MO ) in PBS , 4°C and then rinsed with PBS and washed 3 × 15 min with PBS , 2 mM MgCl2 ( Merck , Darmstadt , Germany ) , 0 . 02% NP-40 ( Sigma-Aldrich ) , 4°C .", "Subsequently , the samples were stained for 10 hr at RT , in dark with 1 mg/ml X-Gal ( Thermo Fischer Scientific , Vilnius , Lithuania ) , 5 mM K3Fe ( CN ) 6 ( Merck , Darmstadt , Germany ) , 5 mM K4Fe ( CN ) 6 ( Merck , Darmstadt , Germany ) , 2 mM MgCl2 , 0 . 1 % NP-40 ( Calbiochem , San Diego , CA ) , 0 . 2% Na-deoxycholate ( Sigma-Aldrich , St . Louis , MO ) in PBS .", "The embryos were washed 3 × 10 min with PBS and fixed with 4% PFA in PBS at RT .", "After imaging , the samples were processed for paraffin blocks using standard protocols and sectioned into 5 µm sagittal sections .", "The sections were deparaffinized and counter stained with Nuclear Fast Red ( Sigma-Aldrich , Steinheim , Germany ) for 5 min , dehydrated and mounted .", "The sections were imaged using Axio Imager M . 2 wide field microscope equipped with Plan-Neofluar 10X/0 . 3 objective and AxioCam HRc camera ( Zeiss ) .", "Primary dermal fibroblasts were extracted from E13 . 5 wild-type NMRI mouse embryos .", "Briefly , the back skin was dissected and treated with 2 . 5 mg/ml Pancreatin ( Sigma-Aldrich ) , 22 . 5 mg/ml Trypsin ( Difco , Sparks , MD ) in Tyrode’s solution for 8 min at RT , followed by an incubation with 10% FBS in DMEM ( Gibco by Life Technologies , Paisley , UK ) for 1 hr at RT .", "The tissues were manually separated; epidermis was discarded and the mesenchyme was gently dissociated by pipetting in 0 . 2% FBS in DMEM .", "For scratch wound healing assay , the fibroblasts were seeded on fibronectin-coated plates ( 1 µg/cm2 , R and D Systems , Minneapolis , MN ) at the density of 125 , 000 /cm2 and cultured for 16 hr in 0 . 2% FBS , 1% penicillin-streptomycin ( Life Technologies , Eugene , OR ) in DMEM .", "Cell cycle inhibition was achieved by a 2 hr treatment with 5 ng/ml aphidicolin ( Sigma-Aldrich , Jerusalem , Israel ) .", "Scratches were induced with a pipette tip and the cells were treated with human recombinant FGF20 ( 200 ng/ml , Peprotech , Rocky Hill , NJ ) , human recombinant FGF9 ( 200 ng/ml , R and D Systems ) , SU5402 ( 20 µM , Calbiochem , Darmstadt , Germany ) or BSA ( 0 . 1% ) .", "4 µg/ml heparin ( Sigma-Aldrich , St . Louis , MO ) was added with the FGF proteins .", "Conditions were carried out in duplicate in five independent experiments .", "Wounds were imaged at 0 , 4 , 8 , and 24 hr using Leica DM IRB phase contrast microscope ( Leica Microsystems ) , and ImageJ ( http://rsbweb . nih . gov/ij/ ) was used to manually measure the open area at the indicated time points in two locations .", "Proportion of closure was determined as the ratio of open area at each time point compared to 0 hr .", "For transwell migration assay , 50 , 000 freshly isolated cells were seeded in 300 µl of DMEM , 1% FBS in cell culture inserts for 24-well plates ( Millicell , Basel , Switzerland ) and cultured overnight .", "The cells were then treated with 200 ng/ml FGF20 , FGF9 or 0 . 1% BSA vehicle control ( baseline ) in DMEM containing 1% FBS and 5 ng/ml aphidicolin; conditions were carried out in duplicate in six independent experiments .", "The proteins were introduced either in the receiving compartment or both receiving and seeding compartments and cells were allowed to migrate for 8 hr .", "The seeding compartment side of the membrane was mechanically cleared of cells; the remaining cells were fixed with methanol 5 min , and the membrane was stained with 0 . 1% Crystal violet ( Sigma-Aldrich , St . Louis , MO ) , 70% ethanol in RO-water .", "Absolute cell number was counted at five positions on each membrane .", "Relative migration was determined as the average number of cells in the duplicate inserts divided by average number of cells in the duplicate inserts .", "E13 . 5 skins were dissected from Fgf20-/- embryos and enzymatically separated using 2 . 5 U/ml Dispase II ( Roche by Godo Shusei , Tokyo , Japan ) in 4°C and 30 min resting in culture medium , followed by mechanical separation .", "Each dermis was cut into two halves along the midline and one half was incubated in FGF20 ( 1 µg/ml ) and the other half was incubated in 0 . 1% BSA vehicle control in 0 . 1% FBS , heparin ( 2 µg/ml ) , 1% penicillin-streptomycin in DMEM .", "The tissues were incubated in hanging drops of the indicated media for 3 hr at 37°C , 5% CO2 .", "For RNAseq , five biological replicates were used .", "The samples were stored in RNAlater ( Qiagen GmbH , Hilden , Germany ) .", "RNA was extracted using RNeasy Plus micro kit ( Qiagen GmbH , Hilden , Germany ) , according to manufacturer’s instructions .", "RNA quality was assessed with 2100 Bioanalyzer ( Agilent , Santa Clara , CA ) and RIN values averaged 9 . 6 .", "The cDNA libraries were prepared with TruSeq Stranded Total RNA with RiboZero ( Illumina , San Diego , CA ) , and sequenced with NextSeq500 ( Illumina , San Diego , CA ) .", "The Illumina-seq reads produced around 35 million reads for each sample .", "The quality of each sample was assessed with FastQC and processed with AfterQC ( Chen et al . , 2017 ) .", "The ribosomal RNAs were filtered out with SortMeRNA ( Kopylova et al . , 2012 ) .", "Thereafter the reads that passed the QC threshold were mapped to mouse genome ( GRCm38/mm10/Ensembl release 79 - March 2015 ) using STAR mapping tool ( Dobin et al . , 2013 ) and on average 81% reads uniquely mapped .", "The expression of genes and differentially expressed genes ( adjusted p value<0 . 05 ) were measured by HTseq-count ( Anders et al . , 2015 ) and DEseq2 ( Love et al . , 2014 ) .", "Access to the data set is found at https://www . ncbi . nlm . nih . gov/geo/query/acc . cgi ?", "acc=GSE110459 .", "For qRT-PCR , 1 µg of RNA was used for cDNA synthesis with the QuantiTect Reverse Transcription Kit ( Qiagen ) , according to manufacturer’s instructions .", "cDNA was diluted to 10 ng/µl and 40 ng was used for one qPCR reaction .", "Probe-based multiplex RT-qPCR reactions were prepared in 1X iTaq Universal Probes Supermix ( Bio-Rad , Hercules , CA ) using a validated combination of gene-specific PrimePCR Probe Assays ( Bio-Rad , Hercules , CA ) in a 20 µl volume .", "The probe combinations are listed in Supplementary file 1 .", "Reactions from all samples were run in triplicate wells with the CFX96 Real-Time System ( Bio-Rad , Hercules , CA ) using the following cycling protocol: Initial denaturation 3 min at 95°C , followed by 45 cycles of denaturation 10 s at 95°C and annealing/extension 50 s at 60°C .", "Fold changes were calculated with the ∆∆CT method ( Livak and Schmittgen , 2001 ) .", "These were normalized to reference genes as follows: Etv5 and Spry4 were normalized to Gapdh , Cdkn1a and Dusp6 were normalized to Hprt , and Spred1 was normalized to Eef .", "Primer-based RT-qPCR reactions were prepared in Fast SYBR Green Master Mix ( Thermo Fisher Scientific ) .", "Bcl6 qPCR primers were designed using template NM_001348026 . 1 , Forward primer: CGCGAACCTTGATCTCCAGT , Reverse primer: CAGGGACCTGTTCACGAGAT .", "Hprt qPCR primers were designed using template NM_013556 . 2 , Forward primer: CAGTCCCAGCGTCGTGATTA , Reverse primer: TCGAGCAAGTCTTTCAGTCCT Embryonic back skin was dissected from E13 . 0 - E14 . 25 embryos as indicated and cultured in a Trowell-type tissue culture setup ( liquid-air interface ) as previously described ( Närhi and Thesleff , 2010 ) .", "For dermis cultures , back skins were obtained after treatment with Pancreatin-Trypsin or Dispase II as described above .", "The culture medium ( DMEM , 1X Glutamax , 10% FBS , 1% penicillin-streptomycin ) was supplemented where indicated with 20 µM SU5402 ( Calbiochem ) , 600 nM or 1 . 5 µM BGJ398 ( Selleckchem . com , Houston , TX ) , 50 nM or 250 nM XL184 ( Selleckchem . com , Houston , TX ) , or with 0 . 1% DMSO only .", "Explants were fixed in 4% PFA for immunostaining ( see below ) .", "Heparin-agarose beads ( Ø=70–100 µm , MCLAB , San Francisco , CA ) were loaded with 100 µg/ml FGF9 or FGF20 or 0 . 1% BSA for 2 hr at room temperature and subsequently washed with PBS .", "Dermises from E13 . 0–13 . 5 wild-type NMRI embryos were pooled for gene expression analysis , cell shape , and EdU incorporation assay .", "Dermises from E13 . 0 – E13 . 5 Fgf20-/- embryos were used in pairs for density analysis to compare treatment with control .", "To label proliferating cells , pregnant dams mated with Fgf20-/- males were given one intraperitoneal injection of 25 mg/kg body weight EdU ( Life Technologies , Eugene , OR ) dissolved in saline 2 hr prior to sacrifice .", "To label cultured dermises , NMRI E13 . 5 dermises cultured with FGF20- or FGF9-loaded beads for 18 hr as described above .", "During the last 2 hr , 10 µM EdU ( Life Technologies , Eugene , OR ) was introduced in the medium .", "The samples were then fixed with 4% PFA and EdU detection was performed with Click-iT kit ( Life Technologies , Eugene , OR ) according to manufacturer’s protocol .", "Briefly , samples were permeabilized with 3% BSA , 0 . 5% Triton X-100 ( MP Biomedicals , Solon , OH ) for 1 hr , stained with Click-iT reaction cocktail containing Alexa488-azide for 2 hr protected from light , washed thoroughly for 2 hr with PBS and mounted in Vectashield and imaged using Lumar . V12 stereomicroscope with 1 . 2x objective and AxioCam ICc camera ( all Zeiss ) .", "The following antibodies and reagents were used: Sox2 ( goat polyclonal , Santa Cruz , SC-17320 , Dallas , TX ) 1:500 for sections and 1:200 for whole-mounts , Sox2 ( rabbit polyclonal , Stemgent , 09–0024 , Glasgow , UK ) 1:300 for whole-mounts , β-galactosidase ( β-gal , rabbit polyclonal , MP Biomedicals , 55976 , Solon , OH ) 1:1500 , β-galactosidase ( β-gal , chicken polyclonal , Abcam , ab9361 , Cambridge , UK ) 1:1500 , EpCAM ( rat monoclonal , BD Pharmingen , 552370 , San Diego , CA ) 1:500 , Keratin 14 ( rabbit monoclonal , Thermo Fisher Scientific , RB-9020-P , Runcorn , UK ) 1:500 , and Cdkn1a ( rabbit monoclonal , Abcam , ab188224 , Cambridge , UK ) 1:1000 .", "Alexa 488 , 568 , or 647-conjugated secondary antibodies ( Life Technologies ) were used at 1:500 for sections and 1:400 for whole-mount samples for staining .", "For labeling of multiple antigens , the primary antibodies and secondary antibodies were incubated simultaneously .", "For immunostaining tissue sections , microscope slides were deparaffinized and washed with PBS for 10 min .", "Antigen retrieval was performed by incubation with 10 mM Na-citrate acid at 100°C for 10 min and the sections were allowed to cool to room temperature slowly .", "For fluorescent labeling , sections were permeabilized with 0 . 1% Triton X-100 , 10 min and blocked with 10% normal donkey serum , 0 . 1% Triton X-100 for 30 min .", "The sections were stained with primary antibodies overnight at 4°C and washed with PBS at room temperature .", "Slides were incubated with Alexa Fluor-conjugated secondary antibodies at room temperature for 2 hr , washed with PBS , and mounted with Vectashield containing DAPI ( Vector Laboratories , Burlingame , CA ) .", "For immunohistochemical labeling , slides were stained using BrightVision Poly-HRP-AntiRB-kit ( DVPR110HRP , ImmunoLogic , Duiven , The Netherlands ) according to manufacturer’s instructions .", "Briefly , slides were blocked using Pre-Antibody Blocking solution , 5 min , RT and washed with PBS .", "Then primary antibody was diluted in Pre-Antibody Blocking and the slides were stained overnight , 4°C , before washing with PBS .", "The slides were then stained with secondary goat , anti-rabbit-Poly-HRP antibody 30 min , RT and washed with PBS .", "The slides were then treated with DAB-solution ( BS04-110 , ImmunoLogic , Duiven , The Netherlands ) according to maunfacturer’s instructions , 8 min , RT , washed in de-ionized water , and mounted with ImmuMount ( ThermoScientific , Kalamazoo , MI ) .", "Images were acquired with Axio Imager M . 2 microscope equipped with Plan-Neofluar 20x/0 . 5 objective and AxioCam HRc .", "For whole-mount confocal microscopy , embryonic skin was spread onto 0 . 1 µm nucleopore filters and fixed for 2 hr at room temperature .", "Tissues were washed in large volumes of PBS and subsequently blocked in 10% normal donkey serum , 0 . 4% Triton X-100 for 1 hr , 4°C .", "Blocking solution was changed directly for primary antibody incubation in blocking solution 12–48 hr .", "The samples were washed in several changes of large volumes of PBS over 6–24 hr .", "Secondary antibody and Hoechst33342 ( Life Technologies , Eugene , OR ) were incubated for 6–24 hr , followed by washing .", "In the case of Fucci reporter samples and cell shape analysis of DC and IF cells , given the differential localization of Sox2 ( nuclear ) and Fgf20-β-Gal ( cytosolic , epithelial ) we used the same secondary Alexa fluor in order to reduce imaging time .", "Otherwise , different fluorophores were used .", "Optical sections of skin were obtained using Leica TCS SP5 confocal microscope equipped with HCX APO 63x/1 . 30 glycerol-immersion objective ( Leica , Wetzlar , Germany ) at 0 . 5 µm intervals for bead-treated dermis and for confocal imaging of in vivo cell shape analysis using Zeiss LSM 780 confocal microscope equipped with 63x/1 . 40 Plan-Apochromat oil-immersion objective at 0 . 5 µm intervals .", "Otherwise , images were acquired using an upright laser scanning confocal microscope LSM700 Axio Imager . M2 ( Zeiss ) using LCI Plan-Neofluar 63x/1 . 30 glycerol-immersion objective at 0 . 4–0 . 5 µm intervals and using Plan-Apochromat 10X/0 . 45 air objective at 0 . 8 µm intervals .", "Z-stacks were analyzed in 3D using Imaris software ( Bitplane , Zurich , Switzerland ) , unless stated otherwise .", "To analyze the number of Sox2-positive cells , co-localization of Fucci and R26R-tomato reporters and their distances to each other and the placode , surfaces of Sox2 nuclei were generated based on Sox2 immunostaining using local intensity measures .", "The parameters were as follows: surface level detail 0 . 5 µm , local contrast background subtraction ( seed point diameter 4 . 25 µm ) .", "In the analyses of interfollicular cells , sub-placodal cells of the Fgf20-/- skin , and the quantification of cell shapes , Hoechst 33342 staining was exploited for surface rendering as above ( surface level detail 0 . 6 µm , local background subtraction ( seed point diameter 5 µm ) .", "To generate surfaces based on cytoplasmic GFP in Sox2-GFP cells , surface detail was 0 . 6 µm with local background substraction ( seed point diameter 8 µm ) .", "All nuclear surfaces were manually corrected .", "In all analyses , center of nuclear surface was used as a marker for cell position .", "For reporter studies , average intensity of the reporter channel within Sox2 or Hoechst 33342 surfaces was used as a measure for a cell’s reporter activity .", "A cut-off value was manually determined , where a cell could be distinctly identified as reporter positive .", "To determine the distance from placode , a placodal surface was created as above , with surface level detail of 1 . 5 µm .", "In description of DC morphogenesis and analysis of Fgfr inhibition on DC cells , number of Sox2+ cells and their distances from the placode were determined from Fgf20+/- and Fgf20-/- skin samples immunostained for β-gal and Sox2 .", "Cell density of the DC was determined from Sox2-EGFP;Fgf20+/- as the number of Sox2+ surfaces inside the area distinguished by Sox2-EGFP reporter surface ( created as above ) , the density of the interfollicular cells and sub-placodal cells of the Fgf20-/- samples was determined as the number of Hoechst33342 surfaces within a corresponding volume of the interfollicular tissue .", "Density of sub-placodal dermal cells of the Fgf20-/- skin was determined as the number of Hoechst33342 surfaces underneath a manually adjusted region of interest underlying the placode ( β-gal ) .", "F-actin intensities in the DC and in the interfollicular dermis were determined from Fgf20+/- skins mount skin samples stained with A568-phalloidin ( LifeTechnologies , Eugene , OR ) and antibodies against Sox2 and β-gal .", "A surface was drawn around the Sox2-positive cells beneath placode marked by β-gal and copied to a Sox2-negative interfollicular area of the dermis at approximately the same depth to measure the mean intensity of A568-phalloidin staining .", "F-actin intensity in DC Sox2-positive cells and non-DC Sox2-positive cells in relation to Sox2-negative dermal fibroblasts was determined from E13 . 75 Sox2-EGFP;Fgf20+/- back skin samples stained with A568-phalloidin and β-gal antibody .", "Sox2-GFP surfaces demarcating individual cells were classified as DC or non-DC based on the proximity to the placode and mean F-actin intensities inside the surfaces measured .", "Dermal fibroblast surfaces were drawn based on phalloidin-staining at locations close to the non-DC Sox2-GFP cells .", "Distance of Sox2+ surfaces to placode , were measured as the shortest distance to β-gal surface .", "Distance to nearest neighbor was determined based on Sox2 surface center coordinated using R software and nabor package ( version 0 . 4 . 7 ) and median distances of each cell group within each placode were analyzed using Mann-Whitney test .", "For lineage tracing analysis , Sox2CreERT/+;R26RtdTomato/+;Fgf20+/- skin immunostained for Sox2 and β-gal were used .", "DC cell number , tdTomato positivity and position were determined by Sox2 surfaces as described above .", "Distance to placode was determined as the distance to a manually designated point on the center of placode surface and the identity of the nearest neighbor was determined based on Sox2 surface coordinates using R software with nabor package ( version 0 . 4 . 7 ) .", "For cell cycle analysis , confocal stacks of Fgf20+/-;Fucci reporter skins immunostained for Sox2 and β-gal were used .", "Sox2 surfaces were used for DC cells and Hoechst33342 surfaces were used for interfollicular cells of Fgf20+/- skin and sub-placodal cells of the Fgf20-/- skin .", "Reporter positivity was determined as above .", "DC and IF nuclear shapes were determined from Hoechst33342 staining using Imaris software sphericity measurement .", "Non-adjacent cells were selected for analysis in order to avoid bias introduction from manual surface editing .", "IF control cells were selected at ~70 µm distance from condensate center .", "Cell shape was also determined using Fgf20+/-;R26RmTmG skins immunostained for Sox2 and β-gal .", "Cell shapes were analyzed with ImageJ software based on membrane-bound tdTomato signal with rolling ball background subtraction ( 5 µm diameter ) , 3D Gaussian filtering ( 0 . 4 µm diameter ) and Gamma correction ( 0 . 6 value ) .", "The mT-negative region containing the cytoplasm was segmented using the Wand-tracing tool in the Segmentation Editor of Image J . The generated objects were automatically detected with the 3D Objects Counter , exported into the 3D ROI Manager ( Ollion et al . , 2013 ) and visualized/animated with the 3D Viewer .", "For studies with FGF-loaded beads , Fgf20-/- dermises were used .", "A region within a 30 µm distance from the bead surface at its midsection was analyzed from confocal stacks for cell density and nuclear shape .", "Cell density was measured by manual counting from optical sections and nuclear shapes were derived from Hoechst33342 surfaces .", "Nuclear surfaces of cells surrounding the middle third of the bead height were used for analysis and cells at 0–15 µm and 15–30 µm distances from the bead surface were analyzed .", "E13 . 5 Sox2-EGFP;Fucci-mKO back skins were explanted into Trowell-type culture with DMEM/F12 ( no phenol red ) , 10% FBS , 1% penicillin-streptomycin as previously described ( Ahtiainen et al . , 2014 ) .", "Briefly , explants were allowed to recover a minimum of 2 hr after dissection and then imaged with Leica SP5 laser scanning confocal microscope using HC PL APO 10x/0 . 4 objective and equipped with an incubation chamber ( LifeImagingServices ) to maintain 5% CO2 humidified atmosphere at 37°C .", "Confocal image stacks were acquired at 3 . 25 µm intervals with 10% laser power , 700 Hz scanning speed , and sub-optimal sampling every 20 min to minimize laser-induced damage .", "Time-lapse videos were analyzed using Imaris software .", "Tissue drift was corrected by manually tracing a minimum of seven non-motile cells over time and using the software’s translational drift correction algorithm .", "Sox2-GFP+ cell movements were manually traced back in time based on Fucci-mKO signal for as long as cell tracking could be reliably done .", "DC center was determined as the center of the Sox2-GFP signal of the DC .", "Similar tracking was performed on interfollicular dermal fibroblasts around an arbitrary migration center .", "Variables of cell movement were as follows: Velocity = track length/track duration Net velocity = track displacement length/track duration Track straightness = displacement length/track lengthcosα=a-b-a-∙b- where α is the escape angle , a- is cell trajectory , and b- is a vector between cell’s starting position and DC center or migration center for DC and IF cells , respectively .", "The normality of distributions of data were analyzed using the Shapiro-Wilk test ( confidence interval 95% ) .", "Normally-distributed data was analyzed using two-tailed Student’s T-test or One-Way Anova , while a non-parametric Mann-Whitney U test was performed to analyze statistical difference of data that failed Shapiro-Wilk test .", "When data was normalized , a one-sample T-test was performed .", "χ2-test was used to analyze randomness of cell neighbor distribution in lineage-tracing analysis .", "Rayleigh’s Z-test was conducted to test normal distribution of cell movement direction , followed by Watson’s U2 test to analyze significance of cell movement direction distributions in live imaging experiments ." ] ]

[ "Mesenchymal condensation is a critical step in organogenesis , yet the underlying molecular and cellular mechanisms remain poorly understood .", "The hair follicle dermal condensate is the precursor to the permanent mesenchymal unit of the hair follicle , the dermal papilla , which regulates hair cycling throughout life and bears hair inductive potential .", "Dermal condensate morphogenesis depends on epithelial Fibroblast Growth Factor 20 ( Fgf20 ) .", "Here , we combine mouse models with 3D and 4D microscopy to demonstrate that dermal condensates form de novo and via directional migration .", "We identify cell cycle exit and cell shape changes as early hallmarks of dermal condensate morphogenesis and find that Fgf20 primes these cellular behaviors and enhances cell motility and condensation .", "RNAseq profiling of immediate Fgf20 targets revealed induction of a subset of dermal condensate marker genes .", "Collectively , these data indicate that dermal condensation occurs via directed cell movement and that Fgf20 orchestrates the early cellular and molecular events ." ]

[ "All mammal hair springs from hair follicles under the skin .", "These follicles sit in the dermis , beneath the outermost skin layer , the epidermis .", "In the embryo , hair follicles develop from unspecialized cells in two tissues , the epithelium and the mesenchyme , which will later develop into the dermis and epidermis , respectively .", "As development progresses , the cells of these tissues begin to cluster , and signals passing back and forth between the epithelium and mesenchyme instruct the cells what to do .", "In the mesenchyme , cells called fibroblasts squeeze up against their neighbors , forming patches called dermal condensates .", "These mature into so-called dermal papillae , which supply specific molecules called growth factors that regulate hair formation throughout lifetime .", "Fibroblasts in the developing skin respond to a signal from the epithelium called fibroblast growth factor 20 ( Fgf20 ) , but we do not yet understand its effects .", "It is possible that Fgf20 tells the cells to divide , forming clusters of daughter cells around their current location .", "Or , it could be that Fgf20 tells the cells to move , encouraging them to travel towards one another to form groups .", "To address this question , Biggs , Mäkelä et al . examined developing mouse skin grown in the laboratory .", "They traced cells marked with fluorescent tags to analyze their behavior as the condensates formed .", "This revealed that the Fgf20 signal acts as a rallying call , triggering fibroblast movement .", "The cells changed shape and moved towards one another , rather than dividing to create their own clusters .", "In fact , they switched off their own cell cycle as the condensates formed , halting their ability to divide .", "A technique called RNA sequencing revealed that Fgf20 also promotes the use of genes known to be active in dermal condensates .", "Dermal papillae control hair growth , and transplanting them under the skin can form new hair follicles .", "However , these cells lose this ability when grown in the laboratory .", "Understanding how they develop could be beneficial for future hair growth therapy .", "Further work could also address fundamental questions in embryology .", "Condensates of cells from the mesenchyme also precede the formation of limbs , bones , muscles and organs .", "Extending this work could help us to understand this critical developmental step ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "chromosomes and gene expression", "cancer biology" ]

[ [ "The molecular changes underlying human myeloid malignancies remain difficult to unravel , posing major obstacles to the development of effective countermeasures .", "Although the silencing of tumor suppressor genes by chromosomal deletions , point mutations , or other mechanisms is an acknowledged factor in myeloid cell transformation , the specific involvement of gene dosage is not well understood .", "In broadest terms , single-copy loss of a suppressor gene can be sufficient to modify gene function and promote tumorigenesis ( classical haploinsufficiency ) , while in other tumors , the loss of two alleles is required ( two-hit paradigm of Knudson ) ( Knudson , 1971 ) .", "Recent evidence indicates that more subtle reductions in suppressor gene function may contribute importantly to myeloid malignancy ( Rosenbauer et al . , 2004; Liu et al . , 2007 ) , leading to the need for faithful animal models to establish that such changes are truly involved in tumorigenesis .", "Loss of an interstitial segment of chromosome 20q ( 20q CDR ) is detected in about 4% of myelodysplastic syndromes ( MDS ) ( Haase et al . , 2007 ) , and this region is variably affected in different types of myeloproliferative neoplasms ( MPN ) , including polycythemia vera ( 10% ) and primary myelofibrosis ( 12% ) , and less commonly in acute myeloid leukemia ( AML; 1% ) ( Bench et al . , 2000 ) .", "Notably , only heterozygous deletions have been found in studies of myeloid malignancies with loss of chromosome 20q , without any evidence of homozygous deletion or mutations of a gene within the affected region ( Heinrichs et al . , 2009; Huh et al . , 2010 ) .", "These findings implicate a gene within the 20q CDR that is essential for cell viability , but whose tumor suppressor function is strongly dose-dependent and does not follow the classical Knudson model ( Knudson , 1971 ) , which predicts biallelic gene inactivation .", "Instead , monoallelic loss , with or without additional epigenetic or microRNA ( miRNA ) -mediated downregulation of the remaining allele , may reduce gene expression levels sufficiently to promote myeloid cell transformation .", "Thus , we sought to identify candidate tumor suppressor genes within the 20q CDR on the basis of their reduced expression in malignant myeloid progenitor cells , as we have reported previously for CTNNA1 in myeloid malignancies with deletions of chromosome 5q ( Liu et al . , 2007; Ye et al . , 2009 ) .", "Here we identify MYBL2 , which encodes a transcription factor with functions in checkpoint control of the G2 cell cycle phase , as a key tumor suppressor gene in over one half of all MDS cases , whether characterized by a 20q deletion or a normal karyotype .", "Reductions of MYBL2 dosage to levels below those commensurate with single-copy loss conferred a competitive advantage to hematopoietic progenitor cells in both primary and secondary transplantation assays and were associated with histopathologic changes typical of myeloid neoplasia .", "These findings implicate aberrantly low levels of MYBL2 expression as a central mechanism in the development of clonal dominance in MDS and other myeloid malignancies ." ], [ "We first studied the gene expression profiles of CD34+ hematopoietic progenitor cells from eight MDS cases with cytogenetically evident aberrations of chromosome 20q , as compared to CD34+ cells from normal individuals ( Figure 1—figure supplement 1A ) .", "We found that MYBL2 , which resides near the center of this region ( Figure 1—figure supplement 2 ) , was among the top-scoring genes downregulated in MDS cells compared to normal CD34+ controls .", "MYBL2 encodes a highly conserved transcription factor that acts as a major component of the dREAM complex , which controls the G2-to-M phase transition within the cell cycle ( Korenjak et al . , 2004; Lewis et al . , 2004 ) .", "The mean expression level of MYBL2 ( 39% ) was less than that of normal CD34+ cells ( Figure 1—figure supplement 1B ) , suggesting that mechanisms beyond the deletion of one allele might affect the remaining allele .", "Thus , on the assumption that some MDS cases with a normal karyotype might also have reduced expression of a gene or genes within the 20q CDR , we undertook an expression analysis of CD34+ cells from 18 MDS cases with a normal karyotype and the eight cases with a 20q aberration ( Supplementary file 1A ) , compared to CD34+ cells from four normal bone marrow samples .", "MYBL2 was the top-scoring gene among 108 differentially expressed genes ( Figure 1A ) .", "Further analysis showed that in 17 ( 65% ) of 26 MDS patients ( Figure 1—figure supplement 3A ) , MYBL2 expression levels in CD34+ cells were reduced to ≤45% of normal CD34+ cells levels ( mean 32 . 8% ) .", "These microarray data were confirmed by QRT-PCR analysis of MYBL2 expression levels in 10 MDS cases and eight normal CD34+ bone marrow samples , including four additional normal control CD34+ samples that were not part of the original microarray analysis ( Figure 1B ) .", "Importantly , MYBL2 levels measured by QRT-PCR in MDS samples were highly correlated ( r = 0 . 966 ) with those determined by microarray analysis ( Figure 1—figure supplement 3B ) .", "Hence , MYBL2 appears to be expressed at reduced levels in nearly two-thirds of MDS cases , including those with a normal karyotype , suggesting that it functions as a dose-dependent tumor suppressor gene . 10 . 7554/eLife . 00825 . 003Figure 1 . Gene expression analysis of CD34+ cells from MDS patients with a 20q aberration or a normal karyotype .", "( A ) Heat map showing the top-scoring 25 genes that were either up- or downregulated in CD34+ cells from 26 MDS patients vs normal donors ( see Supplementary file 1A for patient characteristics ) .", "Genes were considered differentially expressed if they met two criteria: q ≤ 0 . 006 and fold-change ≥2 . 0 ( 108 genes ) .", "( B ) Validation of microarray-based MYBL2 gene expression data for selected clinical samples ( upper panel ) by qRT-PCR analysis ( lower panel ) , including four additional samples of normal CD34+ bone marrow cells .", "Relative MYBL2 expression results by qRT-PCR analysis were normalized to three control genes . DOI: http://dx . doi . org/10 . 7554/eLife . 00825 . 00310 . 7554/eLife . 00825 . 004Figure 1—figure supplement 1 . Gene expression analysis comparing CD34+ MDS cells with a 20q aberration to normal CD34+ cells .", "( A ) Heat map showing the top 25 downregulated genes .", "A two class comparison was performed using q ≤ 0 . 006 and fold-change ≥2 . 0 as criteria .", "Asterisks indicate genes located within the 20q CDR .", "( B ) Bar chart illustrating MYBL2 expression levels corresponding to row 2 of the heat map . DOI: http://dx . doi . org/10 . 7554/eLife . 00825 . 00410 . 7554/eLife . 00825 . 005Figure 1—figure supplement 2 . Genomic map of the chromosome 20 CDR . The boundaries are based on the markers D20S465 and D20S17 according to Bench et al . ( 1998 ) and the location of MYBL2 is highlighted by a red circle .", "All regions delineated in the publications below included the MYBL2 genomic locus .", "The map was generated from the hg19 assembly of the UCSC genome browser ( http://genome . ucsc . edu ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 00825 . 00510 . 7554/eLife . 00825 . 006Figure 1—figure supplement 3 . MYBL2 expression levels .", "( A ) MYBL2 expression was determined by microarray analysis .", "Normalized fluorescence intensity values are displayed as percentages of the mean expression levels of MYBL2 in CD34+ cells from normal bone marrow .", "( B ) Correlation analysis of qRT-PCR and microarray expression data .", "The Pearson correlation coefficient is 0 . 966 . DOI: http://dx . doi . org/10 . 7554/eLife . 00825 . 006 To address whether reduced levels of MYBL2 expression actively contribute to the MDS phenotype or merely constitute a compensatory response to other abnormalities , we asked if reduced MYBL2 transcriptional activity is reflected in the MDS gene expression signature .", "To model gene dose insufficiency by RNA interference ( RNAi ) , we used a set of eight shRNA-expression vectors in the MDS/AML cell line SKM1 , which reduced MYBL2 expression to 5–30% of the endogenous MYBL2 levels ( Figure 2A ) , approximating the endogenous levels we had identified in CD34+ cells from patients ( Figure 1B ) .", "To identify the genes most highly correlated with decreased MYBL2 expression , we introduced our shRNA-expression vectors into normal primary human CD34+ cells and measured gene expression at 48 hr post-transduction .", "Nearly all differentially expressed genes were downregulated after MYBL2 knockdown ( 81 of 89 genes; Figure 2B and Figure 2—figure supplement 1 ) .", "Because these results are based on eight different MYBL2-specific shRNAs , each with different knockdown efficiency , this analysis ensures the robustness of the data and eliminates potentially confounding off-target effects by individual shRNAs .", "Gene set enrichment analysis ( GSEA ) ( Subramanian et al . , 2005 ) identified mitosis as the key biological process reflected by the MYBL2 gene signature , specifically the G2-to-M phase transition ( Figure 2—figure supplement 2 ) ( Whitfield et al . , 2002; Zhu et al . , 2004; Shepard et al . , 2005 ) .", "Individual genes and their functions included the G2 cell cycle regulator cyclin B ( CCNB1 and CCNB2 ) , the early mitotic regulator FBXO5 and the coregulator of chromosome architecture CDCA2 ( Supplementary file 1B ) . 10 . 7554/eLife . 00825 . 007Figure 2 . Identification of an MYBL2-regulated gene expression signature and its enrichment in CD34+ cells from MDS patients .", "( A ) MYBL2 knockdown in SKM1 cells by a series of shRNAs ( control shRNAs are labeled B and C; MYBL2-specific shRNAs are labeled 1–8 ) .", "MYBL2 expression levels were measured by qRT-PCR normalized to three control genes ( upper panel ) and by Western blotting with tubulin as a normalization control ( lower panels ) .", "( B ) Heat map of the top 25 genes whose expression levels positively correlated ( score ≥ 0 . 8 ) with those of MYBL2 .", "Gene expression in normal CD34+ cells was measured by microarray in one unperturbed sample ( ‘no shRNA’ ) , four control shRNA-expressing samples ( shRNAs A–D ) and eight MYBL2-specific shRNA-expressing samples ( shRNAs 1-8 ) .", "( C ) Gene set enrichment analysis ( GSEA ) of the MYBL2 gene signature ( top 81 positively correlated genes ) within the ranked gene expression results for CD34+ cells from MDS patients , compared to CD34+ cells from normal bone marrow .", "Enrichment of the MYBL2 signature in MDS bone marrow cells ( see Figure 1A ) is apparent .", "Genes are ranked by score and plotted on the x-axis; hits are indicated by vertical black bars and the enrichment score is plotted in red .", "( D ) Heat map corresponding to the GSEA showing the 25 top-scoring genes .", "One sample , indicated by an ‘x’ , shows enrichment of the MYBL2 signature , but relatively normal MYBL2 expression suggesting a measurement problem . DOI: http://dx . doi . org/10 . 7554/eLife . 00825 . 00710 . 7554/eLife . 00825 . 008Figure 2—figure supplement 1 . Gene expression analysis upon MYBL2 knockdown . After exclusion of all nonexpressed genes , the remaining genes ( 7194 ) were rank-ordered according to their correlation score ( Pearson ) relative to MYBL2 expression .", "Using a cut-off score of ±0 . 8 ( blue lines ) , 8 genes were found to be negatively correlated with MYBL2 knockdown ( score < 0 . 8 ) , while 81 were found to be positively correlated ( score > 0 . 8 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 00825 . 00810 . 7554/eLife . 00825 . 009Figure 2—figure supplement 2 . Gene set enrichment analysis ( GSEA ) of the expression profile of CD34+ cells upon MYBL2 knockdown . The highest scoring subset using the ‘gene ontology processes’ gene sets of MSigDB ( v3 . 0 ) was the ‘mitosis’ gene set ( left panel ) .", "Gene sets reflecting distinct phases of the cell cycle were used to determine the prominent cycle phase , and the gene set ‘G2/M’ had the highest score ( right panel ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 00825 . 009 We finally performed GSEA to assess whether this MYBL2 gene expression signature ( 81 genes , Supplementary file 1B ) was evident in MDS samples with intrinsically low expression of this gene .", "This analysis revealed robust enrichment of the MYBL2 signature in MDS CD34+ cells ( Figure 2C ) .", "Furthermore , the expression levels of MYBL2-regulated genes corresponded closely with those of MYBL2 itself ( Figure 2D ) .", "Together , these findings indicate that MYBL2 downregulation in CD34+ cells is reflected in the aberrant gene expression profile of a large fraction of MDS cases .", "To determine which MDS patients with a normal karyotype have an ‘MYBL2-low’ expression signature , we first analyzed the expression levels of MYBL2-regulated genes in the CD34+ cells from our 18 normal-karyotype MDS cases , using a k-nearest neighbor ( KNN ) classifier and Euclidean distance ( k = 3 ) to predict the class label by a majority vote .", "10 of these cases ( 56% ) were classified as MYBL2 low , while the remaining eight cases had an MYBL2 signature that was statistically similar to that of normal CD34+ controls ( Figure 3—figure supplement 1 ) .", "To validate our classification algorithm using a separate dataset , we then interrogated the normal karyotype cases ( n = 94 ) reported by Pellagati et al . ( Pellagatti et al . , 2010 ) , identifying a MYBL2-low signature in 36 cases ( 38% ) , most of which ( 30 cases ) were identified at a confidence level of 1 . 0 .", "58 cases ( 62% ) had a normal MYBL2 signature ( Figure 3 ) .", "Thus , we were able to confirm in an independent cohort that the ‘MYBL2 low’ signature is present in CD34+ cells from a substantial fraction of normal-karyotype MDS cases . 10 . 7554/eLife . 00825 . 010Figure 3 . KNN classification for normal karyotype patient samples of an independent data set ( Pellagatti et al . , 2010 ) .", "A k-nearest-neighbor ( KNN ) classification model for patient samples was trained on our gene expression data set of CD34+ cells with or without MYBL2 knockdown by RNAi .", "Upper panel: KNN classification for the presence ( negative values ) or absence ( positive values ) for the MYBL2 signature using Euclidean distance ( k = 3 ) to predict the class label by a majority vote .", "Lower panel: gene expression profile of MYBL2 signature genes .", "Note that some gene profiles ( INCEP , GTSE1 , PRR11 , HIST1H1B , HIST1H2AL , HIST1H2BE , HIST2H4A , HN1 ) appear not to match the overall signatures most likely due to platform inconsistencies . DOI: http://dx . doi . org/10 . 7554/eLife . 00825 . 01010 . 7554/eLife . 00825 . 011Figure 3—figure supplement 1 . KNN classification for normal karyotype patient samples . A k-nearest-neighbor ( KNN ) classification model for patient samples was trained on our gene expression data set of CD34+ cells with or without MYBL2 knockdown by RNAi .", "Upper panel: KNN classification for the presence ( negative values ) or absence ( positive values ) for the MYBL2 signature using Euclidean distance ( k = 3 ) to predict the class label by a majority vote .", "Lower panel: gene expression profile of MYBL2 signature genes . DOI: http://dx . doi . org/10 . 7554/eLife . 00825 . 011 To begin to clarify the mechanism of reduced MYBL2 activity or aberrantly low MYBL2 expression in MDS , we resequenced DNA samples from 144 patients , identifying two cases with heterozygous somatic mutations within the coding sequence that were verified by analysis of patient-specific germline DNA collected from buccal swabs ( Figure 4A ) .", "Both mutations , an amino acid substitution and a nonsense mutation , were located within the C-terminus .", "To test whether these mutations inactivate the MYBL2 protein , we replaced endogenous MYBL2 in SKM1 cells with MYBL2 carrying one of the mutations , using a knockdown/rescue approach .", "SKM1 cells died upon complete knockdown of MYBL2 with an shRNA targeting the 3′UTR ( Figure 4B ) .", "This phenotype was rescued by re-expression of the wild-type MYBL2 cDNA and the cDNA from patient 28 ( Figure 4A ) , but not by re-expression of the mutated , truncated MYBL2 cDNA of patient 27 , which failed to restore the viability of the SKM1 cells .", "Thus , we have documented a somatically acquired gene-specific inactivating mutation that targets MYBL2 in one case of MDS .", "This specific case is highly informative because the inactivating mutation targets MYBL2 directly , and not an adjacent gene on 20q . 10 . 7554/eLife . 00825 . 012Figure 4 . MYBL2 is mutated at a low frequency in MDS .", "( A ) MYBL2 exons were resequenced in the DNA of mononuclear bone marrow cells of 144 MDS patients .", "The results indicated a P-to-L aminoacid substitution ( top , mutation 1 , patient MDS28 ) and a nonsense mutation ( bottom , mutation 2 , patient MDS27 ) .", "( B ) Growth curves of SKM1 cells after transduction with a MYBL2 shRNA .", "SKM1 cells were transduced with an empty vector ( circles ) , a vector expressing wildtype MYBL2 cDNA ( squares ) , mutant MYBL2 cDNA ( mut . 1 , triangles ) or mutant MYBL2 cDNA ( mut . 2 , diamonds ) .", "Upon transduction with a control ( solid symbols ) or a MYBL2-specific ( open symbols ) shRNA-expressing vector growth was monitored for 6 days . DOI: http://dx . doi . org/10 . 7554/eLife . 00825 . 01210 . 7554/eLife . 00825 . 013Figure 4—figure supplement 1 . Analysis of Mybl2 expression in context with mir-29a and mir-30e expression in selected patient samples and controls .", "( A ) Confirmation of microarray-based MYBL2 gene expression data ( white bars ) by qRT-PCR analysis ( black bars ) .", "Here , the same cDNA reaction was used for mRNA and miRNA expression analysis .", "( B ) Relative expression levels of mir-30e and mir-29a determined by qRT-PCR analysis .", "Normalization was performed to three control genes . DOI: http://dx . doi . org/10 . 7554/eLife . 00825 . 013 Given that a substantial number of MYBL2-low cases lacked either a 20q deletion or gene-specific inactivating mutation , we hypothesized that additional mechanisms of MYBL2 downregulation exist in MDS .", "A potential candidate is aberrant DNA methylation , especially in view of the 921 base-pair CpG island that spans the proximal promoter and the first exon of the MYBL2 gene .", "However , analysis of this site using bone marrow cell DNA from 30 MDS patients failed to detect any methylated cytosines , indicating that DNA methylation is not a frequent mechanism for MYBL2 downregulation in MDS ( JP Issa , personal communication , 2012 ) .", "This finding is supported by a recently published study that did not identify methylation affecting the MYBL2 gene in cells from 83 MDS cases ( Del Rey et al . , 2012 ) .", "We also considered that the upregulation of one or more miRNAs targeting MYBL2 might explain our results .", "We pursued this notion in a subset of patient samples ( seven with low MYBL2 expression and two with normal MYBL2 expression , together with three controls ) by analyzing the expression levels of mir-29a and mir-30e .", "Both of these miRNAs target MYBL2 ( Han et al . , 2010; Martinez et al . , 2011 ) , with aberrant expression of mir-29a leading to clonal dominance and acute myeloid leukemia ( Han et al . , 2010 ) .", "Our results show upregulation of either mir-29a or mir-30e in three cases with MYBL2 downregulation , but not in controls or cases with normal MYBL2 expression ( Figure 4—figure supplement 1 ) .", "Thus , overexpression of miRNAs targeting MYBL2 affords an attractive mechanism by which MYBL2 expression levels are lowered in a substantial fraction of MDS cases .", "If sub-haploinsufficient decreases in MYBL2 dosage contribute to MDS pathogenesis , a technique such as RNAi knockdown with a series of shRNA hairpins of graded potencies should provide an experimental model that can be used to demonstrate a clonal advantage within hematopoietic progenitor cells and implicate the level of Mybl2 knockdown that produces the maximum effect .", "Thus , using five independent , specific shRNA-expressing vectors to target Mybl2 in primitive hematopoietic cells , we established a competitive reconstitution assay in mice .", "These vectors downregulated Mybl2 expression to levels that ranged from 13% ( M1 ) to 33% ( M5 ) of those expressed by control shRNA-expressing vectors in 32D cells , a murine myeloid cell line ( Figure 5—figure supplement 1 ) .", "After transducing Lin− mononuclear bone marrow cells in separate vials with these five vectors , which coexpressed a green fluorescent protein ( GFP ) , or with two different control shRNA vectors coexpressing a red fluorescent protein ( RFP ) ( Strack et al . , 2008 ) , we washed the cells , pooled the five Mybl2-specific shRNA knockdown cell aliquots and the two control aliquots , and transplanted equal numbers of these two cell pools into lethally irradiated mice ( Figure 5A ) .", "A second group of mice were transplanted with pooled GFP+ and RFP+ cells expressing control shRNAs only , generating control/GFP vs control/RFP recipients ( Figure 5—figure supplement 2A ) .", "Analysis of GFP and RFP levels in peripheral blood at 12 weeks ( Figure 5B ) showed a strikingly higher median frequency of GFP+ vs RFP+ cells in the peripheral blood of the mice reconstituted with GFP+/Mybl2-shRNA vs RFP+/control shRNA ( 30 . 9% vs 2 . 6% , p<0 . 0001 ) .", "By contrast , in mice injected with control/GFP vs control/RFP cells , the median frequencies of both RFP- and GFP-expressing cells were low ( 1 . 5% and 3 . 4%; Figure 5—figure supplement 2B ) . 10 . 7554/eLife . 00825 . 014Figure 5 . Competitive in vivo reconstitution assay testing expansion capacity of cells with low Mybl2 expression .", "( A ) Mononuclear bone marrow cells were collected from donor mice , enriched for lineage-negative cells and transduced in separate wells with specific Mybl2 shRNA/GFP vectors and control shRNA/RFP vectors .", "Equal numbers of cells were combined to obtain a pool with specific shRNA-expressing cells and a pool of control shRNA-expressing cells .", "Both pools were combined in equal parts and transplanted into lethally irradiated mice ( n = 25 ) in two independent experiments with transduction efficiencies of 5–8% .", "( B ) Analysis of peripheral blood mononuclear cells for GFP and RFP positivity at 12 weeks post-transplant as an indicator of long-term reconstitution ( Morrison and Weissman , 1994 ) ( GFP+ vs RFP+ , p<0 . 0001 by paired t-test; horizontal bars denote median values ) .", "( C–F ) , Analysis of bone marrow samples from mice 6–7 months after competitive reconstitution .", "Flow cytometric analysis of mononuclear bone marrow cells from five experimental mice ( E1–E5 ) transplanted with Mybl2-specific shRNA/GFP cells and control shRNA/RFP cells showed marked overrepresentation of GFP+ cells by comparison with results for control mice ( C1 , C2 ) transplanted with control shRNA/RFP- and shRNA/GFP-transduced cells .", "The fractions of GFP ( green ) , RFP ( red ) and unlabeled cells ( gray ) are shown for the total cell populations ( C ) , B220-positive population ( D ) , Gr1/Mac1-positive population ( E ) , and CD3-positive population ( F ) .", "( G ) Respective analysis of bone marrow erythropoiesis by CD71/Ter119 staining .", "Frequencies of Ter119-positive cells are shown for all animals , and representative plots presented for mouse C1 ( H ) and mouse E1 ( I ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 00825 . 01410 . 7554/eLife . 00825 . 015Figure 5—figure supplement 1 . Mybl2 knockdown in 32D cells by a series of shRNAs . Mybl2 knockdown in 32D cells by a series of shRNAs ( control shRNAs are labeled B and C; Mybl2-specific shRNAs are labeled M1–M5 ) .", "Mybl2 expression levels were measured by qRT-PCR normalized to three control genes ( upper panel ) and by Western blotting with tubulin as a normalization control ( lower panel ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 00825 . 01510 . 7554/eLife . 00825 . 016Figure 5—figure supplement 2 . Control experiment for the competitive in vivo reconstitution assay testing expansion capacity of cells with low Mybl2 expression .", "( A ) Mononuclear bone marrow cells were collected from donor mice , enriched for lineage-negative cells and transduced in separate wells with control shRNA/GFP vectors and control shRNA/RFP vectors .", "Equal numbers of cells were combined to obtain a pool of control shRNA-expressing cells and transplanted into lethally irradiated mice ( n = 13 ) in two independent experiments with transduction efficiencies of 5–8% .", "( B ) Analysis of peripheral blood mononuclear cells for GFP and RFP positivity at 12 weeks post-transplant as an indicator of long-term reconstitution . DOI: http://dx . doi . org/10 . 7554/eLife . 00825 . 01610 . 7554/eLife . 00825 . 017Figure 5—figure supplement 3 . Analysis of bood samples from mice 6–7 months after competitive reconstitution . Flow cytometric analysis of blood samples from five experimental mice ( E1–E5 ) transplanted with Mybl2-specific shRNA/GFP cells and control shRNA/RFP cells demonstrating marked overrepresentation of GFP+ cells in comparison to results for control mice ( C1 , C2 ) transplanted with control shRNA/RFP and shRNA/GFP transduced cells ( GFP+ ( green ) , RPF+ ( red ) and unlabeled cells ( gray ) ) DOI: http://dx . doi . org/10 . 7554/eLife . 00825 . 017 The possibility that these results were influenced by the use of pooled cells representing different shRNA sequences was excluded by transducing the Lin− cells with single hairpins specific for Mybl2 .", "For this experiment ( Figure 6A ) we transduced Lin− bone marrow cells with either the M3 shRNA vector ( n = 7 mice ) or the M4 shRNA vector ( n = 8 mice ) .", "These two shRNAs were chosen because QPCR analysis of the mice injected with pools of cells transduced with each of the five shRNAs ( Figure 5A ) showed that M3 and M4 were integrated into the DNA of the reconstituted cells that exhibited clonal dominance ( Figure 6—figure supplement 1 ) .", "The results of transplanting cells transduced with single Mybl2-specific hairpins clearly show clonal dominance by cells transduced with either of these vectors ( Figure 6B ) .", "In a ‘color-swap’ experiment using the M3 shRNA , the bone marrow cells with Mybl2 knockdown became clonally dominant , whether the red or green fluorophore was used ( Figure 6C ) .", "Together , these competitive reconstitution studies firmly establish the surprising finding that downregulation of Mybl2 levels to ∼30% of normal levels using two different Mybl2 shRNAs imparts a strong clonal advantage to hematopoietic progenitor cells , one that becomes reflected in the analysis of circulating white blood cells as early as 12 weeks after transplantation . 10 . 7554/eLife . 00825 . 018Figure 6 . Analysis of peripheral blood and bone marrow samples from mice transplanted with single Mybl2-targeting shRNA vectors .", "( A ) Mononuclear bone marrow cells were collected from donor mice , enriched for lineage-negative cells and transduced in separate wells with a single , specific Mybl2 shRNA vector or control shRNA vector with an efficiency of <10% .", "The vectors allowed for co-expression of RFP or GFP .", "Equal numbers of control shRNA-expressing cells and Mybl2-specific shRNA-expressing cells were combined and transplanted into lethally irradiated mice in two independent experiments .", "( B ) Experiment 1: Analysis of peripheral blood mononuclear cells for GFP and RFP positivity at 12 weeks post-transplant as an indicator of long-term reconstitution .", "Significant expansion of GFP-positive cells with use of either shRNA M3 ( left , p=0 . 0004 by paired t-test ) or M4 ( right , p=0 . 0005 ) .", "Horizontal bars denote median values .", "( C ) Experiment 2 , ‘color-swap’ experiment: Mybl2-specific shRNA M3 coupled with GFP ( right ) or RFP ( left ) expression associated with a significant expansion of Mybl2-downregulated cells ( p=0 . 007 and p=0 . 001 , respectively ) .", "( D ) Flow cytometric analysis of mononuclear bone marrow cells from eight experimental mice ( E6–E13 ) transplanted with cells expressing Mybl2-specific M3 shRNA/GFP and control shRNA/RFP cells ( E6–E9 ) or Mybl2-specific M3 shRNA/RFP cells and control shRNA/GFP cells ( E10–E13 ) .", "Mybl2-RNAi cells were markedly overrepresented by comparison to results for control mice ( C3 , C4 ) transplanted with control shRNA/RFP- and shRNA/GFP-transduced cells .", "The fractions of GFP ( green ) , RPF ( red ) and unlabeled cells ( gray ) are shown .", "( E ) Analysis of bone marrow erythropoiesis by CD71/Ter119 staining with frequencies of Ter119-positive cell frequencies shown for all animals .", "( F ) Analysis of Mybl2 levels of flow sorted cells by Western-blotting ( upper panel ) and qRT-PCR ( lower panel ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 00825 . 01810 . 7554/eLife . 00825 . 019Figure 6—figure supplement 1 . Analysis of genomic DNA for the presence of specific Mybl2-targeting shRNA sequences .", "( A ) Analysis of the relative abundance of shRNAs M1 to M5 in the peripheral blood of seven mice and the bone marrow of one mouse 12 weeks after reconstitution .", "( B ) Analysis of the bone marrow of six animals more than 6 months after reconstitution . DOI: http://dx . doi . org/10 . 7554/eLife . 00825 . 01910 . 7554/eLife . 00825 . 020Figure 6—figure supplement 2 . Cell cycle analysis . Cell cycle phase distribution was determined by standard Propidium Iodide staining in control bone marrow cells and in sorted , RFP-negative cells ( no Mybl2 RNAi ) and in sorted , RFP-positive cells ( Mybl2 knockdown ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 00825 . 020 The marked competitive expansion of Mybl2 knockdown cells could represent either a benign process or a myeloid malignancy .", "Thus , we sacrificed 17 mice ( nine transplanted with pooled cells [E1–E5 , PD1–PD5] plus three controls and eight transplanted with cells transduced with a single vector [E6-E13] plus two controls ) at 6 to 7 months post-transplantation , and studied their bone marrow cells by flow cytometry .", "The results confirmed expansion of the Mybl2-knockdown cell population compared to control bone marrow cells ( Figures 5C and 6D; Figure 8—figure supplement 1C ) , which surpassed even the percentage of GFP+ cells in the blood ( Figure 5—figure supplement 3 ) .", "Lineage analysis showed that this expansion included lymphocytes as well as myeloid cells ( Figure 5D–F ) , indicating clonal dominance originating from transduced multilineage long-term repopulating cells .", "Cells of the myeloid lineage were most prominently affected , as the frequency of GFP+ cells in the Gr1+/Mac1+ population exceeded 90% in three of five experimental animals .", "Bone marrow erythropoiesis was reduced by Mybl2 knockdown , as indicated by the decreased percentages of Ter119+ cells ( Figures 5G–I and 6E; Figure 8—figure supplement 1D ) .", "As expected , analysis by Western blotting and QRT-PCR of bone marrow cells expressing the M3 Mybl2-shRNA as a single hairpin showed that Mybl2 expression levels were reduced to approximately 20–30% of control levels after long-term reconstitution ( Figure 6F ) .", "We did not observe any effect of this level of Mybl2 knockdown on the cell cycle-phase distribution of bone marrow cells from mice 6–7 months post-transplantation ( Figure 6—figure supplement 2 ) , indicating that this level of decreased Mybl2 expression does not induce a delay in the G2-to-M-phase transition .", "We also evaluated the circulating blood counts in 13 of the 17 mice at the time of euthanasia 6–7 months after transplant .", "Reduced hemoglobin levels indicating anemia were observed in 12 of the 13 mice ( Figure 7A ) , indicative of the ineffective erythropoiesis in the bone marrow observed by flow cytometry .", "Circulating white blood cell and platelet counts were in the normal range .", "Spleen weight was increased in 12 of the 13 mice , reflecting extramedullary hematopoiesis .", "A few of the mice showed hunched posture , weight loss and lethargy at the time of euthanasia ( 6–7 months post-transplant ) , but the majority of the recipient mice appeared well with no evidence of overt hematologic disease . 10 . 7554/eLife . 00825 . 021Figure 7 . Analysis of blood counts and spleen weight ( A ) , spleen ( B–E ) and bone marrow ( F–K ) samples from mice 6-7 months after competitive reconstitution .", "( A ) Analysis of peripheral blood parameters showing anemia of animals that received transplants of specific Mybl2-shRNA engineered cells: white blood cell count ( WBC ) , red blood cell count ( RBC ) , hemoglobin ( HB ) , hematocrit ( HCT ) , and platelet count ( PLT ) .", "Spleen weights are shown as well .", "Grey areas indicate the normal range for each value .", "( B and C )", "Normal spleen histology at different magnification of control mouse C1 showing normal lymphoid-rich white pulp ( 50% spleen volume ) and red pulp ( 50% ) .", "About 10% of the spleen consists of extramedullary hematopoiesis , which is in the red pulp and normal for mice .", "( D and E )", "Spleen histology of mouse E1 with specific Mybl2-knockdown at different magnification shows a marked depletion of white pulp .", "About 90% consists of red pulp , which is almost entirely extramedullary hematopoiesis .", "( F–K )", "Normal bone marrow histology ( F and G ) and cytology ( H ) of control mouse C1 , compared to corresponding results for mouse E1 with specific Mybl2-knockdown ( histology I , J; cytology K ) .", "Note marked shift to myeloid elements with loss of erythropoiesis and occasional dysplastic myeloid cells ( white arrow: hypolobate megakaryocyte , black arrow: dysplastic neutrophil ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 00825 . 02110 . 7554/eLife . 00825 . 022Figure 7—figure supplement 1 . Erythroid leukemia associated with Mybl2 knockdown . Flow cytometric analysis for erythroid differentiation markers of bone marrow ( A ) and spleen ( B ) cells of a diseased animal .", "Histologic and cytological analysis of the bone marrow ( C and E ) and the spleen ( D and F ) shows dominance of erythroblasts ( arrows ) .", "More than 90% of the immature spleen CD71+ are GFP+; the spleen weight was 792 mg ( not shown ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 00825 . 02210 . 7554/eLife . 00825 . 023Figure 7—figure supplement 2 . Flow cytometric analysis of the LSK population in the bone marrow of mice upon reconstitution with Mybl2 shRNA-expressing cells . The analysis was performed in control animals ( left panel ) and in animals with Mybl2 downregulation ( middle and right panel ) .", "Vertical bars designate median values .", "Populations were labeled according to the following markers: CD150+/CD48- ( HSC ) , CD150-/CD48- ( MPP ) and CD150-/CD48+ ( LRP ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 00825 . 023 Histopathologic analysis of the spleen ( Figure 7B–E ) and the bone marrow ( Figure 7F–K ) at 6 to 7 months post transplantation showed marked changes in the overall tissue architecture and in specific cellular constituents .", "The bone marrow in experimental mice was hypercellular and contained more than 95% mature myeloid cells at the expense of erythroid cells ( <5% ) , while 2% of the cells had a dysplastic morphology .", "The spleens were frequently enlarged ( Figure 7A ) , the white pulp was virtually absent , and the whole organ showed evidence of extramedullary hematopoiesis .", "Notably , one mouse developed an erythroid leukemia characterized by a block of differentiation at the CD71-positive stage at 6 months post-transplantation ( Figure 7—figure supplement 1 ) .", "In summary , the histopathologic findings in these mice indicate a clonal myeloproliferative disorder with dysplastic features .", "To identify the cells principally involved in the clonal dominance associated with this disorder , we analyzed the Lin−/Sca1+/Kit+ ( LSK ) bone marrow cells of the affected mice ( n = 12 ) in comparison to controls .", "Within the LSK population of GFP+ or RFP+ Mybl2-knockdown bone marrow cells , HSCs ( CD150+/CD48− ) were diminished , MPPs ( CD150−/CD48− ) were maintained and LRPs ( CD150−/CD48+ ) were increased compared to unlabeled , non-transduced cells .", "Thus , the clonal dominance due to low Mybl2 levels is first evident as an increased percentage of cells in the LRP fraction .", "( Figure 7—figure supplement 2 ) .", "We next performed secondary transplantation experiments to determine if self-renewing neoplastic cells were involved .", "We first analyzed four primary donors in detail and confirmed the presence of clonally dominant GFP+/Mybl2-RNAi cells in the peripheral blood and bone marrow of these mice ( Figure 8—figure supplement 1A , C ) .", "Moreover , the mice were anemic based on low RBC and low hemoglobin levels ( Figure 8—figure supplement 1B ) and had markedly reduced erythropoiesis , as shown by low percentages of CD71neg/Ter119+ cells compared to that in a control transduced mouse ( Figure 8—figure supplement 1D ) .", "For the secondary transplants , we injected 15 mice with bone marrow cells and eight with spleen cells that originated from these four individual donors ( no pooling ) .", "Analysis of blood samples collected from secondary recipients at 10 weeks revealed a marked clonal advantage for GFP+/Mybl2-RNAi cells transplanted from each of the four mice with primary disease , regardless of whether the cell source was spleen or bone marrow ( Figure 8A ) .", "In addition , there was evidence of anemia ( low RBC and low Hb levels ) and thrombocytopenia in 9 of 13 animals ( Figure 8B ) .", "The bone marrow of secondary recipients was also dominated by GFP+/Mybl2-RNAi cells ( Figure 8C ) and showed decreased levels of erythropoiesis ( Figure 8D ) as well as histologic evidence of a myeloproliferative disorder with dysplasia .", "Overall , the phenotypic consequences of Mybl2 knockdown were much more pronounced in secondary compared to primary recipients . 10 . 7554/eLife . 00825 . 024Figure 8 . Secondary transplants: In vivo reconstitution assay testing capacity of cells with low Mybl2 expression to engraft in lethally irradiated mice .", "( A ) Analysis of peripheral blood mononuclear cells for GFP and RFP positivity at 10 weeks post-transplant showing engraftment of cells originating from spleen ( p<0 . 0001 , left panel ) or bone marrow cells ( p<0 . 0001 , right panel ) .", "Horizontal bars denote median values .", "( B ) Analysis of peripheral blood parameters showing anemia of animals that received secondary transplants of specific Mybl2-shRNA engineered cells: white blood cell count ( WBC ) , red blood cell count ( RBC ) , hemoglobin ( HB ) , hematocrit ( HCT ) , and platelet count ( PLT ) .", "Spleen weights are shown as well .", "Grey areas indicate the normal range for each value .", "( C ) Flow cytometric analysis of mononuclear bone marrow cells 3 months after transplantation from 13 mice ( S1–S13 ) demonstrating strong engraftment and expansion of cells with a specific Mybl2 knockdown ( color code as in Figure 5 ) .", "( D ) Analysis of bone marrow erythropoiesis by CD71/Ter119 staining showing the frequencies of CD71−/Ter119+ cells and revealing a strong reduction of Ter119+ cells in all experimental animals in comparison to a wild-type control animal . DOI: http://dx . doi . org/10 . 7554/eLife . 00825 . 02410 . 7554/eLife . 00825 . 025Figure 8—figure supplement 1 . Analysis of the peripheral blood and bone marrow 8 months after transplantation of lineage-negative bone marrow cells transduced with Mybl2-targeting shRNA expression vectors ( primary transplant donors ) .", "( A ) Analysis of peripheral blood mononuclear cells for GFP and RFP positivity showing engraftment and competitive expansion GFP-positive cells expressing shRNAs targeting Mybl2 in competition to non-transduced cells ( unlabeled , grey ) or control shRNA expressing cells ( RFP-positive , red ) .", "PD1-4: primary donor animals ( GFP: Mybl2-specific shRNA vectors , RFP: control shRNA vectors ) ; C3: control animal ( GFP: control shRNA vectors , RFP: control shRNA vectors ) ( B ) Analysis of peripheral blood parameters showing anemia of the animals transplanted with specific Mybl2-shRNA engineered cells: white blood cell count ( WBC ) , red blood cell count ( RBC ) , hemoglobin ( HB ) , hematocrit ( HCT ) , and platelet count ( PLT ) .", "Spleen weights are shown as well .", "Grey areas indicate the normal range for each value .", "( C ) Analysis of the bone marrow mononuclear cells as in A . ( D ) Analysis of bone marrow erythropoiesis by CD71/Ter119 staining showing the frequencies of Ter119-positive cells . DOI: http://dx . doi . org/10 . 7554/eLife . 00825 . 025 Thus , our secondary transplantation assays show that either spleen or bone marrow cells from mice with myeloid neoplasia due to shRNA-mediated reduced Mybl2 expression can efficiently transmit the blood disorder to secondary recipients .", "This indicates that the disorder is cell autonomous and that hematopoietic progenitor cells with reduced Mybl2 expression can self-renew after transplantation and undergo sustained expansion leading to clonal dominance ." ], [ "Recent molecular studies of MDS have uncovered a number of previously unrecognized genes whose aberrant inactivation through mutation or other means can disrupt hematopoietic cell development , leading to ineffective hematopoiesis and a myelodysplastic phenotype ( Shih et al . , 2012 ) .", "The picture beginning to emerge from these investigations is that a broad array of molecular changes have the potential to drive the biology and clinical phenotypes of MDS in unique ways .", "Yet , the disease remains very heterogeneous and none of the molecular lesions that have been convincingly linked to MDS pathogenesis affect more than a fourth of cases overall , with a prevalence of 1–15% being much more common ( Bejar et al . , 2011; Shih et al . , 2012 ) .", "In this study , we identified MYBL2 as a dosage-dependent tumor suppressor gene that was reduced in expression to sub-haploinsufficient levels in 17 ( 65% ) of 26 human MDS cases .", "This result is somewhat surprising given that MYBL2 has been implicated as a gene that is typically overexpressed ( rather than downregulated ) in several types of cancer and whose elevated expression correlates with a poor prognosis in human breast cancer and other malignancies ( Amatschek et al . , 2004; Paik et al . , 2004 ) .", "However , no direct evidence demonstrating oncogenic activity by overexpressed MYBL2 has been reported to date .", "High levels of expression of this transcription factor in other tumor types may reflect the greater proliferative rate of the tumor cell population compared to resting cells , so that the apparently levels of high MYBL2 activity may be a consequence rather than a cause of malignant transformation .", "By contrast , the proliferative fraction of cells in myeloid neoplasia is generally quite low .", "Our findings demonstrate the surprising finding that abnormally low levels of MYBL2 expression have the unique property of imparting a clonal advantage to multipotent hematopoietic progenitors , leading rapidly to the clonal dominance of such cells within the myeloid , and B- and T-lymphocyte cell lineages .", "The persistence of clonal dominance in recipient mice for more than 6 months after bone marrow cell transplantation , and in recipients of secondary transplants , indicates that the affected multipotent hematopoietic cells have both self-renewal and long-term repopulating ability ( Morrison and Weissman , 1994 ) .", "Hence , our finding establishes MYBL2 as a key tumor suppressor gene of the 20q CDR affected in human MDS and MPD .", "However , we cannot exclude the possibility that haploinsufficiency for other genes within the 20q CDR cooperates with aberrantly low expression of MYBL2 to move the cells toward malignant transformation , as has been shown for the CDR associated with the 5q-syndrome , where haploinsufficiency of the gene RPS14 as well as loss of expression of an miRNA ( Ebert et al . , 2008; Barlow et al . , 2010; Starczynowski et al . , 2010 ) are critical steps in MDS pathogenesis .", "Most intriguing is our finding of decreased MYBL2 expression in MDS cases with a normal karyotype , which suggests that interstitial deletion of chromosome 20q is not the only mechanism by which MYBL2 activity can be reduced in MDS .", "Indeed , low levels of MYBL2 were apparent in 10 ( 56% ) of 18 of our normal-karyotype MDS cases , and 38% of those reported by Pellagatti et al . ( Pellagatti et al . , 2010 ) .", "We were unable to implicate aberrant methylation of the MYBL2 genomic locus as an additional mechanism of gene-dosage reduction; instead , we found elevated expression levels of mir-29a and mir-30e , which are known to target MYBL2 ( Martinez et al . , 2011 ) , in CD34+ cells from patients with MDS and low levels of MYBL2 expression .", "In addition , others have shown that overexpression of mir-29a in murine bone marrow cells leads to acute myeloid leukemia ( Han et al . , 2010 ) .", "Combined with our results , this observation suggests that mir-29a may be oncogenic , at least in part , because it targets Mybl2 transcripts .", "Finally , we identified a case with heterozygous missense mutation in MYBL2 that led to the loss of gene function , analogous to the effect of monoallelic gene deletion .", "Thus , our results indicate that the MYBL2 transcription factor gene is targeted by diverse molecular events in MDS , including not only deletion within the 20q chromosomal region , but also heterozygous loss-of-function mutations and the repression of gene expression by dysregulated miRNAs .", "Clarke et al . ( Clarke et al . , 2012 ) have also described an association between MYBL2 expression levels and the occurrence of hematologic disorders , including MDS , in aged Mybl2 haploinsufficient mice .", "These authors were able to show that haploinsufficiency for the Mybl2 gene increases susceptibility to age-related myeloid neoplasia .", "Our data indicate that more pronounced gene dosage reduction of Mybl2 might be even more advantageous for clonal dominance .", "Although our findings do not rule out a causal role for straight-forward haploinsufficiency in myeloid malignancies , they strongly suggest that the oncogenic effects of reduced MYBL2 dosage are more pronounced as the dosage level falls to near 30% of normal , in keeping with the continuum model of tumor suppression ( Berger et al . , 2011 ) .", "We would also stress that in the range of MYBL2 dosage reductions in this study were modeled based on those found in CD34+ cells from MDS clinical samples , in which the reductions of MYBL2 gene dosage were predominately in the range of 20–30% of normal levels , and thus were considerably lower than the 50% predicted by classical haploinsufficiency .", "Importantly , expression levels lower than 20% appear incompatible with cell growth ( Figure 4B ) , consistent with the known function of MYBL2 as a key regulator of G2-to-M transition and the fact that biallelic inactivation of this gene causes early embryonic lethality in the mouse due to a block in inner cell mass formation during embryonic development ( Tanaka et al . , 1999 ) .", "Our results implicating a transforming role for sub-haploinsufficient levels of MYBL2 build upon earlier studies implicating progressive inactivation of CTNNA1 and the graded reduction of PU . 1 expression in the molecular pathogenesis of myeloid malignancies ( Rosenbauer et al . , 2004; Liu et al . , 2007 ) .", "Our approach , which tested the effects of graded levels of Mybl2 knockdown of hematopoietic progenitors in vivo , provides compelling evidence for the importance of reductions of tumor suppressor gene levels below the 50% level associated with classical haploinsufficiency in the molecular pathogenesis of myeloid malignancies ." ], [ "Bone marrow samples represented 28 MDS patients ( Supplementary file 1A ) seen at the MD Anderson Cancer Center ( MDACC ) .", "The diagnosis and classification of MDS followed criteria of the World Health Organization ( WHO ) , the French-American–British Cooperative Group ( FAB ) and the International Prognostic Scoring System ( IPSS ) .", "Mononuclear cells were isolated from bone marrow aspirates by Ficoll centrifugation , and further purified by magnetic beads ( Miltenyi Biotec , Auburn , CA ) to obtain the CD34+ fraction .", "Normal CD34+ cells were processed in exactly the same manner .", "Mononuclear cells were isolated from the femurs and tibias of syngeneic mice ( C57BL/6 ) , and were depleted of lineage-marked cells using sheep anti-rat IgG-coupled Dynabeads ( Life Technologies , Carlsbad , CA ) together with affinity-purified rat anti-mouse antibodies ( eBioscience , San Diego , CA ) against CD3 , CD5 , B220 , Gr1 , Mac1 , Ter119 .", "Cells were cultured for 24 hr in StemSpan SFEM ( Stem Cell Technologies , Vancouver , BC , Canada ) supplemented with 100 U/ml penicillin/streptomycin , 2 mM glutamine , 80 ng/ml Scf , 40 ng/ml TPO and 40 ng/ml Flt3l .", "Cells were aliquoted and transduced in separate wells with single shRNA- or control shRNA-expressing vectors , extensively washed and cultured separately for an additional 24 hr before transplantation .", "Single vector transduced cells were pooled according to the scheme in Figure 5 just before transplantation .", "Recipient C57BL/6 mice were lethally irradiated ( 9 . 5 Gy ) at a dose rate of approximately 1 Gy/min , delivered as a single dose .", "The next day , mice were reconstituted by retro-orbital venous sinus injection of 2 × 105 lentivirus-transduced cells mixed with a 1 × 105 freshly isolated whole bone marrow cells for radioprotection .", "RNA was extracted from Trizol ( Life Technologies , Carlsbad , CA ) according to the manufacturer’s instructions , and 500 ng were used for reverse transcription ( Quantitect kit , Qiagen , Hilden , Germany ) .", "Real-time PCRs were performed on an ABI PRISM 7700 system using SYBR green-based assays with AmpliTaq Gold ( Life Technologies ) .", "All reactions were performed in triplicate .", "Quantitative data were calculated from the Ct-values for each reaction using the mean reaction efficiency for each primer pair .", "Data were normalized to expression levels of PAPOLA , UBQLN1 and VPS39 ( housekeeping genes ) and scaled to the mean of the controls .", "Sequences of primer sets are available in Supplementary file 1C .", "Total RNA was isolated from cells lysed in Trizol ( Life Technologies ) .", "100–500 ng of each sample was converted into fragmented , biotinylated cDNA hybridized to a microarray chip ( Gene 1 . 0 ST ) and fluorescently labeled according to the standard protocol ( Affymetrix , Santa Clara , CA ) at the Dana-Farber microarray core facility .", "Raw data were processed in Expression Console ( Affymetrix ) using RMA normalization .", "Expression values for each gene were annotated by mapping all probe sets to the human genome version hg19 .", "Data complexity was reduced to one canonical transcript per gene , resulting in a single identifier per gene ( 17 , 982 total ) .", "The expression data were processed in Gene Pattern ( Reich et al . , 2006 ) ( Broad Institute , Cambridge , MA ) .", "Non-expressed genes were filtered out , and the resulting expression matrix was analyzed with the comparative marker module ( two class comparison with 10 , 000 permutations ) or the gene neighbor module in Gene Pattern .", "GSEA ( Broad Institute ) was performed as described previously ( Subramanian et al . , 2005 ) .", "Data are available at Gene Expression Omnibus , accession number GSE43401 .", "A k-nearest neighbor ( KNN ) algorithm was used to classify normal-karyotype MDS cases by using Euclidean distance and k = 3 to predict the class label by a majority vote .", "To generate a training set , the 89 genes most correlated with MYBL2 ( Pearson correlation coefficient >0 . 80; 81 positively correlated and 8 negatively correlated ) based on the short hairpin knock-down experimental data were reduced to a 59 gene signature positively correlated with MYBL2 by removing 14 genes which hierarchically clustered with the 8 negatively correlated genes and 9 genes having low expression ( ≥3 out of 4 controls ) in the CD34+ control samples in the 30 patient samples arrays .", "This signature was also examined in an independent cohort of 94 normal karyotype patients using KNN .", "Protein extracts were prepared by SDS lysis and normalized by total protein content ( BCA assay ) .", "Total protein ( 5–25 µg ) was separated by SDS polyacrylamide gel electrophoresis and blotted on PVDF membranes .", "Specific proteins were visualized by chemiluminescence using antibodies for MYBL2 ( ab12296 , Abcam , Cambridge , United Kingdom ) or Tubulin ( B-5-1-2 , Sigma , St . Louis , MO ) .", "Cryopreserved human bone marrow CD34+ cells were obtained from Lonza and cultured in StemSpan SFEM ( Stem Cell Technologies ) supplemented with 100 U/ml penicillin/streptomycin , 2 mM glutamine , 50 µg/ml lipoproteins ( EMD Millipore , Billerica , MA ) , 80 ng/ml SCF , 40 ng/ml TPO , 40 ng/ml FLT3L , 10 ng/ml IL3 and 10 ng/ml IL6 .", "SKM1 cells were obtained from the DSMZ ( Braunschweig , Germany ) and maintained in RPMI medium supplemented with 10% fetal bovine serum .", "Oligonucleotides encoding shRNAs were cloned into pLKO1 .", "pLKO1 derivatives were obtained by replacing the puromycin resistance marker with EGFP or DsRedExpress2 ( Strack et al . , 2008 ) .", "Lentiviral RNAi vectors are available on request .", "The MYBL2 cDNA including a 3xFLAG tag was cloned into derivate of the pLenti6 . 3 ( Life Technologies ) vector under control of the SFFV promoter .", "Lentiviral particles were produced in 293T cells by cotransfection of pMD2G , pCMV-dR8 . 9 and the lentiviral vector .", "Cells were spin-transduced in the presence of polybrene ( 4 µg/ml ) and selected at 24 hr post-transduction with puromycin ( 1 . 5 µg/ml ) or blasticidin ( 3 µg/ml ) .", "Growth curves were obtained with the CellTiter-Glo luminescent assay ( Promega , Madison , WI ) .", "A FACSCanto II machine ( BD Biosciences , San Jose , CA ) equipped with a 407 nm , 488 nm and 633 nm laser was used .", "Staining was performed with the following antibodies ( clone id in parentheses ) , all purchased from eBioscience: Mac1 ( M1/70 ) , Gr1 ( RB6-8C5 ) , B220 ( RA3-6B2 ) , CD3 ( 145-2C11 ) , CD71 ( R17217 ) and Ter119 ( Ter119 ) .", "C57BL/6 mice , purchased from Jackson Laboratories , were housed in individually ventilated cages in the Dana-Farber Cancer Institute animal facility in accord with guidelines of the Institutional Animal Care Use Committee .", "Transplanted mice received Baytril-containing water ( Bayer , Robinson Township , PA ) to reduce the probability of infection from opportunistic pathogens in first 30 days after transplantation .", "For peripheral blood analysis , tailvein blood was collected and RBCs were lysed with ammonium chloride buffer prior to staining for flow cytometry .", "For bone marrow analysis , cells were isolated from femurs and tibias with RBCs lysis buffer used for all subsequent procedures except analysis of erythropoiesis by Ter119/CD71 flow cytometry .", "Cytospin slides were prepared from isolated mononuclear bone marrow cells and stained with Wright-Giemsa stain .", "Bone and spleen samples were fixed in 10% formalin , washed with PBS and stored to 80% ethanol .", "Sections were stained with hematoxylin and eosin .", "All data were analyzed with GraphPad Prism , version 5 . 04 for Windows ( http://www . graphpad . com ) .", "p-values ( two-tailed ) for competitive reconstitution were calculated by a paired t-test ." ] ]

[ "A common deleted region ( CDR ) in both myelodysplastic syndromes ( MDS ) and myeloproliferative neoplasms ( MPN ) affects the long arm of chromosome 20 and has been predicted to harbor a tumor suppressor gene .", "Here we show that MYBL2 , a gene within the 20q CDR , is expressed at sharply reduced levels in CD34+ cells from most MDS cases ( 65%; n = 26 ) , whether or not they harbor 20q abnormalities .", "In a murine competitive reconstitution model , Mybl2 knockdown by RNAi to 20–30% of normal levels in multipotent hematopoietic progenitors resulted in clonal dominance of these ‘sub-haploinsufficient’ cells , which was reflected in all blood cell lineages .", "By 6 months post-transplantation , the reconstituted mice had developed a clonal myeloproliferative/myelodysplastic disorder originating from the cells with aberrantly reduced Mybl2 expression .", "We conclude that downregulation of MYBL2 activity below levels predicted by classical haploinsufficiency underlies the clonal expansion of hematopoietic progenitors in a large fraction of human myeloid malignancies ." ]

[ "Blood cells are produced within bone marrow by specialized stem cells and progenitor cells .", "Abnormalities in this process lead to a group of diseases known as myeloid malignancies , which include acute myeloid leukaemia—in which the bone marrow produces abnormal white blood cells—and myelodysplastic syndromes , which are caused by too few mature blood cells being produced .", "Many individuals affected by these disorders possess a shortened form of chromosome 20 that lacks a number of genes .", "This deletion is only ever seen in one of their two copies of the chromosome—suggesting that at least some of these genes are essential for survival—but the identity of the gene ( s ) that are associated with the increased risk of myeloid malignancies is unknown .", "Now , Heinrichs et al . have uncovered a key tumor suppressor among those genes frequently lost on chromosome 20 .", "The gene , which is called MYBL2 , encodes a transcription factor that helps to control the cell division cycle .", "Myeloid malignancy patients lacking one copy of this gene showed levels of MYBL2 expression that were less than 50% of those in healthy individuals .", "This suggests that additional mechanisms must be acting to reduce expression of their remaining copy of the gene .", "Surprisingly , MYBL2 levels were also reduced in myeloid malignancy patients who possessed two intact copies of chromosome 20 , indicating that loss of a single copy represents only one mechanism to reduce MYBL2 expression , i . e . , the ‘tip-of-the-iceberg’ .", "Hence , this finding reveals a more general role for MYBL2 as it indicates that more patients are likely to be affected by altered expression of this gene .", "To confirm their findings from studies in patients , Heinrichs et al . used gene silencing techniques to reduce the expression of MYBL2 in mice and showed that this induced symptoms of myeloid malignancies in the animals .", "Moreover , injection of modified cells from these animals into healthy mice also induced symptoms in the recipients .", "The modified cells are able to expand more robustly than normal cells , and this dominance induced by downregulation of the tumor suppressor increases the risk of malignancy .", "In addition to revealing a new tumor suppressor gene and its contribution to myeloid malignancies , the study by Heinrichs et al . highlights the importance of gene dosage in mediating the effects of tumor suppressors ." ]